Group Title: Malaria Journal 2009, 8:159
Title: Optimally timing primaquine treatment to reduce Plasmodium falciparum transmission in low endemicity Thai-Myanmar border populations
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Title: Optimally timing primaquine treatment to reduce Plasmodium falciparum transmission in low endemicity Thai-Myanmar border populations
Series Title: Malaria Journal 2009, 8:159
Physical Description: Archival
Creator: Lawpoolsri S
Klein EY
Singhasivanon P
Yimsamran S
Thanyavanich N
Maneeboonyang W
Hungerford LL
Maguire JH
Smith DL
Publication Date: 40009
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/

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Research

Optimally timing primaquine treatment to reduce Plasmodium
falciparum transmission in low endemicity Thai-Myanmar border
populations
Saranath Lawpoolsri*1,2, Eili Y Klein3, Pratap Singhasivanon2,
Surapon Yimsamran2, Nipon Thanyavanich2, Wanchai Maneeboonyang2,
Laura L Hungerford', James H Maguire' and David L Smith4,5


Address: 'Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA,
2Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 3Department of Ecology and Evolutionary
Biology, Princeton University, Princeton, New Jersey, USA, 4Department of Biology, University of Florida, Gainesville, FL, USA and 5Emerging
Pathogens Institute, University of Florida, Gainesville, FL, USA
Email: Saranath Lawpoolsri* saranatlaw@yahoo.com; Eili Y Klein eklein@princeton.edu; Pratap Singhasivanon tmpsh@mahidol.ac.th;
Surapon Yimsamran tmsys@mahidol.ac.th; Nipon Thanyavanich tmnty@mahidol.ac.th;
Wanchai Maneeboonyang tmwmn@mahidol.ac.th; Laura L Hungerford lhungerf@epi.umaryland.edu;
James H Maguire jmaguire@partners.org; David L Smith davesmith@ufl.edu
* Corresponding author



Published: 15 July 2009 Received: 17 May 2009
Malaria journal 2009, 8:159 doi: 10. 186/1475-2875-8-159 Accepted: 15July2009
This article is available from: http://www.malariajournal.com/content/8/1/159
2009 Lawpoolsri et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Effective malaria control has successfully reduced the malaria burden in many
countries, but to eliminate malaria, these countries will need to further improve their control
efforts. Here, a malaria control programme was critically evaluated in a very low-endemicity Thai-
Myanmar border population, where early detection and prompt treatment have substantially
reduced, though not ended, Plasmodium falciparum transmission, in part due to carriage of late-
maturing gametocytes that remain post-treatment. To counter this effect, the WHO recommends
the use of a single oral dose of primaquine along with an effective blood schizonticide. However,
while the effectiveness of primaquine as a gametocidal agent is widely documented, the mismatch
between primaquine's short half-life, the long-delay for gametocyte maturation and the proper
timing of primaquine administration have not been studied.
Methods: Mathematical models were constructed to simulate 8-year surveillance data, between
1999 and 2006, of seven villages along the Thai-Myanmar border. A simple model was developed
to consider primaquine pharmacokinetics and pharmacodynamics, gametocyte carriage, and
infectivity.
Results: In these populations, transmission intensity is very low, so the P. falciparum parasite rate
is strongly linked to imported malaria and to the fraction of cases not treated. Given a 3.6-day half-
life of gametocyte, the estimated duration of infectiousness would be reduced by 10 days for every
10-fold reduction in initial gametocyte densities. Infectiousness from mature gametocytes would
last two to four weeks and sustain some transmission, depending on the initial parasite densities,
but the residual mature gametocytes could be eliminated by primaquine. Because of the short half-
life of primaquine (approximately eight hours), it was immediately obvious that with early


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administration (within three days after an acute attack), primaquine would not be present when
mature gametocytes emerged eight days after the appearance of asexual blood-stage parasites. A
model of optimal timing suggests that primaquine follow-up approximately eight days after a clinical
episode could further reduce the duration of infectiousness from two to four weeks down to a few
days. The prospects of malaria elimination would be substantially improved by changing the timing
of primaquine administration and combining this with effective detection and management of
imported malaria cases. The value of using primaquine to reduce residual gametocyte densities and
to reduce malaria transmission was considered in the context of a malaria transmission model; the
added benefit of the primaquine follow-up treatment would be relatively large only if a high fraction
of patients (>95%) are initially treated with schizonticidal agents.
Conclusion: Mathematical models have previously identified the long duration of P. falciparum
asexual blood-stage infections as a critical point in maintaining malaria transmission, but
infectiousness can persist for two to four weeks because of residual populations of mature
gametocytes. Simulations from new models suggest that, in areas where a large fraction of malaria
cases are treated, curing the asexual parasitaemia in a primary infection, and curing mature
gametocyte infections with an eight-day follow-up treatment with primaquine have approximately
the same proportional effects on reducing the infectious period. Changing the timing of primaquine
administration would, in all likelihood, interrupt transmission in this area with very good health
systems and with very low endemicity.


