Citation
U.S. Army Medical Department journal

Material Information

Title:
U.S. Army Medical Department journal
Alternate title:
United States Army Medical Department journal
Alternate Title:
AMEDD journal
Running title:
Army Medical Department journal
Abbreviated Title:
U.S. Army Med. Dep. j.
Creator:
United States -- Army Medical Department (1968- )
Place of Publication:
Fort Sam Houston, TX
Publisher:
U.S. Army Medical Department
Publication Date:
Frequency:
Quarterly[<Oct.-Dec. 2001->]
Bimonthly[ FORMER Sept.-Oct. 1994-]
quarterly
regular
Language:
English
Physical Description:
volumes : illustrations ; 28 cm

Subjects

Subjects / Keywords:
Medicine, Military -- Periodicals -- United States ( lcsh )
Military Medicine ( mesh )
Medicine ( mesh )
Medicine, Military ( fast )
United States ( mesh )
United States ( fast )
United States
Genre:
Electronic journals.
Periodicals.
Periodicals. ( fast )
Fulltext.
Government Publications, Federal.
Internet Resources.
serial ( sobekcm )
federal government publication ( marcgt )
periodical ( marcgt )
Electronic journals ( lcsh )
Periodicals ( mesh )
Periodicals ( fast )
Fulltext
Government Publications, Federal
Periodicals
Internet Resources

Notes

Dates or Sequential Designation:
Sept.-Oct. 1994-
General Note:
Title from cover.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
This item is a work of the U.S. federal government and not subject to copyright pursuant to 17 U.S.C. §105.
Resource Identifier:
32785416 ( OCLC )
98642403 ( LCCN )
1524-0436 ( ISSN )
ocm32785416
Classification:
RC970 .U53 ( lcc )
616.9/8023/05 ( ddc )
W1 JO96 ( nlm )

Related Items

Preceded by:
Journal of the US Army Medical Department.

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Digital Military Collection

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July–September 2015Perspective 1MG Steve JonesBaseline Susceptibility to Pyrethroid and Organophosphate Insecticides 3 in Two Old World Sand Fly Species (Diptera: Psychodidae)Andrew Y. Li, PhD; Adalberto A Perez de Leon, DVM, PhD; Kenneth J. Linthicum, PhD; Seth C. Britch, PhD; MAJ Joshua D. Bast; Mustapha Debboun, PhDEf cacy of Permethrin Treated Bed Nets Against Leishmania major 10 Infected Sand FliesTobin Rowland; MAJ Silas A. Davidson; Kevin Kobylinski, PhD; Claudio Menses; Edgar Rowton, PhDControlled Human Malaria Infection at the Walter Reed Army Institute of Research: 16 The Past, Present, and Future From an Entomological PerspectiveLindsey S. Garver, PhD; Megan Dowler; MAJ Silas A. DavidsonMosquito Fauna of Lao People’s Democratic Republic, with Special Emphasis 25 on the Adult and Larval Surveillance at Nakai District, Khammuane ProvinceLeopoldo M. Rueda, PhD; Khamsing Vongphayloth, MD; James E. Pecor; LCDR Ian W. Sutherland, USN; Jeffrey Hii, PhD; Mustapha Debboun, PhD; Paul T. Brey, PhDRecords and Distribution of New World Phlebotomine Sand Flies 33 (Psychodidae, Diptera), With Special Emphasis on Primary Types and Species DiversityLeopoldo M. Rueda, PhD; Desmond H. Foley, PhD; David Pecor; Matthew WolkoffDevelopment of Air Force Aerial Spray Night Operations: 47 High Altitude Swath CharacterizationLt Col Karl A. Haagsma, USAFR; Lt Col Mark S. Breidenbaugh, USAFR; Kenneth J. Linthicum, PhD; Robert L. Aldridge; Seth C. Britch, PhDToxicity Testing in the 21st Century: Re ning the Army’s Toolbox 60LTC Erica Eggers Carroll; Mark S. Johnson, PhD, DABTThe New US Military Role in the European Union’s Import Program: 64 Strategic Implications Ensuring Safe Food for the European TheaterMAJ Michael McCown; Jacob L. Hall, II; Megan McCormick, DVM, MPH; Lt Col Henry H. Triplett, III, USAFEvaluation of Postdeployment Cancers Among Active Duty Military Personnel 68Jessica M. Sharkey, MPH; Joseph H. Abraham, ScDA Preliminary Analysis of Noise Exposure and Medical Outcomes 76 for Department of Defense Military MusiciansCindy Smith; Sharon Beamer, AuD; Shane Hall, MS; Thomas Helfer, PhD; Timothy A. Kluchinsky, Jr, PhDThe Bene ts of Deploying Health Physics Specialists to Joint Operation Areas 83LTC Scott Mower; MAJ Joshua D. Bast; MAJ Margaret MyersForensic and Ethical Issues in Military Behavioral Health A New Volume in the Borden Institute Textbook of Military Medicine Series 89 Reviewed by LTC Daniel E. Banks and COL (Ret) Edward Lindeke J J O U R N A L OURNAL THE UNITED STATES ARMY MEDICAL DEPARTMENT FORCE HEALTH PROTECTION: EVOLVING CHALLENGES AND SOLUTIONS

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J OURNAL A Prof essional Publication of the AMEDD Community THE UNITED STATES ARMY MEDICAL DEPARTMENT Online issues of the AMEDD Journal are available at http://www.cs.amedd.army.mil/amedd_journal.aspx July September 2015 US Army Medical Department Center & School PB 8-15-7/8/9 The Army Medical Department Journal [ISSN 1524 0436 ] is published quarterly 3630 Stanley RD STE B 0204 78234 6100 The Army Medical Department Journal are listed and Journal s CORRESPONDENCE: (210) 221-6301, DSN 471-6301 DISCLAIMER: The AMEDD Journal in the AMEDD Journal AMEDD Journal CONTENT: AMEDD Journal OFFICIAL DISTRIBUTION: By Order of the Secretary of the Army: GERALD B. OKEEFE Secretary of the Army Raymond T. Odierno 1503606 LTG Patricia D Horoho MG Steve Jones

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July – September 2015 1When the Army Medical Department was organized on July 27, 1775, it was formed as a team. The act of Congress establishing a hospital for an Army of twenty thousand appointed the following of cers and attendants: one director general and chief physician, four surgeons, one apothecary, twenty surgeon’s mates, one clerk, two storekeepers and one nurse for every ten sick. Army Medicine had been serving our Soldiers for almost a year when our nation declared independence—we are the country’s rst, and arguably best healthcare team. Our accomplishments over the past 240 years have been signi cant and have been the result of a team effort. Major Walter Reed discovered the mosquito was the vector that carried Yellow Fever, but it was Colonel William Gorgas who led the campaign to control mosquitoes that allowed construction of the Panama Canal. Today, casualty survival rates are at historic levels because of the teamwork between AMEDD staff, operational commanders, Soldiers, and Families. AMEDD doctrine has traditionally described ten medical battle eld operating systems; the major healthcare functions that are employed to provide care in theater. This was a useful concept for planners and helped ensure critical functions were not forgotten. It was aligned with Army doctrine which described battle eld operating systems as the elements of combat power, but created an arti cial distinction between care in theater and care in garrison. It does not portray the fact that casualty care occurs every day in our xed hospitals, and that every member of the AMEDD makes important contributions to the effort. It also does not adequately re ect recent changes in how we provide healthcare while deployed and at home. The February 27, 2008, edition of Field Manual 3-0: Operations was a fundamental departure from prior versions. Just as the 1976 edition of Field Manual 1005: Operations brought the Army from the rice paddies of Vietnam to the battle elds of Western Europe, the 2008 edition took the Army into 21st century con icts among people. It replaced the older battle eld operating systems with six war ghting functions as the elements of combat power: Mission Command Fires Movement and Maneuver Sustainment Intelligence Protection A seventh war ghting function, Engagement, has since been added. War ghting functions are de ned as a group of tasks and systems (people, organizations, information, and processes) united by a common purpose that commanders use to accomplish missions. It is a useful concept that reinforces the ideas of mutual support and ghting as combined arms teams. Field Manual 4-02: Army Health System lists the ten medical functions: Medical Mission Command Medical Treatment Hospitalization Medical Evacuation Dental Services Preventive Medicine Services Combat and Operational Stress Control Veterinary Services Medical Logistics Medical Laboratory Services It de nes two missions for the AMEDD: Health Service Support and Force Health Protection. The Health Service Support Mission comprising casualty care, medical evacuation, and medical logistics falls under the Sustainment War ghting Function. Force Health Protection comprising preventive medicine, veterinary services, preventive aspects of combat and operational stress control, dental and services, and laboratory services falls under the Protection War ghting Function. The full breadth of AMEDD support to Uni ed Land Operations and the continuum of casualty care are far greater than what is described in AMEDD doctrine. Efforts to keep Soldiers healthy and t allow them to perform better while deployed and recover more quickly if injured. Medical treatment facilities at home station TIME FOR A REVOLUTION IN AMEDD DOCTRINEPerspectiveCOMMANDER’S INTRODUCTIONMG Steve Jones

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2 http://www.cs.amedd.army.mil/amedd_journal.aspxprovide specialty care through reach-back support and telemedicine. Casualty care does not stop after evacuation to the continental United States, but continues with de nitive care and rehabilitation of the wounded, the operation of Warrior Transition Units, and the support provided to families. Our approach to healthcare has changed signi cantly since Vietnam. Primary healthcare is now provided in Soldier-Centered Medical Homes who partner with line unit staff and commanders. The Brigade Healthcare Team now includes a physical therapist and behavioral health provider and nurse in addition to medics, physician assistants, dentists, and surgeons. The Combat Lifesaver and 68W (Health Care Specialist) programs, tactical combat casualty care, damage control resuscitation, and damage control surgery have signi cantly improved prehospital care. Critical care ight paramedics and nurses on tactical medevac aircraft have increased survival rates of critically injured patients. These patients no longer spend weeks in theater hospitals but are evacuated to large medical centers within days, with care continuing in hospitals along the way. Patient outcomes are monitored closely and data collected by the Joint Theater Trauma System forms the basis of a robust performance improvement system. Regular after action conferences with returning units and research teams also provide information that is used to improve care. This approach to care and the hard work of the entire AMEDD team has raised the survival rate of casualties wounded on the battle eld from 76% in Vietnam to 92% today. Updating our doctrine will allow the AMEDD to better communicate the many ways it supports Uni ed Land Operations every day. It will lead to a better understanding of new concepts in casualty care, the contributions of all members of the team, and of how units and leaders combine capabilities across medical functions to accomplish their mission. It should start with the premise that casualty care begins with the maintenance of a healthy and t force, extends from the point of injury through de nitive care and rehabilitation, and includes the operation of Warrior Transition Units. After action reviews from our xed hospitals should be regularly conducted and included in lessons learned programs. Gaps in the provision of de nitive care and rehabilitation should be identi ed and included in the requirements process to drive development of new capabilities. Medical functions should be updated to re ect how we provide care rather than listing capabilities, and should include: Medical Mission Command Primary Care Prehospital Care Evacuation and En Route Care Hospital Care De nite Care and Rehabilitation Force Health Protection Medical Sustainment Medical Engagement Medical Information Research and Innovation Several of these functions are new. The addition of Medical Engagement as a function recognizes the signi cant support the AMEDD provides Combatant Commanders in shaping the environment. Including Research and Innovation recognizes the important contributions of these members of the team. Medical Information includes not only health threats but capabilities, treatment considerations, military, political, economic, and cultural considerations that affect healthcare operations. It includes the information needed to develop and sustain a high degree of situational understanding while operating in complex environments against determined, adaptive enemy organizations and emerging health threats. Like con ict, healthcare is a complex, risk lled human endeavor. Army healthcare is more complex, has greater risk, and is lled with even more uncertainty and emotion. We often deliver care under austere and extreme conditions, sometimes under re, and always under the scrutiny of Congress and the media. We understand we are part of a bigger team with a bigger mission because we serve our nation. Our mission is to keep Soldiers healthy and ready to ght, giving them con dence with our presence on the battle eld and comfort knowing we are caring for their families back home. We should re ect this reality in our doctrine. PERSPECTIVE

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July – September 2015 3Phlebotomine sand ies are vectors of Leishmania species protozoan parasites that cause various forms of leishmaniasis and the sand y fever viruses ( Phlebovirus ; Bunyaviridae) in humans and other animals in tropical and subtropical regions of the world.1 Leishmania parasites are transmitted by the bite of infected female sand ies.2 An estimated 1.3 million new human cases of leishmaniasis and 20,000 to 30,000 deaths occur worldwide annually, which represents a major public health problem in affected regions including the Mediterranean basin, Central Asia, Southwest Asia, Southeast Asia, East Africa, Afro-Eurasia, and the Americas.3 This sand y-borne disease with no approved vaccines and lengthy and costly treatment causes signi cant social and economic burden to civilian populations in endemic regions. Phlebotomine sand ies also pose a signi cant threat to US military personnel who are deployed to those regions, particularly the Middle East, Southwest Asia, and Africa where sand y Leishmania vectors are endemic.4 During Operation Iraqi Freedom, the US military reported over 1,000 con rmed cases of cutaneous leishmaniasis among deployed personnel between May 2003 and November 2004.5 Various insecticides and application technologies were tested at a US airbase in southern Iraq during that period with only limited success in reducing sand y populations.6 The reasons for failure in completely eliminating or substantially reducing sand y populations by frequent insecticide applications remain poorly understood.6Chemical insecticides have been used for indoor and outdoor residual applications and bed net treatments to reduce sand y populations in endemic regions.1,7-10 The insecticide dichlorodiphenyltrichloroethane (DDT) has been used to control sand ies after World War II.11,12 DDT resistance was reported in Phlebotomus papatasi (Scopoli) populations in Iran in a eld survey conducted during 1985-1988, although the use of DDT had been discontinued since 1969.13 DDT resistance was similarly reported in both P papatasi and P argentipes (Annandale and Brunetti) in India, Nepal, and Sri Lanka.10,14-17 Populations of P papatasi were shown to be susceptible to pyrethroid and organophosphate insecticides in laboratory studies of eld-collected sand y samples from North Africa and the Middle East.18,19 However, multiple resistances to DDT, organophosphate, and pyrethroid insecticides were detected in India and more recently in Sudan.14,20 One P papatasi population collected from the Surogia village in Sudan demonstrated signi cant resistance to malathion and propoxur in laboratory bioassays, and the observation was also supported by reduced inhibition of acetylcholinesterase by these organophosphate Baseline Susceptibility to Pyrethroid and Organophosphate Insecticides in Two Old World Sand Fly Species (Diptera: Psychodidae) Andrew Y. Li, PhD Seth C. Britch, PhD Adalberto A. Prez de Len, DVM, PhD, MS MAJ Joshua D. Bast, MS, USA Kenneth J. Linthicum, PhD Mustapha Debboun, PhDABSTRACTPhlebotomine sand ies are blood-feeding insects that transmit Leishmania parasites that cause various forms of cutaneous and visceral leishmaniasis and sand y fever viruses ( Phlebovirus ; Bunyaviridae) in humans. Sand ies pose a signi cant threat to US military personnel deployed to Leishmania -endemic and sand y fever endemic regions which include Europe, the Mediterranean basin, Middle East, Central Asia, Southwest Asia, and Africa. A research project supported by the Department of Defense Deployed War ghter Protection Program was initiated to evaluate the susceptibility of 2 Old World sand y species, Phlebotomus papatasi and P duboscqi to a number of commonly used pyrethroid and organophosphate insecticides. A new glass vial bioassay technique based on the CDC bottle assay was developed for this study. The exposure time-mortality relationship at a given insecticide concentration was determined for each insecticide, and their relative toxicity against the 2 sand y species was ranked based on bioassay results. This study validated the new bioassay technique and also generated baseline insecticide susceptibility data to inform future insecticide resistance monitoring work.

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4 http://www.cs.amedd.army.mil/amedd_journal.aspxinsecticides.20 Compared to mosquitoes and other vector species, there is a general lack of understanding of prevalence of insecticide resistance in sand ies.1,21 Only a few studies have been reported in the literature on possible biochemical or molecular mechanisms of insecticide resistance in sand ies.21-23Previous sand y insecticide susceptibility/resistance tests were carried out following a World Health Organization (WHO) standard procedure that was originally designed for mosquitoes in which insects were exposed to an insecticide-impregnated lter paper 24,25, or the Centers for Disease Control and Prevention (CDC) bottle assay 3,26 that exposes adult sand ies to an insecticide-treated surface inside the glass bottle. The WHO assay requires sand ies to be exposed to insecticide for one hour and subsequent transfer of sand ies to clean containers for incubation under appropriate temperature and humidity conditions for 24 hours before mortality can be assessed. The CDC bottle assay is more rapid and the time-mortality relationship of an insecticide concentration can be established by checking mortality every 15 minutes during a 2-hour exposure period.3,21 In most sand y studies, one or two diagnostic concentrations were tested for resistance detection.18,19,27,28 Dif culties in collecting suf cient numbers of live sand ies for bioassays and the lack of a standard sand y bioassay technique may have impeded progress in insecticide resistance studies in sand ies. In this study we developed a simpli ed glass vial bioassay technique based on the CDC bottle assay to test exposure time–mortality relationships at selected insecticide concentrations. This test used fewer sand ies and allowed rapid determination of sand y susceptibility. The objective of this study was to evaluate this new bioassay technique and determine baseline susceptibility to a number of commonly used pyrethroid and organophosphate insecticides in 2 sand y species, P papatasi and P duboscqi (Neveu-Lemaire), which are relevant to US military operations in the Middle East and Africa. MATERIALS AND METHODSSand Flies. Adult male P papatasi and P duboscqi were used in this study. Adult P papatasi were from a laboratory colony maintained at the US Department of Agriculture Agricultural Research Service Knipling-Bushland US Livestock Insects Research Laboratory, Kerrville, TX. The colony was established using pupae from a long-established Israeli strain of P papatasi maintained at the Division of Entomology, Walter Reed Army Institute of Research, Silver Spring, MD. Adult females were blood-fed using an in vitro membrane feeding system (Figure 1A). Larvae were fed with a sand y larval diet consisting of a composted mixture of rabbit feces and rabbit food.29 Male and female sand ies in the cage were fed daily with 30% sucrose water after emergence and maintained at 26 2 C and a relative humidity of 85%2% in an environmental chamber. Adult P duboscqi were from a laboratory colony maintained at the US Army Medical Research Unit-Kenya (USAMRU-K) eld station located at the Kenya Agricultural Research Institute Marigat Field Station.Insecticides. Technical-grade permethrin (92.2% active ingredient) and coumaphos (97.4% active ingredient) were obtained from FMC (Philadelphia, PA) and Bay Vet (Shawnee, KS), respectively. All other technical-grade insecticides were obtained from Chem Service (West Chester, PA). Formulated etofenprox (Zenivex E4 RTU, 4%) was a product of Wellmark International (Schaumburg, IL). Dilutions of technical insecticide in acetone were made to generate test concentrations ranging from 0.0001% to 0.1% (Figure 1B). To treat the inside surface of glass vials with insecticide, a volume of 0.2 mL of the test solution was added to a 20 mL glass scintillation vial (Fisher Scienti c, Pittsburgh, PA), which was placed on a hotdog roller (Figure 1C) for 1 hour to allow evaporation of solvent and uniform insecticide coating of the glass surface. The treated vials were recapped and stored at room temperature after drying and used within 2 days for study of P papatasi or 4 to 7 days for study of P duboscqi to allow for travel to the Kenya eld location. Dilution of formulated etofenprox was made using bottled water at the eld station in Kenya. Etofenprox-treated vials were set at ambient room temperature ( 25 C to 30 C) with frequent rolling by hand for 3 hours to allow solvent to evaporate. Three vials were prepared for each test concentration.Bioassays. All bioassays with P papatasi were done using adult males of mixed ages (3 to 10 days) for reasons noted below under normal laboratory conditions (Temperature 23 2 C). Five to 7 treated vials, each representing one concentration, and the control vial that was treated with acetone only were placed on the counter (Figure 1D, 1E). Adult male sand ies were directly aspirated from the holding cage using a mouth aspirator and brie y knocked down with CO2 before being placed in the vials (10 sand ies/vial). Flies woke up in about 30 seconds. Fly mortality in each vial was checked every 10 minutes, according to the order in which ies were added to each vial, for up to 3 hours, or until all ies in each vial were dead (Figure 1F). Each experiment was repeated 3 times so each test concentration had a total of 3 replicates. Bioassays with P duboscqi were conducted following the same protocol using adult males of mixed ages ( 3 to 10 days) kept at room temperature in the BASELINE SUSCEPTIBILITY TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES IN TWO OLD WORLD SAND FLY SPECIES (DIPTERA: PSYCHODIDAE)

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July – September 2015 5THE ARMY MEDICAL DEPARTMENT JOURNAL USAMRU-K eld station in Kenya (Figure 2). Room temperature in the laboratory ranged between 25 C and 30 C. All glass vials were pretreated with insecticides at the USDA laboratory in Texas, except for etofenprox which was prepared on site.Data Analysis. Probit analysis of time-mortality data for each insecticide and concentration tested were conducted using POLO PLUS software.30 The LT50 and LT90 (exposure time at which 50% and 90% ies died) were generated and used to compare the relative toxicity of insecticides tested in this study. RESULTS AND DISCUSSIONResults with P papatasi. Adult males began to die after 30 to 40 minutes of exposure to the lowest concentration (0.00001% or 0.5 ng/cm2) of pyrethroid insecticides (Figure 3), and it took over 120 minutes to reach 100% mortality. Both the time at which sand ies started to die and the time when 100% mortality was reached decreased with increasing insecticide concentrations (0.0001, 0.001, and 0.01%). The time (minutes) it took to kill 50% of the treated ies (LT50) for each of 3 concentrations of 6 pyrethroid insecticides tested are listed in Table 1. Based on LT50 data, prallethrin and -cyhalothrin were most toxic to sand ies, followed in order by deltamethrin, cy uthrin, cypermethrin, and permethrin. Compared to pyrethroid insecticides, organophosphate insecticides were generally less toxic (Figure 4). At the lowest concentration (0.00001%) tested, organophosphate insecticides were slow in killing sand ies. Sand ies started to die only after over 60-minute exposure (diazinon) or even after 120 minutes (chlorpyrifos). Similarly, higher concentrations of organophosphate insecticide caused ies to die more rapidly. Based on LT50 data listed in Table 2, the order of relative toxicity of the 4 organophosphate insecticides was diazinon chlorpyrifos > coumaphos > dichlorvos.Results with P duboscqi. Toxicity bioassay results for 6 pyrethroid and 3 organophosphate insecticides against adult males are listed in Table 2. Prallethrin was again the most toxic pyrethroid insecticide. Susceptibility of P duboscqi sand ies to permethrin was similar to that of P papatasi The order of relative toxicity was: prallethrin > -cyhalothrin > deltamethrin > cypermethrin > permethrin > etofenprox. A limited data set was obtained for the 3 organophosphate insecticides tested against P duboscqi sand ies. Chlorpyrifos appeared to be more toxic than malathion and carbaryl (Table 2). B C D F A E Figure 1: (A) Membrane feeding of adult sand ies; (B,C) treatment of glass vials with insecticide; (D,E,F) determination of sand y mortality. Figure 2. Sand y bioassays were conducted at the US Army Medical Research Unit-Kenya eld station in Marigat, Kenya.

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6 http://www.cs.amedd.army.mil/amedd_journal.aspxThe bioassay technique involving a glass vial to measure exposure time-mortality relationships has been used previously to assess sand y susceptibility to a number of insecticides.28 Based on data obtained in this study, we veri ed that our modi ed version of the CDC bottle bioassay technique was sensitive and reliable. We were able to demonstrate a time-mortality relationship at different concentrations of the same insecticide, as well as compare sand y susceptibility to different insecticides at particular test concentrations. Based on LT50 data, we were able to rank relative toxicity of pyrethroid and organophosphate insecticides to both P papatasi and P duboscqi sand y species. The susceptibility data obtained from this study will help military entomologists in the eld with the selection of insecticides for sand y control. The results from this study could also be used as baseline susceptibility data for comparative purposes in resistance monitoring of eld-collected sand y populations. Additionally, having reference susceptibility data to several conventional pyrethroid and organophosphate insecticides facilitates future work involving the screening of new insecticides, including essential oils and other natural products31 for sand y control. Like mosquitoes, only female sand ies are blood feeders. Many insecticide studies use females as test subjects. Because we were at the early stage of establishing the P papatasi colony, females were reserved for maintaining and propagating the sand y colony. Therefore, we used adult males for toxicity bioassays. Male sand ies are slightly smaller than females, and are known to be more sensitive than females to insecticides.32 However, our results indicate that relative toxicity of insecticides against sand ies could be assessed using males in bioassays. Depending on the collection technique employed in the eld, sand y samples can include adult males and females. Therefore, future laboratory studies will involve experiments to ascertain if males are as susceptible to insecticides as females. Although P duboscqi is one of the major sand y species transmitting leishmaniasis in Kenya and has been the target of control efforts,33,34 little is known about insecticide susceptibility in this sand y species. To the best of our knowledge, this is the rst report of laboratory testing of P duboscqi susceptibility to commonly used pyrethroid and organophosphate insecticides. Due to the dif culty in collecting large numbers of live sand ies in the eld, having enough ies to run a dose-response bioassay, which requires a large number of sand ies, is impractical. While the standard CDC bottle assay uses 250 ml Wheaton glass bottles, the 10 ml glass vials we used in this study were smaller, required fewer sand ies to run a test, and are amenable for eld deployment. Treatment of glass vials with technical insecticides in a resourceconstrained environment may be dif cult. Pretreatment of glass vials at a standard laboratory equipped with a simple roller and similar devices would allow the use of pretreated glass vials in eld locations at least for 7 days posttreatment, as demonstrated in this study. Diagnostic concentrations are recommended for a number BASELINE SUSCEPTIBILITY TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES IN TWO OLD WORLD SAND FLY SPECIES (DIPTERA: PSYCHODIDAE) 20406080100120140160 20 0 40 60 80 100 0.00001% 0.0001% 0.001% 0.01%% Mortality (SE)Time (minutes) Permethrin Time (minutes) 0.00001% 0.0001% 0.001% 0.01% 20406080100120 20 0 40 60 80 100 Cyfluthrin % Mortality (SE)Figure 3. Exposure time-mortality responses of male P papatasi to different concentrations of representative pyrethroid (permethrin, cy uthrin) insecticides.

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July – September 2015 7THE ARMY MEDICAL DEPARTMENT JOURNAL of insecticides against mosquitoes.25 Although several concentrations have been tested in susceptibility studies, no standard diagnostic insecticide concentrations are available for most insecticides to detect resistance in sand y species.19,28 Collaboration among sand y researchers is necessary to coordinate efforts to develop standards for insecticide resistance monitoring in sand ies. This study provides baseline susceptibility data that can inform future insecticide resistance monitoring in sand ies. ACKNOWLEDGMENTSWe thank Darci Burchers, DeEsta Hyatt (USDA Agricultural Research Service Knipling-Bushland US Livestock Insects Research Laboratory), Francis Ngere, Daniel Ngonga, and Clifford Chepchieng (USAMRU-Kenya) for technical assistance during this study. This work was funded by US Department of Agriculture appropriated funds (USDA-ARS CRIS project 6205-32000-033000D) and Deployed War ghter Protection Research Program (Project number 6201-32000033017R) of the US Department of Defense through the Armed Forces Pest Management Board.REFERENCES1. Alexander B, Maroli M. Control of phlebotomine sand ies. Med Vet Entomol 2003;17(1):1-18. 2. Killick-Kendrick R. The biology and control of phlebotomine sand ies. Clinic Dermatol 1999;17:279-289. 3. World Health Organization. Leishmaniasis Fact Sheet No. 375 [internet]. February 2015. Available at: http://www.who.int/mediacentre/factsheets/fs375/ en/. Accessed April 15, 2015. 4. Cope SE, Strickman DA, White GB. The Deployed War ghter Protection Research Program: nding new methods to vanquish old foes. US Army Med Dept J October-December 2012:9-20. 5. Lay JC. Leishmaniasis among US armed forces, January 2003-November 2004. MSMR 2004;10:2-5. 6. Coleman RE, Burkett DA, Sherwood V, et al. Impact of Phlebotomine sand ies on United States military operations at Tallil Air Base, Iraq: 6. Evaluation of insecticides for the control of sand ies. J Med Entomol. 2011;48(3):584-599. 7. Maroli M, Majori G. Permethrin-impregnated curtains against phlebotomine sand ies (Diptera: Psychodidae): laboratory and eld studies. Parassitologia. 1991;33(suppl):399-404. 8. Morsy TA, Aboul-Ela RG, El-Gozamy BM, Salama MM, Ragheb DA. Residual effects of four insecticides applied for indoor control of Phlebotomus papatasi (Scopoli). J Egyptian Soc Parasitol. 1993;23(2):485-492. 9. Kasili S, Kutima H, Mwandawiro C, Ngumbi PM, Anjili CO. Laboratory and semi eld evaluation of long-lasting insecticidal nets against leishmaniasis vector, Phlebotomus duboscqi in Kenya. J Vec Borne Dis 2010;47(1):1-10. Table 1. Results of glass vial bioassays of pyrethroid and organophosphate insecticides against the Israeli strain of P papatasi maintained at USDA-ARS-KBUSLIRL in Kerrville, Texas.Solution Concentration (%) Surface Concentration Slope 2 (n) LT50 (95% CI) Pyrethroids Cypermethrin0.00015 ng/cm26.513.9 (14)30.0 (26.9-32.9) 0.00150 ng/cm24.417.4 (10)21.1 (16.1-26.2) 0.010.5 g/cm2//<20Deltamethrin0.00015 ng/cm22.619.6 (21)29.9 (24.8-35.0) 0.00150 ng/cm2//<20 0.010.5 g/cm2n/t Cyfluthrin0.00015 ng/cm26.17.4 (17)32.1 (28.9-35.2) 0.00150 ng/cm24.94.9 (9)16.5 (14.0-19.) 0.010.5 g/cm2n/t -cyhalothrin0.00015 ng/cm22.46.5 (14)12.8 (8.1-16.5) 0.00150 ng/cm2//<10 0.010.5 g/cm2n/t Permethrin0.00015 ng/cm25.011.6 (45)85.9 (80.1-91.8) 0.00150 ng/cm25.113.1 (17)34.2 (30.5-37.8) 0.010.5 g/cm24.46.9 (8)14.2 (11.6-16.7)Prallethrin0.00015 ng/cm24.94.1 (8) 13.8 (11.4-16.0) 0.00150 ng/cm2//<10 0.010.5 g/cm2n/t Organophosphates Chlopyrifos0.00150 ng/cm28.726.1 (21)53.1 (49.1-57.2) 0.010.5 g/cm27.55.5 (14)28.3 (25.6-30.9) 0.15 g/cm2n/t Coumaphos0.00150 ng/cm212.216.3 (28)77.6 (74.3-81.2) 0.010.5 g/cm25.46.5 (17)34.7 (31.2-38.2) 0.15 g/cm2n/t Diazinon0.00150 ng/cm212.113.7 (20)53.4 (50.5-56.2) 0.010.5 g/cm210.19.6 (10)25.5 (23.4-27.6) 0.15 g/cm2n/t Dichlovos0.00150 ng/cm23.310.2 (58)122.0 (112.4-254.8) 0.010.5 g/cm26.69.1 (32)58.4 (54.3-62.4) 0.15 g/cm2n/tLT50 indicates exposure time (minutes) at which 50% of the flies had died. n/t indicates not tested.

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8 http://www.cs.amedd.army.mil/amedd_journal.aspx10. Chowdhury R, Akhter S, Huda MM, et al. The Indian and Nepalese programmes of indoor residual spraying for the elimination of visceral leishmaniasis: performance and effectiveness. Ann Trop Med Parasitol 2011;105(1):31-45. 11. Jacusiel F. Sand y control with DDT residual spray eld experiments in Palestine. Bull Entomol Res 1947;38(3):479-488. 12. Wilamowski A, Pener H. Ef cacy of microencapsulated insecticides against the sand y, Phlebotomus papatasi Scopoli. J Vector Ecol. 2003;28:229-233. 13. Rashti MA, Panah HY, Mohamadi HS, Jedari M. Susceptibility of Phlebotomus papatasi (Diptera: Psychodidae) to DDT in some foci of cutaneous leishmaniasis in Iran. J Am Mosq Control Assoc. 1992;8(1):99-100. 14. Amalraj DD, Sivagnaname N, Srinivasan R. Susceptibility of Phlebotomus argentipes and P papatasi (Diptera: Psychodidae) to insecticides. J Comm Dis. 1999;31(3):177-180. 15. Kishore K, Kumar V, Kesari S, Bhattacharya SK, Das P. Susceptibility of Phlebotomus argentipes against DDT in endemic districts of North Bihar, India. J Comm Dis. 2004;36(1):41-44. 16. Singh RK, Mittal PK, Dhiman RC. Insecticide susceptibility status of Phlebotomus argentipes a vector of visceral leishmaniasis in different foci in three states of India. J Vec Borne Dis 2012;49(4):254-257. 17. Dinesh DS, Das P, Das ML, et al Insecticide susceptibility of Phlebotomus argentipes in visceral leishmaniasis endemic districts in India and Nepal. PLoS Negl Trop Dis 2010;4(10):e859. 18. Tetreault GE, Zayed AE-BB, Hana HA, Beavers GM, Zeichner BC. Susceptibility of sand ies to selected insecticides in North Africa and the Middle East. J Am Mosq Control Assoc 2001;17(1):23-27. 19. Faraj C, Ouahabi S, Adlaoui EB, EL Elkohli M, Lakraa L, El Rhazi M, Ameur B. Insecticide susceptibility status of Phlebotomus (Paraphlebotomus) sergenti and Phlebotomus (Phlebotomus) papatasi in endemic foci of cutaneous leishmaniasis in Morocco. Parasit Vectors. 2012;5(1):51.20. Hassan MM, Widaa SO, Osman OM, Numiary MSM, Ibrahim MA, Abushama HM. Insecticide resistance in the sand y, Phlebotomus papatasi from Khartoum State, Sudan. Parasit Vectors. 2012;5(1):46. 21. Brogdon WG, McAllister JC. Insecticide resistance and vector control. Emerging Infect Dis. 1998;4(4):605-613. 22. Surendran SN, Karunaratne SHPP, Adams Z, Hemingway J, Hawkes NJ. Molecular and biochemical characterization of a sand y population from Sri Lanka: Evidence for insecticide resistance due to altered esterases and insensitive acetylcholinesterase. Bull Entomol Res. 2005:95 (4):371-380. 23. Temeyer KB, Brake DK, Tuckow AP, Li AY, Prez de Len AA. Acetylcholinesterase of the sand y, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties. Parasit Vectors 2013;6:31.BASELINE SUSCEPTIBILITY TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES IN TWO OLD WORLD SAND FLY SPECIES (DIPTERA: PSYCHODIDAE)Figure 4. Exposure time-mortality responses of male P papatasi to different concentrations of representative organophosphate (diazinon, chlorpyrifos) insecticides. 20406080100120140160180200 20 0 40 60 80 100Time (minutes) Diazinon 0.00001% 0.0001% 0.001% 0.01%% Mortality (SE)Time (minutes)% Mortality (SE) 220 20406080100120140160180200 20 0 40 60 80 100 0.00001% 0.0001% 0.001% 0.01% Chlorpyrifos

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July – September 2015 9THE ARMY MEDICAL DEPARTMENT JOURNAL24. World Health Organization. Instructions for determining the susceptibility or resistance of adult black ies, sand ies and biting midges to insecticides. Geneva, Switzerland: World Health Organization; 1981. WHO/VBC/81.810. 25. World Health Organization. Vector resistance to pesticide. Geneva, Switzerland: World Health Organization; 1992. Tech Rep. Ser 818. 26. Centers for Disease Control and Prevention. Guideline for evaluating insecticide resistance in vectors using the CDC bottle bioassay [internet]. Available at: http://www. cdc.gov/malaria/resources/pdf/fsp/ ir_manual/ir_cdc_bioassay_en.pdf. Accessed March 3, 2015. 27. Aboul Ela RG, Morsy TA, el-Gozamy BM, Ragheb DA. The susceptibility of the Egyptian Phlebotomus papatasi to ve insecticides. J Egyp Soc Parasitol 1993;23(1):69-94. 28. Maroli M, Cianchi T, Bianchi R, Khoury C. Testing insecticide susceptibility of Phlebotomus perniciosus and P papatasi (Diptera: Psychodidae) in Italy. Ann 1st Super Sanita 2002;38(4):419-423. 29. Young, DG, Perkins P, Endris RG. A larval diet for rearing phlebotomine sand ies (Diptera, Psychodidae). J Med Entomol. 1981;18(5):446-446. 30. Robertson JL, Russell RM, Savin NE. PoloPlus Probit and Logit Analysis User’s Guide Berkeley, CA: LeOra Software;2007. 31. Dinesh DS, Kumari S, Kumar V, Das P. The potentiality of botanicals and their products as an alternative to chemical insecticides to sand ies (Diptera: Psychodidae): a review. J Vector Borne Dis 2014;51(1):1-7. 32. Alexander B, Barros VC, de Souza SF, et al. Susceptibility to chemical insecticides of two Brazilian populations of the visceral leishmaniasis vector Lutzomyia longipalpis (Diptera: Psychodidae). Trop Med Int Health. 2009;10;1272-1277. 33. Beach R, Hendricks L, Oster C, Kiilu G, Leeuwenburg J. Cutaneous leishmaniasis in Kenya: transmission of Leishmania major to man by the bite of a naturally infected Phlebotomus duboscqi Trans Royal Soc Trop Med Hyg 1984;78(6):747-751. 34. Britch SC, Linthicum KJ, Walker TW, et al. Evaluation of ULV applications against Old World sand y (Diptera: Psychodidae) species in equatorial Kenya. J Med Entomol 2011;48(6):1145-1159.AUTHORSDr Li is a Research Entomologist, USDA-ARS Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, MD. Dr Prez de Len is Director and Research Leader, USDAARS Knipling-Bushland US Livestock Insects Research Laboratory, Kerrville, TX. Dr Linthicum is Director, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL. When this article was written, MAJ Bast was assigned to the US Army Medical Research Unit, Nairobi, Kenya. Dr Britch is a Research Entomologist, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL. Dr Debboun is Director, Mosquito Control Division, Harris County Public Health & Environmental Services, Houston, TX. Table 2. Results of glass vial bioassays of pyrethroid and organophosphate insecticides against the Israeli strain of P duboscqi strain maintained at USAMRU-K eld station in Marigat, Kenya.Solution Concentration (%) Surface Concentration Slope 2 (n) LT50 (95% CI) Pyrethroids Cypermethrin0.00015 ng/cm22.834.3 (19)109.2 (74.7-503.2) 0.00150 ng/cm26.08.3 (13)24.1 (21.0-27.0) 0.010.5 g/cm2//<10Deltamethrin0.00015 ng/cm24.058.2 (19)57.1 (43.6-77.8) 0.00150 ng/cm210.56.8 (10)20.2 (17.8-22.0) 0.010.5 g/cm2//<10Etofenprox0.00015 ng/cm25.834.6 (13)25.0 (18.9-30.7) 0.00150 ng/cm23.436.8 (22)32.5 (23.5-40.8) 0.010.5 g/cm25.65.0 (6)17.9 (14.3-21.2) -cyhalothrin0.00015 ng/cm22.08.1 (22)65.3 (52.9-91.5) 0.00150 ng/cm2//<20 0.010.5 g/cm2//<5Permethrin0.00015 ng/cm23.111.2 (25)80.8 (69.8-101.4) 0.00150 ng/cm24.919.4 (23)33.1 (29.4-36.5) 0.010.5 g/cm2//<20Prallethrin0.00015 ng/cm22.410.5 (13)22.4 (17.8-27.0) 0.00150 ng/cm2//<10 0.010.5 g/cm2//<5Organophosphates Carbaryl0.00150 ng/cm28.1141.1 (19)32.2 (21.8-40.3) 0.010.5 g/cm27.5270.4 (13)/ 0.15 g/cm211.43.9 (10)26.8 (24.8-28.8)Chlopyrifos0.00150 ng/cm26.233.1 (13)21.4 (16.6-25.5) 0.010.5 g/cm2//<10 0.15 g/cm2//<10Malathion0.00150 ng/cm2n/t0.010.5 g/cm26.38.6 (7)13.1 (10.6-15.5) 0.15 g/cm2 n/tLT50 indicates exposure time (minutes) at which 50% of the flies had died. n/t indicates not tested.

