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HIV-1 Subtype distribution in morocco based on national sentinel surveillance data 2004-2005
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Permanent Link: http://ufdc.ufl.edu/AA00010686/00001
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Title: HIV-1 Subtype distribution in morocco based on national sentinel surveillance data 2004-2005
Series Title: AIDS Research and Therapy
Physical Description: Archival
Language: English
Creator: Akrim, Mohammed
Lemrabet, Sanae
Elharti, Elmir
Gray, Rebecca R.
Tardy, Jean Claude
Cook, Robert L.
Salemi, Marco
Andre, Patrice
Azarian, Taj
El Aouad, Rajae
Publisher: BioMed Central
Publication Date: 2012
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Subjects / Keywords: HIV-1
subtypes
phylogeny
Morocco
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Abstract: Background: Little is known about HIV-1 subtype distribution in Morocco. Some data suggest an emergence of new HIV subtypes. We conducted phylogenetic analysis on a nationally representative sample of 60 HIV-1 viral specimens collected during 2004-2005 through the Morocco national HIV sentinel surveillance survey. Results: While subtype B is still the most prevalent, 23.3% of samples represented non-B subtypes, the majority of which were classified as CRF02_AG (15%). Molecular clock analysis confirmed that the initial introduction of HIV-1B in Morocco probably came from Europe in the early 1980s. In contrast, the CRF02_AG strain appeared to be introduced from sub-Saharan Africa in two separate events in the 1990s. Conclusions: Subtype CRF02_AG has been emerging in Morocco since the 1990s. More information about the factors introducing HIV subtype-specific transmission will inform the prevention strategy in the region. Keywords: HIV-1, subtypes, phylogeny, Morocco
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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution.
Resource Identifier: doi - 10.1186/1742-6405-9-5
System ID: AA00010686:00001

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RESEARCH OpenAccessHIV-1Subtypedistributioninmoroccobasedon nationalsentinelsurveillancedata2004-2005MohammedAkrim1,SanaeLemrabet1,ElmirElharti2,RebeccaRGray3,JeanClaudeTardy4,RobertLCook5*, MarcoSalemi6,PatriceAndre4,TajAzarian5andRajaeElAouad1,2AbstractBackground: LittleisknownaboutHIV-1subtypedistributioninMorocco.Somedatasuggestanemergenceof newHIVsubtypes.Weconductedphylogeneticanalysisonanationallyrepresentativesampleof60HIV-1viral specimenscollectedduring2004-2005throughtheMorocconationalHIVsentinelsurveillancesurvey. Results: WhilesubtypeBisstillthemostprevalent,23.3%ofsamplesrepresentednon-Bsubtypes,themajorityof whichwereclassifiedasCRF02_AG(15%).MolecularclockanalysisconfirmedthattheinitialintroductionofHIV-1B inMoroccoprobablycamefromEuropeintheearly1980s.Incontrast,theCRF02_AGstrainappearedtobe introducedfromsub-SaharanAfricaintwoseparateeventsinthe1990s. Conclusions: SubtypeCRF02_AGhasbeenemerginginMoroccosincethe1990s.Moreinformationaboutthe factorsintroducingHIVsubtype-specifictransmissionwillinformthepreventionstrategyintheregion. Keywords: HIV-1,subtypes,phylogeny,MoroccoIntroductionHIV-1variabilityremainsaformidablechallengefor designingaprotectivevaccineoraneffectivecure.The HIV-1isdividedinto4groups:M,N,OandP.Group Misresponsibleforthecurrentpandemicandincludes morethan49circulatingrecombinantforms(CRFs),9 subtypes,5sub-subtypes,anduniquerecombinant forms(URFs)[1,2].HIVgeneticdiversityisgenerated bythehighrateofvirusmutation,rapidviralturnover andfrequentrecombination eventsbetweensubtypes [3].Furthermore,thereisanu nequalgeographicdistributionofHIV-1subtypesandCRFsaroundtheworld characterizedbydifferentepidemicbehavioursand growthrates[4].Forinstance,inwesternEuropeand NorthAmerica,subtypeBisthemostprevalentwhereas insub-SaharanAfricasubtypesA,C,DandCRF02_AG predominate[5-7].ThisgeographicdistributionofHIV1subtypescouldresultfrommigration,travel,orgeographicaccessibility.Thes efactorsmaycontributeto