Background
Plasmodium falciparum is endemic in 87 countries and
approximately 2.4 billion persons are at risk [1,2]. As pop-
ulations in South Asia and South East Asia regions have
grown, so have the number of people at risk [3-5]. Now,
approximately one billion people live in areas at very low
risk of P. falciparum [2,4]. Recently, the WHO Global
Malaria Programme has called for countries with low and
moderate transmission areas to eliminate malaria trans-
mission from their entire territory [6]. In many of these
areas, malaria transmission is suppressed through a com-
bination of insecticide-treated nets, indoor residual spray-
ing, and prompt, effective treatment with anti-malarial
drugs [1]. However, despite implementation of all the
control measures, in some areas, such as the border region
with Myanmar in Thailand, malaria transmission persists.
This suggests a need to critically re-examine malaria epide-
miology and the parasite life cycle to identify new control
points.

One of the components of an elimination programme is
to reduce transmission from malaria patients by making
the infectious period as short as possible [6]. Prompt treat-
ment of clinical cases is important, but in humans, mature
gametocytes are the only infective stage of malaria para-
site, and most drugs do not kill mature gametocytes [7]. In
addition, in the P. falciparum life-cycle, unlike in other
Plasmodium spp., the appearance of infective gametocytes
is delayed with respect to the erythrocytic-schizogony
cycle, resulting in a delayed appearance of mature P. falci-
parum gametocytes in the peripheral blood about 7-15
days after the initial acute attack [7,8]. While, an untreated


infection lasts about six months, on average, this duration
can be cut short to a few days after the incubation period
[7]. Thus, in areas where asexual parasitaemia is cut short
by effective treatment with anti-malarial drugs and game-
tocyte production is, therefore, limited, lingering gameto-
cytes can maintain malaria transmission [9-11]. This issue
is particularly important in areas with good access to med-
ical clinics.

Although artemisinin derivatives have been shown to
reduce gametocyte carriage by eliminating asexual para-
sites and immature gametocytes, only the 8-aminoquino-
lines, such as primaquine, are lethal to the mature
gametocytes [7,12]. Primaquine, which has been shown
to be effective against mature P. falciparum gametocytes is
rapidly absorbed with peak plasma concentrations
reached about two hours after administration [13,14].
However, it has a drug elimination half-life of approxi-
mately eight hours, so it only remains active against para-
sites for, at most, a few days [12,15]. The WHO
recommends the use of a single oral dose of primaquine
along with an effective blood schizonticide to reduce
transmission, particularly in low endemic areas [12]. In
areas where an early detection and treatment programme
is highly effective, patients generally receive treatment one
or two days after the acute attack, or approximately three
to five days before gametocyte maturation [16]. While the
effectiveness of primaquine as a gametocidal agent is
widely documented, the mismatch between primaquine's
short half-life, the long-delay for gametocyte maturation,
and the proper timing of primaquine administration have
not been studied.


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Here, a mathematical model was constructed to describe
P. falciparum transmission dynamics in seven hamlets
along the Thai-Myanmar border, an area with low, sea-
sonal transmission, and a highly effective health system.
The transmission model was extended to investigate the
timing of follow-up primaquine administration on the
duration of gametocyte carriage and the implications for
malaria transmission in the area. The models were devel-
oped with the goal of critically evaluating malaria trans-
mission and tailoring control measures in areas with
extremely good health systems, where elimination of
malaria is feasible.

Methods
Study site
A malaria transmission model was developed to closely
track the observed transmission intensity over an eight-
year period of seven hamlets in the Tanaosri subdistrict,
Suanphung district in Ratchaburi Province, a mountain-
ous area along the Thai-Myanmar border. The study site
covered an area of around 50 km2 with approximately
3,500 inhabitants in about 500 households. Free diagno-
sis and treatment of malaria has been provided for people
in the area since 1997 by the Rajanagarindra Tropical Dis-
ease International Center (RTIC), operated by Mahidol
University, Thailand, the only malaria clinic in this area.
Individuals commonly receive treatment for febrile
malaria within two days of an acute attack. After deploy-
ment of the early detection and treatment programme, the
peak incidence of clinical malaria has gradually decreased
from approximately four cases per 100 persons in 1999 to
about two cases per 100 persons from 2003 through
2005. A single dose of mefloquine had been used as the
first-line drug for treatment of uncomplicated falciparum
malaria until the year 2005; the standard regimen was
then changed to a two-day artesunate-mefloquine combi-
nation therapy, as recommended by the Thai government,
because of the increased mefloquine resistance in the area


[17]. A single dose of 30 mg primaquine is given to all P.
falciparum positive patients on the last day of the treat-
ment course. In Thailand, the haemolysis after pri-
maquine administration among glucose-6-phosphate
dehydrogenase (G6PD) deficiency patients is relatively
mild [18,19]. A test for G6PD deficiency is not required
before a single dose primaquine administration.

Malaria transmission in this area is markedly seasonal
with a peak of transmission during the rainy season (April
to July), mainly due to the fluctuation of the mosquito
population. However, malaria transmission does con-
tinue at a low level during the dry season. The P. falci-
parum parasite rate (PfPR) in the area is very low,
according to regular active surveillance surveys that have
sampled more than half of the study population since
2003 (Table 1). Parasites were not detected in three sur-
veys in 2005. Although some infections with low parasite
densities might have gone undetected, the low PfPR and
yearlong continuous transmission suggest that asexual
blood-stage infection is rare and thus gametocyte carriage
in the absence of asexual parasites must play an important
role in maintaining mosquito infection and the persist-
ence of malaria transmission in the area.