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10 http://www.cs.amedd.army.mil/amedd_journal.aspxLeishmaniasis is caused by parasitic protozoa in the genus Leishmania that are vectored by Phlebotomine sand ies. The disease manifests in cutaneous, mucocutaneous, or visceral forms. There are an estimated 12 million people in tropical and subtropical regions currently infected with some form of this disease.1 The cutaneous form of the disease was frequently observed among US service members deployed in Iraq and Afghanistan with 1,670 con rmed cases reported from 2003 to 2014.2 However, the actual number of cases was probably much larger. Initial efforts by the US military to control sand ies relied on insecticides and proved largely unsuccessful due to harsh environmental conditions and the cryptic nature of sand ies.3 Current guidelines for managing sand ies endorse an integrated approach and stress the importance of personal protective measures that include using topical repellents on the skin, treating uniforms with permethrin, and sleeping under insecticide treated nets (ITNs).4Insecticide treated nets are recognized by the global health community as an important tool to help control sand ies and prevent Leishmaniasis.5,6 They have been most frequently used and are most well known for their use in malaria control programs where there is a current emphasis to switch to long lasting insecticide nets that do not require periodic retreatment with an insecticide.7 Sand ies cannot be adequately controlled with standard mosquito nets because most species are small enough to pass through the mesh, and decreasing the mesh size restricts air ow and makes the nets uncomfortable to use in hot environments. Therefore, protection is only observed after the addition of an insecticide.8 Insecticide treated nets have been compared to baited traps where sand ies are attracted to host odors emanating from inside the net and then contact the insecticide treated fabric while trying to enter.5 Even if sand ies are able to pass through ITNs, the nets are capable of killing sand ies or changing their feeding behavior.9,10One important question that has not been investigated is whether ITNs provide the same level of protection against Leishmania infected sand ies as they do for noninfected sand ies. It is well documented that Leishmania infection causes many changes to sand y feeding behavior and host seeking.11-13 During normal feeding, noninfected sand ies will land on a host, search for a suitable location, begin probing, and then usually obtain a full blood meal within 2 to 3 minutes. In contrast, infected sand ies will often probe multiple times and for several minutes and never successfully obtain a blood meal. They are also more persistent and are more Efficacy of Permethrin Treated Bed Nets Against Leishmania major Infected Sand Flies Tobin Rowland MAJ Silas A. Davidson, MS, USA Kevin Kobylinski, PhD Claudio Menses Edgar Rowton, PhDABSTRACTInsecticide treated nets (ITNs) are a potential tool to help control sand ies and prevent Leishmaniasis. However, little is currently known about the response of Leishmania infected sand ies to ITNs. In this study, Phlebotomus duboscqi sand ies were infected with the parasite Leishmania major. Infected and noninfected sand ies were then evaluated against permethrin treated and untreated bed nets in a laboratory assay that required sand ies to pass through suspended netting material to feed on a mouse serving as an attractive host. The number of sand ies passing through the nets and blood feeding was recorded. There was not a signi cant difference in the ability of infected or noninfected sand ies to move through treated or untreated nets. Fewer sand ies entered the permethrin treated nets compared to the untreated nets, indicating that permethrin creates an effective barrier. The results show that in addition to reducing the nuisance bites of noninfected sand ies, ITNs also protect against Leishmania infected sand ies and therefore can play in key role in reducing the rates of Leishmaniasis. This study is important to the Department of Defense as it continues to develop and eld new bed nets to protect service members.

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July – September 2015 11likely to return and feed if interrupted.13 This behavior likely enhances transmission of Leishmania parasites, but it is not known if it makes sand ies more likely to enter ITNs and seek a blood meal. In this laboratory study, the ability of Leishmania major infected Phlebotomus duboscqi to pass through a permethrin treated net and take a blood meal was compared to noninfected ies. Permethrin was selected because it is the insecticide used by the military to treat bed nets and belongs to the pyrethroid class of insecticides which is preferred by most global health organizations.14 The sand y P duboscqi is a major vector of cutaneous leishmaniasis in Africa. The parasite L major is found in Africa and the Middle East and was the leading cause of cutaneous Leishmaniasis among service members in Iraq.4MATERIALS AND METHODSSand FliesPhlebotomus duboscqi from Mali were obtained from the National Institute of Health and reared in the insectary at the Walter Reed Army Institute of Research by the methods described in Modi and Rowton.15 The sand ies were maintained at 26C and 80% relative humidity.Infection with LeishmaniaLeishmania major RYN strain parasites were used for infections. A membrane feeding apparatus and water bath circulator were used to infect sand ies.16 De brinated rabbit blood was spiked with L major promastigotes and placed in the feeding apparatus. The feeding apparatus had a chicken skin membrane attached to the lower opening and was placed on a screened carton holding sand ies. The sand ies were allowed to blood feed to repletion. Dissections on day 13 postinfection and immediately after experimental manipulation revealed 90% to 100% infection rates. Noninfected control sand ies were blood fed in the same way, but L major promastigotes were not added to the blood meal.Insecticide Treated NetsUntreated white, 196-mesh polyester netting material was obtained from Vestergaad Frandsen, Inc (Lausanne, Switzerland). This material was selected because it was known that sand ies could pass through. The fabric was cut into 232.4 cm2 squares before treatment. A wooden stand was used to hold an aerosol can of Repel Permanon (0.5% permethrin) (United Industries Corp, Middleton, WI) with the nozzle 20 cm from the center of the material. The fabric was then sprayed for 5 seconds on each side. This method of permethrin application was selected because it simulates how bed nets are currently treated by the US military.14 The treated material was allowed to dry for 48 hours in a chemical fume hood and then stored individually in plastic bags at room temperature until use. Untreated netting material was cut in similar squares and hung in a separate chemical hood for 48 hours.AssaysAssays were conducted using a Grieco module.17 The module consists of 2 Plexiglass cylinders each 15.9 cm long and 10.2 cm in diameter (Figure 1). A Te on linking section (4.4 cm thick, 10.2 cm diameter) connects the 2 cylinders and contains a butter y valve (5.5 cm diameter) that allows movement between the 2 modules. Netting material was stretched tightly between the 2 clear chambers with the butter y door closed. With the door open, the only way for sand ies to move between the cylinders is through the netting material. The assays were performed in a glove box with the temperature maintained between 20C and 24C and 70% to 80% relative humidity. The sand ies used in the assays were approximately 15 to 17 days postemergence and had been water and sugar starved for 12 hours prior to use. A single ICR (Institute for Cancer Research) mouse (Charles River Laboratory, Frederick, MD) was anesthetized and placed in one chamber to serve as a host for sand ies. Twenty female P duboscqi were placed in the opposite chamber and were given 3 minutes to acclimate. The lights were turned off making the room completely dark and the butter y door was then opened allowing the sand ies to move about undisturbed. After 20 minutes, the sand ies from each side of the module Figure 1. Grieco module used to conduct laboratory assays with insecticide treated nets.

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12 http://www.cs.amedd.army.mil/amedd_journal.aspxwere counted. The sand ies were then dissected to verify blood feeding with any trace of blood considered a blood fed sand y. The midguts were also checked for metacyclic promastigotes to con rm infection.Study DesignThere were 4 experimental groups based on the combination of netting material (permethrin treated, untreated) and sand ies (infected, noninfected). Each combination was replicated 6 times. Data was collected on the number of sand ies passing through the nets and blood feeding for all combinations. The data were analyzed by Fisher exact tests, which were conducted using a 2-sided alpha of 0.05. RESULTSThere was not a signi cant difference in the movement of Leishmania infected or noninfected sand ies through treated or untreated nets as shown by Figure 2. For the treated netting material, 27 of 73 (37%) of the infected sand ies passed through and 23 of 77 (30%) of the noninfected sand ies passed through ( P=. 6245, 2=0.4267). Results for the untreated material were similarly not signi cant with 42 of 78 (54%) of infected sand ies passing through and 49 of 81 (60%) of the noninfected sand ies passing through ( P=. 394, 2=0.988). There was a signi cant difference when comparing the 2 types of netting material. Fewer sand ies passed through the permethrin treated net compared to the untreated net for both infected ( P=. 0369, 2=4.9784) and noninfected sand ies ( P=. 0002, 2=14.6701). This indicates that the treated netting material was more effective than the untreated net at preventing sand ies from moving through the mesh. It was noted for the permethrin treated net that most of the sand ies that passed through the netting material were knocked down or dead after 20 minutes. All of the sand ies exposed to the untreated net were still active. There were no blood-fed sand ies in modules containing the permethrin treated net. When the untreated netting material was used, 7% of the infected sand ies took a blood meal and 25% of the noninfected sand ies took a blood meal as shown in the Table. COMMENTThis is the rst study to assess the ability of Leishmania infected sand ies to pass through ITNs. Both infected and noninfected sand ies passed through permethrin treated netting material at similar rates. Infections with very high levels of parasites did not lead to changes in behavior,11-13 and thus allow infected sand ies to bypass the protection of ITNs. The results suggest that ITNs likely play an important role in reducing the transmission of Leishmaniasis in addition to reducing the nuisance bites of sand ies. Permethrin treated nets reduced vector host interactions for both infected and noninfected sand ies. This shows that permethrin is effective in lowering the movement of sand ies into bed nets. However, a small percentage of sand ies were still able to pass through treated nets. This may have been an artifact of the testing module since it was small and enclosed, and, without the ability to escape, some sand ies may have found their way through. It is worth noting that in this study even when sand ies did pass through the permethrin treated net, none of them took a blood meal and most were knocked down. This corresponds to other studies showing that exposure to permethrin alters sand y feeding behavior even if it does not kill them directly.9,10 There have been a Blood feeding results for sand ies that were able to pass through the netting material to host side of the module.Permethrin Treated Net Untreated Net Leishmania infected sand flies0/27 (0%)3/42 (7%)NonInfected sand flies0/23 (0%)12/49 (24%) Proportion of P duboscqi on Host Side of Net P =.0002 P =.0369Treated Net Treated Net Untreated Net Untreated Net0.8 0.0 0.6 0.4 0.2 Uninfected P duboscqi L major infected P duboscqiFigure 2. Proportion of Phlebotomus duboscqi that passed through treated or untreated bed net material. EFFICACY OF PERMETHRIN TREATED BED NETS AGAINST LEISHMANIA MAJOR INFECTED SAND FLIES

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July – September 2015 13THE ARMY MEDICAL DEPARTMENT JOURNAL few unpublished studies at the Walter Reed Army Institute of Research (WRAIR) where blood feeding was observed after sand ies contacted permethrin treated nets, and contact with permethrin may not always prevent blood feeding. In this study, sand y probing behavior could not be observed because the assays were conducted in the dark. It is possible that some sand ies moved through the net and probed before being killed or knocked down. The results showed that infected sand ies were less effective at taking blood meals than the noninfected sand ies. This corresponds to other studies showing that Leishmania infection inhibits blood feeding.11,12 Overall, the blood feeding rates of sand ies in this study were lower than previous studies conducted at WRAIR. Those unpublished studies used noninfected ies that never received a blood meal and feeding rates were 60% to 80%. The lower rates in this study were likely a result of the sand ies receiving a blood meal during the infection process and being gravid at the time of the assay. It was not possible to have nongravid females in this study, since a blood meal was required to become infected and females die in the laboratory immediately after ovipositing their eggs. Many large scale studies have evaluated the effectiveness of ITNs against sand ies and most have shown that they are bene cial. In a study in Brazil, ITNs were associated with reduced indoor human landing rates and high sand y mortality.18 Visceral leishmaniasis rates in Sudan were reduced following the widespread distribution of ITNs.19 In Syria, the rates of cutaneous leishmaniasis dropped by 85% after the distribution of ITNs.20 A large World Health Organization effort to eliminate visceral leishmaniasis in India, Bangladesh, and Nepal has shown both positive and negative results. Communitywide distribution of ITNs reduced the indoor density of sand ies by 25% in India and Nepal,21 and the use of ITNs reduced indoor sand y densities by 60% to 85% in Bangladesh.22,23 However, in one large study in India, ITNs did not lead to lowered sand y densities.24 In another study in India and Bangladesh, the mass distribution of ITNs only slightly lowered sand y biting rates based on serological data among communities.25The most important result of this study is that it justi es using noninfected sand ies to evaluate the effectiveness of new ITNs. There have been many unpublished evaluations of ITNs at WRAIR using noninfected sand ies, and other published studies have also used noninfected sand ies.10 This study indicates that results from noninfected sand ies are most likely similar and applicable for Leishmania infected sand ies. It is much easier to use noninfected sand ies in laboratory assays since they can be produced in greater numbers, at Figure 4. Pop-up style bed net (NSN 3740-01-516-4415 ). This net is factory treated with permethrin and the mesh size is small enough to physically exclude sand ies. Female sand y included for size comparison. Figure 3. Standard mosquito net (NSN 7210-00-266-9736/ 9740). This net is not treated with an insecticide and the mesh is too large to physically exclude sand ies. Female sand y included for size comparison.

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14 http://www.cs.amedd.army.mil/amedd_journal.aspx cheaper costs and are safer to handle. Research with Leishmania infected sand ies must be performed in a Biosafety level 2 laboratory.26 The Department of Defense will continue to rely upon ITNs to protect service members from sand y bites and diseases. There are two bed nets currently available. The standard mosquito net (NSN 7210-00-266-9736/9740), shown in Figure 3, is untreated and its mesh size is too large to physically exclude sand ies. The mesh becomes more permissive to sand ies as the nets become older and receive more wear.4 It is important to treat these nets with permethrin if they are to provide protection against sand ies. The recommended method is to spray with an aerosol can as described in this study.14 Pop-up style bed nets (Figure 4) that are now available (NSN 3740-01-516-4415) have a mesh size small enough to exclude sand ies and are issued factory treated with permethrin.4 These nets are the preferred option for the prevention of sand y bites. The results from this study will be useful as the Department of Defense continues to develop and evaluate new ITNs and ensure that they provide protection from both infected and noninfected sand ies. ACKNOWLEDGMENTSThanks to Mr John Paul Benante for providing pictures of bed nets. This research was funded by a grant from the USDA Deployed War-Fighter Protection program. Research was conducted in compliance with the Animal Welfare Act, other federal statutes and regulations relating to animals, and in accordance with principles stated in the Guide for the Care and Use of Laboratory Animals, MRC Publication, 1996 edition.REFERENCES1. Desjeux P. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 2004;27:305-318. 2. Armed Forces Health Surveillance Center. Leishmaniasis. MSMR 2014;21(9):19. 3. Coleman RE, Burkett DA, Sherwood V, et al. Impact of phlebotomine sand ies on United States military operations at Tallil Air Base, Iraq: 6. Evaluation of insecticides for the control of sand ies. J Med Entomol 2011;48(3):584-599. 4. Technical Guide No. 49. Sand Flies (Diptera: Psychodidae: Phlebotominae): Signi cance, Surveillance, and Control in Contingency Operations Silver Spring, MD: Armed Forces Pest Management Board; 2015. Available at: http://www.afpmb.org/ sites/default/ les/pubs/techguides/TG49/TG49.pdf. Accessed June 2, 2015. 5. Alexander B, Maroli M. Control of phlebotomine sand ies. Med Vet Entomol 2003;17:1-18. 6. Warburg A, Faiman R. Research priorities for the control of phlebotomine sand ies. J Vector Ecol 2011;36(suppl 1):S10-S16. 7. World Health Organization. Long Lasting Insecticide Nets for Malaria Prevention: A Manual for Malaria Programme Managers [Trial Edition]. Geneva, Switzerland: World Health Organization; 2013. Available at: http://www.who.int/manage ment/programme/LongLastingInsecticidalNets Malaria.pdf. Accessed June 2, 2015. 8. Ostyn B, Vanlerberghe V, Picado A, et al. Vector control by insecticide-treated nets in the ght against visceral leishmaniasis in the Indian subcontinent, what is the evidence?. Trop Med Int Health 2008;13:1073-1085. 9. Maroli M, Marjori G. Permethrin-impregnated curtains against phlebotomine sand ies (Diptera: Psychodidae): laboratory and eld studies. Parassitologia 1991;33(suppl):399-404. 10. Kasili S, Kutima H, Mwandawiro C, Ngumbi PM, Anjili CO, Enayati AA. Laboratory and semi eld evaluation of long-lasting insecticidal nets against leishmaniasis vector, Phlebotomus (Phlebotomus) duboscqi in Kenya. J Vector Borne Dis 2010;47:1-10. 11. Beach R, Kiilu G, Leeuwenburg J. Modi cation of sand y biting behavior by Leishmania leads to increased parasite transmission. Am J Trop Med Hyg 1985;34:278-282. 12. Vaidyanathan RR. Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects. J Invertebr Pathol 2004;87:123-128. 13. Rogers ME, Bates PA. Leishmania manipulation of sand y feeding behavior results in enhanced transmission. PLoS Pathog 2007;3:e91. 14. Technical Guide No. 36. Personal Protective Measures Against Insects and Other Arthropods of Military Signi cance Silver Spring, MD: Armed Forces Pest Management Board; 2009. Available at: http://www.afpmb.org/sites/default/ les/pubs/ techguides/tg36.pdf. Accessed June 2, 2015. 15. Modi GB, Rowton ED. Laboratory maintenance of phlebotomine sand ies. In: Maramorosch K, Mahmood F, eds. Maintenance of Human, Animal, and Plant Pathogen Vectors En eld, NH: Science Pub Inc; 1999:109-121. 16. Rowton ED, Dorsey KM, Armstrong KL. Comparison of in vitro (chicken-skin membrane) versus in vivo (live hamster) blood-feeding methods for maintenance of colonized Phlebotomus papatasi (Diptera: Psychodidae). J Med Entomol 2008;45:9-13.EFFICACY OF PERMETHRIN TREATED BED NETS AGAINST LEISHMANIA MAJOR INFECTED SAND FLIES

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July – September 2015 15THE ARMY MEDICAL DEPARTMENT JOURNAL17. Grieco JE, Achee NL, Sardelis MR, Chauhan KR, Roberts DR. A novel high-throughput screening system to evaluate the behavioral response of adult mosquitoes to chemicals. J Am Mosq Control Assoc 2005;21:404-411. 18. Courtenay O, Gillingwater K, Gomes PAF, Garcez LM, Davies CR. Deltamethrin-impregnated bednets reduce human landing rates of sand y vector Lutzomyia longipalpis in Amazon households. Med Vet Entomol 2007;21:168-176. 19. Ritmeijer K, Davies C, van Zorge R, Wang SJ, Schorscher J, Dongu’du SI, Davidson RN. Evaluation of a mass distribution program for nemesh impregnated bednets against visceral leishmaniasis in eastern Sudan. Trop Med Int Health 2007;12:404-414. 20. Jalouk L, Al Ahmed M, Gradoni L, Maroli M. Insecticide-treated bednets to prevent anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria: results from two trials. Trans R Soc Trop Med Hyg 2007;101:360-367. 21. Picado A, Das ML, Kumar V, et al. Effect of village-wide use of long-lasting insecticidal nets on visceral leishmaniasis vectors in India and Nepal: a cluster randomized trial. PLoS Negl Trop Dis 2010;4:e587. 22. Mondal D, Chowdhury R, Huda MM, et al. Insecticide-treated bed nets in rural Bangladesh: their potential role in the visceral leishmaniasis elimination programme. Trop Med Int Health. 2010;15:1382-1389.AUTHORSMr Rowland is the Sand Fly Lab Manager, Entomology Division, Walter Reed Army Institute of Research, Silver Spring, Maryland. MAJ Davidson is the Chief of Vector & Parasite Biology, Entomology Division, Walter Reed Army Institute of Research, Silver Spring, Maryland. Dr Kobylinski is a National Research Council postdoctoral fellow, Entomology Department, AFRIMS, Bangkok, Thailand. Mr Menenses is a Research Assistant in the Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, National Institute for Allergy and Infectious Disease, Rockville, Maryland. Dr Rowton was formerly a Senior Scientist, Entomology Division, Walter Reed Army Institute of Research, Silver Spring, Maryland.

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16 http://www.cs.amedd.army.mil/amedd_journal.aspxMalaria remains one of the greatest infectious disease burdens worldwide, with 200 million cases and 600,000 deaths reported in 2013.1 Though not endemic to the United States, malaria incidence is reported in 97 countries, indicating this threat to global health is also a threat to travelers and deployed military personnel. Resistance to drugs that kill Plasmodium parasites (the etiological agent of malaria) is common and spreads rapidly upon introduction. There is no currently available vaccine. Therefore, development and testing of antimalarial vaccines and new drugs has been a top priority for infectious disease research within the Department of Defense for decades. Plasmodium parasites are delivered to humans by the bite of an Anopheles mosquito; as a female mosquito takes blood from a human host, she deposits the sporozoite stage of the parasite into the host’s skin along with her saliva. Sporozoites navigate to the liver where they invade hepatic cells, shift to a new form called the merozoite, and multiply, eventually being released into circulation where they can continue an invade-multiplyrelease cycle, now dependent on erythrocytes. Since the mosquito only deposits about 10 to 100 sporozoites per bite2-4 and the ensuing life cycle involves exponential multiplication, the pre-erythrocytic sporozoite stage represents a bottleneck in the parasite population that is vulnerable to vaccine and drug activity. Interventions that speci cally target the pre-erythrocytic stage have been in the pipeline since it was rst shown that sporozoites elicit an immune response capable of preventing subsequent infection.5,6 However, as these vaccine and drug candidates were showing ef cacy in animal models, it became apparent to medical entomologists that clinical testing of such interventions would require a method of mimicking the natural acquisition of sporozoites by humans via mosquito bite. Previous methods used human gametocyte donors to infect mosquitoes intended to deliver sporozoites to vaccines,7 but this method was unpredictable and dependent on the availability of people naturally infected with malaria as gametocyte sources. A more controlled, reproducible method was needed; thus, an experimental human malaria infection, later coined as controlled human malaria infection (CHMI),8 was developed at the Walter Reed Army Institute of Research (WRAIR). The CHMI method encompasses the entirety of a purposeful human malaria infection, from mosquito bite to parasite detection in the blood, to resolution by drug administration. The entomological part of CHMI is considered the “challenge”: the transmission of malaria parasites as mosquitoes bite a human volunteer in a safe, reliable, and reproducible way. IDENTIFYING PARASITES AND VECTORS CAPABLE OF INFECTING HUMANSTo develop a controlled challenge model, entomologists at WRAIR tested the feasibility of arti cially infecting lab-reared Anopheles mosquitoes with lab-cultured Plasmodium. A reliable culture method for growing P falciparum in vitro was nally published in 1976.9 This system became critical for the development of a malaria challenge since it enabled manufacture of parasite Controlled Human Malaria Infection at the Walter Reed Army Institute of Research: The Past, Present, and Future From an Entomological Perspective Lindsey S. Garver, PhD Megan Dowler MAJ Silas A. Davidson, MS, USAABSTRACTThirty years ago, the Entomology Branch at the Walter Reed Army Institute of Research (WRAIR) performed the rst controlled human malaria infection, in which lab-reared mosquitoes were infected with lab-cultured malaria parasites and allowed to feed on human volunteers. The development of this model was a turning point for pre-erythrocytic malaria vaccine research and, through decades of re nement, has supported 30 years of ef cacy testing of a suite of antimalarial vaccines and drugs. In this article, we present a historical overview of the research that enabled the rst challenge to occur and the modi cations made to the challenge over time, a summary of the 104 challenges performed by WRAIR from the rst into 2015, and a prospective look at what the next generation of challenges might entail.

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July – September 2015 17lines with known origin and drug sensitivity, followed a somewhat consistent schedule, and used blood and sera of known type that could be tested for pathogens. The downfall of in vitro parasite growth was (and still is) that most parasite lines adapted to grow well asexually in vitro infect mosquitoes poorly, if at all. WRAIR and the Naval Medical Research Institute (NMRI), predecessor to the Naval Medical Research Center, collaboratively tweaked the Trager-Jensen method to grow the best lines for infecting mosquitoes.10 Foreseeing the need for a compatible parasite-vector pair on which to base the malaria challenge, WRAIR entomologists exposed various anopheline species to multiple P falciparum parasite lines, both lab-adapted and patient-derived, to assay for successful mosquito infection. Although the screening was exhaustive, infection rates were often disappointing, sometimes yielding months of no infectiousness to mosquitoes. By 1983, the 7G8 strain (chloroquine resistant) was cloned from a Brazilian patient sample and, in regular production at WRAIR, showed low numbers of oocysts and sporozoites but with greater consistency than any other strain. In 1985, WRAIR received the Africanderived, chloroquine sensitive NF54 P falciparum strain from NMRI which had received it from collaborators in Nijmegen, The Netherlands.11 This quickly became the primary culture in production. In 1987, WRAIR subsequently received 3D7, a strain cloned from NF54 by NIH researchers,12 from NMRI. Based on the mosquito infectivity studies done at WRAIR, NMRI, and elsewhere, 7G8, NF54, and 3D7 would become the worldwide standards for cultured parasites suitable for infecting mosquitoes and nearly the only strains of P falciparum used in malaria challenges as of 2015. For vector selection, the breadth of available parasite strains were fed to a variety of potential vectors, including An stephensi An freeborni An balabacensis An albimanus An quadrimaculatus and others. The studies showing 7G8, NF54, and 3D7 were infectious to mosquitoes also showed that An stephensi was a robust mosquito, amenable to mass rearing with hearty feeding propensity, widely used by other mosquito biologists and displayed excellent susceptibility to both P falciparum and P berghei a pre-clinical rodent model for infection. Therefore, it is not surprising that An stephensi is the primary colony supported within WRAIR and, to date, 3 challenges used An freeborni but the rest have used An stephensi INITIAL DEVELOPMENT OF THE WRAIR CHALLENGE MODELDuring vector-parasite compatibility experiments in 1982, exposure of a laboratory worker to an escaped infectious mosquito resulted in accidental transmission of cultured 7G8 P falciparum by lab-reared An freeborni to a human.13 While this study highlighted the acute need for a safety regimen to safeguard workers’ health, it also showed for the rst time that parasites grown in culture and capable of infecting a mosquito could also retain infectiousness to humans, inadvertently paving the way for CHMI. The rst CHMI was performed in 1985 as a proof-ofconcept trial to assess whether 6 volunteers would develop malaria after being bitten by 5 mosquitoes infected with NF54.14 Collectively, WRAIR, NMRI, and NIH contributed An freeborni and An stephensi that were given a blood meal containing cultured parasites in donor blood; only blood-fed (and therefore potentially infected) mosquitoes were retained for possible use. At appropriate times, subpopulations were dissected and numbers of oocysts and sporozoites were quanti ed in midgut and salivary glands, respectively. Mosquitoes determined to likely be infectious were sorted into cups of 5 and allowed access to a volunteer’s arm for 5 minutes. Mosquitoes were then checked for the presence of a blood meal (con rmed they fed on the volunteer) and the presence of sporozoites on a 0 to 4 quanti cation scale (rating of 2 or greater con rmed infectiousness) and, if fewer than all 5 satis ed those criteria, the volunteer was exposed to more mosquitoes until 5 infectious bites were con rmed. This process was performed on a rolling basis—volunteers were called when mosquitoes were ready and not all on the same day. All 6 of the volunteers came down with malaria. This method was independently repeated at the University of Maryland15 with success (4 of 4 volunteers infected) and the fundamentals of the process are largely how challenges are performed today. Questions were raised about the validity of using 5 mosquito bites for a challenge. In nature, people are typically infected by the bite of one mosquito; could vaccinederived immunity be overwhelmed by a 5-bite dosage? And, if so, would a vaccine that would be ef cacious against a natural 1 or 2 bite dose be erroneously perceived as ineffective in a 5-bite challenge? Also, how does sporozoite load affect dosage? Compared to natural conditions, laboratory conditions can load mosquito salivary glands with a much heavier burden of sporozoites16; however, the number of sporozoites successfully deposited in the skin is orders of magnitudes lower than in the salivary glands and highly variable.3,17 Direct enumeration of sporozoites put into each volunteer is ethically impossible, so 2 challenges were performed by WRAIR for the Navy to assess the feasibility of a 1or 2-bite challenge. Three out of 5 volunteers receiving a

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18 http://www.cs.amedd.army.mil/amedd_journal.aspxsingle bite became malaria positive, while 2 of 5 receiving 2 bites became positive.18 A third 2-bite challenge was performed by WRAIR for Johns Hopkins University with only 1 of 3 volunteers becoming malaria positive.19 Therefore, a 5-bite challenge has been standard since about 1990. Later studies show that 3 bites from aseptically reared An stephensi can result in 100% infectivity,20,21 but the consistency and the theoretical advantages of this model have yet to be demonstrated, so WRAIR continued to provide a 5-bite challenge. In 2012, a series of meetings were held to generate a consensus of all CHMI-capable centers, ultimately agreeing on the WRAIR challenge model of 5 bites from An stephensi using a 0 to 4 rating scale as the global standard.8After 24 years, a second parasite species was introduced to the 5-bite challenge. In 2009, the Armed Forces Research Institute of Medical Sciences (AFRIMS) in Bangkok, Thailand, sent 2 challenges using P vivax in An dirus to WRAIR for infectivity studies. Since P vivax cannot be easily cultured in vitro, the lab-reared mosquitoes were infected with blood from a human gametocytemic patient in Thailand, then shipped to WRAIR for challenge administration. All 12 volunteers from these studies became infected, demonstrating that the challenge model has a measure of exibility. VARIATIONS ON THE TRADITIONAL CHALLENGEOff-site ChallengesShipping or hand-carrying infected mosquitoes to perform a challenge overseas was initially tested for feasibility in 2000. A batch of prepared mosquitoes was own from Washington, DC, to London as a mock challenge test of transport and mosquito viability in anticipation of challenges performed by WRAIR personnel for collaborators from Oxford University. This validated the feasibility of a “traveling” challenge that, with slight variations that defer to site-speci c clinical trial centers, is performed similarly to in-house challenges. This includes not just the supply of infectious mosquitoes but of dissectors, entomologists, quality assurance/quality control, standard operating procedures, and challenge day methodology that has produced success in the past. This still requires the receiving facility to have minimal insectary infrastructure for mosquito storage but requires no entomological experience, parasite culture, or mosquito rearing on the part of the receiver.Mosquito Bites as VaccinesSoon after the debut of CHMI, a second mosquito-biting-humans method was developed, in which volunteers were exposed to hundreds or even thousands of bites from mosquitoes infected with attenuated parasites. At rst, this was radiation-attenuated sporozoites as a natural progression from the animal studies and few human studies that already demonstrated this produced a protective immune response.22 These studies, performed on a rolling basis over several years, would collectively use 23,279 mosquitoes. Later, sporozoites would also be genetically attenuated,23 but the role of entomology remained the same, differing from traditional challenges in that many more mosquitoes were required and realtime dissections were not necessary. These trials culminated with a traditional challenge to test the ef cacy of the mosquito-delivered “vaccine” and/or investigate the immune response generated. Eventually, production of mosquitoes that functioned as a vaccine would be considered by the Federal Drug Administration (FDA) to be a manufacturing process (reviewed later in this article), instituting a sum of regulatory requirements that would impose the greatest modi cation of the challenge process since inception.Challenge in a BottleNot surprisingly, challenges can be expensive, timeconsuming, and require specialized facilities and entomological expertise. Innovations in sporozoite cryopreservation by Sanaria, Inc (Rockville, MD)24 initiated an effort to overcome these limitations by vialing aseptic, cryopreserved sporozoites into an FDA-regulated product called PfSPZ Challenge, colloquially referred to as “challenge in a bottle.” This mosquito-free challenge delivers sporozoites by needle inoculation and is capable of reasonable infectivity rates at a dose of 3,500 sporozoites per vial. This type of challenge is most useful in eld settings or locations where facilities cannot support insect maintenance; however, in bypassing the skin, it does not fully mimic the natural route of sporozoite inoculation by mosquito.25 This means it also bypasses immune responses elicited by skin-deposited parasites in the dermis and draining lymph nodes, and may affect the degree of protection observed.26,27MEETING THE INCREASING NEEDS OF THE CHALLENGEBy 1989, demand for infected mosquitoes, stemming from both clinical and preclinical vaccine research, shifted Entomology into a production role. Every aspect of producing infected mosquitoes, from obtaining enough blood and serum to rearing enough mosquitoes to having the tools and infrastructure to safely handle so many infectious mosquitoes, was reexamined and retooled to meet the needs of CHMI. General rearing rooms were out tted with specialized equipment to improve insect production and increase ef ciency, while smaller equipment such as aspirators to transfer mosquitoes, water CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH: THE PAST, PRESENT, AND FUTURE FROM AN ENTOMOLOGICAL PERSPECTIVE

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July – September 2015 19THE ARMY MEDICAL DEPARTMENT JOURNAL jacketed membrane feeders, and mosquito containment devices underwent multiple rounds of innovation to comply with increased demand and increased safety precautions. Mosquito rearing conditions and parasite culture methods were optimized and Entomology personnel began to routinely record data on prevalence and intensity of mosquito infection, no longer as basic research but as indicators of mosquito quality for use in CHMI. The biggest physical innovation in mosquito production occurred in the late 1990s as WRAIR moved from downtown Washington, DC, to the Forest Glen Annex in Silver Spring, MD. The insectary facilities in that building were speci cally designed to meet the needs of the challenge. The challenge suite exists separate from general insect rearing and consists of (1) an empty vestibule to discourage accidental mosquito release as doors are opened, (2) a main room where volunteers and noninsectary personnel are stationed on challenge day, (3) an adjacent room that houses both walk-in and reach-in incubators for infected mosquitoes, and (4) a separate adjacent room for real-time dissection of mosquito salivary glands. Doors with screens allow personnel to communicate with one another but also contain any escaped mosquitoes in work areas away from the main challenge room where visitors are permitted. Incubator set-up facilitates scale production depending on the sizes and numbers of clinical trials in progress and enable segregation of mosquitoes infected with different parasite lines. The dissection room is designed for the comfort, safety, and ef ciency of up to 5 dissectors. A person must pass through 5 doors and a downward air current to get from infected mosquito housing to the main corridor, ensuring the safety of all who work in the building. Two distinct labs speci c for parasite culture exist separately from the insectary and other lab space, isolating challenge-speci c cultures from general lab work while simultaneously enabling segregation of different P falciparum strains destined for challenges. The 1990s also ushered in an extensive suite of methodological innovations, transitioning the orientation of challenge preparation from academic to production. Extensive screens for fail-proof stocks of NF54, 3D7, and 7G8 were undertaken, mass sporozoite harvesting methods were adopted, and individualized ne-tuning of each round of parasite culture/mosquito infection was abandoned in favor of a scheduled, standardized culture/ rearing/infection regimen used for every round of production (Figure 1). This was also highly in uenced by the advent of new regulatory requirements as discussed in the next section. REGULATORY INFLUENCE ON THE CHALLENGEUntil 1993, challenges were performed with mosquitoes infected as they would be for routine laboratory experiments. At that time, the FDA became interested in the challenge as a systemized and monitored part of a clinical trial and introduced a wave of new regulatory requirements, exponentially increasing the labor and planning required to carry out each successful trial. A batch master le was created in the fall of 1994 and, within one year, entire cell banks comprised of 140, 110, and 75 vials of NF54, 3D7, and 7G8, respectively, were manufactured under good manufacturing practices (GMP) conditions at the Pilot Bioproduction Facility also located on the Forest Glen Annex. These cell banks, derived from blood collected from clinical trial volunteers, have provided the seed parasites for every WRAIR challenge through 2015, though a new bank was created for 3D7 in 2014 as the original lot dwindled. Every cell bank creation was preceded by months of methodical selection of line isolates that gave robust infection in An stephensi mosquitoes. The use of infected mosquito bites as a vaccine (reviewed in previous section) precipitated the need to treat infected mosquitoes as an investigational product and to treat anything related to culture, husbandry, and feeding Figure 1. Work ow diagram describing the regimen for producing infected mosquitoes for CMHI. Strain 3D7: 9 Week Production Run Strain NF54: 11 Week Production Run Spz Counts1 Week Oocyst Counts1 Week Infectious Feeds1 Week Thaw and Recover3-10 Days Acclimate to Culture Conditions2 Weeks Culture Expansion2-3 Weeks Rear Mosquitoes for Infection1.5-2 Weeks Challenge Generate Eggs1 Week

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20 http://www.cs.amedd.army.mil/amedd_journal.aspxas a manufacturing process. By 2009, production of infected mosquitoes was performed as close to GMP standards as possible for a population of live insects: a library of standard operating procedures were written; the batch master le for parasite production was improved; raw materials and equipment were tracked and certi ed; forms were added; and each step of the process was documented, reviewed by quality assurance/ quality control (QA/QC) personnel, and led. These methods were extended to traditional challenges and have become standard. SUMMARY OF CHALLENGES PERFORMEDOne hundred and four challenges or immunizations by mosquito bite have been performed or are planned through the end of 2015, resulting in over 2200 volunteer mosquito exposures (VME) (some volunteers are counted more than once due to rechallenges or cumulative immunizations on the same person). About half of all challenges have been with the 3D7 strain of P falciparum and another 20.5% were with NF54. 7G8, P vivax and genetically attenuated parasites with an NF54 background comprise the remainder (Figure 2). All data is summarized from recordkeeping within the Mosquito Biology/Vector and Parasite Biology department within the Entomology Branch at WRAIR. While the number of challenges performed by year did not remarkably increase until about 2009, the number of VME per year displays a growth trend throughout the 30 year time line. The average number of VME per year for the 1980s is 9.8, for the 1990s is 43.6, for the 2000s is 64.8, and for 2010 to 2015 is 183, with particularly active years in 2014 and 2015 (recorded and projected) (Figure 2). Increase in demand for challenges re ects, rst, advancement of vaccine and drug interventions to clinical trials and, second, tentative success of several vaccine candidates leading to follow-up trials to re ne dosage, schedule, and durability. This escalation in activity parallels the advent of many organizations with the mission of controlling malaria, such as the Roll Back Malaria Partnership in 1998, the PATH-Malaria Vaccine Initiative in 1999, The Global Fund in 2002, and the President’s Malaria Initiative in 2005. Funding from Figure 2. Volunteer mosquito exposures involved in malari a challenges performed by the WRAIR Entomology Department from 1985 through 2015 † and distribution of strains of parasites used for those challenges. Genetically attenuated parasites. † Actual and projected volunteer mosquito exposures shown for 2015. Volunteer Mosquito Exposures 7G8 P vivax NF54 3D7 GAP*1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014†2015350 300 250 200 150 100 50 0 CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH: THE PAST, PRESENT, AND FUTURE FROM AN ENTOMOLOGICAL PERSPECTIVE

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July – September 2015 21THE ARMY MEDICAL DEPARTMENT JOURNAL PATH-MVI in particular has directly in uenced the increased demand for the WRAIR challenge model to test their sponsored vaccines. Vaccines as a malaria control intervention has been by far the most common use for the WRAIR malaria challenge model with 61% of all challenges administered for that reason. Another 14% have used the immunization-by-mosquito-bite, primarily by repeated exposure to radiation attenuated parasites. Veri cation that the model (or modi cation) is infectious comprised 10% of all challenges, a prudent step before new parasites or changes are made to the model for testing interventions or immunity. As shown in Figure 3, 9% of challenges have been used for research into experimental therapeutics, and several challenges either served a purpose unknown or unique (eg, test of transport and viability overseas). To date, WRAIR has performed 28 off-site challenges (27%), both domestically and overseas. The remaining 73% were performed in the WRAIR insectary suite as described in previous sections. Exclusive of challenges performed for Oxford University, nearly 65,000 mosquitoes have been used in the CHMIs summarized here (through February 2015). Over 23,000 were used for the irradiated sporozoite vaccinations in the 1990s and over 27,000 were used in irradiated sporozoite vaccination studies in 2014. These numbers denote the numbers of mosquitoes actually exposed to human volunteers. Exponentially greater numbers of mosquitoes are prepared for QA/QC and to ensure mosquito availability is not a limiting factor for CHMI success. Furthermore, up to 10,000 mosquitoes are produced weekly by the WRAIR insectary to support clinical and preclinical malaria research. CURRENT AND FUTURE DIRECTIONS OF THE WRAIR CHALLENGE MODELAlthough the core of the challenge model has not changed much since the 1980s, the model has improved signi cantly with new scienti c knowledge, applications, technology, and varying needs of users. The next generation of WRAIR challenges anticipates the following variations:Heterologous ChallengeAs vaccine candidates display ef cacy against homologous parasites that meets or exceeds the levels called for by the target product pro le, demand for heterologous challenges is increasing. NF54 and its derivative, 3D7 are of African origin and serve as the template typically used when designing vaccines. 7G8, a Brazilian isolate, displays a high degree of polymorphism compared to NF54 and 3D728 and is, therefore, an excellent heterologous parasite. However, as observed by labs from multiple institutions, 7G8 is unreliably infectious to mosquitoes. An intradepartmental effort at WRAIR to develop new heterologous strains has evaluated a plethora of eld-isolated parasite strains for in vitro cultivation and mosquito infection and, to date, has found none to be dually suitable. Ideally, Entomology would possess a library of heterologous strains from around the world such that parasites with different genetic backgrounds could be tested against vaccines, and those with different drug susceptibility pro les could be tested against candidate therapeutics.Additional speciesConcurrently, the need for challenges using non-falciparum Plasmodium species is rising. Entomology anticipates at least one challenge using P vivax within the next 2 years and more to follow. This encompasses not only late-stage testing of the breadth of protection offered by P falciparum vaccines, but also P vivax -specific vaccines currently in research and development. Despite extensive efforts, P vivax in vitro culture is nearly impossible and existing workarounds (such as constant addition of puri ed reticulocytes29) are incompatible with the challenge model. P vivax -infected mosquitoes can be sourced from AFRIMS, but, as this process uses gametocyte donors, it is not nearly as exible as what exists for P falciparum Nonhuman primate challenges Vaccine61% Infectivity10% Drug 9% Unknown 4% Other4% Immunization14% Figure 3. Proportions of challenges performed for indicated purposes.