Transmission model
A deterministic model of the infection dynamics of
human and mosquito populations in the study area was
developed. In the models, the human population was
divided into five compartments: state variables tracked the
proportion that were susceptible (Sh), liver-stage only
(Li), asymptomatic infection with gametocyte carriage
(Ah), clinical episode (Ch), and gametocyte carriage only
(Gh). In this low malaria transmission setting, malaria
immunity and super-infection are rare, so an immune
population was not included in the model. In the model
for the mosquito population, seasonal mosquito popula-
tion dynamics was considered, so the model tracked the


Table I: Plasmodium falciparum parasite rate (PfPR) according to active surveillance surveys in the study area between 2003 and 2005.


Census Survey month

3059 June
August
October
November
December
2906 February
May
June
September
November
2990 January
March
May
July


Number of blood samples


# P. falciparum positive


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Year 2003




Year 2004




Year 2005


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population density of susceptible (M), infected (Y), and
infectious (Z) mosquitoes (Figure 1). The human and
mosquito infection dynamics were connected by human
blood feeding of uninfected mosquitoes on infectious
humans, and of infectious mosquitoes on uninfected
humans. The dynamics of malaria infections in the two
host populations are, thus, described by the following dif-
ferential equations.

Equations for the human population:

Sh = -baZSh + oGhp pSh + rAh
L, = baZSh -vLh
C17 = PvL1 C (1)
Ah = (1 P)vL, + pSh rAh
G;E = Cth -o o o o
Equations for the mosquito population:


S((1 + sin(2zt / 365)) gM
ac(A, + Gh)(M Y Z) (q + g)Y
qY gZ


Figure I
Schematic illustration of the malaria transmission
model. The diagram shows the compartments of human
population (Top) and mosquito population (Bottom) and
transmission processes. For human population: Sh = suscepti-
ble; Lh = liver-stage only; Ah = asymptomatic infection with
gametocyte carriage; Ch = clinical episode; and Gh = gameto-
cyte carriage only. For mosquito population: M = susceptible;
Y = infected; and Z = infectious.


Susceptible humans become infected when exposed to
infectious mosquitoes at the rate baZ, where a is the
human feeding rate and b is the probability of transmis-
sion [20,21]. A large proportion of infected patients (P)
become symptomatic at the rate v, an inverse of the latent
period [22], these symptomatic individuals are subse-
quently detected and treated by passive surveillance at the
clinic. Because of the delayed development of P. falci-
parum gametocytes, the rate that patients become infec-
tious ( ) after an acute attack depends on the duration of
the gametocyte maturation process [7,8]. Individuals nor-
mally remain infectious for a period of time even if the
infection is treated and cured. Infections with gametocytes
lose their infectivity to mosquitoes at the rate o, which
depends in part on the different treatment regimens
[13,23].

Asymptomatic asexual blood-stage infections are occa-
sionally observed in low-endemic malaria areas [24].
Therefore, a small proportion of infected individuals (1-
P) was assumed to become asymptomatically infected. In
addition, asymptomatic cases imported from neighboring
areas occur at the rate p. These asymptomatic patients are
likely to remain untreated and infectious for a long
period, until the gametocytes are naturally eliminated at
rate r [7]. The imported rate of asymptomatic cases was
assumed to be equal to the exported rate of susceptible
individuals, so that the population size remains constant.

For the mosquito population, adult mosquitoes emerge
from larval habitat, which is modeled with a sinusoidal
function for seasonal forcing, X(1+sin(27xt/365). Mos-
quito infection occurs at rate ac, where c is the probability
of transmission when they feed on infectious humans
[21]. These infected mosquitoes subsequently become
infectious at the rate q. A constant death rate, g, was
applied to all mosquito classes.

All parameter estimates were obtained from published lit-
erature and unpublished data from the study area; except
for the average mosquito birth rate, 2, which was esti-
mated by fitting the model to the observed malaria occur-
rence data, and the waiting time to clear gametocytes
under different treatment regimens, which was computed
using another model (see below). Details and value esti-
mates of parameters in the models are described in Table
2.

Model for gametocyte cycle
The duration of infectivity under different drug regimens
was computed using a within-host model of gametocy-
togenesis (Figure 2). The dynamics of the model are
described by the following differential equations.


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Table 2: Description of parameters and parameter estimated used in the transmission model.