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22 http://www.cs.amedd.army.mil/amedd_journal.aspx(NHP) have also been provided by WRAIR using P knowlesi -infected mosquitoes sourced from partners.30 In the future, full in-house NHP challenges with P cynomolgi cycled from NHP to mosquito and back are expected at WRAIR. No plans for P ovale or P malariae challenges are in place. Each new tweak to a challenge model requires an investigation into which mosquito species (and strain) is the best to vector the target parasite and how well sporozoites can be recovered, both in terms of prevalence and intensity of infection.Transmission Blocking InterventionsVaccines and drugs with transmission-blocking potential should be investigated for ef cacy at the clinical level, inciting a need for an inverse challenge: a controlled human-to-mosquito malaria transmission that elevates the standard membrane feeding assay to natural transmission dynamics. Arms of volunteers who received a transmission blocking intervention (TBI) or placebo and who acquire malaria would be offered to mosquitoes for feeding and the ef cacy of transmission blocking assessed by Plasmodium prevalence and intensity in those mosquitoes. Currently, most TBIs are still in development, but at least one is moving on to Phase I trials employing this type of methodology.Dengue Human Infection Model (and Others)Just as entomologists in the 1980s foresaw the need for a way to test candidate malaria vaccines against natural routes of transmission, it is now obvious that virologists will soon need such a way to test candidate dengue vaccines. CHMI has the distinct advantage of using a pathogen that is susceptible to available drugs and can be completely cured by a simple dosing regimen. This is not a characteristic of other vector-borne diseases that need a CHMI-like challenge to properly test vaccine candidates. A controlled challenge for dengue is the most pressing need, but it would present ethical considerations (ie, if the vaccine is not protective, you can only provide supportive care, not cure, to a volunteer). From the entomological point of view, CHMI presents an excellent template in which to substitute other mosquito-borne pathogens but with careful consideration of where the processes differ biologically. Dengue human infection model (DHIM) requires a different mosquito species, Aedes aegypti which displays high variability in vector competence across strains that is often dependent on the speci c virus strain used.31,32 A suitable Ae aegypti strain would have to be validated for every viral strain desired in challenges. Dengue virus prevalence and intensity cannot be determined in real-time similar to the con rmation of malaria sporozoites via light microscopy, so DHIM would rely on either preor postscreening of mosquitoes for positive infection. Additionally, the number of bites optimal for guaranteeing dengue transmission while avoiding overwhelming the immune response would require investigation. The feasibility of such a challenge and some theoretical design elements were reviewed by Mores et al.33SUMMARYControlled human malaria infection is a powerful tool in antimalarial testing that requires or bene ts from mimicking the natural route of infection. All of the leading pre-erythrocytic vaccines have been tested using this model, and even after 30 years its utility is still increasing. Preparing infected mosquitoes for a challenge is a task that forces a complex and tenuous biological interaction into a manufacturing-style operation of precision and predictability. The challenge portion of CHMI as it exists at WRAIR today is the result of decades of research and re nement. Such cumulative effort is re ected not only in how well the challenge has performed historically but also in the ways that it can adapt to answer new questions about malaria and vector-borne disease. ACKNOWLEDGMENTSWe thank Dr Imogene Schneider, Dr Jack L. Williams, Dr Claudia Golenda, and MAJ Jittawadee Murphy for their leadership in the Mosquito Biology/Vector and Parasite Biology Department during the development and execution of the WRAIR malaria challenge model. We are very grateful to the dozens of Entomology researchers and staff members who have carried out the 30 years of work described here. We also thank Dr. Frank Klotz for fruitful and engaging discussion about past CHMIs.REFERENCES1. World Health Organization. World Malaria Report 2014 Geneva, Switzerland: World Health Organization; December 2014. Available at: http://www. who.int/malaria/publications/world_malaria_re port_2014/en/. Accessed May 11, 2015. 2. Ponnudurai T, Lensen AH, van Gemert GJ, Bolmer MG, Meuwissen JH. Feeding behavior and sporozoite ejection by infected Anopheles stephensi Trans R Soc Trop Med Hyg. 1991;85(2):175-180. 3. Medica DL, Sinnis P. Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes. Infect Immun. 2005;73(7):4363-4369. 4. Jin Y, Kebaier C, Vanderberg J. Direct microscopic quanti cation of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice. Infect Immun 2007;75(11):5532-5539.CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH: THE PAST, PRESENT, AND FUTURE FROM AN ENTOMOLOGICAL PERSPECTIVE

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July – September 2015 23THE ARMY MEDICAL DEPARTMENT JOURNAL5. Mulligan HW, Russell PF, Mohan BN. Speci c agglutinogenic properties of inactivated sporozoites of Plasmodium gallinaceum J Malar Inst Ind. 1941;4:25. 6. Nussenzweig RS, Vanderberg J, Most H, Orton C. Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei. Nature. 1967;216:160-162. 7. Clyde DF, Most H, McCarthy VC, Vanderberg J. Immunization of man against sporozoite-induced falciparum malaria. Am J Med Sci 1973;266(3):169-177. 8. Laurens MB, Duncan CJ, Epstein JE, et al. A consultation on the optimization of controlled human malaria infection by mosquito bite for evaluation of candidate malaria vaccines. Vaccine 2012;30(36):5302-5304. 9. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193(4254):673-675. 10. Burkot TR, Williams JL, Schneider I., Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans R Soc Trop Med Hyg. 1984;78:339-341. 11. Ponnudurai T, Leeuwenberg AD, Meuwissen JH. Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture. Trop Geogr Med 1981;33(1):50-54. 12. Walliker D, Quakyi IA, Wellems TE, et al. Genetic analysis of the human malaria parasite Plasmodium falciparum Science 1987;236(4809):1661-1666. 13. Williams JL, Innis BT, Burkot TR, Hayes DE, Schneider I. Falciparum malaria: accidental transmission to man by mosquitoes after infection with culture-derived gametocytes. Am J Trop Med Hyg 1983;32(4):657-659. 14. Chulay JD, Schneider I, Cosgriff TM, et al. Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum Am J Trop Med Hyg 1986;35(1):66-68. 15. Herrington DA, Clyde DF, Murphy JR, et al. A model for Plasmodium falciparum sporozoite challenge and very early therapy of parasitaemia for ef cacy studies of sporozoite vaccines. Trop Geogr Med 1988;40(2):124-127. 16. Davis JR, Murphy JR, Clyde DF, et al. Estimate of Plasmodium falciparum sporozoite content of Anopheles stephensi used to challenge human volunteers. Am J Trop Med Hyg 1988;40(2):128-130. 17. Menard R. The journey of the malaria sporozoite through its hosts: two parasite proteins lead the way. Microbes Infect 2000;2:633-642. 18. Rickman LS, Jones TR, Long GW, et al. Plasmodium falciparum -infected Anopheles stephensi inconsistently transmit malaria to humans. Am J Trop Med Hyg. 1990;43(5):441-445. 19. Fries LF, Gordon DM, Schneider I, et al. Safety, immunogenicity, and ef cacy of a Plasmodium falciparum vaccine comprising a circumsporozoite protein repeat region peptide conjugated to Pseudomonas aeruginosa Toxin A. Infect Immun 1992;60(5):1834-1839. 20. Lyke KE, Laurens M, Adams M, et al. Plasmodium falciparum malaria challenge by the bite of aseptic Anopheles stephensi mosquitoes: results of a randomized infectivity trial. PLoS One 2010;5(10):e13490. 21. Laurens M, Billingsley P, Richman A, et al. Successful human infection with P falciparum using three Anopheles stephensi mosquitoes: a new model for controlled human malaria infection. PLoS One 2013;8(7):e68969. 22. Hoffman SL, Goh LML, Luke TC, et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis 2002;185(8):1155-1164. 23. Spring M, Murphy J, Nielsen R, et al. First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers. Vaccine 201331(43):4975-83. 24. Roestenberg M, Bijker EM, Sim BK, et al. Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites. Am J Trop Med Hyg 2013;88(1):5-13. 25. Vaughan JA, Scheller LF, Wirtz RA, Azad AF. Infectivity of Plasmodium berghei sporozoites delivered by intravenous inoculation versus mosquito bite: implications for sporozoite vaccine trials. Infect Immun. 1999;67(8):4285-4289. 26. Sinnis P, Zavala F. The skin: where malaria infection and the host immune response begin. Semin Immunopathol 2012;34(6):787-792. 27. Sack BK, Miller JL, Vaughn AM, et al. Model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum. Infect Immun 2014;82(2):808-817. 28. Jiang H, Yi M, Mu J, et al. Detection of genome wide polymorphisms in the AT rich Plasmodium falciparum genome using a high density microarray. BMC Genomics 2008; 9: 398. 29. Golenda CF, Li J, Rosenberg R. Continuous in vitro propagation of the malaria parasite Plasmodium vivax Proc Natl Acad Sci USA. 1997;94:6786-6791. 30. Murphy JR, Weiss WR, Fryauff D, et al. Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies. Malar J 2014;13:215.

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24 http://www.cs.amedd.army.mil/amedd_journal.aspx31. Bennett KE, Olson KE, de Lourdes Munoz M, et al. Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States. Am J Trop Med Hyg 2002;67(1): 85-92. 32. Sim S, Jupatanakul N, Ramirez JL, et al. Transcriptomic pro ling of diverse Aedes aegypti strains reveals increased basal-level immune activation in dengue virus-refractory populations and identi es novel virus-vector interactions. PLoS Negl Trop Dis 2013;7(7):e2295. 33. Mores CN, Christofferson RC, Davidson SA. The role of the mosquito in a dengue human infection model. J Infect Dis 2014;209(suppl 2):S71-S78.AUTHORSDr Garver is a Malariologist in the Vector and Parasite Biology Department, Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland. Ms Dowler is a Biologist in the Vector and Parasite Biology Department, Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland. MAJ Davidson is Chief, Vector and Parasite Biology Department, Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland.CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH: THE PAST, PRESENT, AND FUTURE FROM AN ENTOMOLOGICAL PERSPECTIVE

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July – September 2015 25Laos People’s Democratic Republic (Lao PDR) (18 00 N; 105 00 E; area 236,800 km2) is a landlocked Southeastern Asian country, surrounded by 5 countries: Burma, Cambodia, China, Thailand, and Vietnam.1 These 5 countries, together with the Lao PDR, formed the Greater Mekong Subregion (GMS), which have a combined population of 92 million. Vector-borne diseases have a signi cant effect on morbidity in these countries, and of these diseases, malaria causes more deaths in remote and border areas.2,3 In addition to malaria and high heterogeneity in Plasmodium falciparum (Welch) risk,4 dengue, scrub typhus, Japanese encephalitis,5 and lariasis6 are common insect-borne diseases in the GMS. However, their effects on human populations are poorly characterized and the taxonomic identities of most vectors should be studied and clari ed. The mosquito fauna of the Lao PDR are not well known, except for several scattered reports .7-14 In this study, we updated the records and checklist of mosquito species from the Lao PDR based on the literature, specimens deposited at the US National Mosquito Collections (USNMC), National Museum of Natural History (NMNH), Smithsonian Institution, Washington, DC, and our latest specimen collections from Khammuane Province, particularly at the Phou Hin Poun National Biodiversity Conservation Area (PHP NBCA). This area, which has a human population of approximately 30,000, is located in a limestone tower karst region of the Annamite Range in Khammuane Province. It is composed mainly of rugged caves, porous karst terrain, and dry evergreen forest and scrubland. It is also the home to a number of rare or newly discovered species of animals.15-17 We are in the process of con rming the identi cation of several species of mosquitoes and sand ies, and possibly describing new species from our recent collections in the area. MATERIALS AND METHODSMosquito Field Collection, Museum Specimens and IdentificationSpecimen collections were conducted from May 1 to May 31, 2012, and from February 21 to March 10, 2014, from various areas in the PHP NBCA (17.99524 N, 104.82108 E), Ban Natan, Nakai District, Khammuane Province (Figure 1). Adults were collected using modi ed Centers for Disease Control and Prevention traps (Figure 2A, B) with light attractants, and were suspended about 1.3 m above ground level on selected sites and inside the caves. Larvae were collected using a standard larval dipper (350 ml, 13 cm diameter: BioQuip, Rancho Dominguez, CA) (Figure 2C, D) from various habitats including water pockets along edges of rivers, rock holes, temporary pools in between rocks, caves, etc (Figures 2, 3, and 4). They were individually link-reared to adult stage, as morphological voucher specimens for this work. Emergent adults were pinned on paper points, each given a unique collection number, properly labeled, and identi ed using diagnostic morphological characters.18-23 Voucher specimens were deposited at the USNMC NMNH, Smithsonian Institution, Washington, DC, USA, and at the Entomology Laboratory, Institut Pasteur du Laos, Vientiane, Lao PDR. In addition, old mosquito specimens at the NMNH repository were examined, and their collection data were recorded.Mosquito Fauna of Lao People’s Democratic Republic, With Special Emphasis on the Adult and Larval Surveillance at Nakai District, Khammuane Province Leopoldo M. Rueda, PhD Jeffrey Hii, PhD Khamsing Vongphayloth, MD Mustapha Debboun, PhD James E. Pecor, BS Paul T. Brey, PhD LCDR Ian W. Sutherland, USNABSTRACTThis article includes the distribution records and updated checklist of mosquitoes (Culicidae, Diptera) from the Lao People’s Democratic Republic (PDR), based on the literature, specimens deposited at the US National Museum of Natural History mosquito collections, and our recent eld collections from the Nakai District, Khammuane Province. Ten of 101 species in the updated checklist of mosquitoes are new records for the Lao PDR.

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26 http://www.cs.amedd.army.mil/amedd_journal.aspxRESULTSThe summary of mosquito collections from the PHP NBCA, Khammuane Province is presented in Table 1. Figure 1 shows the map of the Lao PDR, with 10 of 16 provinces, Vientiane (capital city) and PHP NBCA (all with asterisks as shown in the map) where adult and larval mosquitoes were collected or reported in the literature. In the PHP NBCA, mosquito habitats included water pockets along edges of rivers, rock holes, temporary pools along the edges of rivers, in between rocks, and in caves (Figures 2-4). A total of 43 mosquito taxa were collected from PHP NBCA in 9 genera ( Aedes Anopheles Culex Heizmannia Mansonia Orthopodomyia Topomyia Toxorhynchites Tripteroides ). Among the 3 genera examined, Aedes (19 species) had the greatest number of species, followed by Culex (8 species) and Anopheles (7 species). Only 18 species out of 43 (42%) were morphologically identi ed, while the rest (25 species; 58%) need further analyses (including molecular techniques) to clarify their taxonomic identities. Known or potential vectors of human infectious diseases were also collected from PHP NBCA, including Aedes vexans (Meigan), Ae albopictus (Skuse), and several uncon rmed species of Anopheles (Anopheles) An (Cellia ), Culex (Culex) and Mansoni a. An updated checklist of mosquitoes in the Lao PDR (Table 2) includes a total of 101 species. They are in 16 genera, namely Aedes (22 species), Anopheles (33), Armigeres (14), Coquillettidia (2), Culex (12), Ficalbia (1), Heizmannia (1), Hodgesia (1), Mansonia (4), Mimomyia (2), Orthopodomyia (1), Topomyia (1), Toxorhynchites (2), Tripteroides (2), Uronotanea (2), Verrallina (1). About 80 of 101 species were reported in the Walter Reed Biosystematics Unit (WRBU) catalog,23 2 species found from the Smithsonian/NMNH collections, 17 species from current PHP NBCA collections, and the remaining species from the literature. About 10 species of mosquitoes are new records for the Lao PDR. They include 9 species under 7 subgenera of the genus Aedes and one species in the genus Orthopodomyia (Table 2). COMMENTThe Lao PDR, like other countries comprising the GMS, has a high biodiversity of vector species, a great number of mosquito species complexes, enormous spatial heterogeneity in distribution patterns, and extensive behavioral plasticity both between and within species2. In 19347 and 1938,8 Anopheles mosquitoes were reported in the Laos PDR (Table 2). In December 1999, malaria vector surveys were carried out by Vythilingam et al11 in 7 provinces, namely Borikhamxay, Champasak, Luangprabang, Saravane, Savannakhet, Xayaboury, and Sekong, and in the capital city of Vientiane in the Lao PDR. Using bare leg collections from indoors and outdoors from 6 PM to 5 PM, a total of 438 Anopheles mosquitoes belonging to 19 species were obtained. Of these, only 3 species were found infected with oocysts, namely An maculatus Theobald, An dirus Peyton and Harrison, and An minimus Theobald. Anopheles aconitus Doenitz was the predominant species in the 1999 collection, but its vectorial status was unknown. The prevalence of Anopheles and epidemiology of malaria were also reported in the provinces of Xekong12 and Attapeu.13,14 In 2014, Hii and Rueda2 listed 3 species in Anopheles (Anopheles) and 20 species in Anopheles (Cellia) in the Lao PDR including known and potential MOSQUITO FAUNA OF LAO PEOPLEÂ’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE Figure 1. Map of Lao PDR showing 16 provinces and the national capital Vientiane. Key to Provinces: AU=Attapu O U=Oudomxai BO=Bokeo PH=P hongsali BL=Borikhamxay S A=Saravane CH=Champasak S V=Sav annakhet HO=Houaphan V I=Viangchan KH=Khammuane X A=Xay aboury LO=Louangnamtha X E=Sekong LU=Luangprabang X I=Xiangkhouang VT indicates national capital city area of V ientiane. P indicates Phou Hin Poun National Biodiversity Conservation Area, Nakai District where recent collections were conducted. Provinces or localities where mosquitoes were collected or reported in the literature. BO* LO PH OU XA* LU* HO VI XI BL* KH* P* SV* SA* XE* AU* CH* VT*

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July – September 2015 27THE ARMY MEDICAL DEPARTMENT JOURNAL malaria vectors in countries of the Mekong Subregion. While there are numerous examples of An dirus mostly feeding outdoors and much earlier in the evening,24,25 Vythilingam et al13 reported an unusual stereotypical nocturnal indoor and late feeding behavior in Attapeu province, Laos PDR. In 2002, Tsuda et al10 conducted an ecological survey of Aedes dengue vectors in the central part of the Lao PDR. A new hydroelectric project, Nam Theun 2, created ideal conditions for Aedes aegypti (Linnaeus) breeding in water storage jars and tires, and Ae albopictus was abundant.26The present study indicates the species diversity of mosquitoes in the Lao PDR. The dif culty in doing morphological comparisons among species warrants further molecular analysis to ascertain taxonomic identities and to clarify hierarchic classi cations. With the diversity of the habitats, particularly the caves and surrounding areas, we expect that more unknown species will be collected and described in the near future. Deforestation, water resources and management,27,28 conventional agricultural practices, and unregulated destruction of many habitats are major human activities that may adversely affect the oral and animal fauna of the Lao PDR, including the creation or elimination of suitable breeding sites of mosquitoes and other arthropods. While habitats in some government protected areas are not hugely damaged yet, continuous inventories of arthropod fauna, particularly those groups (mosquitoes, sand ies, ticks, mites, etc) with known disease vectors, should be conducted to accumulate much needed data for developing strategies to manage and control infectious human diseases. Proper vector surveillance, including ecological surveys, should be performed in areas where human diseases (malaria, dengue, tick-borne viruses, lariasis, etc) are common and severely affect the local human populations. The updated checklist of mosquitoes in this article (including several vector species) may help health personnel in mapping out some risk areas for infectious diseases in the Lao PDR. ACKNOWLEDGMENTWe express sincere gratitude and appreciation to Lea Thutkhin for collecting, processing, mounting and pinning specimens, and staff of the Institut Pasteur du Laos for assistance; to Sonexay Ounekham of the Entomology Unit, Center for Malaria, Parasitology, and Entomology, Vientiane, for help in collecting specimens in Khammuane Province. We are Table 1. Summary of collected mosquito adults and larvae in Phou Hin Poun NBCA, Ban Natan, Nakai District, Khammuane Province, Lao PDR (17.99524 N, 104.82108 E), from May 1 thru May 31, 2012, and February 21 thru March 10, 2014.Species Sex*Collection no.Aedes (Aedimorphus) alboscutellatus (Theobald)3FLN-048, 050, 060Aedes (Aedimorphus) sp 1FLN-012Aedes (Aedimorphus) vexans (Meigen)1FLN-047Aedes (Bothealla) eldridgei Reinert3F†LN-002,041, 068Aedes (Bothealla) sp 3F, 1MLN-018, 022,023, 069Aedes (Collessius) sp 1FLN-013Aedes (Downsiomyia) ganapathi Colless1FLN-024Aedes (Downsiomyia) harinasutai Knight1FLN-001Aedes (Downsiomyia) sp 1MLN-008Aedes (Fredwardsius) vittatus (Bigot)2F, 1MLN-015,065, 066Aedes (Hulecoeteomyia) chrysolineatus (Theobald)1FLN-035Aedes (Hulecoeteomyia) formosensis Yamada1F, 1MLN-036, 037Aedes (Hulecoeteomyia) sp (near reinerti or formosensis )1F, 1MLN-031, 046Aedes (Kenknightia) dissimilis (Leicester)1FLN-063Aedes (Kenknightia) sp 1FLN-044Aedes (Stegomyia) albopictus (Skuse)1FLN-043Aedes (Stegomyia) pseudoscutellaris (Theobald)1FLN-039Aedes (Tewarius) pseudonummatus Reinert2FLN-003Aedes sp 4F, 2MLN-011, 013, 019,020, 026, 042Anopheles (Anopheles) sp (Barbirostris Group) 1FLN-011Anopheles (Anopheles) sp (Asiaticus Group) 1FLN-049Anopheles (Anopheles) sp 1FLN-046Anopheles (Anopheles) sp (Culiciformis Group)1FLN-062Anopheles (Cellia) pseudowillmori Theobald1FLN-045Anopheles (Cellia) sp (Leucosphyrus Group)1FLN-005Anopheles (Cellia) sp 2FLN-009, 010Coquillettidia (Coquillettidia) ochracea (Theobald)1FLN-004Culex (Culex) sp (Vishnui Complex) 1FLN-052Culex (Culex) sp (Sitiens Group) 1FLN-054Culex (Culex) sp 3FLN-028,055, 071Culex (Culex) tritaeniorhynchus Giles1FLN-053Culex (Culiciomyia) nigropunctatus Edwards2F, 1MLN-064, 074,075Culex (Culiciomyia) sp 1F, 1MLN-067, 070Culex (Eumelanomyia) sp (Temipalpus Complex)1F, 2MLN-017, 072, 073Culex (Lophoceraomyia) sp 1F, 1MLN-007, 016 Heizmannia sp 3FLN-025, 038, 059Mansonia (Mansonioides) uniformes (Theobald)1F LN-057Mansonia sp 1FLN-051Orthopodomyia albipes Leicester1MLN-033Orthopodomyia sp 1FLN-032Topomyia sp 1FLN-030Toxorhynchites sp 3M†LN-076, 077, 078Tripteroides sp 2F, 1MLN-021, 040, 058 *F indicates female adult, M indicates male adult.†Larvae collected using plastic larval dipper.

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28 http://www.cs.amedd.army.mil/amedd_journal.aspxMOSQUITO FAUNA OF LAO PEOPLEÂ’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE Table 2A. Updated checklist of mosquito species from Lao PDR.SpeciesReferenceaAedes (Aedimorphus) alboscutellatus (Theobald)14, 23, X Aedes (Aedimorphus) pipersalatus (Giles)14, 23Aedes (Aedimorphus) vexans (Meigen)14, 23, X Aedes (Bothaella) eldridgei Reinert b X Aedes (Collessius) macfarlanei (Edwards)23Aedes (Diceromyia) iyengari Edwards14, 23Aedes (Downsiomyia) ganapathi Colless b X Aedes (Downsiomyia) harinasutai Knight b X Aedes (Downsiomyia) niveus (Ludlow)23Aedes (Fredwardsius) vittatus (Bigot)10, X Aedes (Hulecoeteomyia) chrysolineatus (Theobald)14, 23, X Aedes (Hulecoeteomyia) formosensis Yamada b X Aedes (Hulecoeteomyia) reinerti Rattanarithikul and Harrison b X Aedes (Kenknightia) dissimilis (Leicester) b X Aedes (Neomelaniconion) lineatopennis (Ludlow)19Aedes (Paraedes) ostentatio (Leicester)19Aedes (Phagomyia) prominens (Barraud) b X Aedes (Stegomyia) albopictus (Skuse)14, X Aedes (Stegomyia) aegypti (Linnaeus)10, 23, M Aedes (Stegomyia) pseudalbopictus Borel14, 23Aedes (Stegomyia) pseudoscutellaris (Theobald) b X Aedes (Tewarius) pseudonummatus Reinert b X Anopheles (Anopheles) albotaeniatus (Theobald)11Anopheles (Anopheles) argyropus (Swellengrebel)2Anopheles (Anopheles) baileyi Edwards23Anopheles (Anopheles) barbirostris Van der Wulp2, 8, 11, 14, 23Anopheles (Anopheles) donaldi Reid2, 14, 23Anopheles (Anopheles) sinensis Wiedemann3, 8Anopheles (Anopheles) umbrosus (Theobald)12Anopheles (Cellia) aconitus Doenitz2, 7, 11, 14, 23Anopheles (Cellia) annularis Van der Wulp2Anopheles (Cellia) culicifacies Giles2, 7, 23Anopheles (Cellia) dirus Peyton and Harrison2, 10, 11, 12, 23Anopheles (Cellia) dravidicus Christophers2, 14, 23Anopheles (Cellia) harrisoni Harbach and Manguin23Anopheles (Cellia) inde nitus (Ludlow)2, 23Anopheles (Cellia) jamesii Theobald2, 23Anopheles (Cellia) jeyporiensis James2, 7, 8, 12, 23Anopheles (Cellia) karwari (James)2, 11, 12, 23Anopheles (Cellia) kochi Donitz2, 8, 11, 14, 23Anopheles (Cellia) maculatus Theobald2, 7, 8, 11, 12, 14, 23Anopheles (Cellia) minimus Theobald2, 8, 11, 12, 14, 23Anopheles (Cellia) nivipes (Theobald)11, 12, 14, 23Anopheles (Cellia) notanandai Rattanarithikul and Green2, 14, 23Anopheles (Cellia) pallidus Theobald11, 12a X indicates observed, eld collection; M indicates observed, Smithsonian/National Museum of Natural History museum collection.b New record for Lao PDR. Table 2B. Updated checklist of mosquito species from Lao PDR (continued).SpeciesReferenceaAnopheles (Cellia) pampanai Buttiker and Beales11 14 23Anopheles (Cellia) philippinensis Ludlow2 8 11 12 14 23Anopheles (Cellia) pseudowillmori Theobald2 14 23 X Anopheles (Cellia) rampae Harbach and Somboon29Anopheles (Cellia) sawadwongporni Rattanarithikul and Green14 23Anopheles (Cellia) splendidus Koidzumi11 12 14 23Anopheles (Cellia) subpictus Grassi2 11Anopheles (Cellia) sundaicus (Rodenwaldt)2Anopheles (Cellia) tessellatus Theobald2 11 14 23Anopheles (Cellia) vagus Donitz2 7 8 11 12 14Anopheles (Cellia) varuna Iyengar2 11 12 14 23Armigeres (Armigeres) aureolineatus (Leicester)23Armigeres (Armigeres) durhami (Edwards)23Armigeres (Armigeres) kuchingensis Edwards23Armigeres (Armigeres) laoensis Toma and Miyagic 23 M Armigeres (Armigeres) moultoni Edwards23Armigeres (Armigeres) setifer Del nado14 23Armigeres (Armigeres) subalbatus (Coquillett)14 23Armigeres (Armigeres) theobaldi Barraud14 23Armigeres (Leicesteria) annulitarsis (Leicester)23Armigeres (Leicesteria) dolichocephalus (Leicester)23Armigeres (Leicesteria) avus (Leicester)23 M Armigeres (Leicesteria) longipalpis (Leicester)23Armigeres (Leicesteria) magnus (Theobald)23Armigeres (Leicesteria) pectinatus (Edwards)23Coquillettidia (Coquillettidia) crassipes (Van der Wulp)14 23Coquillettidia (Coquillettidia) ochracea (Theobald)23 X Culex (Culex) fuscocephala Theobald14 23Culex (Culex) gelidus Theobald23Culex (Culex) hutchinsoni Barraud14 23Culex (Culex) pseudovishnui Colless14 23Culex (Culex) quinquefasciatus Say14 23 M Culex (Culex) tritaeniorhynchus Giles14 23 X Culex (Culex) vishnui Theobald14 23Culex (Culex) whitmorei (Giles)14 23Culex (Culiciomyia) nigropunctatus Edwards14 23 X Culex (Oculeomyia) bitaeniorhynchus Giles14 23Culex (Oculeomyia) pseudosinensis Colless14 23Culex (Oculeomyia) sinensis Theobald14 23Ficalbia minima (Theobald)23Heizmannia (Heizmannia) complex (Theobald)23Hodgesia malayi Leicester23a X indicates observed, eld collection; M indicates observed, Smithsonian/National Museum of Natural History museum collection.c Holotype male, 1 paratype female, 4 females, 3 whole larvae, and 3 larval exuviae, deposited in the Smithsonian/National Museum of Natural History museum collection.

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July – September 2015 29THE ARMY MEDICAL DEPARTMENT JOURNALFigure 2. Nakai District cave showing mosquito adult wall resting areas (A, D) and larval habitats inside the cave (B, C). A mo di ed light trap hung from the cave wall (D at arrow). Samples were obtained from the cave water pocket (C at arrow) using a larv al dipper (C inset). A B D C Table 2C. Updated checklist of mosquito species from Lao PDR (continued).SpeciesReferenceaSpeciesReferenceaMansonia (Mansonioides) annulifera (Theobald)14, 23Toxorhynchites (Toxorhynchites) albipes (Edwards)23Mansonia (Mansonioides) dives (Schiner)14Toxorhynchites (Toxorhynchites) kempi (Edwards)23Mansonia (Mansonioides) indiana Edwards14, 23Tripteroides (Rachionotomyia) aranoides (Theobald)23Mansonia (Mansonioides) uniformes (Theobald)14, 23, X Tripteroides (Rachionotomyia) ponmeki Miyagi and Toma9, 23Mimomyia (Mimomyia) chamberlaini Ludlow23Uranotaenia (Pseudo calbia) nivipleura Leicester23, M Mimomyia (Mimomyia) hybrida (Leicester)23Uranotaenia (Pseudo calbia) novobscura Barraud23, M Orthopodomyia albipes LeicesterbX Verrallina (Verrallina) dux (Dyar and Shannon)23Topomyia (Topomyia) gracilis Leicester23a X indicates observed, eld collection; M indicates observed, Smithsonian/National Museum of Natural History museum collection.b New record for Lao PDR. grateful to Yiau-Min Huang, Jeffrey Clark, and Belen P. Rueda for reviewing this manuscript and for their valuable comments. Special thanks go to the staff of the Public Health Of ce, Nakai District, Khammuane Province for cooperation and eld assistance. Partial funding was provided by the US Naval Medical Research Center-Asia through the Global Emerging Infections Surveillance and Response System, a Division of the US Armed Forces Health Surveillance Center, Silver Spring, MD. Institut Pasteur du Laos and the Lao Ministry of Health also contributed to support this project. This research was performed under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organizations.

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30 http://www.cs.amedd.army.mil/amedd_journal.aspxREFERENCES1. Central Intelligence Agency. Laos. The World Factbook (internet). Available at: https://www.cia. gov/library/publications/the-world-factbook/geos/ la.html. Accessed January 15, 2015. 2. Hii J, Rueda LM. Malaria vectors in the Greater Mekong Subregion: overview of malaria vectors and remaining challenges. Southeast Asian J Trop Med Public Health. 2013;44(suppl 1):73-165. 3. Hewitt S, Delacollette C, Chavez I. Malaria situation in the Greater Mekong Subregion. Southeast Asian J Trop Med Public Health. 2013;44(suppl 1):46-72. 4. Jorgensen P, Nambanya S, Gopinath D, et al. High heterogeneity in Plasmodium falciparum risk illustrates the need for detailed mapping to guide resource allocation: a new malaria risk map of the Lao PeopleÂ’s Democratic Republic. Malaria J 2010;9:59. 5. Mayxay M, Castonquay-Vanier J, Chansamouth V, et al. Causes of non-malarial fever in Laos: a prospective study. Lancet Glob Health [serial online]. 2013;1(1):46-54. Available at: http://www.malaria journal.com/content/9/1/59. Accessed April 27, 2015. 6. Knight Y, Chanthavisouk C, Nakhonosesid-Fish V, Chindavongsa K, Michael E, Aratchige P. A survey of lymphatic lariasis using ICT test in Attapeu Province, Lao PDR. Int J Infect Dis 2010;14(suppl 1):e276. Available at: http://dx.doi.org/10.1016/j. ijid.2010.02.2100. Accessed April 27, 2015. 7. Gaschen H. Prospection entomologique au Laos. B ull Soc Medico-chirurgicale Indochine 1934;12(5):1-5. 8. Lefebvre M. Recherches sur la faune Anophelinae au Laos. Bull Soc Pathol Exot Filiales 1938;31:381-386. 9. Miyagi I, Toma T. Tripteroides (Rachionotomyia) ponmeki (Diptera: Culicidae): a new species from Khammouane Province, Lao PDR. Med Entomol Zool 2001;52(4):269-277.MOSQUITO FAUNA OF LAO PEOPLEÂ’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCEFigure 3. Nakai District river showing mosquito larval habitats along river edge (A), and in rock holes (B, C, D). B D C A

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July – September 2015 31THE ARMY MEDICAL DEPARTMENT JOURNAL10. Tsuda Y, Kobayashi J, Nambanya S, Miyagi I, Toma T, Phompida S, Manivang K. An ecological survey of dengue vectors in Central Lao PCDR. Southeast Asian J Trop Med Public Health 2002;33(1):63-67. 11. Vythilingam I, Keokenchanh K, Phommakot S, Nambanya S, Inthakone S. Preliminary studies of Anopheles mosquitoes in eight provinces in Lao PDR. Southeast Asian J Trop Med Public Health. 2001;32(1):83-87. 12. Vythilingam I, Phetsouvanh R, Keokenchanh K, et al. The prevalence of Anopheles (Diptera: Culicidae) mosquitoes in Sekong Province, Lao PDR in relation to malaria transmission. Trop Med Int Health. 2003;8(6):525-635. 13. Vythilingam I, Sidavong B, Chan ST, et al. Epidemiology of malaria in Attapeu Province, Lao PDR in relation to entomological parameters. Trans R Soc Trop Med Hyg. 2005;99(11):833-839. 14. Vythilingam I, Sidavong B, Thim CS, Phonemixay T, Phompida S, Jeffrey J. Species composition of mosquitoes of Attapeu Province, Lao’s People Democratic Republic. J Am Mosq Control Assoc. 2006;22:140-143. 15. Wikipedia [online]. Phou Hin Poun National Biodiversity Conservation Area. Available at: http:// en.wikipedia.25es of Thailand. II. Genera Culex and Lutzia Southeast Asian J Trop Med Public Health. 2005;36(suppl 2):1-97. 16. Lao National Tourism Administration. Phou Hin Poun NBCA. Ecotourism Laos web site. Available at: http://www.ecotourismlaos.com/directory/pro tected_areas/phouhinpoun.htm. Accessed January 15, 2015. 17. Musser GG, Smith AL, Robinson MF, Lunde DP. Description of a new genus and species of rodent (Murinae, Muridae, Rodentia) from the Khammouan Limestone National Biodiversity Conservation Area in Lao PDR. American Museum Novitates 2005;3497:1-31. Available at: http://digi tallibrary.amnh.org/dspace/handle/2246/5666. Accessed April 28, 2015.Figure 4. Nakai District river and tributaries showing typical mosquito larval habitats including river edge with oating grasses (A, B), water pocket (C inset), and temporary water pool (D). A C B D

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32 http://www.cs.amedd.army.mil/amedd_journal.aspx18. Rattanarithikul R, Harbach RE, Harrison BA, Panthusiri P, Jones JW, Coleman RE. Illustrated key to the mosquitoes of Thailand. II. Genera Culex and Lutzia Southeast Asian J Trop Med Public Health. 2005;36(suppl 2):1-97. 19. Rattanarithikul R, Harbach RE, Panthusiri P, Peyton EL, Coleman RE. Illustrated key to the mosquitoes of Thailand. III. Genera Aedeomyia, Ficalbia, Mimomya, Hodgesia, Coquillettidia, Mansonia and Uranotaenia Southeast Asian J Trop Med Public Health. 2006;37(suppl 1):1-85. 20. Rattanarithikul R, Harrison BA, Harbach RE, Panthusiri P, Coleman RE. Illustrated key to the mosquitoes of Thailand. IV. Anopheles Southeast Asian J Trop Med Public Health. 2006;37(suppl 2):1-128. 21. Rattanarithikul R, Harbach RE, Harrison BA, Panthusiri P, Coleman RE. Illustrated key to the mosquitoes of Thailand. V. Genus Orthopodomyia, Kimia, Malaya, Topomyia, Tripteroides, and Toxorhynchites Southeast Asian J Trop Med Public Health. 2007;38(suppl 2):1-65. 22. Rattanarithikul R, Harbach RE, Harrison BA, Panthusiri P, Coleman RE, Richardson JH. Illustrated key to the mosquitoes of Thailand. VI. Tribe Aedini. Southeast Asian J Trop Med Public Health. 2010;41(suppl 1):1-225. 23. Walter Reed Biosystematics Unit. Keys to the Medically Important Mosquito Species [database online]. Suitland, MD: Walter Reed Biosystematics Unit, Smithsonian Institution; 2015. Available at: http://www.wrbu.org/command_aors_MQkeys. html. Accessed January 15, 2015. 24. Obsomer V, Defourny P, Coosemans M. The Anopheles dirus complex; spatial distribution and environmental factors. Malaria J 2007;6:26. 25. Killeen GF. Characterizing, controlling and eliminating residual malaria transmission. Malaria J 2014;13:330. 26. Hiscox A, Kaye A, Vongphayloth K, et al. Risk factors for the presence of Aedes aegypti and Aedes albopictus in domestic water-holding containers in areas impacted by the Nam Theun 2 hydroelectric project, Laos. Am J Trop Med Hyg 2013;88:1070-1078. 27. Erlanger TE, Sayasone S, Krieger GR, et al. Baseline health situation of communities affected by the Nam Theun 2 hydroelectric project in central Lao PDR and indicators for monitoring. Int J Env Health Res. 2008;18:223-242. 28. Ziegler AD, Petney TN, Grundy-Warr C, et al. Dams and disease triggers on the lower Mekong river. PLoS Negl Trop Dis. 2013;7:e2166.AUTHORSDr Rueda is a Research Entomologist, Principal Investigator, and former Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. Dr Vongphayloth is a Medical Doctor and Entomologist, Institut Pasteur du Laos, Vientiane, Laos PDR Mr Pecor is a Museum Specialist at the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. LCDR Sutherland is the Chief of Entomological Sciences, US Naval Medical Research Center – Asia located at the U.S. Navy Region Center, Sembawang, Singapore. Dr Hii, formerly a WHO Malaria Scientist, is an Adjunct Principal Research Fellow in the School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University based in Bangkok, Thailand. Dr Debboun is the Director, Mosquito Control Division, Harris County Public Health & Environmental Services, Houston, TX. Dr Brey is a Research Entomologist and Director of the Institut Pasteur du Laos, Vientiane, Laos PDR.MOSQUITO FAUNA OF LAO PEOPLE’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE ErratumIn the article “A Heart Gripping Case: Carcinoid Heart Disease” published on pages 93-96 of the January-March 2015 issue of the AMEDD Journal the byline entry “Capt John P. Magulik” is incorrect. The correct byline entry is “Capt John P. Magulick.” The article “Performance Differences Between Male and Female Marines on Standardized Physical Fitness Tests and Combat Proxy Tasks: Identifying the Gap” appearing on pages 12-21 in the print edition of the April-June 2015 issue of the AMEDD Journal has been retracted by the authors. The article does not appear in the online digital version of that issue, nor does data describing the article appear in the PubMed MEDLINE record database.

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July September 2015 33Phlebotomine sand ies (Subfamily Phlebotominae, Family Psychodidae, Order Diptera) are of major health importance because they are capable of transmitting pathogens, including protozoans ( Leishmania ), bacteria ( Bartonella ), and viruses (Phleboviruses, sand y fever).1 Like mosquitoes, only female sand ies, particularly species of Phlebotomus and Lutzomyia suck blood, including humans. Species of Sergentomyia species primarily feed on reptiles, and rarely bite man.2 Of approximately 900 sand y species, only about 70 species are capable of transmitting protozoan Leishmania parasites that cause visceral leishmaniasis (kala-azar) and various forms of cutaneous leishmaniasis (oriental sore, espundia, etc.) in man.3,4 A few sand y species have been associated with Phlebovirus and other viruses,3-6 and only one, Lutzomyia verrucarum (Townsend) sensu lato can transmit the bacterium Bartonella bacilliformis (Strong, Tyzzer, Brues, Sellards and Gastiaburu) causing bartonellosis (Oroya fever, Carrion’s disease) in the Andean Region of South America.7,8 Ready9 reviewed the biology of Phlebotomine sand ies as vectors of disease agents, including the transmission cycles of human leishmaniasis both in the Old and New Worlds, mostly in rural communities. Additional Phlebotomine reviews also focused on sand y biology,10 and emphasis on leishmaniasis control.11Leishmaniasis has a great impact on military operations, particularly those of the United States.12 Since World War II, more than 1,000 US service personnel were infected with cutaneous leishmaniasis.13 In Afghanistan (Operation Enduring Freedom, OEF) and Iraq (Operation Iraqi Freedom, OIF), more US soldiers have been exposed to signi cant leishmaniasis risk than any time since World War II. During the disease surveillance period from 2001-2006, there were 1,287 incident diagnoses/reports of leishmaniasis, both cutaneous (1,283 cases) and visceral (4 cases) forms, among OEF/ OIF deployers.13 Furthermore, in an effort to establish the Leishmaniasis Control Program (LCP) during OIF, US military entomologists conducted comprehensive phlebotomine sand y surveillance at Tallil Air Base (TAB), Iraq from April 2003-November 2004. They determined the biology and temporal distribution of sand ies at TAB, and noted the impact of sand y vectors on military operations, including the leishmanial threat to deployed troops in Iraq.14-16The phlebotomine sand ies are found between 50N and 40S, with the majority distributed in the tropics and subtropics, and none reported on Paci c Islands or in New Zealand. In the Old World, the anthropophilic Phlebotomus sand ies (and principally Leishmaniasis transmission) are con ned in the subtropics (particularly in dry, semiarid areas), with a few human biting species in Africa south of the Sahara and none in Southeast Asia (although Phlebotomus species are found). In the New World (Nearctic and Neotropical Regions), the Records and Distribution of New World Phlebotomine Sand Flies (Psychodidae, Diptera), With Special Emphasis on Primary Types and Species Diversity Leopoldo M. Rueda, PhD Desmond H. Foley, PhD David Pecor, BS Matthew Wolkoff, BAABSTRACTThis article includes the records and distribution of Phlebotomine sand ies (Psychodidae, Diptera) in the New World based on the specimen collections housed in 2 repositories, the US National Museum of Natural History and the Museum of Entomology, Florida State Collection of Arthropods. Approximately 128 species have primary types housed in the 2 repositories, including holotypes (47 species, 3 subspecies), “types” (7 species), allotypes (52 species, 6 subspecies), lectotypes (4 species), paratypes (93 species, 10 subspecies), and neoallotype (1 species), mounted on slides, with a total of 1,107 type slides. For species diversity, collection data from 24 countries in the sand y database were analyzed according to the number of species present, specimen records, decade of collections, and countries where collections were conducted.

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34 http://www.cs.amedd.army.mil/amedd_journal.aspxtransmission of leishmaniasis is mainly in the tropics (particularly in the forests and savanna areas) of South America.2In this article, we examine the types and related specimens of New World Phlebotomine sand ies housed in the US National Museum of Natural History (USNMNH), and those borrowed from the Museum of Entomology, Florida State Collection of Arthropods (MEFSCA). We record the collection data of sand ies, including their geographical distribution, past and present taxonomic arrangement and related information. The species occurrence and diversity of these sand ies, according to the number of collections for each country over certain periods, were analyzed and reported. Other collection or occurrence data of sand y specimens (including nontypes, from the Nearctic and Neotropical Regions) from the 2 repositories (USNMNH and MEFSCA) were also examined and recorded, and will be posted later to the Walter Reed Biosystematics Unit (WRBU)/VectorMap RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITYFigure 1. Phlebotomine sand y collection sites on the New World, based on specimens deposited in the USNMNH and MEFSCA.

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July September 2015 35THE ARMY MEDICAL DEPARTMENT JOURNAL website (www.vectormap.org).17 They may be helpful in developing world sand y taxonomic catalogs, and in creating sand y vector risk maps and prediction distribution models for WRBU/VectorMap. In addition to increasing the knowledge of sand y distribution, the collection holdings in these repositories, particularly the primary types, will assist future phlebotomine researchers in their taxonomic and related studies. MATERIALS AND METHODSSpecies Types and Related SpecimensNew World Phlebotomine sand y specimens used in this study are either housed in the USNMNH repository in Suitland, MD, or were borrowed from the MEFSCA in Gainesville, FL. The slide mounted specimens (about 10,000 slides in more than 300 slide boxes) were examined and their collection data were recorded. All collection data from both repositories were entered into the USNMNH/MEFSCA database. They were processed and used for analyses in this article. The primary types (holotypes, allotypes, neoallotype, paratypes, metatypes) of sand ies were examined for collection records and related information. Other sand y slides (more than 3,000 slides, mainly from Afrotropical and Palearctic Regions) housed in 5 other repositories were also examined and their collection or occurrence data were also recorded in separate databases. Those sand y repositories included Institut de Reserche pour le Developpement, Montpellier, France; Institut Pasteur, Paris, France; Museum National d’Historie Naturelle, Paris, France; and Royal Museum for Central Africa, Tervuren, Belgium. However, data from the above 5 repositories were not included in this article, but may be processed for another report.Species DiversityThe number of sand y species according to each country in the New World was compiled in MS Excel and maps were constructed in ArcMap 10.1 (ESRI, Redlands, CA). Georeferences for individual specimens were determined and uncertainty calculated using the point-radius method.18-20 Label data from each specimen was recorded verbatim and entered into an Excel spreadsheet. These text descriptions were then assigned coordinates using a web-based gazetteer.21 For named places, the geographic center of the locality was used as the latitude and longitude anchor. Once the coordinates were established, a measurement of uncertainty was calculated for each point. This measurement is de ned as the radius of a circle surrounding the coordinate anchor, indicating that the collection site is within this circle. The uncertainty measurement takes into consideration errors involving the extent of the named place, the geographic datum, map scale, and imprecision of collectors’ location descriptions. All information including the verbatim locality description, gazetteer results and geo-referencing calculations were recorded and will be available for user review via VectorMap.17 The sand y data from our database (USNMNH and MEFSCA) were sorted and ranked according to: ( a ) number of species per country, ( b ) number of collection records per species, and ( c ) number of records by decade of collections. RESULTSSpecies TypesThe list of New World Phlebotomine species with type specimens housed in the USNMNH and MEFSCA is shown in Table 1, using the new taxonomic arrangements.22,23 The number of slides for each type and species and the country of type origin are also included in Table 1 at the end of this article. A comparison of the new22,23 and old4 generic and subgeneric classi cations of types at both repositories is shown in Table 2 at the end of this article. About 139 species have primary types housed in those 2 repositories, including holotypes (49 species, 3 subspecies), “types” (8 species) allotypes (51 Figure 2. Map of the New Word showing the number of New World Phlebotomine sand y species in the USNMNH and MEFSCA sand y database according to country of collection. Number of Species 20-44 45-167 2 3 4 5-7 8-10 11-12 13-15 16-19

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36 http://www.cs.amedd.army.mil/amedd_journal.aspxspecies, 6 subspecies), paratypes (93 species, 10 subspecies), lectotypes (4 species), neoallotype (1 species), and metatype (1 species), mounted on slides, with a total of 1,113 type slides. The number of paratypes (103) ranged from 1 to 53/species or subspecies, with a total of 917. Those specimens on slides labeled “Type” could be considered as holotypes, but proper designations should be done later. Attempts to check for any additional primary types housed in the MEFSCA are still ongoing, and they will be listed in separate reports, if any types are found later. Species, without type specimens, will be posted later on the VectorMap website.17Species DiversityThe total number of specimen slides considered in this database numbered 2,743. Depending on the amount of location detail, georeference uncertainty ranged from highest (country only information recorded) through to lowest (country, province and village information recorded), with an average of 234,678 m (n=2,573). Twenty-four countries of the New World were represented in our database of species collection record. The maps showing the collections sites in the New World countries, based on the specimens housed in the USNMNH and MEFSCA, are presented in Figures 1 and 2. They include (from smallest to largest number of species occurring in the collection database): Belize=Cuba= Haiti=Jamaica=PuertoRico(2species)
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July September 2015 37THE ARMY MEDICAL DEPARTMENT JOURNAL the LUCID24 interactive keys for the New World Neotropical Phlebotomine sand ies, particularly Neotropical Region (South and Central America, Southern Command, SOUTHCOM). Twenty-four morphological keys for males and females of the Neotropical Region (South and Central America), were created by L. M. Rueda with assistance from the WRBU staff, particularly J. Stoffer for the Automontage images which are now posted at the WRBU website,25 namely: Phlebotomine Sand Fly Genera, Neotropical (SOUTHCOM): Females, Males Phlebotomine Sand Flies, Subgenera, Neotropical (SOUTHCOM): Females, Males Subgenus Dampfomyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Evandromyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Helcocyrtomyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Lutzomyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Nyssomyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Pintomyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Psathyromyia Sand Flies, Adults, Neotropical (SOUTHCOM): Males Subgenus Psychodopygus Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Sciopemyia Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Species Grp. Verrucarum Sand Flies, Adults, Neotropical (SOUTHCOM): Females, Males Subgenus Trichophoromyia Sand Flies, Adults, Neotropical (SOUTHCOM): MalesThe old arrangement of sand y taxa by Young and Duncan4 was followed in the above keys. Other interactive keys for Africa and Central, Eastern, and Southwest Asia are still in preparation by the WRBU staff. Concerning species diversity, a latitudinal biodiversity gradient was observed for mosquitoes, with species richness increasing toward the equator.26 For mosquitoes, the total number of species increases with geographic area, according to a linear log-log relationship, and island Figure 4. New World Phlebotomine sand y species ranked according to the number of records present in the USNMNH and MEFSCA sand y database. The top 3 most common species (and number of records) in the database are identi ed. Psychodopygus geniculatus (122) Nyssomyia ylephiletor (116) Lutzomyia panamensis (111) Species Rank Number of Records050100200300 250 150 20 0 40 60 80 100 120 140 First Year of Decade Number of Records 0 100 200 300 400 500 600 700 800 900 1940195019601970198019902000 19001910192019300 1 1 1 1410 551 97 252 648 831Figure 5. Number of New World Phlebotomine sand y specimen records in the USNMNH and MEFSCA sand y database according to decade of collection.

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38 http://www.cs.amedd.army.mil/amedd_journal.aspx countries are more species-rich and have a higher number of endemic species than do mainland countries. With 17 sand y taxa in the USNMNH/MEFSCA sand y database, Trinidad-Tobago is the most species-rich. Foley et al26 also found that this country is species-rich for mosquitoes, even in comparison with other island nations. As in mosquitoes, there appears to be little relationship (ie, no shared species) between the sand y fauna in the database for Trinidad-Tobago and Venezuela (the closest continental area), despite these countries having been joined during the Pleistocene.27 Separating the effects of sampling effort, taxonomic output and species richness may be dif cult, as a species-rich or endemic area will initially result in higher numbers of new species per sampling effort, and may attract the greatest sampling effort. According to Foley et al,26 Brazil, Panama, French Guiana, and Costa Rica had the highest number of mosquito species, including endemic species. With the exception of French Guiana, this pattern is also seen for sand ies from the USNMNH/MEFSCA database. The list of New World countries with above average species-level mosquito taxonomic output (type locations, taxonomic publications) included El Salvador, Venezuela, Brazil, Ecuador, Guatemala, Costa Rica, Panama, French Guiana, Belize, and Trinidad-Tobago, while Haiti and Uruguay were below average.26 The numbers of sand y species in the USNMNH/MEFSCA database from Haiti and Uruguay were also low, possibly re ecting a similar lack of taxonomic output. A number of assumptions and limitations are inherent in the present study. For example, Hijmans et al28 identi ed 4 types of bias that could apply in the present case, namely species bias (eg, oversampling species of sand ies due to greater abundance); speciesarea bias (eg, oversampling island endemics compared with mainland species); hotspot bias (eg, oversampling areas where previous studies indicated a high species richness); and infrastructure bias (eg, oversampling near roads and towns). Recently, about 12,000 slides of Phlebotomine sand ies were donated by retired COL Philip Lawyer to the USNMNH for safekeeping. These slides were temporarily mounted using HoyerÂ’s medium, and should be remounted permanently. Their locality data from slide labels and collection sheets will be retrieved and recorded. Additional collection data from other regions of the world (including Old World countries) will be loaded into VectorMap to enable further analysis of species diversity, and to create sand y vector distribution models that will be useful for leishmaniasis risk assessments. ACKNOWLEDGMENTSWe are very grateful to Richard Wilkerson and Thomas Gaf gan for help in loaning and retrieving sand y slides from the MEFSCA; to Gary J. Steck, Curator of Diptera, MEFSCA, for facilitating the loan of Phlebotomine sand y specimens to WRBU; to David Levin; Tracy Brown; Victoria Adeboye; Lori Makauskas; and WRBU interns, students, and volunteers for help in retrieving collection data from sand y slides; and to James Pecor for maintaining the sand y collections. This research was performed under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by the Armed Forces Health Surveillance CenterÂ’s Global Emerging Infections Surveillance and Response System Operations Division and the Military Infectious Diseases Research Program.REFERENCES1. Bayer HealthCare AG. Sand Fly-Borne Diseases. Companion Vector-borne Diseases Web site. Available at: http://www.cvbd.org/en/sand y-bornediseases/. Accessed March 24, 2015. 2. Lane R. Sand ies (Phlebotominae). In: Lane RP, Crosskey RW, eds, Medical Insects and Arachnids London, UK: Chapman & Hall; 1993:78-119. 3. Seccombe AK, Ready PD, Huddleston LM. A catalogue of Old World Phlebotomine sand ies (Diptera: Psychodidae, Phlebotominae). The Natural History Museum (London) Occasional Papers on Systematic Entomology 1993;8:1-58. 4. Young DG, Duncan MA. Guide to the Identi cation and Geographic Distribution of Lutzomyia Sand Flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae) Gainesville, FL: American Entomological Institute. 1994. Available at: http://www.amentinst.org/memoirs. php. Accessed May 1, 2015. 5. Depaquit J, Grandadam M, Fouque F, Andry PE, Peyre tte C. Arthropod-borne viruses transmitted by phlebotomine sand ies in Europe: a review. Eurosurveillance. 2010;15(10):1-8. 6. Tesh RB. The genus Phlebovirus and its vectors. Annu Rev Entomol. 1998;33:169-181. 7. Cohnstaedt LW, Beati L, Caceres AG, Ferro C, Munstermann LE. Phylogenetics of the phlebotomine sand y group Verrucarum (Diptera: Psychodidae: Lutzomyia ). Am J Trop Med Hyg. 2011;84:913-922. 8. Hambuch TM, Handley SA, Ellis B, Chamberlin J, Romero S, Regnery R. Population genetic analysis of Bartonella bacilliformis isolates from areas of Peru where CarrionÂ’s disease is endemic and epidemic. J Clin Microbiol. 2004;42:3675-3680. 9. Ready PD. Biology of Phlebotomine sand ies as vectors of disease agents. Annu Rev Entomol. 2013;58:227-250. RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

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July September 2015 39THE ARMY MEDICAL DEPARTMENT JOURNAL10. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sand ies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27:123-147. 11. Bates, PA, Depaquit J, Galati EAB, et al. Recent advances in phlebotomine sand y research related to leishmaniasis control. Parasit Vectors 2015;8:131. 12. Claborn DM. The biology and control of leishmaniasis vectors. J Glob Infect Dis. 2010;2(2):127-134. 13. Aronson NE. Leishmaniasis in relation to service in Iraq/Afghanistan, US Armed Forces, 2001-2006. MSMR 2007;14:2-5. 14. Coleman RE, Burkett DA, Putnam JL, et al. Impact of phlebotomine sand ies on US military operations at Tallil Air Base, Iraq: 1. Background, military situation, and development of a “Leishmaniasis Control Program”. J Med Entomol. 2006;43(4):647-642. 15. Coleman RE, Burkett DA, Sherwood V, et al. Impact of phlebotomine sand ies on US military operations at Tallil Air Base, Iraq: 2. Temporal and geographic distribution of sand ies. J Med Entomol. 2007;44(1):29-41. 16. Coleman RE, Hochberg LP, Swanson KI, et al. Impact of phlebotomine sand ies on US military operations at Tallil Air Base, Iraq: 4. Detection and identi cation of leishmania parasites in sand ies. J Med Entomol. 2009;46(3):649-663. 17. Walter Reed Biosystematics Unit. VectorMap Website. Available at: http://www.vectormap.org/. Accessed March 25, 2015. 18. Wieczorek J, Guo Q, Hijmans R. The point-radius method for geo-referencing locality descriptions and calculating associated uncertainty. Int J Geogr Inf Sci. 2004;18(8):745-767. 19. Wieczorek J, Bloom D. Georeferencing calculator [internet] Berkeley, CA: University of California; 2008. Available at: http://manisnet.org/gci2.html. Accessed March 23, 2015. 20. Foley DH, Wilkerson RC, Rueda LM. Importance of the “what,” “when,” and “where” of mosquito collection events. J Med Entomol. 2009 ; 46(4):717-722 21. Wick M. GeoNames [internet]. Available at: http:// www.geonames.org/. Accessed March 23, 2015. 22. Walter Reed Biosystematics Unit. Catalog of subfamily Phlebotominae (Diptera: Psychodidae) [internet]. Available at: http://www.sand ycatalog. org//. Accessed March 25, 2015. 23. Galati EAB. 2. Morfologia e taxonomia, 2.1. Classi cacao de Phlebotominae, 2.2. Morfologia, terminologia de adultos e identi cacao dos taxons da America. In: Rangel EF, Lainson R, eds. Flebotomineos do Brazil Rio de Janeiro, Brazil: Editora Fiocruz; 2003:23-175. 24. LUCID. Tools for identi cation and diagnosis (LUCID v. 3.5 software). Brisbane, Australia: University of Queensland. Available at: http://www.lucid central.com/. Accessed March 25, 2015. 25. Walter Reed Biosystematics Unit. Keys to medically important species of sand ies, SOUTHCOM [internet]. Available at: http://www.wrbu.org/south com_SFkeys.html. Accessed March 11, 2015. 26. Foley D, Rueda LM, Wilkerson RC. Insight into global mosquito biogeography from country species records. J Med Entomol 2007;44(4):554-567. 27. Koopman KF. Land bridges and ecology in bat distribution on islands off the northern coast of South America. Evolution. 1958;12:429-439. 28. Hijmans RJ, Garrett KA, Huaman Z, et al. Assessing the geographic representativeness of gene bank collections: the case of Bolivian wild potatoes. Conserv Biol 2000;14:1755-1765.AUTHORSDr Rueda is a Research Entomologist, Principal Investigator and former Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. Dr Foley is a Research Entomologist of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. Mr Pecor is a VectorMap technical assistant of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. Mr Wolkoff is an intern of the College Student Leadership Program, Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland.

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40 http://www.cs.amedd.army.mil/amedd_journal.aspxRECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY Table 1A. Types of New World sand ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued through 1B, 1C, 1D). SpeciesRepository Type (No. of Slides) Country of Type Origin†Bichromomyia olmeca olmeca (Vargas and Najera 1959)F: P(1) Brazil Bichromomyia olmeca bicolor (Fairchild and Theodor 1971)F: H, A, P(44) Panama Bichromomyia olmeca nociva (Young and Arias 1982)F: A, P(14); U: P(10) Brazil Brumptomyia galindoi (Fairchild and Hertig 1947) F: HPanama Brumptomyia hamata (Fairchild and Hertig 1947)F: H, A, P(1) Panama Brumptomyia leopoldoi (Rodriguez 1953)F: P(3) Panama Dampfomyia (Coromyia) steatopyga (Fairchild and Hertig 1958)F: P(2); U: P(1) Mexico Dampfomyia (Coromyia) vesicifera (Fairchild and Hertig 1947)F: H, A, P(8); U: P(2) Panama Dampfomyia (Coromyia) vespertilionis (Fairchild and Hertig 1947)F: H, A, P(24) Panama Dampfomyia (Coromyia) viriosa (Fairchild and Hertig 1958)F: P(1); U: P(1) Costa Rica Dampfomyia (Coromyia) zeledoni Young and Murillo 1984F: H, P(1) Costa Rica Dampfomyia (Dampfomyia) anthophora (Addis 1945)U: P(1) United States Dampfomyia (Dampfomyia) rosabali (Fairchild and Hertig 1956)F: P(2) Panama Dampfomyia (Incertae sedis) caminoi (Young and Duncan 1994)F: H,A,P(1) Mexico Evandromyia (Aldamyia) sericea (Floch and Abonnenc 1944) U: HBrazil Evandromyia (Aldamyia) williamsi (Damasceno, Causey and Arouck 1945) U: HBrazil Evandromyia (Evandromyia) begonae (Ortiz and Torres 1975)U: P(1) Brazil Evandromyia (Evandromyia) inpai (Young and Arias 1977)F: P(11) Brazil Evandromyia (Evandromyia) wilsoni (Damasceno and Causey 1945) U: HBrazil Hertigia hertigi Fairchild 1949F: ACosta Rica Lutzomyia (Helcocyrtomyia) botella (Fairchild and Hertig 1961)F: H, P(6) Panama Lutzomyia (Helcocyrtomyia) cirrita Young and Porter 1974F: A, P(5) Colombia Lutzomyia (Helcocyrtomyia) hartmanni (Fairchild and Hertig 1957)F: H, P(10) Panama Lutzomyia (Helcocyrtomyia) imperatrix (Alexander 1944) U: HPeru Lutzomyia (Helcocyrtomyia) noguchii (Shannon 1929) U: TPeru Lutzomyia (Helcocyrtomyia) peruensis (Shannon 1929) U: TPeru Lutzomyia (Helcocyrtomyia) pescei (Hertig 1943) U: LPeru Lutzomyia (Helcocyrtomyia) strictivilla Young 1979F: A, P(6) Colombia Lutzomyia (Helcocyrtomyia) tortura Young and Rogers 1984F: AEcuador Lutzomyia (Incertae sedis) tanyopsis Young and Perkins 1984F: P(1) United States Lutzomyia (Lutzomyia) battistinii (Hertig 1978)U: L, P(2) Peru Lutzomyia (Lutzomyia) lichyi (Floch and Abonnenc 1950)F: A, P(6) Panama Lutzomyia (Tricholateralis) carvalhoi (Damasceno, Causey and Arouck 1945) U: HBrazil Lutzomyia (Tricholateralis) cruciata (Coquillett 1907) U: TGuatemala Lutzomyia (Tricholateralis) diabolica (Hall 1936) U: TUnited States Lutzomyia (Tricholateralis) falcata Young, Morales and Ferro 1994F: P(7) Brazil Lutzomyia (Tricholateralis) marinkellei Young 1979F: A, P(3) Colombia Martinsmyia gasparviannai Martins, Godoy and Silva 1962F: P(1) Brazil Martinsmyia waltoni Arias, Freitas and Barrett 1984F: P(2); U: P(2) Brazil Micropygomyia (Coquillettimyia ) apache (Young and Perkins 1984)F: A, P(2) United States Micropygomyia (Coquillettimyia) stewarti (Mangabeira and Galindo 1944)F: H; U: P(1) United States Micropygomyia (Coquillettimyia) vexator (Coquillett 1907) U: HUnited States Micropygomyia (Micropygomyia) cayennensis cayennensis (Floch and Abonnenc 1941)F: P(3) Guatemala Micropygomyia (Micropygomyia) cayennensis hispaniolae (Fairchild and Trapido 1950)U: P(2) Dominican Republic Micropygomyia (Micropygomyia) cayennensis jamaicensis (Fairchild and Trapido 1950)F: H, A, P(1) Jamaica Micropygomyia (Micropygomyia) cayennensis maciasi (Fairchild and Hertig 1948)F: (P1); U: (P1) Mexico*Key to Repository Type: H=holotype (1 specimen) A=allotype (1 specimen) F=MEFSCA P=paratype (1 or more specimens) N=neoallotype (1 specimen) T=Type (1 specimen) U=USNMNH † Based on types and repositories, as listed in adjacent column.

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July September 2015 41THE ARMY MEDICAL DEPARTMENT JOURNAL Table 1B. Types of New World sand ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued).SpeciesRepository Type (No. of Slides) Country of Type Origin†Micropygomyia (Micropygomyia) cayennensis puertoriciensis (Fairchild and Hertig 1948) F: A, P(5); U: P(1) Puerto Rico Micropygomyia (Micropygomyia) cayennensis viequesensis (Fairchild and Hertig 1948) F: H, A, P(4); U: P(2) Puerto Rico: H, A, P(4); Panama: P(2) Micropygomyia (Micropygomyia) cubensis (Fairchild and Trapido 1950)F: H, A, P(5); U: P(1) Cuba Micropygomyia (Micropygomyia) duppyorum (Fairchild and Trapido 1950)F: A, P(6); U: P(2) Jamaica Micropygomyia (Micropygomyia) hispaniolae (Fairchild and Trapido 1950)F: A, P(8)Dominican Republic: A, P(5); Haiti: P(3) Micropygomyia (Micropygomyia) pilosa (Damasceno and Causey 1944) U: HBrazil Micropygomyia (Micropygomyia) xerophila (Young, Brener, and Wargo 1983)F: A, P(10); U: P(1) United States Micropygomyia (Sauromyia) atroclavata (Knab 1913)U: P(1) Trinidad and Tobago Micropygomyia (Sauromyia) ferreirana (Barretto, Martins, and Pellegrino 1956) U: HBrazil Micropygomyia (Sauromyia) quechua (Martins, Llanos, and Silva 1975)F: A, P(1) Peru Micropygomyia (Sauromyia) quinquefer (Dyar 1929) U: T, AArgentina Migonemyia (Blancasmyia) cerqueirai (Causey and Damasceno 1945) U: HBrazil Migonemyia (Blancasmyia) gorbitzi (Blancas 1959)F: A, P(50) Panama Nyssomyia anduzei (Rozeboom 1942) U: HVenezuela Nyssomyia trapidoi (Fairchild and Hertig 1952)F: H, A, P(29); U: P(2) Panama Nyssomyia ylephiletor (Fairchild and Hertig 1952)F: H, P(36) Panama Nyssomyia yuilli Young & Porter 1972U: H, A; F: P(20) Colombia Oligodontomyia oligodonta (Young, Prez, and Romero 1985)F: A, P(9) Peru Pintomyia (Pifanomyia) andina Osorno, Osorno-Mesa, and Morales 1972U: P(1) Colombia Pintomyia (Pifanomyia) boliviana (Velasco and Trapido 1974) U: HBolivia Pintomyia (Pintomyia) christenseni Young and Duncan 1994F: H, A, P(34)Panama: H, A P(20); Colombia: P(12); Brazil (P2) Pintomyia (Pifanomyia) christophei (Fairchild and Trapido 1950)F: H, A, P(3) Dominican Republic Pintomyia (Pifanomyia) gruta Ryan 1986F: P(1) Brazil Pintomyia (Pifanomyia) moralesi Young 1979F: P(4) Colombia Pintomyia (Pifanomyia) odax (Fairchild and Hertig 1961)F: A, P(17) Panama Pintomyia (Pifanomyia) oresbia (Fairchild and Hertig 1961)F: A, P(2) Panama Pintomyia (Pifanomyia) orestes (Fairchild and Trapido 1950)F: H, P(1) Cuba Pintomyia (Pifanomyia) pia (Fairchild and Hertig 1961)F: H, A, P(10) Panama Pintomyia (Pifanomyia) torvida Young, Morales, and Ferro 1994F: A, P(1) Colombia Pintomyia (Pifanomyia) youngi Feliciangeli and Murillo 1985F: P(2) Venezuela Pressatia camposi (Rodriguez 1952)F: A, P(31) Panama Pressatia dysponeta (Fairchild and Hertig 1952)F: A, P(53) Panama Pressatia trispinosa (Mangabeira 1942)F: H, A, P(7) Colombia Psathyromyia (Incertae sedis) ignacioi (Young 1972)F: P(1) Venezuela Psathyromyia (Forattiniella) barrettoi barrettoi (Mangabeira 1942)U: P(1) Panama Psathyromyia (Forattiniella) barrettoi majuscula (Young 1979)F: A, P(15); U: P(2)Panama: A, P(11); Colombia: P(3); Costa Rica: P(1); Ecuador (P=1); Nicaragua: P(1) Psathyromyia (Forattiniella) carpenteri (Fairchild and Hertig 1953)F: H, A, P(36) Panama Psathyromyia (Forattiniella) runoides (Fairchild and Hertig 1953)F: H, A, P(28) Panama Psathyromyia (Forattiniella) texana (Dampf 1938) U: TUnited States*Key to Repository Type: H=holotype ( 1 specimen) A=allotype ( 1 specimen) F=MEFSCA P=paratype ( 1 or more specimens) N=neoallotype ( 1 specimen) T=Type ( 1 specimen) U=USNMNH † Based on types and repositories, as listed in adjacent column.

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42 http://www.cs.amedd.army.mil/amedd_journal.aspxRECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY Table 1C. Types of New World sand ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued).SpeciesRepository Type (No. of Slides) Country of Type Origin†Psathyromyia (Psathyromyia) campbelli (Damasceno, Causey, and Arouck 1945) U: HBrazil Psathyromyia (Psathyromyia) cratifer (Fairchild and Hertig 1961)F: H, P(1) Mexico Psathyromyia (Psathyromyia) dasymera (Fairchild and Hertig 1961)F: H, A, P(45)Panama: H, A, P(42); Mexico: P(1); Nicaragua: P(2) Psathyromyia (Psathyromyia) guatemalensis Porter and Young 1986F: A, P(1) Guatemala Psathyromyia (Psathyromyia) shannoni (Dyar 1929)U: L, P(1); F: P(2)Argentina: H; Panama: L, P(2); Peru (P1) Psathyromyia (Psathyromyia) soccula (Fairchild and Hertig 1961)F: P(2) Panama Psathyromyia (Psathyromyia) souzacastroi (Damasceno and Causey 1944) U: HBrazil Psathyromyia (Psathyromyia) undulata (Fairchild and Hertig 1950)U: P(1) Guatemala Psathyromyia (Psathyromyia) volcanensis (Fairchild and Hertig 1950)F: N, P(3) Panama Psathyromyia (Xiphomyia) aclydifera (Fairchild and Hertig 1952) F: APanama Psychodopygus amazonensis (Root 1934)U: (L); F: P(1)Peru: L; French Guyana: P(1) Psychodopygus ayrozai (Barretto and Coutinho 1940)F: P(2) Panama Psychodopygus bispinosus (Fairchild and Hertig 1951)F: H, A, P(4) Panama Psychodopygus carrerai carrerai (Barretto 1946)F: P(1) Panama Psychodopygus carrerai thula (Young 1979)F: A, P(27)Panama: A, P(17); Colombia: P(10) Psychodopygus davisi (Root 1934)U: P(1) Brazil Psychodopygus fairchildi Barretto 1966F: H,A, P(4) Colombia Psychodopygus fairtigi (Martins 1970)F: H, P(1) Colombia Psychodopygus nocticolus (Young 1973)F: A, P(7) Colombia Psychodopygus panamensis (Shannon 1926) U: TPanama Psychodopygus recurvus (Young 1973)F: A, P(12) Colombia Sciopemyia nematoducta Young and Arias 1984F: A, P(23); U: P(8) Brazil Sciopemyia pennyi Arias and Freitas 1981F: P(2); U: P(1) Brazil Sciopemyia preclara Young and Arias 1984F: P(1) Peru Sciopemyia servulolimai (Damasceno and Causey 1945) U: HBrazil Sciopemyia sordellii (Shannon and Del Ponte 1927) U: LArgentina Trichophoromyia castanheirai (Damasceno, Causey, and Arouck 1945) U: HBrazil Trichophoromyia dunhami (Causey and Damasceno 1945) U: HBrazil Trichophoromyia gibba Young and Arias 1994F: P(1) Brazil Trichophoromyia lopesi (Damasceno, Causey, and Arouck 1945) U: HBrazil Trichophoromyia loretonensis (Llanos 1964)F: P(1) Peru Trichophoromyia meirai (Causey and Damasceno 1945) U: HBrazil Trichophoromyia melloi (Causey and Damasceno 1945) U: HBrazil Trichophoromyia napoensis Young and Rodgers 1984F: A, P(12) Ecuador Trichophoromyia pabloi (Barreto, Burbano, and Young 2002)F: P(1) Colombia Trichophoromyia reburra (Fairchild and Hertig 1961)F: H, A, P(2) Panama Trichophoromyia ruii Arias and Young 1982F: P(31) Brazil Trichophoromyia sinuosa Young and Duncan 1994F: H, P(1) Peru Trichopygomyia elegans Martins, Falcao and Silva 1976U: P(1) Peru Trichopygomyia ferroae (Young and Morales 1987)F: H, A, P(1) Colombia Trichopygomyia martinezi Young and Morales 1987F: H, A, P(1) Colombia Trichopygomyia ratcliffei Arias, Ready, and Freitas 1983U: P(5) Brazil Trichopygomyia triramula (Fairchild and Hertig 1952)F: H, A, P(28) Panama Trichopygomyia wagleyi (Causey and Damasceno 1945) U: HBrazil*Key to Repository Type: H=holotype ( 1 specimen) A=allotype ( 1 specimen) F=MEFSCA P=paratype ( 1 or more specimens) N=neoallotype ( 1 specimen) T=Type ( 1 specimen) U=USNMNH † Based on types and repositories, as listed in adjacent column.

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July September 2015 43THE ARMY MEDICAL DEPARTMENT JOURNAL Table 1D. Types of New World sand ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued).SpeciesRepository Type (No. of Slides) Country of Type Origin†Trichopygomyia wilkersoni Young and Rodgers 1984F: A, P(1) Ecuador Trichopygomyia witoto Young and Morales 1987F: H, P(1) Colombia Viannamyia fariasi (Damasceno, Causey, and Arouck 1945) U: HBrazil Warileya nigrosaccula Fairchild and Hertig 1951F: HPanama Warileya phlebotomanica Hertig 1948F: HPeru Warileya rotundipennis Fairchild and Hertig 1951F: H, A, P(6) Panama Warileya yungasi Velasco and Trapido 1974F: P(1); U: H, P(1) Bolivia*Key to Repository Type: H=holotype ( 1 specimen) A=allotype ( 1 specimen) F=MEFSCA P=paratype ( 1 or more specimens) N=neoallotype ( 1 specimen) T=Type ( 1 specimen) U=USNMNH † Based on types and repositories, as listed in adjacent column. Table 2A. Types of New World sand ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and new generic and subgeneric classi cations (continued through 2B, 2C, 2D).New ArrangementOld Arrangement†Bichromomyia olmeca olmeca (Vargas and Najera 1959) Lutzomyia (Nyssomyia) olmeca olmeca Bichromomyia olmeca bicolor (Fairchild and Theodor 1971) Lutzomyia (Nyssomyia) olmeca bicolor Bichromomyia olmeca nociva (Young and Arias 1982) Lutzomyia (Nyssomyia) olmeca nociva Brumptomyia galindoi (Fairchild and Hertig 1947) Brumptomyia galindoi Brumptomyia hamata (Fairchild and Hertig 1947) Brumptomyia hamata Brumptomyia leopoldoi (Rodriguez 1953) Brumptomyia leopoldoi Dampfomyia (Coromyia) steatopyga (Fairchild and Hertig 1958) Lutzomyia (Coromyia) steatopyga Dampfomyia (Coromyia) vesicifera (Fairchild and Hertig 1947) Lutzomyia (Coromyia) vesicifera Dampfomyia (Coromyia) vespertilionis (Fairchild and Hertig 1947) Lutzomyia (Coromyia) vespertilionis Dampfomyia (Coromyia) viriosa (Fairchild and Hertig 1958) Lutzomyia (Coromyia) viriosa Dampfomyia (Coromyia) zeledoni Young and Murillo 1984Lutzomyia (Coromyia) zeledoni Dampfomyia (Dampfomyia) anthophora (Addis 1945) Lutzomyia (Dampfomyia) anthophora Dampfomyia (Dampfomyia) rosabali (Fairchild and Hertig 1956) Dampfomyia (Dampfomyia) rosabali Dampfomyia (Incertae sedis) caminoi (Young and Duncan 1994) Lutzomyia caminoi Evandromyia (Aldamyia) sericea (Floch and Abonnenc 1944) Lutzomyia sericea Evandromyia (Aldamyia) williamsi (Damasceno, Causey, and Arouck 1945) Lutzomyia williamsi Evandromyia (Evandromyia) begonae (Ortiz and Torres 1975) Lutzomyia (Evandromyia) begonae Evandromyia (Evandromyia) inpai (Young and Arias 1977) Lutzomyia inpai Evandromyia (Evandromyia) wilsoni (Damasceno and Causey 1945) Lutzomyia wilsoni Hertigia hertigi Fairchild 1949Hertigia hertigi Lutzomyia (Helcocyrtomyia) botella (Fairchild and Hertig 1961) Lutzomyia (Helcocyrtomyia) botella Lutzomyia (Helcocyrtomyia) cirrita Young and Porter 1974Lutzomyia (Helcocyrtomyia) cirrita Lutzomyia (Helcocyrtomyia) hartmanni (Fairchild and Hertig 1957) Lutzomyia (Helcocyrtomyia) hartmanni Lutzomyia (Helcocyrtomyia) imperatrix (Alexander 1944) Lutzomyia (Helcocyrtomyia) imperatrix Lutzomyia (Helcocyrtomyia) noguchii (Shannon 1929) Lutzomyia (Helcocyrtomyia) noguchii Lutzomyia (Helcocyrtomyia) peruensis (Shannon 1929) Lutzomyia (Helcocyrtomyia) peruensis Lutzomyia (Helcocyrtomyia) pescei (Hertig 1943) Lutzomyia (Helcocyrtomyia) pescei Lutzomyia (Helcocyrtomyia) strictivilla Young 1979Lutzomyia (Helcocyrtomyia) strictivilla Lutzomyia (Helcocyrtomyia) tortura Young and Rogers 1984Lutzomyia (Helcocyrtomyia) tortura Lutzomyia (Incertae sedis) tanyopsis Young and Perkins 1984Lutzomyia tanyopsis Lutzomyia (Lutzomyia) battistinii (Hertig 1978) Lutzomyia (Lutzomyia) battistinii Lutzomyia (Lutzomyia) lichyi (Floch and Abonnenc 1950) Lutzomyia (Lutzomyia) lichyi Lutzomyia (Tricholateralis) carvalhoi (Damasceno, Causey, and Arouck 1945) Lutzomyia (Lutzomyia) carvalhoi*Based on WRBU22 and Galati.23† Based on Young and Duncan4 and various references.

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44 http://www.cs.amedd.army.mil/amedd_journal.aspxRECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY Table 2B. Types of New World sand ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and new generic and subgeneric classi cations (continued).New ArrangementOld Arrangement†Lutzomyia (Tricholateralis) cruciata (Coquillett 1907) Lutzomyia (Lutzomyia) cruciata Lutzomyia (Tricholateralis) diabolica (Hall 1936) Lutzomyia (Lutzomyia) diabolica Lutzomyia (Tricholateralis) falcata Young, Morales and Ferro 1994Lutzomyia (Lutzomyia) falcata Lutzomyia (Tricholateralis) marinkellei Young 1979Lutzomyia (Lutzomyia) marinkellei Martinsmyia gasparviannai Martins, Godoy and Silva 1962Lutzomyia (Lutzomyia) gasparviannai Martinsmyia waltoni Arias, Freitas and Barrett 1984Lutzomyia (Nyssomyia) waltoni Micropygomyia (Coquillettimyia ) apache (Young and Perkins 1984) Lutzomyia apache Micropygomyia (Coquillettimyia) stewarti (Mangabeira and Galindo 1944) Lutzomyia (Helcocyrtomyia) stewarti Micropygomyia (Coquillettimyia) vexator (Coquillett 1907) Lutzomyia (Helcocyrtomyia) vexator Micropygomyia (Micropygomyia) cayennensis cayennensis (Floch and Abonnenc 1941) Lutzomyia (Micropygomyia) cayennensis cayennensis Micropygomyia (Micropygomyia) cayennensis hispaniolae (Fairchild and Trapido 1950) Lutzomyia (Micropygomyia) cayennensis hispaniolae Micropygomyia (Micropygomyia) cayennensis jamaicensis (Fairchild and Trapido 1950) Lutzomyia (Micropygomyia) cayennensis jamaicensis Micropygomyia (Micropygomyia) cayennensis maciasi (Fairchild and Hertig 1948) Lutzomyia (Micropygomyia) cayennensis maciasi Micropygomyia (Micropygomyia) cayennensis puertoriciensis (Fairchild and Hertig 1948) Lutzomyia (Micropygomyia) cayennensis puertoriciensis Micropygomyia (Micropygomyia) cayennensis viequesensis (Fairchild and Hertig 1948) Lutzomyia (Micropygomyia) cayennensis viequesensis Micropygomyia (Micropygomyia) cubensis (Fairchild and Trapido 1950) Lutzomyia (Micropygomyia) cubensis Micropygomyia (Micropygomyia) duppyorum (Fairchild and Trapido 1950) Lutzomyia (Micropygomyia) duppyorum Micropygomyia (Micropygomyia) hispaniolae (Fairchild and Trapido 1950) Micropygomyia (Micropygomyia) hispaniolae Micropygomyia (Micropygomyia) pilosa (Damasceno and Causey 1944) Lutzomyia pilosa Micropygomyia (Micropygomyia) xerophila (Young, Brener and Wargo 1983) Lutzomyia xerophila Micropygomyia (Sauromyia) atroclavata (Knab 1913) Lutzomyia (Micropygomyia) atroclavata Micropygomyia (Sauromyia) ferreirana (Barretto, Martins and Pellegrino 1956) Lutzomyia ferreirana Micropygomyia (Sauromyia) quechua (Martins, Llanos and Silva 1975) Lutzomyia quechua Micropygomyia (Sauromyia) quinquefer (Dyar 1929) Lutzomyia quinquefer Migonemyia (Blancasmyia) cerqueirai (Causey and Damasceno 1945) Lutzomyia (Evandromyia) cerqueirai Migonemyia (Blancasmyia) gorbitzi (Blancas 1959) Lutzomyia gorbitzi Nyssomyia anduzei (Rozeboom 1942) Lutzomyia (Nyssomyia) anduzei Nyssomyia trapidoi (Fairchild and Hertig 1952) Lutzomyia (Nyssomyia) trapidoi Nyssomyia ylephiletor (Fairchild and Hertig 1952) Lutzomyia (Nyssomyia) ylephiletor Nyssomyia yuilli Young & Porter 1972Lutzomyia (Nyssomyia) yuilli yuilli Oligodontomyia oligodonta (Young, Prez and Romero 1985) Lutzomyia oligodonta Pintomyia (Pifanomyia) andina Osorno, Osorno-Mesa and Morales 1972Lutzomyia andina Pintomyia (Pifanomyia) boliviana (Velasco and Trapido 1974) Lutzomyia boliviana Pintomyia (Pintomyia) christenseni Young and Duncan 1994Lutzumyia (Pintomyia) christenseni Pintomyia (Pifanomyia) christophei (Fairchild and Trapido 1950) Lutzomyia christophei Pintomyia (Pifanomyia) gruta Ryan 1986Lutzomyia gruta Pintomyia (Pifanomyia) moralesi Young 1979Lutzomyia moralesi Pintomyia (Pifanomyia) odax (Fairchild and Hertig 1961) Lutzomyia odax Pintomyia (Pifanomyia) oresbia (Fairchild and Hertig 1961) Lutzomyia oresbia Pintomyia (Pifanomyia) orestes (Fairchild and Trapido 1950) Lutzomyia orestes Pintomyia (Pifanomyia) pia (Fairchild and Hertig 1961) Lutzomyia pia Pintomyia (Pifanomyia) torvida Young, Morales and Ferro 1994Lutzomyia torvida Pintomyia (Pifanomyia) youngi Feliciangeli and Murillo 1985Lutzomyia youngi Pressatia camposi (Rodriguez 1952) Lutzomyia (Pressatia) camposi Pressatia dysponeta (Fairchild and Hertig 1952) Lutzomyia (Pressatia) dysponeta Pressatia trispinosa (Mangabeira 1942) Lutzomyia (Pressatia) trispinosa *Based on WRBU22 and Galati.23† Based on Young and Duncan4 and various references.