Parameters Description


a Human feeding rate: Number of bites on human, per mosquito, per day, i.e., the product of the number of bites per
mosquito per day and the proportion of bites on humans.
b Parasite transmission probability of mosquitoes to humans: The probability that an infectious mosquito transmit the
parasite to a human from a single bite.
c Parasite transmission probability of humans to mosquitoes: The probability that a mosquito become infected from a
single bite on an infectious human.
v The rate that an infected human becomes positive for malaria parasite: An inverse of latent period.
P Treatment coverage: The proportion that infected humans receive the malaria treatment
4 The rate that an symptomatic human becomes infectious: An inverse of duration of gametocytogenesis
r The rate that an asymptomatic human lose the infectivity: An inverse of duration of gametocyte carriage in natural
infection
o The rate that an infectious human lose the infectivity with respect to treatment:
ao: An inverse of duration of gametocyte carriage after mefloquine treatment
op: An inverse of duration of gametocyte carriage after ACTs treatment
oQ: An inverse of duration of gametocyte carriage after primaquine follow-up treatment
g Import rate of asymptomatic cases: The proportion of imported case per person per day
g Death rate of mosquitoes: An inverse of expected lifespan of a mosquito
q The rate that an infected mosquito becomes infectious: An inverse of duration of sporogony
k Average recruitment rate of adult mosquitoes


Ag =(1 p)OAg pOAg aAg

i = POA, Ig
(3)
S, = I PS,

Gg = S, -pGg Gg

In P. falciparum, the erythrocytic stage takes approximately
two days, at the end of which each asexual parasite, Ag,



Erythrocytic Visible Sequestered Visible
schizogony Immature-- Immature -+ mature
cycle gametocyte gametocyte gametocyte
Asexual Natural
Parasite Death rate due Death
Death rate due to primaquine rate
Death rate due treatment
to schizonticidal
treatment


Figure 2
Schematic illustration of the model for gametocy-
togenesis process. An asexual parasite produces 16 new
parasites at every erythrocytic schizogony cycle. A propor-
tion of these asexual parasites convert to immature gameto-
cytes that appear in the peripheral blood for a day before
sequestrating on the blood vessel. The gametocytes become
visible again when the maturation process is completed. A
combination of schizonticidal and gametocidal treatment
affects the gametocyte production process by eliminating the
asexual parasites and the mature gametocytes.


produces approximately 16 merozoites (0) [25,26]. In
each cycle of erythrocytic-schizogony, all merozoites were
assumed to have an equal chance of undergoing gameto-
cytogenesis; however, only a proportion, p = 0.02, of
merozoites commit to gametocytogenesis [27]. The mor-
tality rate (a) is applied to the asexual parasite population
due to the schizonticidal treatment. In the model, patients
were assumed to receive treatment immediately after asex-
ual parasite density reached 104/j-L of blood, the level that
normally causes clinical symptoms among people in low-
endemic areas [28-30]. After artesunate-mefloquine com-
bination treatment, the density of asexual parasites is
reduced by a factor of about 1,000 per 2-day schizogony
cycle (a), i.e., the asexual parasites density is dropped
from 104/j-L to 10/iL at the first cycle after treatment [31-
33]. The surviving merozoites at each cycle then either
convert to gametocytes or multiply into newly merozoites
that continue to the next schizogony cycle. Therefore, the
initial density of early stage gametocytes is a product of
the conversion proportion and the net number of mero-
zoites produced with each cycle (pO). The early stage of
immature gametocytes (Ig), which are indistinguishable
from asexual parasites, remain in circulation for about
one day (31), then sequester on blood vessels while con-
tinuing the maturation process (sequestered immature
gametocytes; S,). It takes about 8 days (fi^) for gameto-
cytes to mature and release to the peripheral blood (visi-
ble mature gametocytes; G,). The longevity of a mature
gametocyte in the blood stream is approximately 3.5-4
days (p/) [8].

The mortality rate of mature gametocytes due to pri-
maquine treatment (r) depends on the day ofprimaquine


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Point estimates

0.4 day-1

0.6

0.5

1/18 day-1
0.99
1/7 day-1
1/188 day-1


1/10 day-1
1/6 day-1
1/2 day-1
0.001/365 day-1
1/12 day-1
1/12 day-1
0.33/12 day-1


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administration relative to the day of initial schizonticidal
treatment (asexual parasites reach 104/gL of blood).
Plasma concentration of primaquine was estimated to
decrease at elimination rate 0.53 per day (an 8-hour elim-
ination half-life). Since the plasma concentration of pri-
maquine in vivo is difficult to determine, primaquine was
assumed to remain effective in killing 90% of mature
gametocytes when the plasma concentration was above
10-5 of the maximum concentration, for a net gametocyte
reduction ratio of 100 per 2-day cycle, i.e., gametocyte
density, which is a function of the mature gametocytes
that arise from newly-emerging merozoites at each two-
day cycle, eight days earlier, are reduced by 100-fold. The
untreated gametocytes remaining in the blood over time
were then used to determine the duration of infectious-
ness. Multiple realizations with different timeframes of
primaquine administration were performed to determine
the different durations of individual infectiousness after
primaquine treatment

The duration of infectiousness is related to mature game-
tocyte densities and mature gametocyte longevity. The
duration of infectiousness was computed based on the
density of gametocytes in an individual host, using a log
sigmoid relationship between gametocyte density (G,)
and infectivity to mosquitoes [34].