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July September 2015 45THE ARMY MEDICAL DEPARTMENT JOURNAL Table 2C. Types of New World sand ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and new generic and subgeneric classi cations. (continued).New ArrangementOld Arrangement†Psathyromyia (Incertae sedis) ignacioi (Young 1972) Lutzomyia ignacioi Psathyromyia (Forattiniella) barrettoi barrettoi (Mangabeira 1942) Lutzomyia barrettoi barrettoi Psathyromyia (Forattiniella) barrettoi majuscula (Young 1979) Lutzomyia barrettoi majuscula Psathyromyia (Forattiniella) carpenteri (Fairchild and Hertig 1953) Lutzomyia carpenteri Psathyromyia (Forattiniella) runoides (Fairchild and Hertig 1953) Lutzomyia runoides Psathyromyia (Forattiniella) texana (Dampf 1938) Lutzomyia texana Psathyromyia (Psathyromyia) campbelli (Damasceno, Causey and Arouck 1945) Lutzomyia (Psathyromyia) campbelli Psathyromyia (Psathyromyia) cratifer (Fairchild and Hertig 1961) Lutzomyia (Psathyromyia) cratifer Psathyromyia (Psathyromyia) dasymera (Fairchild and Hertig 1961) Lutzomyia (Psathyromyia) dasymera Psathyromyia (Psathyromyia) guatemalensis Porter and Young 1986Lutzomyia (Psathyromyia) guatemalensis Psathyromyia (Psathyromyia) shannoni (Dyar 1929) Lutzomyia (Psathyromyia) shannoni Psathyromyia (Psathyromyia) soccula (Fairchild and Hertig 1961) Lutzomyia (Psathyromyia) soccula Psathyromyia (Psathyromyia) souzacastroi (Damasceno and Causey 1944) Lutzomyia (Psathyromyia) souzacastroi Psathyromyia (Psathyromyia) undulata (Fairchild and Hertig 1950) Lutzomyia (Psathyromyia) undulata Psathyromyia (Psathyromyia) volcanensis (Fairchild and Hertig 1950) Lutzomyia (Psathyromyia) volcanensis Psathyromyia (Xiphomyia) aclydifera (Fairchild and Hertig 1952) Lutzomyia aclydifera Psychodopygus amazonensis (Root 1934) Lutzomyia (Psychodopygus) amazonensis Psychodopygus ayrozai (Barretto and Coutinho 1940) Lutzomyia (Psychodopygus) ayrozai Psychodopygus bispinosus (Fairchild and Hertig 1951) Lutzomyia (Psychodopygus) bispinosus Psychodopygus carrerai carrerai (Barretto 1946) Lutzomyia (Psychodopygus) carrerai carrerai Psychodopygus carrerai thula (Young 1979) Lutzomyia (Psychodopygus) carrerai thula Psychodopygus davisi (Root 1934) Lutzomyia (Psychodopygus) davisi Psychodopygus fairchildi Barretto 1966Lutzomyia (Psychodopygus) fairchildi Psychodopygus fairtigi (Martins 1970) Lutzomyia (Psychodopygus) fairtigi Psychodopygus nocticolus (Young 1973) Lutzomyia (Psychodopygus) nocticola Psychodopygus panamensis (Shannon 1926) Lutzomyia (Psychodopygus) panamensis Psychodopygus recurvus (Young 1973) Lutzomyia (Psychodopygus) recurva Sciopemyia nematoducta Young and Arias 1984Lutzomyia (Sciopemyia) nematoducta Sciopemyia pennyi Arias and Freitas 1981Lutzomyia (Sciopemyia) pennyi Sciopemyia preclara Young and Arias 1984Lutzomyia (Sciopemyia) preclara Sciopemyia servulolimai (Damasceno & Causey 1945) Lutzomyia (Sciopemyia) servulolimai Sciopemyia sordellii (Shannon and Del Ponte 1927) Lutzomyia (Sciopemyia) sordellii Trichophoromyia castanheirai (Damasceno, Causey and Arouck 1945) Lutzomyia (Trichophoromyia) castanheirai Trichophoromyia dunhami (Causey and Damasceno 1945) Lutzomyia (Trichophoromyia) dunhami Trichophoromyia gibba Young and Arias 1994Lutzomyia (Trichophoromyia) gibba Trichophoromyia lopesi (Damasceno, Causey and Arouck 1945) Lutzomyia (Trichophoromyia) lopesi Trichophoromyia loretonensis (Llanos 1964) Lutzomyia (Trichophoromyia) loretonensis Trichophoromyia meirai (Causey and Damasceno 1945) Lutzomyia (Trichophoromyia) meirai Trichophoromyia melloi (Causey and Damasceno 1945) Lutzomyia (Trichophoromyia) melloi Trichophoromyia napoensis Young and Rodgers 1984Lutzomyia (Trichophoromyia) napoensis Trichophoromyia pabloi (Barreto, Burbano and Young 2002) Lutzomyia (Trichophoromyia) pabloi Trichophoromyia reburra (Fairchild and Hertig 1961) Lutzomyia (Trichophoromyia) reburra Trichophoromyia ruii Arias and Young 1982Lutzomyia (Trichophoromyia) ruii Trichophoromyia sinuosa Young and Duncan 1994Lutzomyia (Trichophoromyia) sinuosa Trichopygomyia elegans Martins, Falcao and Silva 1976Lutzomyia (Trichopygomyia) elegans Trichopygomyia ferroae (Young and Morales 1987) Lutzomyia (Trichopygomyia) ferroae Trichopygomyia martinezi Young and Morales 1987Lutzomyia (Trichopygomyia) martinezi Trichopygomyia ratcliffei Arias, Ready and Freitas 1983Lutzomyia (Trichopygomyia) ratcliffei Trichopygomyia triramula (Fairchild and Hertig 1952) Lutzomyia (Trichopygomyia) triramula Trichopygomyia wagleyi (Causey and Damasceno 1945) Lutzomyia (Trichopygomyia) wagleyi *Based on WRBU22 and Galati.23† Based on Young and Duncan4 and various references.

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46 http://www.cs.amedd.army.mil/amedd_journal.aspxRECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY Table 2D. Types of New World sand ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and new generic and subgeneric classi cations (continued).New ArrangementOld Arrangement†Trichopygomyia wilkersoni Young and Rodgers 1984Lutzomyia (Trichophoromyia) wilkersoni Trichopygomyia witoto Young and Morales 1987Lutzomyia (Trichopygomyia) witoto Viannamyia fariasi (Damasceno, Causey and Arouck 1945) Lutzomyia (Viannamyia) fariasi Warileya nigrosaccula Fairchild and Hertig 1951Warileya nigrosaccula Warileya phlebotomanica Hertig 1948Warileya phlebotomanica Warileya rotundipennis Fairchild and Hertig 1951Warileya rotundipennis Warileya yungasi Velasco and Trapido 1974Warileya yungasi *Based on WRBU22 and Galati.23† Based on Young and Duncan4 and various references.

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July – September 2015 47Since World War II, the US military has used aerial spray to mitigate vector-borne disease in combat situations as well as domestic postemergency scenarios. The bene t of aerial application is that large areas which may be inaccessible from the ground can be covered rapidly, thus quickly breaking the cycle of infection.1 Currently, USAF aerial spray operations for mosquito and other insect-borne disease control are conducted during daylight hours. The application platform for USAF aerial sprays is a C-130 H2 aircraft with a modular aerial spray system (MASS). Current operational parameters use a ight pro le spraying at 150 ft (45.7 m) above ground level (AGL) at 200 knots (370.4 km/hr) groundspeed. These parameters were derived from ef cacy tests coupled with the need to maintain relatively high airspeeds and low altitudes to mitigate the potential effect of ground re hazards.2-4 Prior to the common use of night vision goggles (NVGs) to offset the unacceptable hazards of low ying nighttime applications, daytime aerial application of pesticides was the only viable option. Bene ts of night-spray capability are multiple. Application of pesticides concurrent to night feeding mosquito (or other) vector activity will potentially maximize vector mortality and reduce human pathogen transmission. It is generally considered that the optimal timing for application of a fast-acting pesticide is when the target insect is active and in search of a blood meal or other specialized ight periods.5 Based on this concept, the need for a nighttime aerial spray application capability becomes self-evident. The need for this capability was reinforced when West Nile virus (WNV) quickly spread through the Americas beginning in 1999. Though most of the endemic encephalitides were well documented to be vectored by mosquitos of the genus Culex the introduction of WNV galvanized the public health eld with respect to the importance of controlling populations of these vectors.6 The Culex and some Aedes species responsible for transmitting viruses like WNV to humans are reportedly far more active in the crepuscular and night hours rather than the daytime hours.7 Because of this, a majority of aerial adulticiding conducted by civilian mosquito control agencies in the United States is conducted in the evening and nighttime hours. Bene ts of night spraying also include minimizing exposure of diurnal bene cial insects to pesticides. The potential for pollinator and other bene cial insect mortality is a signi cant concern with aerial spray pesticide application. In the case of honeybees, standard operating procedure for daytime aerial spray application of pesticides includes notifying local beekeepers so that they can cover and isolate their hives prior to any application. Nighttime application does not remove the responsibility from the applicators to inform the beekeepers, but it reduces potentially negative effects of pesticide application (bee kill) as honeybees are normally naturally ensconced in a protected hive at time of application. The same level of protection may not be as well known for other diurnal bene cial insects, and nocturnally active bene cial insects might not be so protected from night pesticide applications.Development of Air Force Aerial Spray Night Operations: High Altitude Swath Characterization Lt Col Karl A. Haagsma, BSC, USAFR Lt Col Mark S. Breidenbaugh, BSC, USAFR Kenneth J. Linthicum, PhD Robert L. Aldridge Seth C. Britch, PhDABSTRACTMultiple trials were conducted from 2006 to 2014 in an attempt to validate aerial spray ef cacy at altitudes conducive to night spray operations using night vision goggles (NVGs). Higher altitude application of pesticide (more than 400 ft (121.9 m) above ground level (AGL)) suggested that effective vector control might be possible under ideal meteorological conditions. A series of lower altitude daytime applications (300 ft (91.4 m) AGL) demonstrated effective and repeatable mortality of target sentinel insects more than 5,000 ft (1,524 m) downwind, and control of natural vector populations. From these results we believe further pursuit of aerial night applications of pesticide using NVGs at 300 ft (91.4 m) AGL by this group is warranted.

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48 http://www.cs.amedd.army.mil/amedd_journal.aspxNight spraying also leverages favorable meteorological conditions that may reduce effects of unwanted pesticide drift. Convective atmospheric activity during daytime aerial pesticide applications has always plagued uniform pesticide application. Differential heating may cause convective surface currents to blow away sections of pesticide application plumes at altitude, thereby potentially leaving signi cant application gaps. Lowered convective activity during nighttime applications may reduce this effect. At night, surface wind currents generally become more laminar in nature and pesticide drift may become more predictable and effective (conversation with H. Thistle, January 2009). Despite these bene ts of nighttime aerial spray application, the obvious signi cant danger to night application is the lack of ability to easily see and identify objects that present collision hazards at low altitude. In addition to natural obstructions, man-made obstructions such as buildings, towers, or antennae present formidable obstacles to low-level aircraft navigation. In the contiguous United States, 68% of all surface obstructions are less than 300 ft (91.4 m) AGL, 75% of all surface obstructions are less than 350 ft (106.7 m) AGL, and 5% of all surface obstructions are greater than 500 ft (152.4 m) AGL (conversation with Lt Col John Kochansky, September 2014). Thus, ying at higher altitudes (300+ ft AGL) presents signi cantly less risk than ying a 150 ft (45.7 m) ground pro le at night. Although NVGs have mitigated some hazards of night ying, they have limitations regarding which objects are visible at night. In an attempt to increase the military spray capability to include nighttime application, the USAF aerial spray ight investigated the feasibility of pesticide application ying on NVGs. This investigation was especially challenging as the ight pro le was outside the parameters of the standard low-level NVG routines which are most exclusively employed by special operations C-130 missions using a modi ed-contour scenario ying at 300 ft (91.4 m) AGL on NVGs. Aerial spray mission requirements for low-level NVG operations required a new, unique set of operating conditions that had not been previously considered. Proposals including a point paper to convince USAF leadership of the bene ts of aerial spray nighttime operation were initiated in 2004. Following a series of inquiries and safety reviews, and with endorsement from agencies including the US Centers for Disease Control and Prevention, the Armed Forces Pest Management Board, the Of ce of the Surgeon General of the Air Force, and the support of several US mosquito and vector control agencies, permission was granted to the USAF aerial spray ight to begin low-level NVG training activities speci c to night-time aerial spray operations in 2014. The potential operational capability by the USAF aerial spray ight to conduct night spray missions must be supported by evidence of functional capability. This would include demonstrations that night pesticide applications were effective against target medically important insect vectors of signi cant disease while operating within an acceptable USAF operational scenario. Herein, we report the results of multiple daytime aerial pesticide application trials conducted to determine pesticide droplet size and effective swath width, including eld mosquito sentinel mortality, at a variety of altitudes and with a variety of spray nozzle sizes to support USAF NVG operations for night-spray capability at 300 ft (91.4 m) AGL. METHODS AND MATERIALSEach of the trials detailed below, from earliest to most recent, was designed as a standalone evaluation, generally without replication. The aerial spray platform for all trials was a USAF C-130 H2 aircraft equipped with a MASS, with a variety of nozzle sizes and con gurations on 2 fuselage booms as detailed below. Meteorological conditions were measured using a Swath Kit weather monitoring station (Droplet Technologies, College Station, PA) (trial sets 1-3) or a Kestrel NV4500 logging portable weather station (Nielsen-Kellerman, Boothwyn, PA) (trial sets 4-6). Trial sets 7 and 8 used 4 Kestrel NV4500 logging portable weather stations placed at various intervals along each sampling line, and meteorological data were averaged between data derived from each station over the course of each test.Trial Set 1The trials were conducted January 13-18, 2006, at Avon Park Training Range, Avon Park, Florida (Figure 1). On January 14, 10 aerosol impingers (spinners) (John Hock Company, Gainesville, FL) were placed in an eastwest orientation on Kissimmee Road. Impingers were positioned in a linear array with each sampling station separated by 2500 ft (762 m). Each impinger was equipped with 2 Te on-coated 25mm by 75mm microscope slides to collect droplets from the aerosol cloud released from the aircraft as the cloud drifted downwind past each sampling station. The fuselage booms were tted with a total of 6 atfan TeeJet 8008 Nozzles (Spraying Systems Company, Wheaton, IL). BVA-13 oil was used to simulate pesticide, applied at 4.5 gallons of material per minute (17.03 L/ min) at an operating system pressure of 83 psi. Three north-south overlapped (crosswind) passes of the aircraft DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS: HIGH ALTITUDE SWATH CHARACTERIZATION

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July – September 2015 49THE ARMY MEDICAL DEPARTMENT JOURNAL were made to increase the droplet concentrations at the sampling stations. The spray-on time for each pass was 40 seconds, with spraying beginning 30 seconds prior to the aircraft’s point of intersection with Kissimmee Road. The aircraft ew directly over the upwind sampling station at 500 ft (152.4 m) AGL. On January 15, 10 impingers were aligned on Frostproof Road (Figure 1). Sampling stations were placed 1,700 ft (518 m) apart, with the rst sampling station located on the northwest point in the road. Three adjacent southwest-northeast passes at 500 ft (152.4 m) AGL were made over the rst sampling station, with each pass commencing and ending spray 30 seconds before and after the aircraft’s intersection with Frostproof Road. The MASS sprayed BVA-13 oil at 4.75 gal/min (17.98 L/ min) at an operating pressure of 56 psi. On January 16, 10 impingers were placed in a northsouth orientation along Van Eegan Road (Figure 1). Sampling stations were positioned 2,500 ft (762 m) apart with the rst sampling station located at the southern point of the road. The BVA-13 oil spray ow rate was 4.7 gal/min. Four overlapped west-east passes were own at 500 ft (152.4 m) AGL, with each pass commencing and ending spray 30 seconds prior to and after intersection with the road at the rst sampling station. Following each set of sprays on the 3 trial days, Te on slides were collected 45 minutes after the last pass and viewed under a compound microscope. Up to 50 droplets (if available) were counted per slide at each one of the sampling stations. Droplets were measured for volume median diameter (VMD), calculated using the Yeomans method.8Figure 1. Map of Avon Park Air Force Range (Florida) with testing locations highlighted. Van Eegan Rd Kissimmee Frostproof Rd

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50 http://www.cs.amedd.army.mil/amedd_journal.aspxTrial Set 2These trials were conducted at the Avon Park Training Range 13-17 February 2006. In general, the methods were similar to those in Trial set 1. All trials were conducted with the aircraft spraying at 500 ft (152.4 m) AGL. The spray booms were tted with 6 at-fan TeeJet 8008 nozzles, three on each boom and in each trial, the MASS delivered approximately 4.5 gal/min (17.03 L/min) of BVA-13 oil with the system operating at approximately 50 psi. In these tests, an optical whitener (Uvitex OB, Ciba Specialty Chemicals, Tarrytown, NY) was added to the BVA-13 oil in an attempt to differentiate between potential contamination of the Te on slides with ambient atmospheric aerosols versus the spray coming from the aircraft. A uorescent compound microscope was used to examine droplet collection slides. The Uvitex in the BVA-13 oil caused droplets collected from the spray mission to uoresce vigorously, making identi cation unambiguous. Four overlapping spray passes for each trial in this series were conducted on a course perpendicular to the impingers, directly over ying the rst sampling station in the series. Each pass totaled 30 seconds of spray-on time, 15 seconds before and 15 seconds following intersection with the road(s) and the rst sampling station. Slides were collected and droplet data were calculated as in Trial Set 1, with the exception that droplet density (droplets/cm2) was also recorded for each of the sampling stations. On February 14, a trial was conducted with impingers set up on a northwest-southeast orientation along Frostproof Road (Figure 1) with sampling stations separated by 1,700 ft (518.1 m). The rst sampling station was located at the southeast end of the road. On February 15, a test was conducted on an east-west orientation on Kissimmee Road (Figure 1) with sampling stations separated by 2,500 ft (762 m). The rst sampling station was at the east end of the road. On February 16, a north-south linear array of sampling stations was installed on Van Eegan Road (Figure 1). Sampling stations were separated by 1,742 ft (531 m) with the rst station located at the junction of Kissimmee and Van Eegan Roads.Trial Set 3Trials were conducted December 4-8, 2006, at the Avon Park Training Range. In these trials, a sentinel mosquito bioassay was used in conjunction with the droplet collection system described above. The fuselage booms were tted with a total of 20 at-fan TeeJet 8005 nozzles, (10 on each side), which produced ow rates approaching 7.2 gal/min (27.25 L/min) at an operating system pressure of 50 psi. The pesticide used for these trials was Dibrom (Naled; AMVAC Chemical Corp., Los Angeles, CA) organophosphate adulticide, applied at 500 ft (152.4 m) AGL. In each test, 10 impinger sampling stations were collocated with 10 bioassay cages consisting of cardboard rings (approximately 3.175 cm by 15.25 cm), with open ends covered with ne plastic mesh. In each cage was placed approximately 25 adult female Culex quinquefasciatus from the laboratory colony at the US Department of Agriculture Agricultural Research Service Center for Medical, Agricultural, and Veterinary Entomology (USDA-ARS CMAVE), Gainesville, FL. Sampling arrays each consisting of an impinger and a sentinel cage were deployed at 0.5 mile (804.6 m) intervals along Van Eegan Road (Figure 1) on December 6 and 7. The rst sampling array locations for both trials were at the intersection of Kissimmee and Van Eegan Roads. Four control sentinel mosquito cages were located approximately 0.5 miles (804.6 m) north (upwind) of the rst sampler/cage and exposed for the duration of each trial to ambient conditions. The aircraft conducted 2 overlapping passes for each trial on a course perpendicular to the sampling stations, ying directly over the rst sampling station. Spray-on time was 30 seconds before and 30 seconds after over ying this location. The aerosol cloud was allowed to drift and settle for 45 minutes, after which time the Te on slides and the bioassay cages were collected. Droplets were measured as described for the previous Trial Sets, and VMD, numerical median diameter (NMD), and droplet density were calculated for each sampling station. Mosquitoes were aspirated from the bioassay cages and transferred to clean cages equipped with cotton balls soaked with a 10% sugar water solution. Mosquito mortality was determined at 2, 12, and 24 hours postspray, and corrected with AbbottÂ’s formula9 relative to mortality in the controls.Trial Set 4Trials were conducted December 6-10, 2009, at the Avon Park Training Range. Ten impinger sampling stations equipped as before were deployed along Van Eegan Road (Figure 1) on December 8 and 9 at 0.25 mile (402.3 m) intervals for the rst mile (1,609.3 m) of the sampling array, followed by 0.5 mile (804.6 m) intervals for the remaining 4.5 mile (7,242 m) sampling line. The spray platform was out tted with 7 at fan TeeJet 8005 nozzles on each spray boom, producing a ow rate of approximately 7.2 gal/min (27.25 L/min) at an operating pressure of 50 psi. BVA-13 oil was used to simulate pesticide, and application altitude was 300 ft (91.4 m) AGL. The aircraft conducted 2 overlapping spray passes per trial perpendicular to the sampling stations, with each pass totaling 60 seconds of spray-on time (30 seconds prior to and 30 seconds after intersection with rst sampling location). DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS: HIGH ALTITUDE SWATH CHARACTERIZATION

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July – September 2015 51THE ARMY MEDICAL DEPARTMENT JOURNAL The rst sampling station was located at the intersection of Kissimmee and Van Eegan Roads. Te on slides were collected as described in the above Trial Sets. One hundred drops (if available) were counted from each slide, and VMD, NMD, and droplet density were recorded.Trial set 5This trial series was conducted at the Avon Park Training Range the week of January 23, 2012. On January 24, 10 impinger sampling stations separated by 0.5 miles (804.6 m) were placed in an east-west orientation on Kissimmee Road (Figure 1). Fuselage spray booms on the aircraft were tted with 8 TeeJet 8005 nozzles, four on each side, for a ow rate of 4.5 gal/min (17.03 L/min) at an operating pressure of 83 psi. Three north-south overlapped perpendicular passes were made by the aircraft beginning directly over the rst sampling station in the array. Spray-on times were 20 seconds prior to and 20 seconds after intersection of aircraft ight path and Kissimmee Road. Altitude of the application was 400 ft (121.9 m) AGL and BVA-13 oil was used to simulate pesticide. On January 25, the January 24 trial was repeated, however, spray booms were out tted with 10 TeeJet 8005 nozzles, 5 on each side, for a ow rate of 4.75 gal/min (17.98 L/min) at an operating pressure of 56 psi. The rst 4 sampling stations were 0.25 miles (402.3 m) apart, with the remainder positioned at 0.5 mile (804.6 m) intervals. Trial parameters from January 25 were repeated on January 26. However, the sampler array was aligned in a north-south orientation along Van Eegan Road (Figure 1) and aircraft spray passes were from west-east. Flow rate during this test was 4.6 gal/min (17.4 L/min) with a pressure of 45 psi. Droplet size and density data were collected as described for the January 24 trial set.Trial Set 6On October 17, 2012, 2 trials were performed in conjunction with an operational aerial spray at Parris Island Marine Corps Recruit Depot (MCRD), South Carolina. In each of these trials, 9 bioassay cages similar to those described in Trial Set 3, each with approximately 20 adult female Cx. quinquefasciatus were placed in a linear array along Wake Blvd. The bioassay cages were clamped to wooden dowels at approximately 2 ft (0.61 m) AGL and tted with cotton balls saturated with 10% sugar solution. The rst 5 cages were separated from each other by 250 ft (76.2 m), with each of the remaining 4 cages separated by 500 ft (152.4 m). The spray booms were out tted with 18 TeeJet 8003 at-fan nozzles, 9 on each boom. In each trial in the set, the aircraft ew a swath perpendicular the bioassay sampling line, dispensing Dibrom (Naled) at 300 ft (91.4 m) AGL. Flow rate and pressure recorded from the MASS were 3.3 gal/min (12.5 L/ min) at approximately 60 psi. Spray-on times were 20 seconds before and 20 seconds following intersection of the aircraft at the rst bioassay station. An additional 3 bioassay cages were included as controls for each trial and exposed to ambient conditions during the trials, approximately 0.5 miles (804.6 m) upwind of the application spray line. Bioassay cages were collected and initial knockdown was recorded approximately 35 minutes after each trial. Mosquitoes were then transferred into clean holding cups tted with cotton balls saturated with 10% sugar water solution. Mortality was recorded at 1, 12, and 24 hours postspray, and all mortality was corrected with Abbott’s formula as described in Trial Set 3.Trial Set 7Two independent bioassay trials were conducted on April 9, 2013, at Parris Island MCRD in conjunction with an operational aerial application of the island, and were the result of a cooperative effort between the USAF aerial spray unit and personnel from USDA-ARS-CMAVE. Two tests were conducted using linear arrays of bioassay cages placed either along Wake Blvd (Figure 2A) or the Causeway (Figure 2B). One dispensing swath was used per test. For each test, fuselage booms were equipped with 14 TeeJet 8003 at fan nozzles, 7 on each boom. For the rst application, 20 bioassay cages identical to those described in Trial Set 6 were placed along Wake Blvd at approximately 500 foot (152.4 m) intervals. The rst bioassay cage was positioned at the southwest terminus of Wake Blvd. The aircraft dispensed Dibrom (Naled) adulticide at 300 ft (91.4 m) AGL perpendicular to the sampling array, with an approximately 300 foot (91.4 m) upwind offset. Spray-on and off times were 20 seconds before and after the aircraft intersection with the rst sampling station. The MASS parameters were 2.7 gal/min (10.2 L/min) at 52 psi. Five sentinel (control) mosquito control cages were placed at the golf course on the southernmost point on the island outside of the treatment area and exposed to ambient conditions for the duration of each spray test. For the second test, 25 bioassay cages identical to those described in Trial Set 6 were deployed along the Causeway at approximately 300 foot (91.4 m) intervals, with the rst bioassay cage at the northwestern terminus of the road and an additional 5 sentinel (control) cages placed in the open at the golf course upwind from the spray application. The aircraft dispensed adulticide at 300 ft (91.4 m) AGL on a swath perpendicular to the causeway, directly over ying the last sampling station.

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52 http://www.cs.amedd.army.mil/amedd_journal.aspxInitial knockdown from the bioassay cages was recorded after a 40 minute hold following each application, and the sentinel cages were placed in ice chests. Sentinel mosquito mortality was subsequently recorded at 4 and 12 hours postspray, and all mortality was corrected using AbbottÂ’s formula as described above.Trial Set 8Two bioassay tests were conducted on October 9, 2014, at the Parris Island MCRD, SC, with parameters similar to those in Trial Set 7. In both tests, 30 sentinel mosquito cages were deployed along Wake Blvd, each separated by approximately 250 ft (76.2 m), with an additional set of 10 sentinel cages for each trial located upwind of the spray area on the southern end of the island. The rst sampling location in both trials was positioned at the southwest terminus of Wake Blvd. Fuselage booms were equipped with 15 at-fan TeeJet 8003 nozzles, 7 on the left boom and 8 on the right boom. The MASS parameters during the tests were a ow rate of 2.8 gal/ min (10.6 L/min) and an operating system pressure of 40 psi in the rst trial and 52 psi in the second trial. Postapplication protocols for tallying mortality data collection were the same as those described in Trial Set 7. In the rst trial, 2 roughly overlapping passes (swaths) were made as the aircraft dispensed adulticide perpendicular to the sampling line over sampling locations 10 and 13 (Figure 3A). Spray-on times were 20 seconds prior to and after intersection of the aircraft with the sampling points on the ground. In the second trial, 2 swaths were applied with similar spray-on times as in the rst test, with the aircraft DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS: HIGH ALTITUDE SWATH CHARACTERIZATION Figure 2A. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for pesticide release April 9, 2013, on sampling array located on Wake Blvd, Parris Island Marine Corps Recruit Depot, SC. Flight path of aircraft is depicted by green arrow.

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July – September 2015 53THE ARMY MEDICAL DEPARTMENT JOURNAL dispensing adulticide while ying over sampling locations 1 and 7, essentially skipping a 1,000 foot (304.8 m) swath (Figure 3B). RESULTS AND DISCUSSIONTrial Set 1No data were collected from the rst test conducted on January 14, because no droplets were visible on the microscope slides. Wind speed and direction at the time of the test was between 9 and 15 knots from 290 degrees measured at ground level. We speculate that the wind speed was too high, and at the height the simulant was applied combined with the 20 degree crosswind component, the material may have missed the sampling array altogether. Wind speed and direction on the January 15 test was 2-4 knots at 310 degrees. Droplet VMD ranged from approximately 32 m 1,700 ft (518.2 m) from the aircraft release point, increasing to approximately 50 m at 3,400 ft (1036 m) and 5,100 ft (1,554.5 m), followed by a roughly linear decrease in VMD at 13,600 ft (4,145.3 m) from release point. Droplets were not detected at sampling stations greater than this distance, or at the sampling location directly beneath the aircraft ight path. Wind speed and direction on the January 16 test was 6-7 knots from 188 degrees. Droplets collected at the sampling location 2,500 ft (762 m) from the release point were approximately 68 m, with the remainder of droplets collected by the samplers ranging from 30-50 m up to 22,500 ft (6858 m) from the release point. Droplet density increased from approximately 0.75 drops/cm2 at the 2,500 foot (762 m) sampling location, to over 2.25 drops/cm2 at 10,000 ft (3,048 m), and then decreased to between 0.5 and 1.0 drops/cm2. For both ofthese tests, no droplets were collected directly Figure 2B. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for pesticide release April 9, 2013, on sampling array located on Causeway, Parris Island Marine Corps Recruit Depot, SC. Flight path of aircraft is depicted by green arrow.

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54 http://www.cs.amedd.army.mil/amedd_journal.aspxbeneath the release point. As evidenced by the data from the January 15 trial, a lighter wind condition may have resulted in a tighter and more normal distribution of droplet sizes. No droplets were collected at the far limits of the sampling array. This is in contrast with the January 16 trial, where increased wind speed apparently carried the simulant cloud past the limits of our detection devices. Thus, in light wind conditions it appears that the material drift can be reasonably de ned, whereas spraying from 500 ft (152.4 m) AGL may not be optimal in terms of controlling or de ning drift when sprayed at higher wind speeds.Trial Set 2The rst test on February 14 yielded no data, possibly due to light and variable winds. Ground wind speed was less than 2 knots and the wind direction shifted more than 180 degrees several times during the test. Conditions were signi cantly better on February 15. Surface winds ranged from 1.5 to 3 knots and remained consistent in direction from approximately 070 degrees. The highest droplet densities (10-14 drops/cm2) were seen at stations 3 and 4 at 4,224 (1,287.5 m) and 6,336 ft (1,931.2 m) downwind from the release point, respectively. Densities dropped dramatically by sampling station 4, and remained low from then on. The VMD of droplets was greatest at sampling station 1, exceeding 50 m, and ranged from 28-42 m at the remainder of the stations. Because higher droplet densities are generally correlated with greater insect mortality, it appears the portion of the swath with the greatest potential for effective mosquito control may in this case be an effective swath of approximately 2,000 ft (609.6 m), beginning approximately 3,000 ft (914.4 m) downwind of the DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS: HIGH ALTITUDE SWATH CHARACTERIZATION Figure 3A. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for multiple pass pesticide release October 8, 2014, on sampling array located on Wake Blvd, Parris Island Marine Corps Recruit Depot, SC.

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July – September 2015 55THE ARMY MEDICAL DEPARTMENT JOURNAL release point. While this test was encouraging, the lack of a concurrent bioassay data renders this observation unsubstantiated. Ground conditions on the February 16 test remained favorable, with surface winds averaging 1.5 to 4 knots, though wind direction was slightly more variable in nature, ranging from 360 to 030 degrees. Winds at altitude were 11 knots at 060 degrees. Droplets were only recovered downwind as far as station 6 (8,712 ft downrange (2655.4 m)). Droplet sizes were greatest at station 2 (3,485 ft (1,062.2 m)), though droplet density in the aerosol cloud was relatively low (less than 5 drops/cm2). Droplet densities were greatest at station 1 (1,742 ft (530.9 m) downrange). Discrepancies in the data from the 2 tests might have been attributed to the signi cant crosswind component at the 500 ft AGL release point, where winds were almost 090 degrees from the theoretical direction that could have led to greater drift and dispersion. In this case it appears only a small leading edge portion of the aerosol cloud was effectively sampled. While both trials effectively indicated that an aerosol cloud generated at 500 ft (152.4 m) AGL can descend to ground level, they also suggest there might be a fairly low tolerance for crosswind components, making spraying from this altitude much more unpredictable.Trial Set 3Ground wind conditions for the December 6 test were 3-5 knots at 045-060 degrees for 30 minutes prior to the test, but wind direction subsequently shifted to 080-092 degrees at the time of application. For this test, postapplication mosquito mortality was 100% at the second sampling station 2,500 ft (762 m) downrange of release point, and then dropped dramatically to approximately 20% at 5,000 ft (1524 m) downrange. Droplet size and Figure 3B. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for skipped swath test October 8, 2014, on sampling array located on Wake Blvd, Parris Island Marine Corps Recruit Depot, SC.

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56 http://www.cs.amedd.army.mil/amedd_journal.aspxdensity remained consistent at both of these sampling locations, with approximate VMDs of 45 and droplet densities of 85-100 drops/cm2. Data from the December 7 test yielded signi cantly different results. Surface winds during the test ranged from 1.2 to 3.5 knots at 340-360 degrees, while winds at altitude during the application averaged 2 knots at 324 degrees. As occurred the day before, insect mortality was 100% at the second sampling station 2,500 ft (762 m) downrange, and dropped to approximately 75% and 20% at sampling stations 3 and 4 (1,524 m, and 2,286 m), respectively. Droplet diameters and droplet densities were very different from the previous test: VMDs at sampling stations 2 and 3 were approximately 11 m, and droplet densities were 20 drops/cm2 and 10 drops/cm2 at the sampling stations 2,500 (762 m) and 5,000 ft (1,524 m) downrange. No droplets were collected past the third sampling station. It appears that in the rst test the wind shift encountered during the application may have resulted in only a small portion of the aerosol cloud coming in contact with the sampling stations and caged mosquitoes. We speculate that perhaps only the leading edge of the spray with the associated larger and faster depositing droplets encountered the sampling array. Conversely, it would appear that wind shift prior to application in the second trial may have resulted in the heavier fraction of the spray moving perpendicular to the sampling array, with only the ner fractions which dispersed by diffusion or entrainment in the wingtip vortices contacting the remainder of sampling stations. Thus, while it appears that application at 500 ft (152.4 m) AGL can result in insect mortality at ground level, it is apparent that the vagaries of environmental conditions might be greatly ampli ed while spraying at higher altitudes.Trial Set 4Surface wind conditions were ideal during the December 8 trial, with wind speed averaging 3.9 knots from a steady 360 degree direction. Droplet size (VMD) ranged from approximately 43 m at the sampling station 2500 ft (762 m) downwind, generally decreasing to 27 m 10,000 ft (3048 m) downrange. Droplet density peaked at the rst sampling point (1,250 ft (381 m) past release point) at 25 drops/cm2. Droplet density decreased at all further sampling stations with the exception of sampling station 3, which was located 3,750 ft (1,143 m) from simulant release point. Trial 2 on December 9 produced generally similar results. Surface winds were again relatively light. Wind speed was 3-5 knots with directions ranging from 340-016 degrees. The highest droplet density was seen at sampling station 2 (2,500 ft (762 m) downrange) with corrected densities of approximately 27 drops/cm2. Droplet densities fell to roughly 15 drops/ cm2 from station 3 (3,750 ft (1,143 m) downrange), to station 6 (7,500 ft (2,286 m) downrange), after which densities fell dramatically. Droplet size was fairly consistent, in the 40-50 m range for the rst 5,000 ft (1,524 m) downwind, after which it tapered off to approximately 28 m at the end of the sampling array. Based on relative similarities and consistencies of these 2 tests, the data would suggest under similar circumstances that a nominal swath width of perhaps up to 5,000 ft (1,524 m) may be warranted when spraying at 300 ft (91.4 m) AGL, although that had not been empirically tested with bioassays at this point. Unfortunately, in these trials, there was no sampling station at the release point, and as such, we cannot speculate on any outcomes from the spray release point out to 13,500 ft (4,114.8 m) downwind. Perhaps the most important point from these trials is that it appears detection levels of pesticide (or simulant) when dispensed at a lower altitude (300 ft (91.4 m) AGL), display a much more concentrated effect in terms of droplet densities and VMD than those previously demonstrated in higher altitude tests. Previous trials dispensing at 500 ft (152.4 m) AGL indicated some detection of pesticide/ simulant past 18,000 ft (5,486.4 m) downwind. These trials suggest that while material may well indeed drift over 2 miles (3.21 km), the potential for effective mosquito control may be within 5,000 ft (1,524 m) downwind or less of the release point.Trial Set 5The surface conditions on January 24 had ground winds of 6-7 knots from 290 degrees. Droplet size ranged from 42 m to approximately 31 m in diameter, with most of the VMD size classes around 40 m out to 4,000 ft (1,219.2 m) downwind of application. Droplet density was greatest at 1,250 ft (381 m) downwind at approximately 5.8 drops/cm2, and then decreased in a semilinear fashion to where droplet densities were almost zero at 5,000 ft (1,524 m) downwind. Conditions on January 25 were surface winds averaging 4 knots from 280 degrees. As expected, VMD was greatest at the rst 2 sampling stations (750 ft (228.6 m) and 1,250 ft (381 m)), and then decreased at 5,000 ft (1,524 m) downrange. Droplet densities were greatest at the rst sampling station at 12.7 droplets/cm2, and decreased to approximately 2.0 drops/ cm2 by sampling station 4 at 2,750 ft (838.2 m) downrange. The January 26 surface conditions were 6-7 knot winds at a direction of 350 degrees. Droplet size (VMD) was greatest at 750 ft (228.6 m) downrange (approximately 60 m) and then dropped until 2,750 ft (838.2 m) downrange where it peaked again at 61 m. Droplet density was maximized at 1,250 ft (381 m) downrange at approximately 14.0 drops/cm2. Droplet density approached zero at 2,750 ft (838.2 m) postrelease point. From these 3 trials at an application altitude of 400 ft (121.9 m) AGL, it appears that the droplets dispensed from the aircraft DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS: HIGH ALTITUDE SWATH CHARACTERIZATION

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July – September 2015 57THE ARMY MEDICAL DEPARTMENT JOURNAL did show a fairly predictable swath in terms of width and droplet size. Though the range of detectability appeared acceptable, we consider that the droplet density was not—all measured statistics were a result of 3 passes in these test iterations versus a single pass as would be used in a normal mosquito control operation calibration trial. Again, we speculate that while a certain amount a material drifts to the ground or is pushed to the ground by the dynamic effects of the aircraft, perhaps much of the material, that is, the smaller fractions of the aerosol cloud, may be simply drifting away. However, this hypothesis has not been substantiated by eld sentinel bioassay data under these conditions.Trial Set 6In this bioassay trial, both tests were conducted on the same day, October 12. Surface conditions on the ground during these eld tests was an average wind speed of 1-5 knots from 050 degrees, and remained consistent for both trials. No swath characteristics were recorded. In the rst trial, 100% mosquito mortality at 1 hr, 12 hr, and 24 hour was witnessed at the sampling station 500 ft (152.4 m) downwind of release point. This was true also at the station located 1,000 ft (304.8 m) downwind, after which mosquito mortality dropped to approximately 20% at 1,500 ft (457.2 m) downwind, and to almost zero thereafter. The second trial indicated similar, but slightly skewed results. Mosquito mortality of 100% was observed 750 ft (228.6 m) downwind of application, and was continued to 1,500 ft (457.2 m) downrange, at which time mortality dropped to approximately 30% at 2,000 ft (609.6 m) downrange. Interestingly, mortality increased signi cantly at stations 2,500 ft (762 m) or more. This result was unexpected, and we have no explanations for the apparent anomaly. In these conditions, both of these tests would suggest an effective swath from 300 ft (91.4 m) AGL release height to be at minimum 1,000 ft (304.8 m), with the effective edge at approximately 500 ft (152.4 m) for these particular conditions. Of particular note, however, is that, under these conditions, mosquito mortality was quite low at least after 2,000 ft (609.6 m) downwind of application. The bene t of these bioassay data from a single-pass application is that we might determine a minimum baseline for functionality of aerial application under these conditions when pesticides are applied at 300 ft (91.4 m) AGL.Trial Set 7Field sentinel cage insect mortality for a bioassay conducted on April 9, 2013, located on Wake Blvd, Parris Island MCRD is shown in Figure 2A. Wind direction was approximately 160 degrees and wind speed ranged from 2-6 knots. There was zero mortality (Abbott corrected) at all stations when examined at 15 minutes postspray. At 4 hours postspray, the rst 5 stations in the sampling array exhibited 100% mortality. The remainder of the stations showed zero to less than 10% corrected mortality. The situation at 12 hours postspray was not signi cantly different than that the 4 hour observation. It appears that in this test our effective swath was approximately 2,000 ft (609.6 m), which is somewhat expected in that due to a last minute wind shift, the prevailing wind was almost parallel to the ight path of the aircraft, as opposed to the theoretically optimal crosswind application condition to maximize drift. What appears to have happened is that the crosswind component (approximately 10-40 degrees) essentially allowed the applied spray to cut the angle and brought the aerosol cloud in contact with the rst few bioassay cages and very little else. The increased altitude may have also caused some signi cant diffusion of the cloud before it actually reached ground level. Figure 2B shows eld sentinel cage mortality data and meteorological data for the sampling array located on Causeway, Parris Island MCRD on April 9, 2013. Winds at spray release were from approximately 190 degrees and ranged from 2-8 knots. When observed at 15 minutes postapplication, no mortality was noted in any of the test cages. When reexamined at 4 hours postapplication, all test cages from station 4 to 24 along the causeway indicated 95% to 100% mortality of caged mosquitos. In this test, no mortality was noted greater than approximately 6,000 ft (1,828.8 m) downwind of aircraft ight path. No mortality was noted at station 25, which was offset from the aircraft ight path by approximately 1,000 ft (304.8 m). No signi cant differences between 4 hour and 12 hour mortality were observed. Wind direction was much more favorable for this trial than the previous trial conducted on Wake Blvd. Direction was much closer to perpendicular to the ight path of the aircraft, and for this test under these conditions suggests that an effective swath in terms of highest mosquito mortality at or near ground level may be up to 4,000 ft (1,219.2 m). Also, at altitude dispensed with relatively moderate wind speeds, an offset of approximately 1,000 ft (304.8 m) may be anticipated when applying in almost direct crosswind conditions. We were encouraged by these results as they provided an indication that good control over fairly wide areas may be achieved when applying pesticide at 300 ft (91.4 m) AGL.Trial Set 8Figures 3A and3B show eld sentinel cage mortality data from the trials conducted on October 8, 2014. These trials were slightly different than those in Trial Set 7 in that it was a combined application of 2 adjacent swaths as opposed to a single swath test. Winds at release were from 200-208 degrees (averaged from

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58 http://www.cs.amedd.army.mil/amedd_journal.aspx4 weather stations), and wind speed varied from 2-8 knots at the time of the test. As before, no mortality was noted in caged mosquitoes 15 minutes after application, however, mortality at 4 hour postspray was substantial among all downwind cages. Figure 3A shows mortality measured 12 hours postapplication, which was not signi cantly different than mortality noted at 4 hours postapplication. Mortality of 90% to 100% was observed in all cages from station 22 to station 43, an approximate distance of 6,000 ft (1,828.8 m) downwind of the rst spray swath own. Unfortunately, our sampling stations did not extend beyond this point, so it is unknown if the effective swath may be greater than 6,000 ft (1,828.8 m). Interestingly, near 100% mortality was noted at cages which were located directly below the aircraft, which was not expected as the previous single-swath trial under similar conditions suggested a 1,000 foot (304.8 m) offset might be expected. Though we have no de nitive explanation for this phenomenon, it is possible that the relative open area associated with the ri e range may have resulted in local unpredictable air movement at the interface of open and wooded areas, or areas dominated by other structures. Field sentinel cage mortality data for the second trial conducted on October 8, 2014, is shown in Figure 3B. This trial was set up similarly to the previous trial, with a 2-pass application, but the second swath was offset 2,000 ft (609.6 m) from the rst. Wind direction averaged from 195-200 degrees, and wind speed ranged from 2-7 knots. Mortality of 90% to 100% was observed at all stations from sampling site 20 to sampling site 43, which encompassed an area approximately 7,000 ft (2,133.6 m) downwind of release of pesticide. Again, it is unknown if the effectiveness of the pesticide may have gone farther downrange, as this was the limit of the sampling array. In this second trial it is interesting to note the apparent lack of ef cacy of the second applied swath, as there was very little mortality noted in sampling locations 13 through 18. We speculate that either the interface of the water and land produced some cryptic wind conditions, perhaps an extremely localized offshore breeze, or conditions were such that the pesticide drifted farther downrange before ultimately settling. Regardless, it again appears that pesticide application at this altitude can have signi cant ef cacy against target insects at or near ground level, which is the optimal outcome of aerial sprays of this type. GENERAL CONCLUSIONSThis article summarizes outcomes from a series of trials using a variety of aerial spray con gurations in an array of meteorological conditions that provide select empirical data to investigate ef cacy of higher altitude pesticide applications that may be required for effective nighttime aerial pesticide missions. In general, based on our swath characterizations and bioassays, we believe that while pesticide applications at altitudes of 500 ft (152.4 m) and 400 ft (121.9 m) AGL may be effective under absolutely ideal meteorological conditions, lower altitude application will achieve more consistent results with regard to target insect mortality and allow more reliable prediction of where pesticide may drift. We believe the results of these trials, in particular the 300 ft (91.4 m) AGL mosquito sentinel tests, provide supporting evidence that nighttime applications of pesticides may be very ef cacious at lower altitudes. Given that we are developing these innovative, emerging techniques, we acknowledge that these data only provide preliminary quanti cation of ef cacy and drift, which may be different when pesticides are applied in nighttime hours as opposed to daylight. Additional future research and testing will be required to fully validate aerial nighttime pesticide application. However, the research trials described here provide the key baseline data to guide future research. ACKNOWLEDGEMENTSThe authors thank Mr Jerry Kerce and colleagues at Camp Blanding Joint Training Center, Starke, FL, Mr Jim Clark and colleagues at US Marine Corps Recruit Depot Parris Island, Beaufort, SC, for study site availability and expert ground support, and Mr Matthew Hazen Brown at USDA-ARS-CMAVE for providing mosquitoes for use in eld bioassay cages.REFERENCES1. Eldridge BF. Strategies for surveillance, prevention, and control of arboviral diseases in Western North America. Am J Trop Med Hyg. 1987;37(suppl.):77S-86S. 2. Mount GA, Lofgren CS, Pierce NW, Baldwin KF, Ford HR, Adams CT. Droplet size, density, distribution and effectiveness in ultra-low volume aerial sprays dispersed with TeeJet nozzles. Mosq News. 1970;30:589-599. 3. Burkett DA, Biery TL, Haile DG. An operational perspective on measuring aerosol cloud dynamics. J Am Mosq Control Assoc. 1996;12:380-383. 4. Cecil PF, Young AL. Operation FLYSWATTER: A war within a war. Env Sci Pollut Res 2007;15:3-7. 5. World Health Organization. Space Spray Application of Insecticides for Vector and Public Health Pest Control: A PractitionerÂ’s Guide Geneva, Switzerland: World Health Organization; 2003. 6. Komar N. West Nile virus: epidemiology and ecology in North America. Adv Virus Res 2003;61:185-234DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS: HIGH ALTITUDE SWATH CHARACTERIZATION

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July – September 2015 59THE ARMY MEDICAL DEPARTMENT JOURNAL 7. Guimares AE. Gentile C, Lopes CM, de Mello RP. Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of So Paulo, Brazil. III. Daily biting rhythms and lunar cycle in uence. Mem Inst Oswaldo Cruz 2000;95(6):753-760. 8. Yeomans AH. Directions for Determining Particle Size of Aerosols and Fine Sprays Washington, DC: US Department of Agriculture Bureau of Entomology and Plant Quarantine; 1945. Available at: https://archive.org/stream/ directionsfordet00unit#page/2/mode/2up. Accessed June 4, 2015. 9. Healey MJR. A table of Abbott’s Correction for natural mortality. Ann Appl Biol 1952;39:211-212.AUTHORSLt Col Haagsma is Research Entomologist, Air Force Aerial Spray Unit, 757th Airlift Squadron, 910th Airlift Wing, Youngstown Air Reserve Station, Vienna, OH. Lt Col Breidenbaugh is Chief Entomologist, Air Force Aerial Spray Unit, 757th Airlift Squadron, 910th Airlift Wing, Youngstown Air Reserve Station, Vienna, OH. Dr Linthicum is Director, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL. Mr Aldridge is Biological Sciences Technician, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL. Dr Britch is Research Entomologist, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL. Announcing The 2015 Spurgeon Neel Annual Award Competition The Army Medical Department Museum Foundation is pleased to announce the 2015 Spurgeon Neel Annual Award competition for a paper of 5,000 words or less that best exempli es the history, legacy, and traditions of the Army Medical Department. Named in honor of Major General (Retired) Spurgeon H. Neel, rst Commanding General of Health Services Command (now US Army Medical Command), the award competition is open to all federal employees, military and civilian, as well as nongovernmental civilian authors. More information about MG (Ret) Neel can be found at h p://en.wikipedia.org/wiki/Spurgeon_Neel. The AMEDD Museum Foundation will present a special medallion award and a $500 monetary prize to the winner at a Foundation-sponsored event early in 2016. The winning submission will be published in the AMEDD Journal during 2016. All manuscripts must be submi ed to the AMEDD Museum Foundation by September 30, 2015. At the time of submission, a manuscript must be original work and not pending publication in any other periodical. It must conform to the Writing and Submission Guidance of the AMEDD Journal and must relate to the history, legacy, and/or traditions of the Army Medical Department. Manuscripts will be reviewed and evaluated by a six-member board with representatives from the AMEDD Museum Foundation, the AMEDD Center of History and Heritage, and the AMEDD Journal. The winning manuscript will be selected and announced in December 2015. Submit manuscripts to amedd.foundation@att.net Additional details concerning the Spurgeon Neel Annual Award may be obtained by contacting Mrs Sue McMasters at the AMEDD Museum Foundation, 210-226-0265.

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60 http://www.cs.amedd.army.mil/amedd_journal.aspxIn 2007, the National Academy of Sciences (NAS) published a comprehensive report, “Toxicity Testing in the 21st Century: A Vision and a Strategy”1 in response to a US Environmental Protection Agency request to the National Research Council to develop a plan using innovative methods to advance toxicity testing. Toxicity determination in the previous half-century required animal testing of all new chemicals (medicine, food additives, industrial, consumer, and agricultural chemicals) for potential to cause cancer, birth defects, and other adverse health effects. Animal testing is slow, expensive, uses many animals, and often requires assumptions and controversial extrapolations.1The NAS vision discussed harnessing technologies developed in emerging elds such as systems biology (eg, use of computational models fused with in vitro laboratory data) and bioinformatics (computational models to analyze massive data sets), with high throughput screening assays of effects of chemicals on human cells, cellular components, and tissues, at low levels, which are typically more relevant.1 The report recommended identi cation not only of changes at the molecular level, but of signal transduction and other pathways that, when perturbed, lead to adverse effects. Study of these in uences may lead to improved predictability of health effects in human populations. Mapping such “toxicity pathways” and then discerning actual or predicted perturbations by means of computational models and high throughput screening of chemicals:could reduce the backlog of the large number of industrial chemicals that have not yet been evaluated under the current testing system.1(p30)The new approach would most likely reduce animal use as well. However, many of these methods are new; their value remains to be validated in predicting effects or determining safe levels of exposure for humans. In 1985, the Of ce of The Surgeon General (OTSG) designated the Army Institute of Public Health (AIPH), now part of the Army Public Health Command, as lead agent of the Health Hazard Assessment Program.2 In ful llment of that mission, the AIPH Toxicology Portfolio is charged with evaluating the toxicity of speci c military-unique chemicals in materials entering the Army supply system.3 The primary goals have been (1) to identify health hazards associated with exposure to new substances used in military applications, (2) to provide a technical foundation for approvals (or disapprovals) to eliminate or control hazards associated with manufacturing-related exposures and use and disposal of weapons, equipment, clothing, training devices and other materials.3 The Toxicity Clearance (TC) is the instrument used for this evaluation. It provides a technical basis to help the acquisition program manager make important life cycle decisions. Solvents, re extinguishing agents, repellents, fabric nishes, refrigerants, explosives, energetics, propellants, pyrotechnics, hydraulic uids, metals/alloys, and pest control agents are examples of substances used in systems that have been evaluated. Toxicity Clearances are provided for speci c applications and are generally not applicable to systems with different use conditions.2In accordance with the NAS recommendations and Department of Defense mandates to improve ef ciency in research, development, and acquisition, the Toxicology Portfolio has implemented a phased approach to toxicity testing and has expanded its toolbox of in silico in vitro, as well as in vivo methods to better evaluate potential health and environmental threats, ideally, before a new substance is approved for entry into the Army supply chain. Used in a relative manner, these emerging methods can be used in side-by-side comparison to determine which substance is likely to cause health effects from exposure and use and which would present a lower hazard risk. The phased approach to toxicity testing is intended to identify and characterize occupational and environmental human toxicity concerns as early as possible in the science and technology (S&T) phase of research, prior to transition to an advanced developer. Even before a new material has been synthesized, its properties and performance can be estimated and evaluated using computer modeling techniques that allow toxicity and physical properties to be assessed.4 Modi cation, reformulation, or even substitution at the S&T stage of development, generally in budget activity levels (BA) 1 to 3 Toxicity Testing in the 21st Century: Refining the Army’s Toolbox LTC Erica Eggers Carroll, VC, USA Mark S. Johnson, PhD, DABT

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July – September 2015 61would most likely be signi cantly less time-consuming and less costly than comparable changes at BA stages 4 to 8. An example of the phased approach to toxicity testing is underway with the upgrade for the 2.75-inch Hydra rocket:The Hydra is one of the most extensively used munitions in the Army, but environmental concerns are associated with it. The training warhead for the rocket contains perchlorate. The propellant contains lead…The phased approach to environmental safety and occupational health (ESOH) was used to replace components of the Hydra with safer formulations. These replacement compounds are now entering the nal stages of approval and implementation.4Another success story involved the reformulation of the propellant for the M-115/116/177 (whistle, bang, ash) simulators. The original formulation used ammonium perchlorate as the propellant that resulted in signi cant contamination of training ranges where used. The new formulation contained a more traditional black powder mix that was just as effective on the ranges, without resulting in toxic environmental residues, loss of performance, or signi cant addition to cost. Both formulations were evaluated side-by-side and recommendations made using this approach. Rather than waiting for an upgrade, phased toxicity testing should be a prerequisite to reach a given Technology Readiness Level (TRL) for new products or systems in initial development. The type of toxicity assessment would be selected to be compatible with the stage of development. For example, at the risk of repetitiveness, even before a new material has been synthesized, properties and performance of the substance can be initially evaluated using computer modeling techniques to identify potential toxicity. More sophisticated (and more extensive) tools would be used later in development, but still prior to transition to a weapon system or platform. For example, in silico assays that provided reasonable estimates of con dence regarding toxicity would be followed by in vitro assays, including measures of mutagenesis, genotoxicity, and cytotoxicity assays. These measures provide data that help address targets of toxicity and potential mechanisms for extrapolation to soldiers, civilians, and the environment. Focused animal testing would be reserved for those candidates selected as most likely to be ef cacious and safe, in preparation for transition to a program executive of cer or advanced developer like the US Army Medical Materiel Development Activity. Currently, a Programmatic Environmental, Safety, and Occupational Health Evaluation (PESHE) is not required until Milestone B, at which the product must be at TRL 6. New S&T products are often considered for transition about the BA-3 level at roughly TRL 3-4. It is generally accepted that a primary cause of failure to transition includes “lack of technical maturity.” The National Environmental Policy Act of 1969 (Pub L No. 91-90, 83 Stat 852 (1969)) mandates full disclosure of possible impacts, alternatives and environmental mitigation measures. Moreover, if not evaluated alongside S&T, the program manager runs the risk of development of a system that may cause injury to the War ghter or worker, or not lend itself for sustainable use at testing and training ranges. Therefore, it behooves the S&T community to examine the potential for toxicity as part of technology maturity determination, prior to or at least as part of the transition process into an acquisition program of record. Codifying the need for appropriate toxicity assessment in the technology transfer agreement is therefore important. Additionally, since the PESHE is currently the rst requirement addressing toxicity and is not required prior to the product advancement to TRL 6 and Milestone B, some products may require reformulation or replacement due to toxicity. This would be fairly late in the acquisition pipeline, by which time the Army might have already committed hundreds of thousands of dollars to a product. This practice is inconsistent with Executive Order 13514 (“Federal Leadership in Environmental, Energy and Economic Performance, 2009) which required:…minimizing the generation of waste and pollutants… [and] reducing and minimizing the quantity of toxic and hazardous chemicals and materials acquired...5(p3)There is no guidance on what data are needed to help make environmental safety and occupational health (ESOH) decisions for the PESHE or elsewhere regarding toxicity testing, therefore, program managers accept risks based solely on available ESOH data.6 The proposed phased approach:…seeks to make an ESOH evaluation compatible with each stage of the development process by applying appropriate assessment tools…[and] adds a data requirement to each stage for which managers can plan and program,…6(p53)A proposed requirement for appropriate toxicity data at each BA level is illustrated in the Figure. In addition to a phased approach to early toxicity testing, the Toxicology Portfolio has implemented a number of targeted assays to help research, development, test and evaluation (RDT&E) scientists and managers make funding decisions based upon ESOH risks. Moreover, individuals in the Toxicology Portfolio have been working with the Technical Cooperative Program, Key Technical

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62 http://www.cs.amedd.army.mil/amedd_journal.aspxRecommended process using the phased approach in the development of toxicity data to support environmental, safety and occupational health (ESOH) decision making. ? ? X ? X ? X ? X ? X Is ESOH Risk Acceptable?BA4: Advanced Component Development & Prototypes (TRL 6, 7) BA3: Advanced Technology Development (TRL 4, 5, 6) BA2: Applied Research (TRL 2,3) BA1: Basic Research (TRL 1) BA5: System Development & Demonstration (TRL 8) BA7: Operational System Development (TRL 9) A B C Materiel Solution Analysis Technology Development Engineering and Manufacturing Development Production and Deployment Operations and Support Material Development DecisionIDEAL PARADIGM can currently be used for notional new substances in RDT&E where only small amounts are available. High throughput methods can determine if substances have characteristics with a high probability of causing illness in soldiers and workers, or of adversely affecting range sustainment. Computer projections can be made by comparing the molecular structure of a new compound with databases of compounds in which structure has been correlated with toxicity. Such projections typically provide relative measures of con dence in estimates, and can also help identify pathways of toxicity. The latter inform future study design. Af rmation of functionality of a new substance also helps identify potential toxic endpoints. This, in turn, helps industrial hygienists and occupational health physicians conduct meaningful surveillance in the workforce. The AIPH Toxicology Portfolio attempts to assess high priority military-related substances using the above philosophy wherever possible. In Fiscal Year 14 alone, it completed 39 Toxicity Clearances, 21 technical reports (of which 10 are Toxicity Assessments), and 17 peer-reviewed manuscripts. Doctoral level subject matter experts (SMEs) in endocrine disruption, ecotoxicity, developmental toxicity, genotoxicity, Area 4-42 to develop internationally harmonized methods for use in the development of new weapon systems or platforms to ascertain ESOH hazards.7Although currently a “hot topic” in discussions of toxicity testing and foreseen in the NAS vision as a valuable in silico method, most high throughput methods are still in the research stage. Much work remains to validate their ability to determine maximum safe exposure levels. Use of high throughput methods will require re ned dose metrics for calculation of a safe level of exposure using in vitro results. However, these methods TOXICITY TESTING IN THE 21ST CENTURY: REFINING THE ARMY’S TOOLBOX

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July – September 2015 63THE ARMY MEDICAL DEPARTMENT JOURNAL immunotoxicity, and quantitative structure activity relationships make up the 2 branches of the Portfolio (Toxicity Evaluation Program; Health Effects Research Program).6 Technical experts in inhalational toxicity testing, dermal sensitization, mutagenicity and novel methods team with the SMEs to develop and execute good laboratory practices-compliant protocols using rodents and select sentinel species. The Toxicology Portfolio is funded by a combination of core Defense Health Program funds and investments by other Department of Defense organizations in collaborative arrangements. As important as toxicity testing is, the contribution of the AIPH Toxicology Portfolio is not widely known. Years ago, new products and materials were elded based on ef cacy and the ability to meet military operational requirements. Only relatively recently have regulatory guidelines mandated that new military products be not only effective (can it function as designed?), but also safe for Soldiers. Some reports state that human male fertility in certain developed nations declined in the 20th century,8 although others dispute the claim. Regardless, regulatory guidelines for safety assessment of pharmaceuticals and chemicals currently include screens for effects on reproduction and fertility, including stage-aware histopathological examination of the testis, as a sensitive method for detecting disturbances in spermatogenesis.9-11 These are services which AIPH Toxicology Portfolio personnel routinely perform as part of their mission. As the nation grows increasingly motivated to spend taxpayer dollars ef ciently and to protect the environment to preserve our future, the need for phased and targeted toxicity assessment will most likely become a prerequisite for all R&D investments, and it is reasonable to expect that the AIPH Toxicology Portfolio will remain a leader in this effort. REFERENCES1. National Research Council of the National Academies. Toxicity Testing in the 21st Century: A Vision and a Strategy Washington, DC: National Academies Press; 2007. Available at: http://www.nap.edu/ openbook.php?record_id=11970. Accessed May 14, 2015. 2. Mughal MR, Houpt J, Kluchinsky TA. Health hazard assessment and the toxicity clearance process. US Army Med Dep J. July-September 2014:59-60. 3. McCain WC, Salice CJ, Johnson MS, et al. USACHPPM toxicology: maintaining readiness and protecting the environment. US Army Med Dep J. July-September 2004:10-14. 4. Johnson MS. Phased approach to ESOH assessment streamlines search for replacement substances. One Health. Spring, 2012:19-20. 5. Executive Order 13514: Federal Leadership in Environmental, Energy, and Economic Performance. 74 Federal Register 194 (2009). Available at: https://www.whitehouse.gov/assets/docu ments/2009fedleader_eo_rel.pdf. Accessed May 14, 2015. 6. Eck WS, Watts K, Lieb NJ, Johnson MS. Avoiding environmental risk. Army AL&T April-June 2013:50-56. Available at: http://asc.army.mil/web/ wp-content/uploads/2013/04/April-June2013_ army_al.pdf. Accessed May 14, 2015. 7. Brochu S, Hawari J, Monteil-Rivera F, et al. Assessing the Potential Environmental and Human Health Consequences of Energetic Materials: A Phased Approach. The Technical Cooperation Program; April 2014. TTCP Technical Report CP 4-42 [TR-WPN-TP04-15-2014]. 8. Creasy DM. Evaluation of testicular toxicity in safety evaluation studies: the appropriate use of spermatogenic staging. Toxicol Pathol 1997;25(2):119-131. 9. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for human Use. Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products Geneva, SUI: ICH Secretariat; 1994. ICH-S5A. Available at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryIn formation/Guidances/ucm074950.pdf. Accessed May 14, 2015. 10. Organisation for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals: Reproduction/Developmental Toxicity Screening Test. Paris, France: OECD; 1995. OECD Test Guideline 421. 11. Organisation for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screen Test. Paris, France: OECD; 1996. OECD Test Guideline 422.AUTHORSLTC Carroll is Chief, Division of Toxicologic Pathology, Toxicology Portfolio, Army Institute of Public Health, US Army Public Health Command, Aberdeen Proving Ground, Maryland. Dr Johnson is Director, Toxicology Portfolio, Army Institute of Public Health, US Army Public Health Command, Aberdeen Proving Ground, Maryland.

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64 http://www.cs.amedd.army.mil/amedd_journal.aspxIt is known across the Department of Defense (DoD) that the Secretary of the Army is the Executive Agent for DoD Veterinary Public and Animal Health Services, the functional responsibilities for which are assigned to US Army Veterinary Services.1,2 A critical component of the Veterinary Services mission is the assurance of safe food and water for DoD personnel and their dependents assigned throughout the world, even extending to some US embassies and consulates in the most austere environments. However, much less is known about the behind-thescenes, complex mechanisms required to provide those safe food and water sources, achieved only by the consistent and elaborate coordination among military and civilian public health authorities, medical and veterinary personnel, logistics personnel, and foreign customs authorities. It is probably safe to say that the average consumer at an overseas Defense Commissary Agency commissary or an Army and Air Force Exchange Service facility is only vaguely aware of the intensive efforts made on their behalf to ensure constant availability of US food items. The US European Command (USEUCOM) area of responsibility is a prime example of the enormous complexities involved with exporting and shipping foodstuffs to DoD’s multiple locations around the world. As DoD locations span Europe, US government-owned subsistence transits the breadth of the continent, crossing many borders before reaching outlying installations. To support that mission, the processes involved have historically required sound logistics, communication, and teamwork across multiple services. In recent years, however, it has also required strategic diplomacy to integrate those processes into compliance with the European Union (EU) Trade Control and Expert System (TRACES), designed to track animal and animal origin product EU imports. This article highlights the progression of US military’s incorporation into and compliance with TRACES. It outlines the broader strategic implications of United States engagement in that program to ensure food safety and quality assurance of public health for DoD personnel assigned throughout USEUCOM while, at the same time, ensuring, and even strengthening, diplomatic ties and trust with European partners. As the EU was coming together as a fully functioning regulatory and authoritative body representing its member states, around the year 2000 the European Commission Directorate General for Health and Consumers began to pay attention to animal origin products from the US that were entering the EU bound for US military installations. Movement of such products had been ongoing decades, but the backdrop of a number of high impact, economically costly animal disease outbreaks in the EU, combined with emerging awareness of and concerns about the use of growth hormones, antibiotics, and genetically modi ed organisms by the US agricultural industry, served to elevate attention even further. The rst of these animal disease outbreaks in the EU began in 1996 with cases of bovine spongiform encephalopathy (BSE), otherwise known as “mad cow disease,” occurring in Britain. The scienti c community associated human cases of the variant form of Creutzfeld-Jakob disease with the consumption of BSE-contaminated beef, and the resultant economic loss to the United Kingdom (UK) reached an estimated $6.4 billion by early 2001.3 The BSE outbreak was quickly followed by an epidemic of classical swine fever which began with the rst case reported from Germany in January, 1997 and presumably spreading from there to the Netherlands, then on to Italy, Spain and eventually to Belgium.4 In 2001, the UK’s livestock industry was again hit, this time by a foot-and-mouth disease outbreak which crippled exports and cost the UK an estimated £8 to £8.6 billion ($12 to The New US Military Role in the European Union’s Import Program: Strategic Implications Ensuring Safe Food for the European Theater MAJ Michael McCown, VC, USA Jacob L. Hall, II Megan McCormick, DVM, MPH Lt Col Henry H. Triplett, III, USAF

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July – September 2015 65$13 billion at current exchange rates) to resolve.5,6 Collectively, these diseases resulted in devastating effects on health, economic, and consumer con dence. It had become obvious that there was enormous potential economic risk posed by the introduction of foreign animal diseases into the EU. This awareness, undoubtedly, contributed to shaping the comprehensive import regulations currently being developed and implemented. There was, however, another signi cant change as a result of the 1996 association of human disease with the consumption of potentially BSE-contaminated beef. Historically, the US military had procured beef for the European theater’s needs predominantly from US approved European sources (primarily from the UK as it was economically the most competitively priced). But once the protection of human health was at risk according to the best scienti c information available, the US military categorically shifted to exclusively procuring beef from US sources via import, as the US beef industry largely escaped infection with BSE and was considered safe. This monumental change of sourcing was to have far-reaching effects approximately a decade later when EU attention focused on US military meat importation. By 2008, the EU community’s internal implementation and compliance with import regulation had reached maturity and the spotlight shifted to US military animal origin imports which were not in compliance with existing regulations. Historically, US military importation had been permitted with the recognition that its purpose was to supply military forces outside the European economy, so installations were treated as “foreign soil” versus host nation. But that “exemption” was coming under increasing scrutiny, and the United States was approached to initiate an effort to comply with new EU requirements. Initial meetings, panels, working groups, and senior leader engagements failed to make progress toward this end, however, and US representatives gradually concluded that the problem would be more dif cult to solve than originally anticipated, despite the best intentions. Preventing the construction of a solution framework was a signi cant legal hurdle. From the inception of the issue, legal experts in multiple organizations insisted that no governmental organization outside of the Of ce of the Secretary of Defense (OSD) or the Department of State had the authority to initiate any such agreement. Over many years, multiple legal opinions were issued that effectively prevented any work toward an achievable solution. The chief legal concern was that while the United States and each of the individual member states in the EU are themselves sovereign nations, the EU itself, while a legislative body, is not recognized as a sovereign body and thus has no standing from which to negotiate. By 2011, with multiple initials attempts failed and no traction toward compliance in sight, the EU’s patience was wearing thin and US animal origin product imports were being threatened with refusal at ports of entry. It was clear that the US military had not responded rapidly enough and that EUCOM had to step in to reach a resolution. Finally, in the fall of 2012, senior leaders from EUCOM and other agencies including the Principal Deputy Assistant Secretary of Defense for Logistics and Materiel Readiness and the Joint Staff’s J4 and J5 met and concluded that this problem required urgent solution as product procurement was in imminent jeopardy. Ultimately, the OSD determined that because this issue primarily affected forces serving in the EUCOM area of responsibility, the EUCOM Directorate of Logistics could serve as the lead agency to develop and implement policy governing a successful solution. POINT OF ENTRY: EU BORDER INSPECTION POSTSFirst, and most critically, a successful solution was required to address the arrival and acceptance of shipments at EU Border Inspection Posts (BIPs), the importation entry points located at ports, certain borders, and airports. Then, the EU required that US military shipments be tracked from entry to receipt at destination using the EU’s electronic tracking database, TRACES. This system is used, along with the information provided in accompanying animal health certi cates and other import documents, to produce the EU transit health certi cate called a Common Veterinary Entry Document (CVED) which accompanies a shipment. Upon receipt at destination, a shipment is either processed by an exit BIP as having left the EU in the case of transiting goods or, in the case of goods bound for retail operations with consumer sales, its status updated to re ect arrival at its nal tracking destination. Thirty days are allowed from the time a shipment is logged at the entry point until it must be closed out. Failure to meet requirements potentially jeopardizes future shipments for a speci c importer as compliance history is monitored and a poor track record can result in subsequent refusals of entry. PROGRESS TOWARDS SOLUTIONOnce authority was granted for the involved players to engage with the EU toward constructing a workable framework for US compliance, progress was slow, but at least there was movement. While the US would no longer be permitted to import goods with an entirely free rein, it was recognized that shipments intended for US installations were, in fact, not freely entering the EU

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66 http://www.cs.amedd.army.mil/amedd_journal.aspxfood economy as were most other imported goods. So, despite the fact that US military imported animal origin products that often do not meet EU standards (eg, are not from EU approved sources, are not antibiotic free or may contain unapproved growth hormone), the EU level veterinary authority made the determination that US import shipments would be considered to be transiting the EU until they arrived at designated US installations which were to be considered “non-EU” destinations. This decision took advantage of the existing allowances in EU regulations that permitted the legal transit of goods bound for actual non-EU countries, such as from EU ports through the EU, then out via an EU exit BIP where the shipment is closed out of the TRACES tracking system. This decision, however, did require the of cial designation of US installations as exit BIPs and the identi cation and training of personnel at these locations to be able to perform this function. As the oversight and authority for importation of animal origin products falls under the purview of EU veterinarians and the DoD veterinary mission belongs to the US Army, the US Army Public Health Command RegionEurope (PHCR-E) was delegated authority by USEUCOM to provide direct support and program implementation. The PHCR-E controlled most veterinary assets in Europe and its deputy (the senior veterinary of cer) was appointed the Competent Veterinary Authority and granted limited authority to engage the EU directly regarding the importation program, thus ensuring appropriate professional engagement with the European veterinary authorities. A decision was also made regarding prime vendor warehouse facilities located in the EU which could not technically be considered US military installations. They were assessed and categorized as nonconforming warehouses (permitted under EU legislation). They could function to process transiting goods provided they maintained US government owned goods strictly isolated from goods intended for the EU, and they shipped only to nal destinations that included ships’ supply, US military installations, or other locations actually outside the EU, such as those under the US Central Command or Africa Command. European Union veterinarians continued to be involved with these facilities which function as both entry and exit BIPs as subsequent shipments are moved on EU generated transit paperwork (“daughter CVEDs” based on the original CVED), then closed out when they reach their ultimate destinations at US installations. US INTERAGENCY COOPERATIONBefore an export shipment of animal origin product leaves the United States, the Department of Agriculture’s (USDA) Food Safety and Inspection Service (FSIS)* certi es the goods and issues health certi cates con rming their quality. The FSIS is the public health agency within the USDA responsible for ensuring the nation’s commercial supply of meat, poultry, and egg products is safe, wholesome, and correctly labeled and packaged—in other words, safe for human consumption per US standards. Once approved by USDA, a shipment can be loaded at a supplier’s location, then transported to the east coast for transoceanic movement to European BIPs. The USDA Foreign Agricultural Service (FAS)† links US agriculture to the world, enhancing export opportunities and global food security, as well as expanding and maintaining access to foreign markets for US agricultural products by removing trade barriers and ensuring US rights under existing trade agreements. The FAS also works with foreign governments, international organizations, and the Of ce of the US Trade Representative to establish international standards and rules to improve accountability and predictability for agricultural trade. For the DoD, the US Foreign Agricultural Service Mission to the EU, headquartered in Brussels, Belgium, has played an absolutely key role in assisting with issues related to importation of animal origin products to the EU. They were critical in overcoming challenges with the implementation of TRACES and continue to assist with ongoing and emerging situations. APPROACHING A STABLE END STATEIn 2013, the multiyear-long process of US military integration into the EU import program culminated with the release of USEUCOM European Command Instruction 4506.01: USEUCOM Guidance Regarding the EU TRACES, which codi ed the rules of engagement for the working levels of all service components and affected agencies to participate in the European import program. This guidance serves as the cornerstone for ensuring animal origin products including fresh meat and meat products from the US are available at commissaries, dining halls, food courts, schools, day care centers, and other installation food sources. Combatant commands such as EUCOM are organized and structured for strategic military oversight and direction. As such, it is unusual that EUCOM was designated as the lead to pull together multiple service components and both DoD and non-DoD agencies to safeguard the US European theater’s access to US animal origin products. Although progress remains ongoing, it is far enough along to be assessed and considered a success story. *http://www.fsis.usda.gov/About_FSIS/index.asp†http://www.fas.usda.gov/aboutfas.aspTHE NEW US MILITARY ROLE IN THE EUROPEAN UNION’S IMPORT PROGRAM: STRATEGIC IMPLICATIONS ENSURING SAFE FOOD FOR THE EUROPEAN THEATER

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July – September 2015 67THE ARMY MEDICAL DEPARTMENT JOURNAL ACKNOWLEDGEMENTThe research involved in the preparation of this article was supported in part by an appointment to the Knowledge Preservation Program at the US Army Public Health Command administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the Army Public Health Command.REFERENCES1. Department of Defense Directive 6400.04E: DoD Veterinary Public and Animal Health Services Washington, DC: US Dept of Defense; 2013. 2. Army Regulation 40-905: Veterinary Health Services Washington, DC: US Dept of the Army; 2006. 3. Regmi A, ed. Changing Structure of Global Food Consumption and Trade Washington, DC: US Dept of Argiculture Economic Research Service; 2001:60. Agriculture and Trade Report WRS01-1. Available at: http://www.ers.usda.gov/media/293645/wrs011_1_.pdf. Accessed April 13, 2015. 4. Greiser-Wilke I, Fritzemeier J, Koenen F, et al. Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997–1998. Vet Microbiol 2000;77(1-2):17-27. 5. Donaldson A, Lee R, Ward N, Wilkinson K. Five Years On: The Legacy of the 2001 Foot and Mouth Disease Crisis for Farming and the British Countryside. Newcastle, England: Centre for Rural Economy; February 2006:4. Centre for Rural Economy Discussion Paper Series No. 6. Available at: http://www.ncl.ac.uk/cre/publish/discussionpapers/ pdfs/dp6.pdf. Accessed April 22, 2015. 6. National Cattlemen’s Beef Association. Fact Sheet: Industry Economics. FootAndMouthDiseaseInfo. org web site. Available at: http://www.footandmouthdiseaseinfo.org/factsheetindustryeconomics. aspx. Accessed April 22, 2015.AUTHORSMAJ McCown is Chief, Veterinary Services Division, US Army Public Health Command Region-Europe, Landstuhl, Germany. Mr Hall is a Traf c Management Specialist, Air Force Security Assistance and Cooperation Directorate, Wright-Patterson Air Force Base, Ohio. Dr McCormick is the EU TRACES Specialist, US Army Public Health Command Region-Europe, Landstuhl, Germany. Lt Col Triplett is assigned to Headquarters, US European Command, Stuttgart, Germany.

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68 http://www.cs.amedd.army.mil/amedd_journal.aspxSurpassed only by heart disease, cancer is the second highest cause of all deaths, accounting for 1 in every 4 deaths in the United States. According to the American Cancer Society, there will be more than 1.66 million new cancer diagnoses and an estimated 590,000 Americans will die of cancer in 2015.1 These gures are similar to those reported by the Surveillance, Epidemiology, and End Results Program for 2014.2 In its most recent Cancer Trends Progress Report – 2011/2012 Update, the National Cancer Institute reports that death rates for the 4 leading types of cancer as well as all cancers combined have been declining, yet incidence rates of some cancers are on the rise.3 Worldwide, cancer is a leading cause of both morbidity and mortality, with approximately 14 million newly diagnosed cases and more than 8 million deaths attributed to cancer in 2012.4The evidence indicating a connection between occupational and environmental exposures and cancer has been growing in recent years.5 This is of particular concern because such cancers are theoretically avoidable, as measures can be taken to avoid these nongenetic risk factors. The World Health Organization estimates that 19% of all cancers are attributed to environmental factors, accounting for 1.3 million deaths per year.6 The military population presents a unique opportunity to study links between environmental exposures and cancer. Advantageous aspects of studying cancer among military personnel include well characterized person-time, occupation, and, though not always the case, environmental hazards. Access to routine healthcare including recommended cancer screenings at no cost to the service member and robust electronic medical record systems also facilitate assessments of cancer outcomes in the military population. Furthermore, exposures associated with military deployments may in uence cancer risk among military personnel.7 Possible deployment-related exposures have been documented by the Department of Defense,8,9 to include potential carcinogens (eg, industrial solvents, jet fuel, air pollution, radiation). Behavioral changes during deployment, such as increased tobacco use, have also been documented.10 It is thus plausible that military deployment and associated exposures may be risk factors for subsequent cancer among war ghters. CANCER IN THE MILITARYVietnam WarHistorically, there has been concern regarding military service-related hazards and potential long-term health implications following military deployment. Postdeployment cancer risk is often at the forefront of the issue, as was the case after the Vietnam War.11-12 As Richards describes in an article reviewing responses to militaryassociated environmental and occupational exposures:During the latter half of the 20th Century, medical knowledge of and concern about carcinogens grew, and human experimentation guidelines became more stringent. During the Vietnam era, concern for troop exposure to environmental contaminants evolved beyond acute exposures and experimentation to encompass long-term occupational and environmental hazards encountered on the battle eld.13By far, the most prominent exposure in terms of health concern generated during this con ict is the herbicide commonly referred to as Agent Orange. Many veterans of the Vietnam con ict between 1965 and 1972 attribute poor postdeployment health outcomes, including rare cancers, to 2,3,7,8-Tetrachlorodibenzodioxin, an extremely toxic dioxin compound that contaminated one of the compounds used to make the herbicide Agent Orange.14 The scienti c evidence linking postdeployment cancer to Agent Orange exposure during the Vietnam War varies. Some studies have not found higher rates of mortality for outcomes such as soft tissue sarcomas,15 Hodgkin’s disease,16 non-Hodgkin lymphoma, or testicular cancer in Vietnam veterans.17,18 Another study of participants of the Agent Orange Registry had similar results, showing no difference in prevalence for any type of cancer when comparing Vietnam veterans to non-Vietnam veterans.17 However, the CDC Selected Cancer Study reported a higher risk of non-Hodgkin’s lymphoma among Vietnam veterans when compared to other men.19 Frumkin summarized the existing literature on Agent Orange and cancer, reporting consistent Evaluation of Postdeployment Cancers Among Active Duty Military Personnel Jessica M. Sharkey, MPH Joseph H. Abraham, ScD

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July – September 2015 69to fairly consistent negative results for increases of soft tissue sarcomas, Hodgkin’s disease, and gastrointestinal and brain cancers, but inconsistent results of increases in respiratory and prostate cancers among Vietnam veterans.20 Still yet, in the current Institute of Medicine Report of the health effects of herbicides used in Vietnam, Veterans and Agent Orange: Update 2012 ,21 the committee found suf cient evidence of an association between soft tissue sarcomas, non-Hodgkin lymphoma, chronic lymphocytic leukemia, and Hodgkin lymphoma, and limited/suggestive evidence of an association with laryngeal, lung, bronchus, trachea, and prostate cancers as well as multiple myeloma.1991 Gulf WarSimilar to those of the Vietnam con ict, many veterans of the 1991 Gulf War are also concerned about the specter of cancer and possible links to hazards associated with their deployment. Notable hazards of concern to service members during the Gulf War include depleted uranium, petroleum products, pesticides, and chemical and biological warfare agents.22 However, scienti c literature shows mixed ndings regarding potential associations between Gulf War exposure and postdeployment cancer risk. A particular exposure event of interest during the Gulf War was the destruction of chemical munitions at Khamisiyah, Iraq. While Bullman et al indicated an increased risk of brain cancer mortality among US Army Gulf War veterans who were potentially exposed to low-level chemical warfare agents at Khamisiyah when compared to Gulf War veterans who were not exposed,23 a later study by Young et al found no excess in brain cancer.24 In his report on a study on testicular cancer following Gulf War deployment, Levine stated:…testicular cancer was found to be the only signi cantly increased malignancy among deployed Persian Gulf War veterans. The increase became apparent 2 to 3 years after the Persian Gulf War and peaked 4 to 5 years afterward.11Yet, Knoke et al found that although there was an initial increase in testicular cancer immediately following deployment among Gulf War veterans compared to nondeployed Gulf War era veterans, the difference was no longer observed by 4 years postdeployment.25 Kang et al described “very small rate differences (less than 1.0%) between Gulf veterans and non-Gulf veterans” for both skin cancer and other cancers, with higher rates among the Gulf War veterans.26 Kang and Bullman reported...no signi cant excess of overall cancer deaths or deaths from cancer at any speci c site among Gulf veterans compared with non-Gulf veteran controls.27 In a 2005 report, Gulf War and Health an Institute of Medicine committee found suf cient evidence of an association between combustion products and lung cancer and limited/suggestive evidence of an association between combustion products and nasal, oral, laryngeal, and bladder cancers and between hydrazines and lung cancer. There was inadequate/insuf cient evidence to support conclusions regarding potential associations between fuels, combustion products, hydrazines, and nitric acid for numerous types of cancers.28Operations Enduring and Iraqi FreedomDeployment-related exposures are now causing the same concerns regarding cancer among service members following support of Operations Enduring Freedom (OEF) and Iraqi Freedom (OIF). Since 2001, in excess of 2 million US military personnel have deployed to Southwest Asia,29,30 with environmental hazards including but not limited to pollutants from local industry; militaryproduced exhaust from vehicles, machinery, and generators; open air burn pit emissions and fumes from res; high levels of indigenous ambient particulate matter; munitions and weapons; depleted uranium; and radiation.7,31-39 Potential relationships between exposures in theater and cancer diagnoses subsequent to deployment are again a priority for researchers and public health professionals in the military community. BASELINE CANCER RATESIn the population of OIF and OEF veterans, one expects a certain amount of cancer to occur, irrespective of deployment history and associated deployment-related environmental exposures. Understanding baseline rates of cancer in the military population is useful when evaluating whether cancer among service members with a history of deployment in support of OIF and/or OEF occurs at excessive rates. Cancer investigations in military populations typically focus on speci c types of cancer or are speci c to a single service branch. This was the case when Yamane reported on cancer incidence from 1989-2002 among Airmen. In comparison to the general US population, he found standardized incidence ratios for all cancers to be lower than expected among male Air Force service members and as expected among female Air Force service members.40 Zhu et al later compared incidence rates of a select group of cancers (lung, colorectal, prostate, breast, testicular, and cervical cancers) across the military to US civilians. The authors reported lower incidence rates of colorectal, lung, and cervical cancers, and higher rates of prostate and breast cancers.7 Although these comparisons provide valuable information, knowledge of rates across all service branches for all types of cancers is important. In June 2012, the Armed Forces Health Surveillance Center published a report describing incident diagnoses of cancers and cancer-related deaths in active duty