0.8Gg
Infect(Gg) 0G (4)
102+Gg
With a 3.6 day half-life, gametocyte densities decline by
90% every eight days. If gametocyte densities were very
high initially, then there would be virtually no drop-off in
infectivity until the densities reach the S-portion of the
logistic curve (Figures 3A and 3B). The time that a person
remains infective (probability of infectivity more than
zero) differs by about 10 days for every 10-fold difference
in the initial gametocyte density (Figure 3C). Thus, for
every 10-fold reduction in initial gametocyte densities
achieved by curing asexual blood-stage infections alone
there will be about 10-day reduction in the duration of
human infectivity to mosquito. Therefore, a switch from
mefloquine to artemisinin-mefloquine combination ther-
apy (ACT) would reduce infectivity by about 10 days for
every 10-fold reduction in mature gametocyte densities.
Similarly, a two-day delay in appearing at the clinic could
result in a 10-fold increase in gametocyte densities and a
10-day increase in infectiousness. Further reductions can
only be achieved by a follow-up treatment with pri-
maquine to reduce the densities of mature gametocytes
(Figure 3D).


Effect of optimally timing primaquine treatment at the
population-level
The impact of optimally timing primaquine treatment
was assessed by comparing the initial basic reproductive
number for malaria derived from the population when no
intervention was applied (Ro) with the basic reproductive
numbers for malaria at different scenarios of the treat-
ment intervention (Rc). Ro is an estimate of expected
number of hosts infected by a single infectious person
during his or her entire infectious period [35]. The magni-
tude of Ro provides insight into the transmission intensity
of the disease and is often used to justify the effect of inter-
vention programmes [36,37]. The classic formula for Ro of
Ross and Macdonald is shown in equation 5 [35].


Ro = (5)
r

Where V denotes vectorial capacity, detailed definitions of
other parameters are shown in Table 2.

The basic reproductive number for the control pro-
gramme was calculated by modifying the Ross and Mac-
donald formula to consider the proportion treated with
ACT (P), or with the follow-up with primaquine (Q):


R,(P, Q)= bV c(1-P) P(1-Q)cp + PQCQ
r up TQ

(6)
Where c and r indicate the individual infectivity and dura-
tion of gametocyte carriage in natural infections, respec-
tively. Parameters cp and 1/ap indicate the individual
infectivity and duration of gametocyte carriage, respec-
tively, when a schizonticidal drug regimen is applied to a
population; the reductions are due to the clearance of
asexual parasites. The parameters CQ and 1/OQ indicate the
individual infectivity and duration ofgametocyte carriage,
respectively, when a primaquine follow-up regimen is
applied to the population. A product of cp and 1/op, or CQ
and 1/OQ is the cumulative duration of infectiousness for
the two different treatment regimens, which is defined as
a function of gametocyte density in equation 4.

The impact of the follow-up primaquine regimen was rep-
resented by the magnitude of a ratio between Ro and Rc
(Ro/RJ). The vectorial capacity (V) and the transmission
probability from mosquitoes to human (b) were assumed
to be the same regardless of the intervention, so they can-
cel out in the ratio. Therefore, Ro/Rc can be calculated by
the following equation.


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0.8-


0.6-



S0.4-
4-


0.2-



0.0-


0 10 20 30 40 50 60
Decay time (days)


0 10 20 30 40 50 60


Time (days)


1 100 10000
Gametocyte density



















1 100 10000


Initial gametocyte density (Go)


Figure 3
Relationship among initial gametocyte density, gametocyte longevity, and duration of infectiousness. (A)
Changes in gametocyte density over time according to the initial gametocyte density (per ipL): 105 (black line), 104 (blue line),
103 (red line). (B) Infectivity to mosquito as a function of gametocyte density (per piL). (C) Probability of infectivity to mosquito
over time at different initial gametocyte densities. (D) Duration of infectiousness related to initial gametocyte density without
(solid line) and with (dotted line) primaquine follow-up treatment.













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0.8


0.6 -



0.4
4-

0.2



0.0-


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/ /-1
RO -P+P(1 -Q) rcp +pQ
Rc(P,Q) cap coQ C

(7)

The larger the magnitude of R/Rc, the greater the reduc-
tion in potential transmission. In addition, the relation-
ship between the percent coverage with the primary
treatment (P) and with the follow-up treatment (Q) and
the ratio of Ro and Rc was examined.

Finally, the optimal timing of primaquine administration
was computed by finding the timing that produced the
shortest infectious period in the gametocyte model. By
assuming 100% coverage of follow-up primaquine treat-
ment among symptomatic patients, the new infectious
period was replaced in the initial transmission model to
examine the effect of optimal timing of primaquine


o



0



B
c 0.06
o
a-

16 0.05
" 0.04
" 0.03
.1 0.02
g. 0.01
1 0.00


I
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Observed Year Model estimated
Incidence incidence


1999 2000 2001 2002 2003 2004 2005
Year


- Symptomatic


2006 2007 2008 2009


-...--Gametocyte ..... Asymptomatic
Carriage


Figure 4
Plasmodium falciparum incidence and dynamic
changes in proportion of the human population. (A)
Incidence of P.falciparum malaria in seven villages of Ratch-
aburi province. Solid line represents the observed incidence
from 1999 to 2006. The incidence from 1999 to 2009, esti-
mated by the transmission model, is shown by the blue
dashed line. (B) Estimated changes in proportion of popula-
tion in each human compartment over the 10-year period.
Solid line indicates population with clinical symptoms, Ch;
Red dashed line represents population with gametocytaemia,
Gh; Blue dotted line indicates asymptomatic population, Ah.


administration at the population-level. Also the possibil-
ity of malaria elimination in the area was determined
when different elimination strategies were implemented.