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70 http://www.cs.amedd.army.mil/amedd_journal.aspxmilitary personnel from 2000-2011. Results for the 12year surveillance period showed a crude incident rate of 55.2 per 100,000 person-years, with the lowest annual incidence rate of 50.3 per 100,000 person-years occurring in 2003 and the highest annual incidence rate of 60.1 per 100,000 person-years occurring in 2009. The data indicated no apparent increasing or decreasing trends in overall or site-speci c incident cancer diagnoses. Of note, rates of cancer diagnoses among active duty military members have remained stable since 2000.41IDENTIFYING CARCINOGENSMore than 900 agents have been evaluated by the International Agency for Research on Cancer for determination of potential to cause cancer. A group of four different categories is utilized to classify every agent: carcinogenic to humans (Group 1), probably or possibly carcinogenic to humans (Group 2A and Group 2B, respectively), unclassi able as to carcinogenicity in humans (Group 3), and probably not carcinogenic to humans (Group 4). In excess of 125 agents have been classi ed into Group 1.42 It is suspected or known that some of these environmental carcinogens can be found in the deployment environment. IDENTIFYING CANCERSThe concern for postdeployment cancer due to potential exposure environmental carcinogens in theater has been raised by service members and veterans alike, as demonstrated by advocacy groups such as Burnpits360 and Operation Purple Heart, which allow for self-reported cancer diagnoses on website registries.43,44 While these concerns are reasonable and recognized by public health professionals in the military community, they have yet to be supported by epidemiologic studies using appropriate populations and suitable comparison groups. However, there are many factors that should be considered when approaching a study intended to establish whether a history of deployment in support of OIF or OEF is associated with subsequent incidence of postdeployment cancer.AgeAge is an important factor to consider when designing any epidemiologic investigation pertaining to postdeployment cancers among service members and veterans. Incidence rates of many types of cancers are known to increase with age. As pointed out by the Armed Forces Health Surveillance Center, generally speaking, US military personnel are younger than the general population.41 When focused on a chronic disease such as cancer that is known to increase with age, in a younger population, priority should be given to cancers that typically occur with highest incidence falling during the young adult years.Latency PeriodsThe empirical latent period for cancers consists of 2 parts: an induction period ranging from the time between the action of a given component cause (ie, an exposure of interest) and the action of the last causal component (ie, biological onset of the cancer) and a subsequent period ranging from the biological onset of the cancer to the clinical detection of the cancer. Minimum empirical latency periods must be taken into account when deciding which cancers to evaluate in service members and veterans postdeployment, as they must be consistent with study hypotheses. Latency periods vary by different type of cancer of interest, with some cancers having a typical latency period of 15 to 20 years or longer, while some cancers typically have latency periods that are considerably shorter. In the former, these types of cancers would be better suited for postdeployment cancer evaluations among veteran populations of wars that occurred at least that far in the past, such as Vietnam or the rst Gulf War, yet they would not be appropriate for OIF/OEF veterans as that much time has not yet passed since exposure. On the other hand, it would be prudent to study the latter types of cancers in a population of OIF/OEF deployed service members because time since deployment and typical latency periods align.Biologic PlausibilityWhen selecting cancer outcomes of interest, the focus should be on cancers that are biologically plausible. For example, the following cancers were selected for an upcoming collaborative study between the US Army Public Health Command, the Navy and Marine Corps Public Health Center, and the Department of Veterans Affairs: melanoma, leukemia, lymphoma, and brain, thyroid, testicular, and breast cancers. Those cancers have peak incidence during young adult years, which matches the demographics of our service members with potential exposure(s) of interest.45 These selections were also made based on suspected or known occupational or environmental risk factors.46-49 The latent periods of these cancers are also in accordance with investigating the association between in-theater environmental exposures and postdeployment cancer among service members formerly deployed to OIF or OEF.50,51KARSHI-KHANABAD: AN EXAMPLERecent efforts to understand possible associations between environmental exposures in theater and postdeployment cancer diagnoses include an investigation conducted at the US Army Public Health Command, which explored multiple cancer outcomes among service members formerly deployed to Karshi-Khanabad, an air base located in southeastern Uzbekistan used to support missions in neighboring Afghanistan during OEF.39 Active EVALUATION OF POSTDEPLOYMENT CANCERS AMONG ACTIVE DUTY MILITARY PERSONNEL

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July – September 2015 71THE ARMY MEDICAL DEPARTMENT JOURNAL duty members of the US armed forces were located at the Karshi-Khanabad Air Base, also known as K2 or Camp Stronghold Freedom, between 2001 and 2005. General conditions were known to be harsh. Historically, the site was used by the Soviet military to support operations in Afghanistan in the late 1970s. During this time, the Soviet Air Force maintained a eet of various bomber aircraft at K2, which required an underground fuel distribution system. Furthermore, construction of military equipment (including missiles) in the Soviet era used materials such as asbestos and radioactive material. An occupational and environmental survey performed at K2 in November 2001 by the Center for Health Promotion and Preventive Medicine-Europe.found underground jet-fuel plumes and surface dirt contaminated with asbestos and radioactive uranium.38 Periodic high levels of dust and other particulate matter (PM) in the air due to seasonal dust storms was also noted. Although efforts for remediation of the environmental health risks present at K2 were made (eg, covering the contaminated areas with clean soil and declaring these areas “off-limits”), exposure to the toxicants mentioned above during deployment to K2 was plausible. In other settings, exposure to jet fuel plumes, asbestos-contaminated soil, radioactive materials, and/or dust and PM have resulted in documented adverse health outcomes, including both acute and chronic disease. As such, this investigation focused on identifying the frequency of postdeployment medical encounters for health outcomes consistent with exposure to the identi ed toxicants, with an emphasis on cancer due to the various types among personnel previously deployed to K2.52-61At the request of a US Central Command Force Health Protection Of cer, an evaluation of health outcomes among active duty military personnel with a history of deployment to K2 was conducted to address concerns for exposure(s) of health consequence among Army, Air Force, and Marine Corps personnel deployed to the air base. The Army Public Health Command subsequently conducted a comparative health assessment using one year of postdeployment medical follow-up. In the context of the above discussion regarding latency periods for cancer outcomes, the US Army Special Operations Command Surgeon later requested that the original analysis be repeated to incorporate up to 10 years of follow-up, using all available postdeployment medical encounter data. In response to this request, a retrospective cohort study was conducted in order to assess postdeployment health status among service members formerly deployed to K2. This was accomplished by linking K2 deployment rosters from 2001-2005 with postdeployment inpatient and outpatient medical records from 2001-2011. Additionally, a reference group of personnel stationed in South Korea during the same period was selected for comparison. The results are presented in the Table. The results of this analysis are somewhat mixed, with relative risks lower than one for about half of the speci c cancer type outcomes and relative risks higher than one for the other half. The only statistically signi cant ndings were for malignant melanoma and neoplasms of lymphatic and hematopoietic tissues (excluding Non-Hodgkin Lymphoma and Leukemia; highlighted in bluein the Table), indicating a risk approximately 3.7 times greater and 5.6 times greater among those deployed to K2 compared to those stationed in Korea. Concern for postdeployment cancer at K2 is warranted, given the relative risks above one, irrespective of statistical signi cance and the limitations of this particular analysis. Although the environmental hazard risk pro le may differ somewhat between K2 and other OIF/OEF locations, these results bolster the rationale for conducting broader studies to evaluate incidence of cancers following military deployment. CHALLENGES AND LIMITATIONSLong latency periods, low incidence rates of most types of cancer, and appropriate selection of nondeployed controls present challenges for investigators wishing to evaluate postdeployment cancer risk. Only very recently has a suf cient amount of time elapsed in order to assess cancer incidence following OIF and OEF deployments. Given the low incidence rates of most types of cancers, researchers must take care to ensure that study sample sizes are large enough to provide adequate statistical power to detect associations, should they exist. Epidemiologic studies comparing cases to controls with respect to OIF/OEF deployment status presents a challenge due to a high prevalence of deployment for any military personnel serving between 2001 and 2014. As such, a large well-powered study is imperative. Additional challenges include a lack of data on individual environmental exposures over time as well as a lack of exact locations of each service member during military deployments. As a result, deployment in general is typically used as a proxy for deployment-associated exposures. Also limiting to epidemiologic studies such as these is the lack of information on behavioral habits such as smoking, which can have signi cant effects on certain types of cancer. Cancer case de nitions are often based on ICD-9-CM coded medical encounter data from military medical record databases. Using administrative records to

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72 http://www.cs.amedd.army.mil/amedd_journal.aspxascertain cancer cases may result in false positives. For example, not only are some cancers not well de ned, but some require several encounters, sometimes with multiple specialists or requiring different medical procedures, in order to make a de nitive diagnosis. In such circumstances, an ICD-9-CM code may re ect a true case of cancer or the medical encounter may signify that a patient is in the process of ful lling diagnostic evaluations necessary to rule out cancer. Using medical encounter data for case ascertainment presents another limitation of this study: whereas medical encounter data capture is complete for service members who remain in service, the same cannot be said for personnel who leave military service. This becomes particularly problematic when studying chronic health outcomes such as cancer, with the latency periods often years after exposure, beyond the average time of military service. Investigators are currently attempting to establish methodology for linking medical encounter records from military service with medical encounter records from the Veterans Administration (VA) in order to minimize loss of follow up due to attrition from military service. However, this methodology will still fail at perfect case capture, as a certain portion of veterans are not VA bene ciaries or simply choose to obtain healthcare services outside the VA health system. It has been suggested that state cancer registries be used as additional sources of data in postdeployment cancer studies, however, the feasibility of this approach has yet to be explored. Although many challenges are presented to researchers seeking to determine whether or not cancer incidence is elevated among military service members and veterans formerly deployed in support of OIF and OEF relative to personnel without a history of deployment, it is an important topic that is worthy of public health efforts and resources. REFERENCES1. American Cancer Society. Cancer Facts and Figures 2015 [internet]. Available at: http://www.can cer.org/acs/groups/content/@editorial/documents/ document/acspc-044552.pdf. Accessed February 25, 2015. 2. National Cancer Institute. Surveillance, Epidemiology, and End Results Program. SEER Stat Fact Sheets: All Cancer Sites [internet]. Available at: http://seer.cancer.gov/statfacts/html/all.html. Accessed February 25, 2015. 3. National Cancer Institute. Cancer Trends Progress Report – 2011/2012 Update. Available at: http://pro gressreport.cancer.gov/sites/default/ les/archive/ report2011.pdf. Accessed February 25, 2015. Age-Adjusted Relative Risks and Corresponding 95% Con dence Intervals for Cancer Outcomes, Comparing US Military Personnel Deployed to K2 to US Military Personnel Stationed in KoreaOutcome K2Korea Age-Adjusted AgeAge YoungOldYoungOld n%n%n%n%RR95% CI All cancer110.39501.21410.281331.001.230.92-1.65 Brain cancer10.0440.1000.0080.062.040.68-6.09 Cervical cancer00.0000.0010.0100.000-Leukemia 00.0010.0250.0340.030.430.05-3.63 Malignant melanoma 10.0470.1730.0250.043.681.35-10.04 Neoplasm of bone/connective tissue/skin/breast 10.0430.0750.0390.071.060.35-3.22 Neoplasm of colon/rectum 20.0730.0720.0190.071.60.57-4.51 Neoplasm of digestive organs/peritoneum 00.0010.0210.0160.050.480.06-3.95 Neoplasm of female breast 10.0430.0710.0190.071.350.43-4.24 Neoplasm of genitourinary organs 10.0440.1020.0180.061.740.60-5.08 Neoplasm of lip/oral cavity/pharynx 10.0430.0700.0060.052.180.64-7.49 Neoplasm of lung/bronchus 00.0040.1000.0000.00---Neoplasm of lymphatic and hematopoietic tissue 20.0750.1260.0400.005.641.70-18.70 Neoplasm of respiratory/intrathoracic organs 00.0000.0000.0020.020-Neoplasm of testis 10.0420.0580.05120.090.570.17-1.91 Non-Hodgkin lymphoma 00.0030.0740.0380.060.890.25-3.26 Prostate cancer00.0040.1000.00180.140.710.24-2.10 Neoplasm of other and unspecified sites 00.0030.0730.02270.200.330.10-1.09 Neoplasm of uncertain behavior (plasma cells) 00.0000.0000.0000.00---*RR indicates relative risk. CI indicates confidence intervals. EVALUATION OF POSTDEPLOYMENT CANCERS AMONG ACTIVE DUTY MILITARY PERSONNEL

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July – September 2015 73THE ARMY MEDICAL DEPARTMENT JOURNAL4. World Health Organization. Cancer. Fact Sheet No 297 [internet]. 2015. Available at: http://www.who. int/mediacentre/factsheets/fs297/en/. Accessed February 25, 2015. 5. National Cancer Institute President’s Cancer Panel. 2008-2009 Annual Report. Reducing Environmental Cancer Risk: What We Can Do Now [internet]. 2010. Available at: http://deainfo.nci.nih.gov/ad visory/pcp/annualReports/pcp08-09rpt/PCP_Re port_08-09_508.pdf. Accessed February 25, 2015. 6. World Health Organization. Environmental and Occupational Cancers. Fact Sheet No 350 [internet]. 2011. Available at: http://www.who.int/media centre/factsheets/fs350/en/. Accessed February 25, 2015. 7. Zhu K, Devesa SS, Wu H, et al. Cancer incidence in the U.S. military population: comparison with rates from the SEER program. Ca ncer Epidemiol Biomarkers Prev 2009;18(6):1740-1745. 8. Defense Occupational and Environmental Health Readiness System [internet, limited access]. Available at: https://doehrs-ih.csd.disa.mil/Doehrs/. Accessed February 25, 2015. 9. US Army Public Health Command. The Periodic Occupational and Environmental Monitoring Summary (POEMS) – History Intent, and Relationship to Individual Exposures and Health Outcomes. Technical Information Paper 64-002-1110 [internet]. 2014. Available at: http://www.pdhealth.mil/ dcs/deploymentexposure.asp. Accessed June 5, 2015. 10. Smith B, Ryan MAK, Wingard DL, Patterson TL, Slymen DJ, Macera CA. Cigarette smoking and military deployment: a prospective evaluation. Am J Prev Med 2008;35(6):539-546. 11. Levine PH. Is testicular cancer related to Gulf War deployment? Evidence from a pilot populationbased study of Gulf War era veterans and cancer registries. Mil Med 2005;170(2):149-153. 12. Institute of Medicine. Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam Washington, DC: National Academies Press; 1994. Available at: http://www.iom.edu/Reports/1994/ Veterans-and-Agent-Orange-Health-Effects-ofHerbicides-Used-in-Vietnam.aspx. Accessed February 23, 2015. 13. Richards EE. Responses to Occupational and Environmental Exposures in the U.S. Military – World War II to the Present. Mil Med 2011;176(7):22-8. 14. Depa rtment of Veterans Affairs. Agent Orange home page [internet]. Available at: http://www. publichealth.va.gov/exposures/agentorange/index. asp. Accessed February 24, 2015. 15. The Selected Cancers Cooperative Study Group. The association of selected cancers with service in the US military in Vietnam. II. Softtissue and other sarcomas. Arch Intern Med 1990;150(12):2485-2492. 16. The Selected Cancers Cooperative Study Group. The association of selected cancers with service in the US military in Vietnam. III. Hodgkin’s disease, nasal cancer, nasopharyngeal cancer, and primary liver cancer. Arch Intern Med 1990;150(12):2495-2505. 17. Young AL, Cecil PF Sr. Agent orange exposure and attributed health effects in Vietnam veterans. Mil Med 2001;176(7):29-34. 18. Department of Veterans Affairs. Vietnam Veterans and Agent Orange: Independent Study Course [internet]. Available at: http://www.publichealth. va.gov/docs/vhi/VHIagentorange_text508.pdf. Accessed February 24, 2015. 19. The Selected Cancers Cooperative Study Group. The association of selected cancers with service in the US military in Vietnam. I. Non-Hodgkin’s lymphoma. Arch Intern Med 1990;150(12):2473-2483. 20. Frumkin H. Agent Orange and cancer: an overview for clinicians. CA Cancer J Clin 2003;53(4):245-255. 21. Institute of Medicine. Veterans and Agent Orange: Update 2012. Washington, DC: National Academies Press; 2012. Available at: http://www.iom. edu/Reports/2013/Veterans-and-Agent-OrangeUpdate-2012.aspx. Accessed February 23, 2015. 22. Baird C. The basis for and uses of environmental sampling to assess health risk in deployed settings. Mil Med 2011;176(7):84-90. 23. Bullman TA, Mahan CM, Kang HK, Page WF. Mortality in US Army Gulf War veterans exposed to 1991 Khamisiyah chemical munitions destruction. Am J Public Health 2005;95(8):1382-1388. 24. Young HA, Maillard JD, Levine PH, Simmens SJ, Mahan CM. Investigating the risk of cancer in 1990-1991 US Gulf War veterans with the use of state cancer registry data. Ann Epidemiol 2010;20(4):265-272. 25. Knoke JD, Gray GC, Garland FC. Testicular cancer and Persian Gulf war service. Epidemiology 1998;9(6):648-653. 26. Kang HK, Mahan CM, Lee KY, Magee CA, Murphy FM. Illnesses Among United States veterans of the Gulf War: a population-based survey of 30,000 veterans. J Occup Environ Med 2000;42(5):491-501. 27. Kang HK, Bullman TA. Mortality among US veterans of the Persian Gulf war: 7-year follow-up. Am J Epidemiol 2001;154(5):399-405.

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74 http://www.cs.amedd.army.mil/amedd_journal.aspx28. Institute of Medicine Gulf War and Health: Volume 3. Fuels, Combustion Products, and Propellants Washington, DC: National Academies Press; 2005. Available at: http://www.nap.edu/openbook. php?isbn=0309095271. Accessed February 24, 2015. 29. Bonds TM, Baiocchi D, McDonald LL. Army Deployments to OIF and OEF Santa Monica, CA: RAND Corp; 2010. Available at: http://www.rand. org/pubs/documented_brie ngs/DB587.html. Accessed February 23, 2015. 30. Tan M. 2 million troops have deployed since 9/11. Marine Corps Times December 18, 2009. Available at: http://www.marinecorpstimes.com/arti cle/20091218/NEWS/912180312/2-million-troopsdeployed-since-9-11. Accessed February 23, 2015. 31. Teichman R. Exposures of Concern to Veterans Returning from Afghanistan and Iraq. J Occup Environ Med 2012;54(6):677-681. 32. Helmer DA, Rossignol M, Blatt M, Agarwal R, Teichman R, Lange G. Health and exposure concerns of veterans deployed to Iraq and Afghanistan. J Occu p Environ Med 2007;49(5):475-480. 33. Brown KW, Bouhamra W, Lamoureux DP, Evans JS, Koutrakis P. Characterization of particulate matter for three sites in Kuwait. J Air Waste Manag Assoc 2008;58:994-1003. 34. Engelbrecht JP, McDonald EV, Gillies JA, Jayanty RK, Casuccio G, Gertler AW. Characterizing mineral dusts and other aerosols from the Middle East-part 1: ambient sampling. Inhal Toxicol 2009;21:297-326. 35. Mosher DE, Lachman BE, Greenberg MD, Nichols T, Rosen B, Willis HH. Green Warriors: Army Environmental Considerations for Contingency Operations from Planning Through Post-Con ict Santa Monica, CA: RAND Corp; 2008. Available at: http://www.rand.org/pubs/monographs/MG632. html. Accessed June 5, 2015. 36. Weese CB, Abraham JH. Potential health implications associated with particulate matter exposure in deployed settings in southwest Asia. Inhal Toxicol 2009;21:291-296. 37. Abraham JH, Eick-Cost A, Clark LL, et al. A retrospective cohort study of military deployment and postdeployment medical encounters for respiratory conditions. Mil Med 2014;179(5):540-546. 38. Deployment Health Clinical Center, et al. Environmental conditions at Karshi Khanabad [internet]. Available at: http://www.pdhealth.mil/clinicians/ downloads/CIS.02-09-09a.K-2-Provider.pdf. Accessed February 25, 2015. 39. Globalsecurity.org. Khanabad, Uzbekistan, KarshiKanabad (K2) Airbase, Camp Stronghold Freedom [internet]. Available at: http://www.globalsecurity. org/military/facility/khanabad.htm. Accessed February 25, 2015. 40. Yamane GK. Cancer incidence in the U.S. Air Force: 1989-2002. Aviat Space Environ Med 2006;77(8):789-794. 41. Armed Forces Health Surveillance Center. Incident diagnoses of cancers and cancer-related deaths, active component, U.S. Armed Forces, 2000-2011. MSMR 2012;19(6):18-22. 42. International Agency for Research on Cancer. Agents Classi ed by the IARC Monographs. Volumes 1-111 [internet]. 2014. Available at: www. monographs.iarc.fr. Accessed February 27, 2015. 43. Torres R. Burnpits360 [internet]. 2014. Available at: http://www.burnpits360.org/. Accessed February 26, 2015. 44. Stuart R. Operation Purple Heart [internet]. 2014. Available at: http://operationpurpleheart.blogspot. com/. Accessed February 26, 2015. 45. Bleyer A, Barr R, Hayes-Lattin B, Thomas D, Ellis C, Anderson B. The distinctive biology of cancer in adolescents and young adults. Nat Rev Cancer 2008;8(4):288-298. 46. Wrensch M, Bondy ML, Wiencke J, Yost M. Environmental risk factors for primary malignant brain tumors: A review. J Neurooncol 1993;17(1):47-64. 47. Miligi L, Seniori Costantini A, Crosignani P, et al. Occupational, environmental, and life-style factors associated with the risk of hematolymphopoietic malignancies in women. Am J Ind Med 1999;36(1):60-69. 48. Zheng T, Blair A, Zhang Y, Weisenburger DD, Zahm SH. Occupation and risk of non-HodgkinÂ’s lymphoma and chronic lymphocytic leukemia. J Occup Environ Med 2002;44(5):469-474. 49. Mester B, Behrens T, Dreger S, Hense S, Fritschi L. Occupational causes of testicular cancer in adults. Int J Occup Environ Med 2010;1(4):160-170. 50. Armenian HK, Lilienfeld AM. The distribution of incubation periods of neoplastic diseases. Am J Epidemiol 1974;99(2):92-100. 51. Howard J. Minimum latency and types or categories of cancer. centers for disease control and prevention [internet]. 2013. Available at: http://www. cdc.gov/wtc/pdfs/wtchpminlatcancer2013-05-01. pdf. Accessed February 26, 2015. 52. Harris DT, Sakiestewa D, Titone D, Robledo RF, Young RS, Witten M. Jet fuel-induced immunotoxicity. Toxicol Ind Health 2000;16(7-8):261-265. 53. LeVan TD, Koh WP, Lee HP, Koh D, Yu MC, London SJ. Vapor, dust, and smoke exposure in relation to adult-onset asthma and chronic respiratory symptoms: the Singapore Chinese Health Study. Am J Epidemiol 2006;163(12):1118-1128.EVALUATION OF POSTDEPLOYMENT CANCERS AMONG ACTIVE DUTY MILITARY PERSONNEL

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July – September 2015 75THE ARMY MEDICAL DEPARTMENT JOURNAL54. Ling SH, van Eeden SF. Particulate matter air pollution exposure: role in the development and exacerbation of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2009;4:233-243. 55. Marshall AC. Gulf war depleted uranium risks. J Expo Sci Environ Epidemiol 2008;18(1):95-108. 56. Nogueira JB. Air pollution and cardiovascular disease. Rev Port Cardiol 2009;28(6):715-733. 57. O’Toole TE, Conklin DJ, Bhatnagar A. Environmental risk factors for heart disease. Rev Environ Health 2008;23(3):167-202. 58. Periyakaruppan A, Kumar F, Sarkar S, Sharma CS, Ramesh GT. Uranium induces oxidative stress in lung epithelial cells. Arch Toxicol 2007;81(6):389-395. 59. Pleil JD, Smith LB, Zelnick SD. Personal exposure to JP-8 jet fuel vapors and exhaust at air force bases. Environ Health Perspect 2000;108(3):183-192. 60. Polichetti G, Cocco S, Spinali A, Trimarco V, Nunziata A. Effects of particulate matter (PM(10), PM(2.5) and PM(1)) on the cardiovascular system. Toxicology 2009;261(1-2):1-8. 61. Ritchie G, Still K, Rossi J III, Bekkedal M, Bobb A, Arfsten D. Biological and health effects of exposure to kerosene-based jet fuels and performance additives. J Toxicol Environ Health B Crit Rev 2003;6(4):357-451.AUTHORSMs Sharkey and Dr Abraham are Epidemiologists, Environmental Medicine, US Army Public Health Command, Aberdeen Proving Ground, Maryland.

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76 http://www.cs.amedd.army.mil/amedd_journal.aspxBACKGROUNDDuring the fall of 2014, a US medical liaison of cer from the Of ce of The Surgeon General of the Army, stationed in the United Kingdom, made an inquiry to the Army Hearing Program (AHP), US Army Public Health Command (USAPHC) regarding noise induced hearing loss (NIHL) in military musicians (W. Startz, e-mail, September 16, 2014). The USAPHC AHP conducted a literature search for studies on noise exposure and hearing loss in military band members; however, the search yielded limited speci c information on hearing loss in military musicians. As a result, a multidisciplinary team formed at the USAPHC to carry out a preliminary analysis on hearing loss in military band members. Noise exposure is a known occupational health hazard to those serving in the military.1,2 The effect of hazardous noise, however, can vary signi cantly depending on the type of noise (impulse versus steady state), intensity and duration of exposure, and the degree of effort to mitigate the effects. As a result of the variability in noise exposure, some military occupations may be more at risk for NIHL than others. For example, it has been estimated that Soldiers serving in combat arms units have a 30% chance of experiencing a hearing loss.3,4 Previous epidemiology studies have shown that infantry, gun crews, and seamanship specialists are 1.4 to 2 times more likely to suffer a signi cant threshold shift (change in hearing) than other military occupations.5,6Although several military occupations have been identi ed in previous military epidemiology studies, musicians have not been speci cally mentioned. Previous noise measurements collected in rehearsal halls and during performance venues suggest that noise exposure for musicians can range from 83 to 120 A-weighted decibels (dBA).7-14 We presume that military musicians will be at risk for similar noise exposures as their civilian counterparts and may be more at risk for NIHL than other military occupations. Noise exposure in the performance of duties as a military musician varies depending on the type of instrument, composition of the band or orchestra, venue, A Preliminary Analysis of Noise Exposure and Medical Outcomes for Department of Defense Military Musicians Cindy Smith Thomas Helfer, PhD Sharon Beamer, AuD Timothy A. Kluchinsky, Jr, DrPH Shane Hall, MSABSTRACTNoise exposure is a known occupational health hazard to those serving in the military. Previous military epidemiology studies have identi ed military occupations at risk of noise induced hearing loss (NIHL); however, musicians have not been speci cally mentioned. The focus of military NIHL studies is usually on those service members of the combat arms occupations. This project was a preliminary examination of Department of Defense (DoD) active duty military musicians in regard to their noise exposure, annual hearing test rates, and hearing injury rates using available data sources. The analysis concluded that DoD military musicians are an underserved population in terms of hearing conservation efforts. Noise surveillance data extracted from the Defense Occupational and Environmental Health Readiness System-Industrial Hygiene showed that every musician similar exposure group (SEG) with noise survey data from 2009 to 2013 exceeded the occupation exposure level adopted by DoD Instruction 6055.12 However, only a small percentage of all DoD active duty military musicians (5.5% in the peak year of 2012) were assigned to a SEG that was actually surveyed. Hearing test data based on Current Procedural Terminology coding extracted from the Military Health System revealed that the percentage of musicians with annual hearing tests increased over the 5 years studied in all services except the Air Force. During 2013, the data showed that the Navy had the highest percentage of musicians with annual hearing tests at 70.9%, and the Air Force had the lowest at 11.4%. The Air Force had the highest percentage of hearing injuries of those musicians with annual hearing tests for all 5 years analyzed. Although noise surveillance and annual hearing tests are being conducted, they occur at a much lower rate than required for a population that is known to be overexposed to noise.

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July – September 2015 77duration of exposure and proximity to other musicians. Also, as the number of years of military service increases, the likelihood of developing a noise-induced hearing loss increases. There is evidence of NIHL of 15 dB or greater at 4000 or 6000 Hertz in at least one ear in 45% of student (nonmilitary) musicians aged 18 to 25 years. Student musicians who practiced more than 2 hours a day were more likely to exhibit a decrease in hearing at some frequency than those who reported practicing for less hours.15 Another recent study examining incidence of hearing loss among professional musicians in Germany, suggests musicians have 3.51 times higher incidence rate of noise induced hearing loss and 1.45 times higher incidence rate of tinnitus than the general German population.16 The incidence of hearing loss for musicians increases with length of time of exposure.17,18 Professional symphony orchestra musicians in Denmark were found to have better hearing than the general population, but were considered at risk for occupational noise-induced hearing loss after prolonged exposure.19 Brazilian military musicians were found to be 14.54 times more likely to experience hearing loss when compared to their nonexposed counterparts, with a further decline in hearing noted as years of music exposure increases.20 However, among British Army musicians with 8 to 12 years of military service, the risk of developing hearing loss did not appear to be any greater than their nonmusician counterparts.21 Despite con icting results of studies with regard to the effects of music on hearing,11,22 musicians are generally considered to be at risk for NIHL and efforts to prevent hearing loss among this group of military personnel is essential. The military musician serves not only in their chosen occupation but also performs other military duties, such as weapons ring, placing them at even greater risk of hearing loss from noise exposure compared to nonmilitary musicians. PURPOSEA multidisciplinary team consisting of audiologists, industrial hygienists, and a statistician formed to analyze NIHL in military musicians based on a review of available data sources. The purpose of this project was to determine the noise exposure of Department of Defense (DoD) military musicians, the percentage of DoD military musicians receiving annual hearing tests, and the percentage of DoD military musicians that received an annual hearing test and was diagnosed with a hearing injury. This collection of information and its analysis was initiated and completed as a component of operational public health investigations and was not, therefore, subject to review by a human protections board such as an Institutional Review Board. METHODSPopulationThe population used for the analysis consisted of active duty musicians serving in the Air Force, Army, Marine Corps, and Navy as identi ed in Defense Enrollment Eligibility Reporting System (DEERS) by DoD occupational codes 145000 (enlisted) and 271400 (of cer and warrant of cers) during calendar years 2009 to 2013. The DEERS data were extracted using the Military Health System (MHS) Management and Analysis Reporting Tool (M2).Ascertainment of Noise ExposureThe Defense Occupational and Environmental Health Readiness System-Industrial Hygiene (DOEHRS-IH) was queried for the personal noise dosimetry conducted during calendar years 2009 to 2013 by the installation industrial hygiene program. The noise survey results were converted to an 8-hour time weighted average (TWA) using a 3 dB exchange rate as required by the DoD Instruction 6055.1223 to identify personnel who were at risk of occupational exposure to hazardous noise. The DOEHRS-IH personal noise survey results were considered to be representative samples of the similar exposure groups (SEG) in which the sampled musicians were assigned. The use of SEGs is a well-established strategy employed by industrial hygienists to conduct occupational exposure assessments. A SEG is a group of workers who have the same general exposure pro le for an agent, such as noise, because of the similarity and frequency of the tasks they perform.24 The assigned SEG population identi ed in the DOEHRS-IH was con rmed for active duty status, catchment area, and DoD occupational code through DEERS.Ascertainment of Audiology Procedures Data were extracted from the MHS using the M2 for the population using the Current Procedural Terminology (CPT) codes captured during direct care encounters. The CPT codes and their de nitions are listed in Table 1. Table 1. Current Procedural Terminology Codes for Hearing Test SurveillanceCPT Code De nition92552Pure tone audiometry (threshold)92555Speech audiometry threshold 92556Speech audiometry threshold with speech recognition92557Comprehensive audiometry threshold evaluation and speech recognition92559Audiometric test of groups

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78 http://www.cs.amedd.army.mil/amedd_journal.aspxIf one or more of these CPT codes were indicated in the rst 5 procedures for an encounter, the patient was considered to have had an audiogram. Persons with multiple encounters with audiograms within the calendar year were counted once for having an annual hearing test.Ascertainment of Hearing InjuryData for the population were extracted from the MHS direct care via the M2 using the International Classi cation of Diseases, 9th Revision, Clinical Modi cation (ICD-9 CM) codes identi ed as a diagnosis of a hearing injury. The ICD-9 CM codes identi ed for hearing injuries and their corresponding de nitions and hearing injury categories are listed in Table 2. If one of the hearing injury-identi ed ICD-9 CM codes was indicated in the rst 5 diagnoses for the direct care encounter, the diagnosis was identi ed for the person for the calendar year. A person diagnosed with one or more hearing injury ICD-9 CM codes within a calendar year is considered to have a hearing injury and was counted once for the calendar year. A person with one or more hearing injury ICD-9 CM codes within a hearing injury category (NIHL, tinnitus, sensorineural hearing loss (SNHL), and signi cant threshold shift (STS)) is counted once for each category for the calendar year. For this project, hearing injuries were analyzed for those who had an annual hearing test as identi ed through CPT codes. ANALYSISNoise SurveillanceFrom 2009 to 2013, a total 174 personal noise dosimetry samples were taken across the services. These 174 samples were representative of 38 different SEGs for musicians ranging from general characterization such as “band” to more speci c characterization of a speci c musical genera of “rock,” “marching,” and “concert” bands. The population of a SEG ranged from one to 165 personnel. Consistent with SEG assessment strategy, if one of the personal noise dosimetry samples of a given SEG is determined to be over the occupational exposure limit (OEL) of 85 dBA 8-hour TWA adopted by DoD Instruction 6055.12 ,23 then all personnel assigned to the SEG are considered to be over the OEL and at risk of exposure to hazardous noise. All DoD personnel exposed to noise levels greater than the OEL are identi ed on the command’s roster for inclusion in the Hearing Conservation Program (HCP); therefore, requiring personnel to be placed in a hearing testing surveillance program and have an audiogram conducted at least annually.23Hearing Test SurveillanceH earing test data were compared across services within a given year. A chi-square test, followed by the Marascuilo procedure, was used to determine if the Navy, Marines Corps, or Air Force had a signi cantly different proportion of service members with an annual hearing test compared to the Army. Statistical signi cance was de ned as P <.05. The Army was chosen as the reference group because Army Pamphlet 40-50125 requires every Soldier to undergo an annual hearing test that is recorded in both DOEHRS-Hearing Conservation (HC) and the MHS during a direct care encounter. Data from the DOEHRS-HC system is not linked into military treatment data; therefore, this project used the CPT coding as a surrogate for the DOEHRS-HC hearing test data.Hearing InjuryHearing injury data analysis was restricted to those in the population that received an annual hearing test. Hearing injuries were compared across the services and evaluated at the DoD level for total injuries and for each diagnosis. RESULTSNoise SurveillanceAll of the 38 different SEGS had at least one personal noise dosimetry sample over the OEL, resulting in all those assigned to the 38 SEGs being classi ed as being “overexposed.” The percentage of DoD military musicians assigned to the 38 SEGs during the 5-year period represents both those musicians under noise surveillance and those classi ed as being overexposed. Figure 1 shows the percentage of the DoD military musicians by each service that was assigned to SEGs during the noise dosimetry testing. There was limited information available for the personnel assigned to SEGs in 2009 because DOEHRSIH was still being incorporated into DoD-wide Table 2. ICD-9 CM Codes for Hearing InjuryHearing Injury Category ICD-9De nition Noise induced hearing loss388.10Noise effect-ear not otherwise specified (NOS)388.11Acoustic trauma 388.12Hearing loss D/T noise Tinnitus388.30Tinnitus NOS388.31Subjective tinnitus388.32Objective tinnitus Sensorineural hearing loss389.10Sensorineural hearing loss NOS389.11Sensory hearing loss, bilateral389.15Sensorineural hearing loss, unilateral389.16Sensorineural hearing loss, asymmetrical389.17Sensory hearing loss, unilateral389.18Sensorineural hearing loss, bilateral Significant threshold shift794.15Abnormal auditory function study A PRELIMINARY ANALYSIS OF NOISE EXPOSURE AND MEDICAL OUTCOMES FOR DEPARTMENT OF DEFENSE MILITARY MUSICIANS

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July – September 2015 79THE ARMY MEDICAL DEPARTMENT JOURNAL use. From a surveillance perspective, the percentage of musicians being surveyed is far below what is expected given that all SEGs were over the OEL. The 2 services with the highest percentage of personnel in SEGs with noise surveillance were the Air Force and the Army, with peaks of 12% for the Air Force in 2013 and 8% for the Army in 2012. The percentage of military musicians under noise surveillance DoD wide (Figure 2) showed a steady increase from 2009 to 2013; however, the highest percentage in the 5 years was 5.5%. Given that all SEGS within all years were considered overexposed, it appears that regardless of how many SEGs or which SEG the industrial hygienists surveys, they will be classi ed as overexposed.Hearing Test SurveillanceAs shown in Table 3, the Army consistently had the highest (or nearly the highest) proportion (54% to 69%) of musicians with annual hearing tests from 2009 to 2013. In these 5 years, Army had a signi cantly greater proportion of musicians with an annual hearing test compared to the Air Force and Marine Corps ( P <.05). Both the Navy (31% to 71%) and Marine Corps (21% to 51%) had an increased proportion of musicians with annual hearing tests from 2009 to 2013, noting that from 2011 to 2013, the Navy had nearly identical proportions as the Army. The Air Force started with the lowest percentage tested in 2009 (18%) with no improvement observed from 2010 to 2013. The 5-year trend is presented in Figure 3. Although some services, notably the Army and Navy, had higher testing rates, all services were not in compliance with the 100% testing requirement. No service had above 71% of its musicians tested within a given year.Hearing InjuryAs shown in Table 4, hearing injury rates are highest in the Air Force for all 5 years; however, the Air force has the least percentage of musicians with annual hearing tests. The Army showed the second highest injury rates followed by the Marine Corps and Navy, respectively. The most common hearing injury diagnosis among DoD military musicians is SNHL, with tinnitus as the second most common diagnosis. Signi cant threshold shift comes in third and NIHL fourth. The rates of these diagnoses uctuated between 2009 and 2013, but only marginally. The comparison of each diagnosis across the services (Figures 4, 5, 6, 7), revealed that the Air Force had much higher injury rates for NIHL, SNHL, and tinnitus than the other 3 services for all 5 years. Figure 1. Percentage of active duty musicians by military service assigned to SEGs during noise surveillance. 0% 2% 4% 6% 8% 12% 14% 10%Marine Corps Air ForceArmyNavy 2012 2013 2011 2010 2009 Table 3. Number and Percentage by Service of DoD Military Musicians With an Annual Hearing Test from 2009 to 2013Military Service20092010201120122013Counta%Counta%Counta%Counta%Counta%Air Force13417.7%b13618.4%b9513.3%b8913.1%b7811.4%bArmyc107654.6%126861.6%112955.1%117058.5%136369.0%Marine Corps20621.1%b35236.1%b39843.1%b36841.3%b45551.2%bNavy22331.1%b32644.6%b40555.7%b41258.3%49870.9%Data from the Military Health System Management and Analysis Reporting Tool.aCount indicates the number of persons with one or more of the designated CPT codes during the calendar year.bSignificantly different proportion of hearing injuries compared to the reference group (Army).cReference Group Figure 2. Percentage of active duty DoD musicians assigned to SEGS during noise surveillance. 2% 1% 0% 4% 3% 6% 5%20122013 2011 2010 2009

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80 http://www.cs.amedd.army.mil/amedd_journal.aspxThe Air ForceÂ’s injury rate for SNHL was at least 3 times those in the Army. With exception of 2012, the Air ForceÂ’s injury rate for tinnitus was 4 times the Army injury rate. The injury rates for all the services for NIHL, SNHL, and tinnitus had little uctuation during the 5 years. The noise surveillance data from DOEHRS-IH demonstrates that a portion of the DoD musicians are exposed to hazardous occupational noise related to their jobs as musicians in the military. A large percentage of DoD military musicians were not assigned to a SEG and not under noise surveillance; however, the noise surveillance documented shows overexposure. The annual hearing test results do not re ect that which would be expected for a population with known hazardous occupational noise exposure. DoD Instruction 6055.1223 requires personnel in hazardous noise environments to have an annual hearing test. Among the services, the Army and the Navy showed the highest percentage of musicians receiving their annual hearing test; however, even these 2 services are well below 100%. The Air ForceÂ’s injury rates for hearing injuries in general and for NIHL, SNHL, and tinnitus speci cally were higher than the other services. Additionally, the Air Force had the lowest percentage of musicians with annual hearing tests. There are multiple strengths of this analysis. The interdisciplinary team brought different perspectives that enhanced the public health performance evaluation methods promulgated by the Institute of Medicine.26 The data sources used (DEERS, MHS, and DOEHRSIH) provided a broad-spectrum approach using the best available data, which provided an analysis that incorporated multisource data integration techniques including demographic details data, noise exposure data, medical procedures data (as a surrogate for hearing test surveillance), and medical outcome diagnoses data associated with procedure data. There are limitations of the analysis as well. National Guard and Reserve service member data were not included and the injury data do not include purchase care visits. The 5 years of data was not determined to be suf cient to determine trends in the longitudinal data. Since the data set in this analysis is limited, the sample may not truly be representative of the entire population of military musicians; therefore, generalization to the entire population may not be feasible. CONCLUSION AND RECOMMENDATIONSThis article demonstrates that DoD military musicians are an underserved population with regard to hearing conservation efforts. While noise surveillance and annual hearing tests are conducted, they occur Figure 3. Percentage of military musicians within each service with an annual hearing test, 2009 to 2013. 40% 50% 0% 30% 60% 20% 70% 10% 80% 2013 2012 2011 2010 2009 Air Force Marine Corps Navy Army Table 4. Number and Percentage of Injuries by Service and Diagnosis Among DoD Musicians Who Received an Annual Hearing Test from 2009 to 2013Military Service2009a2010a2011a2012a2013aCount%Count%Count%Count%Count%Air Force2518.7%3122.8%2526.3%1719.1%1721.8%Army736.8%917.2%928.1%1018.6%936.8%Marine Corps136.3%164.5%256.3%339.0%337.3%Navy114.9%144.3%194.7%307.3%244.8%DiagnosisNIHL120.7%80.4%100.5%130.6%70.3%SNHL885.4%924.4%904.4%1105.4%1174.9%STS382.3%663.2%723.6%542.6%351.5%Tinnitus744.5%854.1%793.9%1015.0%1064.4%Data from the Military Health System Management and Analysis Reporting Tool.aCount and percentages are representative only of those injuries that occurred among military personnel who had an annual hearing test. Denominator for counts and percentages is the total number of military personnel tested. A PRELIMINARY ANALYSIS OF NOISE EXPOSURE AND MEDICAL OUTCOMES FOR DEPARTMENT OF DEFENSE MILITARY MUSICIANS

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July – September 2015 81THE ARMY MEDICAL DEPARTMENT JOURNAL at a much lower rate than required for a population that is overexposed to noise. Although the focus of this analysis was on a single population that is relatively small in comparison with other military occupations, a systematic approach is recommended to improve the hearing conservation efforts that would affect DoD military musicians. The ultimate responsibility for compliance with annual hearing tests and the requirement for the HCP lie with the commanders. The hearing conservation efforts should be a high-priority item evaluated during all command safety assessments and inspector general inspections. Making hearing conservation efforts a leadership priority will require commanders to engage the professional elds responsible for the different aspects of hearing conservation (industrial hygiene, preventive medicine, occupational health, hearing conservation, audiology, and safety). Command situational awareness and command emphasis that identi es and characterizes hearing health challenges sets a foundation for hearing injury prevention planning and execution at all levels. ACKNOWLEDGMENTSWe thank Kevin Wisniewski and Monica Ahuna-Williams of the Army Institute of Public Health (AIPH) Industrial Hygiene and Medical Safety Management Program, Ralph Rogers and Brian Grace of the AIPH Industrial Hygiene Field Services Program, and Charles Jokel of the Army Hearing Program for their support in this analysis.REFERENCES1. Humes LE, Jollenbeck LM, Durch JS, eds. Noise and Military Service: Implications for Hearing Loss and Tinnitus Washington, DC: National Academy Press. 2006. 2. Government Accountability Of ce. Hearing Loss Prevention: Improvements to DoD Hearing Conservation Programs Could Lead to Better Outcomes GAO Report No. 11-114; 2011. 3. Walden BE, Prosek RA, Worthington DW. The Prevalence of Hearing Loss Within Selected U.S. Army Branches. Washington DC: Walter Reed Army Medical Center; 1975. Available at: http:// www.dtic.mil/dtic/tr/fulltext/u2/a019193.pdf. Accessed June 8, 2015. 4. Ohlin D. Epidemiologic report: hearing evaluation audiometric reporting system (HEARS). MSMR 1996;2(3): 8-9. 5. Helfer T, Beamer SL, Deaver K, Hall S. Active Duty – U.S. Army Noise Induced Hearing Injury Surveillance Calendar Years 2009-2013. Aberdeen Proving Ground, MD: US Army Public Health Command; 2014. Available at: http://phc.amedd. army.mil/Periodical%20Library/USArmyAnnual NIHIsurveillance_CYs_2009_2013.pdf. Accessed June 8, 2015. 6. Helfer TM, Canham-Chervak M, Canada S, Mitchener TA. Epidemiology of hearing impairment and noise-induced hearing injury among U.S. military personnel, 2003-2005. Am J Prev Med 2010;38(suppl 1):S71-S77. 7. Bride D, Gill F, Proops D, Harrington M, Gardiner K, Attwell C. Noise and the classical musician. BMJ 1992;305(6868):1561-1563. 8. Nataletti P, Sisto R, Pieroni A, Sanjus F, Annesi D. P ilot study of professional exposure and hearing functionality of orchestra musicians of a national lyric theatre. G Ital Med Lav Ergon 2007;29(suppl 3):496-498. 9. Emmerich E, Rudel L, Richter F. Is the audiologic status of professional musicians a re ection of the noise exposure in classical orchestral music? Eur Arch Otorhinolaryngol 2008;265(7):753-758. 1.5% 4.0% 3.5% 3.0% 2.5% 2.0% 1.0% 0.5% 0.0% 2012 2013 2011 2010 2009 Marine Corps Air ForceArmyNavy Figure 4. Percentage of DoD military musicians by service with annual hearing test and NIHL diagnosis. Figure 5. Percentage of DoD military musicians by service with annual hearing test and SNHL diagnosis. 0% 2% 4% 6% 8% 12% 14% 16% 18% 10%Marine Corps Air ForceArmyNavy 2012 2013 2011 2010 2009

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82 http://www.cs.amedd.army.mil/amedd_journal.aspx10. Royster,JD, Royster LH, Killion MC. Sound exposures and hearing thresholds of symphony musicians. J Acoust Soc Am 1991;89:2793-2803. 11. Sataloff RT. Hearing loss in musicians. Am J Otol 1991;12(2):122-127. 12. Royster JD, Royster LH, Killion MC. Sound exposures and hearing thresholds of symphony orchestra musicians. J Acoust Soc Am 1991;89(6):2793-2803. 13. Early KL, Hurstman SW. Noise exposure to musicians during practice. Appl Occup Environ Hyg 1996;11(9):1149-1153. 14. Chasin M. Musicians and the Prevention of Hearing Loss San Diego, CA: Singular Publishing Group. 1996 15. Phillips SL, Henrich VC, Mace ST. Prevalence of noise-induced hearing loss in student musicians. Int J Audiol 2010;49: 309-316. 16. Schink T, Kreutz G, Busch V, Pigeot I, Ahrens W. Incidence and relative risk of developing hearing disorders in professional musicians. Occup Environ Med 2014;71(7):472-476. 17. Schmuziger N, Patscheke J, Probst R. Hearing in non-professional pop/rock musicians. Ear Hear 2006;27:321-330. 18. Morais D, Benito JI, Almaraz A. Acoustic trauma in classical music players. Acta Otorhinolaryngol 2007;58:401-407. 19. Schmidt JH, Pederson ER, Paarup HM, Christensen-Dalsqaard J, Andersen T, Poulsen T, Baelum J. Hearing loss in relation to sound exposure of professional symphony orchestra musicians. Ear Hear 2014;35(4):448-460. 20. Giglio de Oliviera Goncalves C, Moreira Lacerda AB, Zeigelboim BS, Marques JM, Luders D. Auditory thresholds among military musicians: conventional and high frequency. Codas 2013;25(2):181-187. 21. Pastil ML, Sadhra S, Taylor C, Folkes SEF. Hearing loss in British Army musicians. Occup Med 2013;63:281-283. 22. Zhao F, Manchaiah VK, French D, Price SM. Music exposure and hearing disorders: an overview. Int J Audiol 2010;49:54-64. 23. Department of Defense Instruction 6055.12: Hearing Conservation Program Washington, DC: US Dept of Defense; 2010. 24. Bullock WH, Ignacio JS. A Strategy for Assessing and Managing Occupational Exposures Farfax, VA: American Industrial Hygiene Association; 2006. 25. Department of the Army Pamphlet 40-501: Army Hearing Program Washington DC: US Dept of the Army; 2015. 26. Perrin EB, Durch JS, Skillman SM, eds. Measuring Health Performance in the Public Sector Washington, DC: National Academy Press; 2015.AUTHORSMs Smith is an Industrial Hygienist with the US Army Public Health Command, Aberdeen Proving Ground, Maryland. Dr Beamer is assigned to the Navy Bureau of Medicine and Surgery, Falls Church, Virginia. Previously she was a Hearing Conservation Consultant with US Army Public Health Command, Aberdeen Proving Ground, Maryland. Mr Hall is a Statistician with the US Army Public Health Command, Aberdeen Proving Ground, Maryland. Dr Helfer is a Hearing Conservation Consultant with the US Army Public Health Command, Aberdeen Proving Ground, Maryland. Dr Kluchinsky is Manager, Health Hazard Assessment Program, US Army Public Health Command, Aberdeen Proving Ground, Maryland.Figure 6. Percentage of DoD military musicians by service with annual hearing test and STS diagnosis. 0% 2% 1% 4% 3% 6% 5%Marine Corps Air ForceArmyNavy 2012 2013 2011 2010 2009 0% 5% 15% 20% 25% 10%Marine Corps Air ForceArmyNavy 2012 2013 2011 2010 2009 Figure 7. Percentage of DoD military musicians by service with annual hearing test and Tinnitus diagnosis. A PRELIMINARY ANALYSIS OF NOISE EXPOSURE AND MEDICAL OUTCOMES FOR DEPARTMENT OF DEFENSE MILITARY MUSICIANS

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July – September 2015 83Historically, health physics support to the Combined Joint Operations Area-Afghanistan (CJOA-A) was administered by a nuclear medical science of cer (NMSO) assigned to Task Force-Medical built around a medical brigade. This of cer was responsible for managing the theater health physics program and had no other health physics experts to serve as assistants. In late spring 2013, the NMSO (military occupational specialty [MOS] 72A) position was eliminated to meet force size reduction requirements. The US Forces Afghanistan (USFOR-A) Safety Of ce initiated a hiring action for a civilian radiation safety professional at this time, but the hiring action was never approved. An NMSO working as the senior medical planner at the International Security Assistance Force Joint Command Headquarters for most of 2013 was available to provide consultation assistance. However, the loss of the dedicated NMSO position left the CJOA-A without a full-time health physics expert when a surge in the health physics mission workload and a corresponding heightening of ionizing radiation exposure risks occurred in early 2014. The reasons for the surge in health physics mission requirements in 2014 were varied. A primary cause was the acceleration of retrograde operations which led to progressively larger quantities of radioactive commodities arriving for processing and shipping at the Bagram Air eld (BAF) and Kandahar Air eld (KAF) Redistribution Property Accountability Team (RPAT) yards. The arrival rates of these commodities far exceeded removal rates, thus overtaxing the RPAT yard’s management capabilities and creating problems in storage of such items. Another leading cause was a dramatic increase in base closures and increase in demolitions of structures. These activities led to the discovery of orphan sources (unwanted and uncontrolled radioactive materials) on the installations and also necessitated radiological surveys of foreign military equipment on some of the closing bases to clear for demilitarization and removal. Additionally, safety concerns about exposures to x-ray and gamma radiation sources in mobile vehicle and cargo inspection systems (MVACISs) found at installation entry control points spurred a requirement to begin monitoring and inspecting the operation of these systems. Filling the health physics capability gap left by the departure of the NMSO forced medical planners in the CJOA-A to devise a solution within the mandated force management level constraints at the time. Under these constraints, reestablishing the lost NMSO billet was not deemed a viable option due to the General Of cer level of approval necessary for such action. The developed solution was to substitute 2 health physics specialists (HPSs) for preventive medicine specialists projected to arrive in June of 2014 with the incoming 172nd Preventive The Benefits of Deploying Health Physics Specialists to Joint Operation Areas LTC Scott Mower, MS, USA MAJ Joshua D. Bast, MS, USA MAJ Margaret Myers, MS, USAABSTRACTPreventive Medicine Specialists (military occupational specialty [MOS] 68S) with the health physics specialist (N4) quali cation identi er possess a unique force health protection skill set. In garrison, they ensure radiation exposures to patients, occupational workers and the public from hospital activities such as radioisotope therapy and x-ray machines do not to exceed Federal law limits and kept as low as reasonably achievable. Maintaining suf cient numbers of health physics specialists (HPSs) to ll authorizations has been a consistent struggle for the Army Medical Department due to the rigorous academic requirements of the additional skill identi er-producing program. This shortage has limited MOS 68SN4 deployment opportunities in the past and prevented medical planners from recognizing the capabilities these Soldiers can bring to the ght. In 2014, for the rst time, HPSs were sourced to deploy as an augmentation capability to the 172nd Preventive Medicine Detachment (PM Det), the sole PM Det supporting the Combined Joint Operations Area-Afghanistan. Considerable successes in bettering radiation safety practices and improvements in incident and accident response were achieved as a result of their deployment. The purposes of this article are to describe the mission services performed by HPSs in Afghanistan, discuss the bene ts of deploying HPSs with PM Dets, and demonstrate to senior medical leadership the importance of maintaining a health physics capability in a theater environment.

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84 http://www.cs.amedd.army.mil/amedd_journal.aspxMedicine Detachment (PM Det). As a stopgap measure, an HPS who had deployed as a Battle Noncommissioned Of cer with the 31st Combat Support Hospital (Task Force 31) in February 2014, was employed to perform crucial health physics mission requirements on a part-time base until arrival of the 172nd PM Det. The substitution of HPSs for PMS was made possible by the fact that HPSs are former preventive medicine specialists. The only difference between the 2 types of specialists from a quali cations standpoint is the HPSs have completed a 20-week course to earn N4 skill identi er. The HPS may be considered MOS quali ed to perform PMS tasks and as such executed these tasks on a routine basis in Afghanistan. However, HPSs are consistently in short supply due to the rigors of their academic training and are usually assigned to health care facilities to support health physics programs. Their small number and high garrison demand have made long-duration deployments rare and deprived them the opportunity to prove their value in the combat theater environment. Therefore, their deployment to the CJOA-A beginning in 2014 was a unique event providing an opportunity for lessons to be learned about how to best employ them. MISSION OVERVIEWThe HPSs assigned to the 172nd PM Det served on PM teams at BAF and KAF. The HPS at BAF supported Regional Command (RC)-North, RC-East, RC-West, and RC-Capital, while the other supported RC-South and RC-Southwest. When not performing health physics missions, they were engaged in standard preventive medicine technician duties such as sanitation inspections and water quality monitoring. The most common health physics and radiation safety missions performed are summarized in the Table. It is important to note these missions could have been executed by a NMSO. The medical imaging oversight mission was performed by a 2-person team comprised of a NMSO and a HPS from US Army Public Health Command (USAPHC) brought in for a 3-week period. The remaining missions shown in the Table were performed by the HPSs assigned to the PM Det.MVACIS InspectionsMany entry control points used MVACIS to image local national vehicles for weapons and explosives prior to permitting their entry onto installations. The Nuclear Regulatory Commission (NRC) regulates the MVACIS radioactive sources in the United States. The Communications-Electronics Command (CECOM) holds the NRC license for these sources before they are shipped overseas. Although the NRC does not have jurisdiction, the MVACIS sources are managed by an Army Radiation Authorization given to USFOR-A by CECOM. Appointments as Radiation Safety Of cers for NRC licenses and Army Radiation Authorizations are routinely held by NMSOs, civilian health physicists, or very specialized trained individuals. United States and International Security Assistance Force (ISAF) uniformed personnel assigned security responsibilities for installations served as the operators of these systems. Many operators had no experience with MVACIS operations prior to their deployments and had never been enrolled in a radiation dosimetry program. Contractors were used by ISAF and USFOR-A to maintain and service these systems. The contractors would position contract service representatives (CSRs) at major bases and designate coverage areas for the representatives to support. The CSRs would travel to the bases to administer safety and operator training, and issue and collect thermoluminescent dosimeters. They also visited entry control points at a speci ed frequency to examine the daily exposure by reading records kept by operators, collect and reissue dosimeters, and troubleshoot any problems. Upon the completion of the visit, they would offer recommendations and advice to the site operators on improving work practices. The effectiveness of their visits was limited since they lacked the authority to hold operators accountable for failing to follow proper safety procedures. THE BENEFITS OF DEPLOYING HEALTH PHYSICS SPECIALISTS TO JOINT OPERATION AREAS Health Physics and Radiation Safety MissionsTypeFrequencyDescription Mobile Vehicle and Cargo Inspection System (MVACIS) inspections QuarterlyQuarterly inspections of MVACIS at entry control points to ensure safe system operation. Radioactive commodity retrograde support ContinuousConsultation on the establishment and operation of a consolidated storage locations, offering radiation safety training to the workforce, leak testing turned-in items, and surveying packaged items prior to shipment. Radiation surveysAs neededSurvey with specialized measurement equipment areas where a suspected radiation release and/or exposure has occurred, followed by an assessment of health risks. Orphan source managementAs neededProvide assistance and consultation on how to manage items unexpectedly found on installations. These discoveries could prompt a radiation survey mission. Medical imaging system oversightAnnuallyInspection of HCF imaging equipment that emits ionizing radiation.

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July – September 2015 85THE ARMY MEDICAL DEPARTMENT JOURNAL In order to address radiation exposure concerns and improve radiation safety operational practices, in mid-2014 USFOR-A issued a directive implementing MVACIS inspection program (Figure 1). The inspection checklist was developed with input from the HPSs, USAPHC health physics experts, and the civilian radiation safety of cer employed by the largest MVACIS contractor in Afghanistan. Prior to starting the quarterly inspections, an initial site assistance visit was completed at each ECP by the HPSs or uniformed PM personnel at bases not supported by the 172nd PM Det. The non-HPS inspectors received training by the CSRs on how to perform these inspections before initiating the initial site assistance visits. The scrutiny and attention garnered by the inspections improved operator adherence to safety policies and procedures. The inspections also improved the completeness of occupational and environmental health site assessments since preventive medicine elements had previously not been populating the Defense Occupational and Environmental Health Readiness System’s (DOEHRS) DoD Deployment Surveillance Portal with information concerning the occupational hazards associated with MVACIS radiation emissions.Radioactive Commodity Retrograde SupportOver the course of 13 years of continuous military operations, signi cant quantities of US equipment containing radioisotopes had been brought into Afghanistan. Examples of some of the more common items included weapon system optics, compasses, and chemical agent detection alarms. The processes and procedures for retrograding items varied based on the manager of the commodity and the type of radioisotope it contained. Many items had to be tested through the collection and submission of wipe tests to the US Army Test, Measurement, and Diagnostic Equipment laboratory in Pirmasens, Germany, to prove they had been surveyed with a radiation detection device and were free of leaks before nal packaging and shipment. Prior to arrival of the HPSs, there was only one contractor in all of Afghanistan stationed at KAF quali ed to perform wipe sampling. When the push to retrograde commodities began, management and shipping processes were not suf ciently mature to handle the in ux of turned-in items. The problem was further magni ed by a lack of trained personnel to support these processes. Unlike the drawdown from Operation Iraqi Freedom, there was no Army Contaminated Equipment Team, a special team deployed by the Army Material Command, to lead the radioactive commodities retrograde effort in Afghanistan. Efforts to overcome personnel shortfalls were further hampered by delays in civilian hiring actions; stringent force management level constraints governing the number of contractors, service members, and Department of Defense employees permitted in country; and dif culties in modifying in exible scopes of work to permit contractors already involved with incountry retrograde operations to participate in radioactive commodity retrograde support activities. By necessity, the HPSs were used to support the retrograde effort even though retrograding activities are doctrinally the responsibility of logistics rather than medical authorities. Their assistance was broad in scope and evolved throughout the deployment as new challenges were identi ed and previous problems were solved. The effect of their assistance was greatly ampli ed by their exceptional knowledge base in health physics, strong oral and written communication skills, and the credibility boost offered by their ranks as noncommissioned of cers. Those areas of assistance where the HPSs had the most meaningful effect on retrograde support were in the administration of safety awareness training to retrosort yard personnel, reviewing and coauthoring pertinent standard operating procedures and policy documents, relieving the wipe sample collection backlog through additional sampling, surveying prepped shipments, participation in installation radiation safety working groups, and providing consultative services to aid the establishment of a consolidated radioactive commodities storage area on BAF.Radiation SurveysDuring the course of their deployment, the HPSs performed surveys of areas and equipment possibly contaminated with radioactive material. Some of these surveys were of an urgent nature and required prompt execution, while others were less time-sensitive. The Figure 1. A nuclear medical science of cer examining a mobile vehicle and cargo inspection systems at Bagram Air eld on March 28, 2015. (Photo courtesy of the authors.)

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86 http://www.cs.amedd.army.mil/amedd_journal.aspxurgent surveys were typically high pro le and garnered intense interest from senior commanders. One such incident resulted after a report was received about possible acute radiation exposures from a malfunctioning X-ray emitting MVACIS received by North Atlantic Treaty Organization (NATO) soldiers manning an entry control point at the ISAF Headquarters in downtown Kabul. The guards were evacuated to a nearby NATO-run hospital and kept under medical observation for symptoms of acute radiation sickness after it was discovered that one of the 3 x-ray tubes of the MVACIS was not functioning properly, and high radiation readings were allegedly read from a radiation detection meter. Within one hour of noti cation of the incident, a HPS was own by air ambulance from BAF to the scene to investigate. Much to the relief of all parties involved, this investigation conclusively proved that no medically signi cant radiation releases had occurred. The malfunctioning x-ray tube was determined to be burned-out, meaning it could not emit any x-ray radiation. The alleged high radiation measurements were due to a 3 order of magnitude instrument reading error. As an additional precautionary measure, the HPS performed a radiation survey of the entry control point and found no radiation readings above normal background levels. Another high-pro le radiation survey was performed at the site of an MI-17 helicopter re within a hangar at the New Kabul Afghanistan International Airport (Figures 2 and 3). The Russian-made helicopter was the property of the Afghanistan Air Force (AAA) and was equipped with instrumentation that contains radioisotopes. The hangar where the helicopter was parked was an important rotary wing aircraft maintenance location and also served as a training area where US military experts administered hands-on training to AAA maintenance recruits. Senior AAA and ISAF leadership wanted to remove the burned out helicopter hulk and resume hangar operations as soon as possible; however, a radiation survey was rst required to assess the dangers posed from any radiation contamination. The survey results from the HPS found the hangar free of contamination, thus clearing the way for recovery operations. Most radiation surveys performed by the HPS were not as spectacular as the two previous examples. The majority of surveys supported retrograde efforts and included surveys checking for contaminated areas at radioactive commodity storage locations and the aforementioned surveys of items prepped for shipment out of the CJOAA. The HPSs also surveyed items, often of foreign make, earmarked for demilitarization to certify those items were radioisotope free.Orphan Source ManagementThere were several instances where the HPSs were called upon to provide assistance and consultative support in dealing with orphan sources discovered on ISAF installations. Examples of such orphan sources included a Soviet-era ice detector containing strontium-90, a beta emitter, found at BAF (Figure 4); thorium nitrate, an alpha emitter, encapsulated in concrete within a 5 gallon bucket in an abandoned building that once served as an East German Pharmaceutical Plant on Camp Phoenix (Figure 5); and 2 ion chamber survey meters improperly discarded, presumably by a contractor, in a dumpster at BAF. Though all the orphan source discovery incidents were judged by the HPS to pose low health risks, they were, nevertheless, documented and archived within DOEHRS. THE BENEFITS OF DEPLOYING HEALTH PHYSICS SPECIALISTS TO JOINT OPERATION AREAS Figure 2. Smoke pours from a burning Afghan MI-17 helicopter inside of a maintenance hang ar at the Kabul International Airport. The helicopter in foreground is another MI-17 not involved in the re. (Photo courtesy of the authors.) Figure 3. Damage inside of the maintenance hangar at the Kabul International Airport follow ing removal of burned Afghan MI-17 helicopter. (Photo courtesy of the authors.)

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July – September 2015 87THE ARMY MEDICAL DEPARTMENT JOURNALMedical Imaging System OversightOne of the basic responsibilities for HPSs in a nondeployed environment is to ensure medical imaging systems (x-rays, uoroscopy machines, and computed tomography scanners) at medical treatment facilities, dental clinics, and veterinary clinics are functioning properly. The highly specialized equipment necessary to perform these checks was not available in Afghanistan. As such, the oversight role of medical imaging systems for HPSs within Afghanistan was limited to checking on TLD wearing and monitoring system operator work practices to determine their adherence to as low as reasonably achievable radiation exposure work practices. In order to satisfy the regulatory requirement for an annual check of imaging systems, a request for assistance was submitted by USFOR-A in collaboration with US Army Central Command to the US Army Medical Command for a team to perform these checks. This team spent 3 weeks in country checking 17 systems at 6 installations. RECOMMENDATIONSThe health physics support provided by the HPSs proved to be of great bene t across multiple staff support areas (safety, logistics, and force health protection). As a rst of its kind deployment, there was initially some confusion as to how best utilize them. The confusion abated as duties and responsibilities became better de ned and bene cial partnerships with other organizations and commands were established. The following paragraphs offer several recommendations on how to best prepare, equip, and employ HPs should they be used for future long-term deployments.PreparationSince HPSs are not assigned to PM Dets in garrison, they should be afforded an opportunity to participate in the PM Det’s predeployment certi cation training exercise. Doing so allows them to meet their deployment teammates, relearn basic PMS tasks, and advance their understanding of health physic mission requirements in the deployed environment before arriving at the deployment destination. The HPSs selected to deploy should be knowledgeable in performing medical imaging system surveys and conducting contamination surveys, and skilled in decontamination practices. Given the strong possibility of being asked to provide retrograde operation support and MVACIS inspections, completion of Class 7 shipment training and familiarization with MVACIS radiation safety fundamentals prior to deployment is advisable. In addition, they should have the necessary rank, experience, and initiative to successfully establish and maintain an effective health physics program in the absence of a NMSO or other of cers well versed in health physics.EquipmentWith the exception of the medical imaging radiation surveys, all other surveys performed by the HPSs required use of an AN/PDR 77 radiac set plus its accessories to measure and detect radiation levels. This radiac set is not an item found in the modi ed table of organization and equipment of a PM Detachment. One of the 2 sets used in Afghanistan by the HPSs was a loaner from a USAPHC, while the other was acquired through logistic channels after submitting an operational needs statement. Had these radiac sets not been available, the scope and the overall effectiveness of the services provided by the HPSs would have been severely diminished. A commercial off-the-shelf portable gamma spectroscopy Figure 4. A Soviet aviation ice warning device containing strontium-90, a beta particle emitter, found at Bagram Air eld. (Photo courtesy of the authors.) F i g u r e Figure 5 5 A m e t a l b u c k e t c o n t a i n i n g t h o r i u m n i t r a t e w h i c h h a d A metal bucket containing thorium nitrate which had b e e n e n c a p s u l a t e d i n c o n c r e t e a t t h e n o w c l o s e d C a m p P h o e been encapsulated in concrete at the now closed Camp Phoen i x i n t h e K a b u l b a s e c l u s t e r ( P h o t o c o u r t e s y o f t h e a u t h o r s ) nix in the Kabul base cluster. (Photo courtesy of the authors.)

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88 http://www.cs.amedd.army.mil/amedd_journal.aspxinstrument will also be useful in a theater of operations since it can identify common radioisotopes, is more sensitive than the AN/PDR-77 in detecting gamma-emitters, and can detect some neutron emissions.EmploymentSince the bulk of the HPS mission services occurred at the retrograde hubs at BAF and KAF, this was logically the best location to station them. While stationed here, they had ample time to gain an understanding of the retrograde processes and learn where their services were needed most to support retrograde efforts. Also, BAF and KAF were installations where the 172nd PM Det had the lead for providing most PM support. This meant the HPSs were able to perform PMS mission work when there were lulls in the health physics missions. Currently, there is only one HPS for the 224th PM Det which replaced the 172nd PM Det. This soldier is located at BAF where the majority of the health physics operations are concentrated. The USFOR-A is preparing to designate an Air Force Industrial Hygienist as their Radiation Safety Of cer (RSO), in absence of an NMSO, and the HPS as the alternate RSO. A NMSO recently arrived in Kuwait and provides long-distance support to Afghanistan and other countries in the area of operations. The NMSO serves as a bridging solution until the safety community develops a permanent solution for the Radiation Safety Program. CONCLUSIONMaintaining a capability to execute health physics missions is critical and likely to grow in urgency and magnitude during the drawdown phase of an overseas military deployment operation. In the absence of an available NMSO, medical planners should give strong consideration to deploying HPSs to assist with lling health physics and radiation safety capability gaps. An ideal place to assign HPSs is within a deploying PM Det. As members of the detachment, they are well-positioned to execute their health physics missions while also being available to perform routine PMS duties. The HPSs deployed to the CJOA-A in 2014 executed a multitude of crucial missions. Some of the missions were extremely high pro le with signi cant diplomatic implications. Based on the notable success of their Afghanistan deployment, the HPSs have proven their worth many times over and should be considered for future deployments. AUTHORSLTC Mower was the International Security Assistance Force Joint Command and US Forces-Afghanistan (USFOR-A) Force Health Protection Consultant from January through October 2014 at North Kabul Afghanistan International Airport. He is an Environmental Science and Engineering Of cer and Registered Environmental Health Specialist assigned to XVIII Airborne Corps, Fort Bragg, North Carolina. MAJ Bast is an Army Medical Entomologist and the Commander of the 172nd Preventive Medicine Detachment, which deployed to Afghanistan from June 2014 through February 2015. He also served as the USFOR-A Force Health Protection Consultant from October 2014 through January 2015. MAJ Myers is currently serving in Kuwait as the Theater Radiation Safety Of cer at US Army Central Command Headquarters (Forward). Her duties require frequent travel to Afghanistan.THE BENEFITS OF DEPLOYING HEALTH PHYSICS SPECIALISTS TO JOINT OPERATION AREASArticles published in the Army Medical Department Journal are indexed in MEDLINE, the National Library of MedicineÂ’s (NLMÂ’s) bibliographic database of life sciences and biomedical information. Inclusion in the MEDLINE database ensures that citations to AMEDD Journal content will be identi ed to researchers during searches for relevant information using any of several bibliographic search tools, including the NLMÂ’s PubMed service.

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July – September 2015 89The US Army has made education of its Soldiers regarding behavioral health one of its foremost considerations. This emphasis has resulted in the Borden Institute’s publication of the fourth Textbook of Military Medicine in the past 20 years to address the behavioral health of the Soldier: Forensic and Ethical Issues in Military Behavioral Health. The writing of the rst two behavioral health books, Military Psychiatry: Preparing in Peace for War (1994) and War Psychiatry (1995) was led by COL (Ret) Franklin D. Jones. These volumes were published soon after the end of the Gulf War in 1991. COL (Ret) Elspeth Ritchie was the Senior Editor for Combat and Behavioral Health published in 2011, as well as this newly published volume. COL Ritchie’s books were published as Operations Iraqi Freedom and Enduring Freedom were winding down. All four of these books present insights into the emotional aspects of war and the role of behavioral health care providers within the context of the US Army. Each volume reinforces the understanding that the stresses of military life can be signi cant. Forensic and Ethical Issues differs from the other books in that it focuses on the practice of forensic psychology and psychiatry and their application to medical issues arising within the military legal system. Covering a broad range of topics, this book provides an easily accessible reference for readers wishing to understand the implications of a Soldier’s behavior and how care providers specializing in psychological and psychiatric care can help Soldiers in trouble. The men and women who serve in the military responded without hesitation to the challenge of terrorism provoked by the events of September 11, 2001. Yet, now that military operations conducted in response are winding down, many Soldiers and their families have been left with the psychological wounds of war. Our military specialists in behavioral health focus on helping Soldiers cope with these mental injuries and associated behaviors. In this book, psychiatrists, psychologists, scientists with expertise in behavioral health, and lawyers who specialize in military law, from all the military services, have coauthored chapters detailing how the sciences of psychology and forensic psychiatry apply to behavioral issues in the context of the legal system. The intent is to show the insight and understanding that forensic specialists add to the military justice system as Soldiers face great challenges in their lives. Throughout the book are descriptions of the very broad roles that behavioral health professionals play in attempting to help jurists better understand why Soldiers commit crimes. The work of these professionals adds an element of fairness to the evaluation of Soldiers in crisis. The content of each chapter re ects topical issues in today’s military world. Suicide, sexual assault, and posttraumatic stress disorder (PTSD) are the primary behavioral health issues facing the military. To begin addressing the rst of these, the authors discuss ways to stop a determined individual from committing suicide. When a suicide occurs, the authors detail the military’s investigative plan, which provides a “psychological autopsy” of the events leading to the act. When suicides are perceived to occur in clusters within a military unit or location, an epidemiologic review of the cases and identi cation of common forces (if any) that drive these events are described. Sexual assaults are discussed in a similarly straightforward manner. The need for the sexual education of men, particularly where it applies to the issue of competent consent, and the underlying psychological environment A New Volume in the Borden Institute Textbooks of Military Medicine SeriesForensic and Ethical Issues in Military Behavioral HealthCOL (Ret) Elspeth Cameron Ritchie, MC, USA Senior Editor LTC Daniel E. Banks, USA COL (Ret) Edward Lindeke, USA

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90 http://www.cs.amedd.army.mil/amedd_journal.aspxleading to a culture in which sexual assaults occur is recognized and addressed. The authors also provide an understanding of the psychiatric diagnosis of PTSD. Flashbacks, sleep disturbances, and a labile mood in the context of previous combat experience form the core of the diagnosis. Yet the chapter proceeds to address unresolved questions about PTSD, with the recognition that it is a very dif cult injury to treat; available therapies are not as effective as we would wish. Not infrequently Soldiers with PTSD come to the attention of care providers because of legal dif culties, during struggles in navigating the Veterans Administration Disability System, or following accusations of malingering. Although there is a recognition that PTSD may invoked as a possible defense for crimes, no clear conclusion is presented to show whether a diagnosis is likely to affect trial outcomes. However, this defense strategy may be a more powerful mitigating factor in a military, as opposed to a civilian, courtroom. Furthermore, care providers continue to struggle with optimal PTSD therapy, and their numerous, widely disparate approaches help us realize how dif cult overcoming this illness can be. Resilience training, cognitive behavioral therapies, and medications are all a part of care. Not infrequently, alternative and complementary methods are also tried. Although PTSD is labelled an anxiety disorder, overlapping therapies of antipsychotics and antidepressants remain the primary approach to medical therapy. The training of forensic psychologists and psychiatrists, both within and outside of the military, is outlined, providing insight into the formal education of these care providers. These experts must be prepared to help Soldiers interact with the sanity board process, help recognize mitigating factors in the defense of a Soldier facing criminal charges, and explain behavioral health issues in a SoldierÂ’s disability hearing by describing how he or she has faced mental health and disciplinary issues. In addition, the role of these care providers in two specialized and speci c criminal arenas is discussed. The rst is the role of the forensic specialist in cases where Soldiers are charged with substance abuse; the second is the rare but intense situation when capital murder is alleged and the death penalty is sought. Another chapter draws a clear line de ning the expected (and required) behavior of psychiatrists in their role as care providers of detainees. A key point is that the psychiatrist or psychologist is not an interrogator, but he or she may act as a behavioral health care provider to the detainee or a as consultant to the interrogators (never serving in both roles). In the latter role, the focus is to help interrogators better recognize features of mental illness and the psychological effects of interrogation techniques. The book also discusses how to design a safe and secure psychiatric facility. A clear description of how to house those with long-term forensic psychiatric illnesses is detailed, as illustrated by the new Saint Elizabeths Hospital in Washington, DC, maximizing both security and therapeutic concerns. The principles presented can be used by all in the mental health eld. The book closes with an intriguing chapter addressing the relationship of me oquine, a drug prescribed to Soldiers for malaria prophylaxis, with development of mood changes, psychosis, and in the context of underlying PTSD. The author recommends that all service members should be screened for the use of me oquine. If the service member has been exposed to the medication and is facing legal charges, the situation should be articulated by the defense. Forensic and Ethical Issues in Military Behavioral Health makes a signi cant contribution to the body of work identifying the lessons learned by Army care providers following the wars in Iraq and Afghanistan. Throughout the book, Dr Ritchie and the contributing authors identify and address many of the issues now in the forefront of care provided by the ArmyÂ’s behavioral medicine specialists. The authors show a strong understanding of the relationship between war and a SoldierÂ’s mental health and help us recognize approaches that must be in place as we go forward to treat service members of the next generation, lessons that will be relevant for many years to come. AUTHORLTC Banks is the Director of the Borden Institute, Joint Base Fort Sam Houston, Texas. COL (Ret) Lindeke is the Assistant Director of the Borden Institute, Joint Base Fort Sam Houston, Texas.FORENSIC AND ETHICAL ISSUES IN MILITARY BEHAVIORAL HEALTH A NEW VOLUME IN THE BORDEN INSTITUTE TEXTBOOKS OF MILITARY MEDICINE SERIES This book is available in PDF and will soon be available in iPad and Kindle formats for download on your electronic reader. The book can be ordered at no cost by those on active duty, in the National Guard, and in the Reserve by following the instructions on the Borden Institute website, http://www.cs.amedd.army.mil/borden/ It will soon be available for purchase from the US Government Printing Of ce.

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July – September 2015 91THE ARMY MEDICAL DEPARTMENT JOURNAL

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92 http://www.cs.amedd.army.mil/amedd_journal.aspx The US Army Health Readiness Center of Excellence The Army Medical Department Center and SchoolENVISION, DESIGN, TRAIN, EDUCATE, INSPIREJoint Base San Antonio Fort Sam Houston, Texas

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SUBMISSION OF MANUSCRIPTS TO THE ARMY MEDICAL DEPARTMENT JOURNALThe United States Army Medical Department Journal is published quarterly to expand knowledge of domestic and international military medical issues and technological advances; promote collaborative partnerships among the Services, components, Corps, and specialties; convey clinical and health service support information; and provide a professional, high quality, peer reviewe d print medium to encourage dialogue concerning health care issues and initiatives.REVIEW POLICYAll manuscripts will be reviewed by the AMEDD Journal ’s Editorial Review Board and, if required, forwarded to the appropriate subject matter expert for further review and assessment.IDENTIFICATION OF POTENTIAL CONFLICTS OF INTEREST1. Related to individual authors’ commitments: Each author is responsible for the full disclosure of all nancial and personal relationships that might bias the work or information presented in the manuscript. To prevent ambiguity, authors must state explici tly whether potential con icts do or do not exist. Authors should do so in the manuscript on a con ict-of-interest noti cation section on the title page, providing additional detail, if necessary, in a cover letter that accompanies the manuscript. 2. Assistance: Authors should identify Individuals who provide writing or other assistance and disclose the funding source for this assistance, if any. 3. Investigators: Potential con icts must be disclosed to study participants. Authors must clearly state whether they have done so in the manuscript. 4. Related to project support: Authors should describe the role of the study sponsor, if any, in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication. If the supporting source had no such involvement, the authors should so state.PROTECTION OF HUMAN SUBJECTS AND ANIMALS IN RESEARCHWhen reporting experiments on human subjects, authors must indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. If doubt exists whether the research was conducted in accordance with the Helsinki Dec laration, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly approve d the doubtful aspects of the study. When reporting experiments on animals, authors should indicate whether the institutional and nat ional guide for the care and use of laboratory animals was followed.INFORMED CONSENTIdentifying information, including names, initials, or hospital numbers, should not be published in written descriptions, photo graphs, or pedigrees unless the information is essential for scienti c purposes and the patient (or parent or guardian) gives written informed consent for publication. Informed consent for this purpose requires that an identi able patient be shown the manuscript to be published. Authors should disclose to these patients whether any potential identi able material might be available via the Internet as well as in print after publication. Patient consent should be written and archived, either with the Journal the authors, or both, as dictated by local regulations or laws.GUIDELINES FOR MANUSCRIPT SUBMISSIONS1. Manuscripts may be submitted either via email (preferred) or by regular mail. Mail submissions should be in digital format ( preferably an MS Word document on CD/DVD) with one printed copy of the manuscript. Ideally, a manuscript should be no longer than 24 double-spaced pages. However, exceptions will always be considered on a case-by-case basis. 2. The American Medical Association Manual of Style governs formatting in the preparation of text and references. All articles should conform to those guidelines as closely as possible. Abbreviations/acronyms should be limited as much as possible. Inclusion of a list of article acronyms and abbreviations can be very helpful in the review process and is strongly encouraged. 3. A complete list of references cited in the article must be provided with the manuscript, with the following required data: • Reference citations of published articles must include the authors’ surnames and initials, article title, publication title, year of publication, volume, and page numbers. • Reference citations of books must include the authors’ surnames and initials, book title, volume and/or edition if appropriat e, place of publication, publisher, year of copyright, and speci c page numbers if cited. • Reference citations for presentations, unpublished papers, conferences, symposia, etc, must include as much identifying information as possible (location, dates, presenters, sponsors, titles). 4. Either color or black and white imagery may be submitted with the manuscript. Color produces the best print reproduction quality, but please avoid excessive use of multiple colors and shading. Digital graphic formats (JPG, TIFF, GIF) are preferred. Editable versions with data sets of any Excel charts and graphs must be included. Charts/graphs embedded in MS Word cannot be used. Prints of photographs are acceptable. If at all possible, please do not send photos embedded in PowerPoint or MS Word. Images submitted on slides, negatives, or copies of X-ray lm will not be published. For clarity, please mark the top of each photographic print on the back. Tape captions to the back of photos or submit them on a separate sheet. Ensure captions and photos are indexed to each other. Clearly indicate the desired position of each photo within the manuscript. 5. The authors’ names, ranks or academic/certi cation credentials, titles or positions, current unit of assignment, and contact information must be included on the title page of the manuscript. Submit manuscripts to: DSN 471-6301 Comm 210-221-6301 Email: usarmy.jbsa.medcom-ameddcs.list.amedd-journal@mail.mil EDITOR, AMEDD JOURNALAHS CDD AMEDDC&S 3630 STANLEY RD STE B0204 JBSA FORT SAM HOUSTON, TX 78234-6100

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