Results
Transmission pattern
The transmission model provided a good estimate of the
dynamics of malaria transmission in the study area. Over
the eight-year period, the estimated incidence and
observed incidence were comparable in all years except
2000, when the model significantly underestimated
malaria incidence (Figure 4A). Malaria transmission in
the area was strongly seasonal. Although the model
assumed that 99% of the infected population received
standard malaria treatment, malaria transmission only
decreased gradually in the first three years before settling
into a lower, stable orbit, with the annual peak incidence
about 1.8 per 100 persons. The switch from mefloquine to
ACT in 2005 resulted in a significant reduction in the inci-
dence in year 2006, to less than one per 100 persons.
However, estimates from the model suggest that malaria
transmission will continue even after the deployment of
ACT.

The changes in transmission dynamics are illustrated in
Figure 4B. As is typical of low malaria transmission set-
tings, the proportion of asymptomatic gametocyte carriers
was low compared with the proportion of symptomatic
individuals, which varied seasonally. While the propor-
tion of people with gametocytaemia almost reached zero
during the dry season, the model predicts that about three
percent of the population remained gametocytaemic
when the environment was suitable for mosquito vectors.
This small proportion of gametocytaemic people could
play an important role in maintaining transmission of the
parasite in the area.

Gametocyte carriage regarding primaquine treatment
Different malaria dynamics were observed when pri-
maquine treatment was given at different times. Changes
in the density of each parasite developmental stage over
time with different primaquine treatment regimens are
shown in Figure 5A. The simulated primaquine plasma
concentration remained above the killing concentration
for up to three days after administration. The duration
and density of individual gametocytaemia differed sub-
stantially according to the timing of primaquine adminis-
tration. When primaquine was given on the same day as
other anti-malarial drugs, there were no mature gameto-
cytes in the blood to be killed by primaquine, and infec-
tiousness of the patient was not changed in comparison
with no primaquine treatment. The duration of individual
infectivity to mosquitoes could be as long as 14 days. In
contrast, duration of infectiousness would be greatly
reduced to two days if the primaquine treatment was


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Primaquine at day 1


.i :

J *
I 1


0 10 20 30 40
Primaquine at day 8










0 10 20 30 40


-- Asexual parasite


Days


Primaquine
Phamacokinetic
curve


3x104-
C-
a)
o 2x104
a)
p 1x10"

0





- 3x104

"s 2x104

2 1x104
a0


Primaquine at day 3









0 10 20 30 40

Primaquine at day 15

II



I
JJ ______


0 10 20 30 40
Days
-- Mature gametocyte Immature gametocyte


2 4 6 8 10 12 14 16 18 20
Timing of primaquine administration
after acute attack (Days)

Figure 5
Gametocyte dynamics and duration of infectiousness with different timings of primaquine administration. (A)
Changes in the density of each parasite developmental stage over time with different timings of primaquine administration.
Pharmacokinetic curve of primaquine (dotted line) related to waves of asexual parasite, A (dashed line), immature gametocytes,
g (grey line), and mature gametocytes, M (solid red line). Initial asexual parasite (Ag) I 0/pL (B) Duration of infectivity of humans
to mosquitoes over different timings of primaquine administration after acute attack.








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3x104
a)
S2x104

2 1x104
a0


3x104

2x104

1x104

0


Malaria Journal 2009, 8:159







http://www.malariajournal.com/content/8/1/159


delayed until the majority of mature gametocytes were cir-
culating in the bloodstream (day 7 or later). On the other
hand, delaying primaquine administration until day 10 or
later results in an increased duration of infectiousness as
mature gametocytes circulate for several days (Figure 5B).

Optimal timing of primaquine treatment and malaria
transmission
The optimal timing of primaquine treatment showed a
substantial effect in reducing malaria transmission in the
area. However, the added value of the follow-up pri-
maquine treatment was strongly correlated with the pro-
portion of patients treated with anti-malarial drugs (P).
The follow-up primaquine treatment showed a greater
effect when a large fraction of clinical malaria was
detected and treated. In an area with an effective detection
and treatment control programme (P = 99%), the basic
reproductive rate (Re) could be reduced up to 35 times by
implementing the optimally timed primaquine treatment
programme, compared with the initial basic reproductive
rate (Ro). More importantly, the proportional reduction in
transmission that was achieved through an 8-day follow-
up with primaquine in 99% of the patients was approxi-
mately equal to the proportional reductions achieved
from the initial treatment. In addition, the follow-up pri-
maquine treatment programme appeared to be effective at


60

50


40

30


20


10

0


2.0


) 1.5
* 0
a)

1.0
0- 0.5


0.0


Year


Figure 7
Estimated P. falciparum malaria incidence for differ-
ent control policies. Solid line represents the incidence
estimated by the initial model when the standard drug regi-
men (2-day ACTs and Primaquine at day 2) was used (solid
line). The incidence significantly decreased when the timing
of primaquine administration was shifted to the eighth day
after initial attack (short-dashed line). The elimination of
malaria transmission can be reached when the combination
of both primaquine follow-up treatment and control of
imported asymptomatic cases was implemented (long-dashed
line).


reducing transmission only when the coverage of the fol-


low-up primaquine treatment was above 90% (Figure 6).
P=1.00
SThe transmission model was re-simulated by replacing the
duration of infectivity (o-) from 10 days to 2 days,
according to the gametocyte model. The model indicated
S0that if all individuals received primaquine at day 8 after
099 initial acute attack, the malaria incidence in the area
would be reduced substantially (Figure 7). However,
changing the primaquine administration regimen alone
/ may not be enough to eliminate P. falciparum malaria in
P=0.95 the area; the incidence of malaria still persisted during the
wet season, largely because of imported malaria. By
-P= 0. including an intervention focused on the effective detec-
P=0.75 tion and treatment of imported asymptomatic infections,
P=0.50 the incidence of malaria in the area could reach zero
0.0 0.2 0.4 0.6 0.8 1.0 within three years after the combination programme was
introduced (Figure 7).
Proportion of follow-up patients (Q)


Figure 6
The R/oRc ratio for ACT and follow-up primaquine
treatment. The relationship between RolRc and the propor-
tion of P. falciparum patients receiving follow-up primaquine
treatment (Q) for different proportions of patients treated
with artesunate-mefloquine combination therapy (P).


Discussion
Mathematical models were constructed to understand the
transmission dynamics ofP. falciparum malaria in an area
where access to health-care has significantly reduced
malaria transmission. Active malariometric surveys sug-
gest that a very high fraction of clinical episodes in the
area, perhaps higher than 97% are promptly treated [38].
Prompt and effective treatment may be a very cost-effec-


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tive strategy for malaria control in low or unstable malaria
transmission settings, because most individuals are likely
to develop acute febrile illness after P. falciparum infection
[39-41]. Significant reductions in mortality and morbidity
of malaria after deploying a strategy of early detection and
prompt treatment has been documented [42]. However,
model simulations find that, despite significant reduc-
tions in incidence, P. falciparum transmission is likely to
continue. This suggests that the elimination of malaria
may be difficult even in an area where malaria health sys-
tems are highly effective.

Given the fact that multiple surveys of large swaths of the
population are unable to find individuals infected asymp-
tomatically, gametocyte carriage in the absence of asexual
parasites seems to be vital for maintaining transmission in
this heavily controlled area [7,24]. Findings of our model
suggest that there is a strong seasonal fluctuation in the
population of residual gametocyte carriers, which is con-
sistent with previous observations in other low transmis-
sion areas [7,9]. An interesting finding is that while the
prevalence of gametocytes is relatively small during the
dry season, it does not drop to zero. Presumably then
these cases are responsible for the source of mosquito
infection at the start of wet season [7,9].

Gametocyte reduction is of great interest for malaria con-
trol, particularly in low-endemic malaria areas. One of the
strategies for the malaria elimination programme, recom-
mended by the WHO, is to identify and treat all malaria
patients as well as to reduce onward transmission caused
by gametocytaemia [6]. The policy change from meflo-
quine treatment to artesunate-mefloquine has been
shown to reduce transmission. Artemisinin combination
therapies can reduce the asexual parasite burden 100
times faster than mefloquine, which can subsequently
inhibit development of more mature gametocytes [31-
33,41]. The gametocyte model indicates that there is still
an added value of the follow-up primaquine treatment
even when the initial gametocyte density is small due to
the switch from mefloquine to ACTs. In addition, in areas
where artemisinin combination therapy has been used,
gametocyte carriage is still common in the 7-21 days fol-
lowing treatment [13,30,41,43-45]. In many countries, a
single oral dose of primaquine is included in the standard
anti-malarial drug regimen with the aim of further reduc-
ing gametocyte carriage, even when artemisinin-based
therapy is used [6,12].

However, while in these areas primaquine can be
extremely effective at clearing gametocytes that persist
after treatment with schizonticidal agents [13,14,43], the
timing and duration of gametocyte carriage and subse-
quent infectiousness have not been considered carefully
when primaquine is deployed as a transmission-blocking


agent. Findings of the gametocyte model indicate that the
effectiveness of primaquine in reducing the duration of
infectiousness depends critically on timing. Primaquine is
most beneficial when the administration is delayed, about
eight days following initial treatment, to coincide with the
release of a large cohort of mature gametocytes into the
blood, which emerged from a large number of merozoites
during an acute attack. The effect of primaquine is signifi-
cantly reduced when the drug is given too early or too late.
Although an immediate primaquine treatment can affect
a small cohort of mature gametocytes that emerge from
the first crop of merozoites that appear in circulation at
the time of an acute attack, primaquine will be cleared
from the system before the largest cohort of gametocytes
mature. If primaquine is given too late, mature gameto-
cytes will be able to circulate and infect mosquitoes until
the drug is administered.

The benefits of optimally timed primaquine are greatest in
those areas where the early treatment programme to cure
asexual blood stage infections is very successful; a high
fraction of clinical malaria episodes are expected to
receive the standard treatment within one to three days
after acute attack. In such areas, optimally timing pri-
maquine administration shows a potential impact on
overall malaria transmission at the population-level. The
current results show that follow-up primaquine treatment
can reduce the duration of infectiousness over the existing
strategy of using artesunate-mefloquine alone, with a
combined total net reduction in transmission of 98%, a
95-fold reduction in Ro. An important observation is that
the added value of optimally timed primaquine can have
relatively large effects on reducing transmission only if a
high fraction of patient infections are treated and cured
with first-line anti-malarial drugs (i.e. when P is high),
suggesting that the first emphasis should be on treating
those with clinical malaria. Because primaquine effec-
tively reduces transmission only in those patients who
have cleared their asexual parasites, and because the aver-
age duration of an asymptomatic infection is approxi-
mately six months, the benefit of reducing the duration of
gametocyte carriage is of little importance unless at least
90% of clinical malaria episodes are effectively treated.
Treatment to clear asexual parasites and prevent asympto-
matic infections can only reduce the duration of infec-
tiousness insofar as the gametocytes are also cleared. In
such situations, primaquine can be very effective at further
reducing the duration of infectiousness, and the added
value of good follow-up with primaquine treatment has
nearly the same proportional effects on potential trans-
mission as does the primary treatment.

In addition, findings from the model indicate that when
all symptomatic P. falciparum patients receive the follow-
up primaquine treatment at day eight, the P. falciparum


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malaria incidence can be reduced nearly to zero. However,
the model suggests that a small number of undetected
imported cases can pose a big threat for malaria elimina-
tion. To reach the elimination goal, vigilance to detect and
cure imported asymptomatic cases may also be required.
These findings support the WHO recommendations for a
malaria elimination programme [6].

Mathematical modeling has been widely used to model
transmission dynamics of malaria, and control interven-
tions [46]. However, findings from the models require a
careful interpretation. This study intends to construct sim-
ple models that provide a valuable insight into the feasi-
bility of malaria elimination in a low malaria
transmission area. Results from the models should be
considered as approximations that are likely to differ
because of the natural variability under field conditions.

In addition, although the model assumed that the level of
individual infectiousness follows the log-sigmoid rela-
tionship with the gametocyte density among non-
immune adults, the duration of infectiousness in the anal-
ysis may be underestimated. Infectivity to mosquitoes is
observed even when gametocyte densities fall below
detection level by microscopy or by a molecular method
[24,47]. However, the trend of duration of infectiousness
over different timings of primaquine administration does
not change when different infectivity levels are applied.
The model also does not take into account the additional
gametocyte carriage from recrudescent infections. Though
gametocytaemia is estimated to be greater in recrudescent
infections than in primary infections, in an area where
artesunate-mefloquine combination therapy is used, few
recrudescent infections are expected [33,48]. Lastly, the
model assumes that most infected individuals develop
clinical symptoms and are treated. In low and unstable
transmission areas this assumption is generally correct;
however pockets of higher transmission may exist, and
the importance of asymptomatic asexual blood-stage
infections in these areas in continuing transmission over
the dry season can be significant. While surveys suggest
that there are basically no asymptomatic carriers in the
region in question, studies in other areas have found that
sub-patent infections can persist for many months
[9,10,49]. Thus, while the models suggest that optimally-
timed primaquine administration can significantly
impact the incidence of malaria in a low-transmission
area well served by health centers, asymptomatic individ-
uals in the area and not just imported carriers may also
play a significant role in sustaining transmission and
should be considered in any elimination plan.

Conclusion
Mathematical models constructed in this study pose an
important and testable hypothesis regarding existing con-


trol programmes in areas with good health systems where
malaria transmission persists. The transmission-blocking
effect of primaquine and the timing of its administration
should be carefully scrutinized. Given the risks associated
with primaquine, it may not be worth giving, except in
areas where the early detection and prompt treatment pro-
gramme is highly effective. In such areas, primaquine
should be administered at an appropriate time, or a long
acting 8-aminoquinoline should be considered, and com-
bined with surveillance to catch imported malaria it could
lead to local malaria elimination. Randomized controlled
trials are recommended to determine the most-effective
timing of primaquine administration in order to decrease
malaria transmission, which is important for planning
malaria elimination programmes in low malaria trans-
mission areas.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
SL and DLS conceived and designed the experiment. SL
analysed the data. PS, SY, NT, and WM managed the data
set. SL, DLS, EK, LLH, and JHM wrote the paper. All
authors read and approved the final manuscript.

Acknowledgements
We would like to thank Dr. Christopher V. Plowe (Department of Medi-
cine, University of Maryland, Baltimore) for helping to review the manu-
script. We also thank all officers and staff at Rajanagarindra Tropical
Disease International Center, Suan Phung, Ratchaburi, Thailand for their
help and support. DLS is supported by a grantfrom the Bill & Melinda Gates
Foundation (#49446) and funding from the RAPIDD program of the Science
& Technology Directorate, Department of Homeland Security, and the
Fogarty International Center, National Institutes of Health.

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