thetransmissionofthesecladesoutsidetheregions wheretheyaremostprevalent[8,9].Theincreasing diversityofHIV-1underscorestheneedfordiagnostics, patientmonitoringtools,andtreatmentoptionsthatare effectiveacrossthefullspectrumofknowngroups,subtypes,andrecombinantforms. ThefirstreportedcaseofHIV/AIDSinMorocco occurredin1986.UptoDecember2010,acumulative totalof2,914personshavebeendiagnosedwithAIDS inMorocco,andestimatessuggestapproximately26,000 personsarelivingwithHIVinthecountry[10].Among them,58%wereidentifiedduringthe6lastyears. Furthermore,morethanhalfofcasesarefrom3 regions:theAgadirregion(22%),theMarrakechregion (16%)andtheCasablancaregion(14%).Youngadults (15-39years)represent64%ofallthecases,andthe proportionofHIVinfectionsinwomenhasincreased from18%(1986-1990)tomorethan40%(2004-2008). HIV-1transmissionisreportedlyattributedtoheterosexualtransmissioninmorethan80%ofindividuals. AnationalHIVsentinelsurveillancenetworkhasbeen implementedinMoroccosince1993[11].Thissurveillanceisbasedonananonymous,unlinkedstudyandis approvedbytheWHOEthicalCommittee.Studied groupsincludepregnantwomen,patientsconsulting *Correspondence:cookrl@ufl.edu5DepartmentofEpidemiologyandEmergingPathogensInstitute,University ofFlorida,Gainesville,USA FulllistofauthorinformationisavailableattheendofthearticleAkrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 2012Akrimetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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healthcarecentreswithSTIs,personswithtuberculosis, prisoners,injectingdrugusers(IDUs)andsexworkers. AstudyofHIVsubtypesinMoroccofrom1997 showedapredominanceofHIV-1subtypeB(93.5%),a patternmoresimilartoEuropethansub-SaharanAfrica [12].Morerecently,ananalysisofHIVsubtypesfroma singleregionofMoroccosuggestsanincreaseinpersonswithHIVCRF02-AG,wh ichhastypicallybeen associatedwithinfectionsfromsub-SaharanAfrica[13]. Sincethemid-1990s,Moroccohasbeenexperiencinga significantimmigrationofpersonsfromsub-Saharan Africa,manyofwhichareattemptingtoenterEurope. Morerecently,SpainandtheEuropeanUnionhave intensifiedtheirborderandcoastalsurveillance.Consequently,Moroccohasshiftedfrombeingatransitcountrytothefinalstationformanymigrants.Other countriesintheMENAregionarealsodemonstratinga morediverseHIVepidemicintermsofHIVsubtype distribution[14]. ThestudyofHIVsubtypedistributionmayrevealepidemiologicalpatternsoftransmissionordistinctnetworksassociatedwithsp ecificriskbehaviours. PhylogeneticanalysiscanfurtherexpandtheidentificationofspecificepidemiologicalclustersofHIVinfection fromacommonorigin[9].Weinvestigatedthepattern ofHIV-1subtypediversityandhigh-resolutionphylogeneticanalysisofarepresentativesampleof60HIVinfectedpersonsidentifiedthroughtheMorocco nationalHIVsentinelsurveillanceprogram.MaterialsandmethodsSamplecollectionSampleswerecollectedaspartofthesentinelsurveillancesystem,anationalH IVepidemicAIDSsurveillancesurveycarriedouteachspringbytheMoroccan MinistryofHealthtoassesstrendsintheHIVepidemic. TheserawerecollectedfromdifferentregionsofMoroccoduring2004and2005.Allofthe60HIV-positive samplesduringthe2004-2005surveywereincludedin thisstudy.SampleswerescreenedHIV-1positiveby Elisa(HIV1/2Genscreenplus,Bio-Rad,France)and confirmedbyWesternblotte st(Genlabs,USA).These sampleswererepresentativeofthedifferentregionsof MoroccoandwereconsistentwiththeageandsexdistributionofreportedHIVcaseswithinthecountry (Table1).PCRandDNAsequencingTostudydiversity,samplesweresequencedin pol gene region[proteasegene(PR)and2/35 regionofthe reversetranscriptasegene(RT)].TheviralRNAwas extractedandPRandRTgeneswereRT-PCRamplified aspreviouslydescribed[15 ].Thefragmentsobtained weresequencedonbothstrandsusinganautomated sequencerBeckmanCEQ2000DNAAnalysisSystem, andsubtypedbyusingtheRegaHIV-1Subtypingtool version2.0(http://dbpartners.stanford.edu/ RegaSubtyping). GenBankaccessionnumbersforthesequences reportedinthisstudyareJQ316543toJQ316600and JQ344156toJQ344204forPRandRTsequences respectively.DatasetassemblingAllsequencesfromMoroccoweredividedintoprotease (PR,N=58)andreversetranscriptase(RT,N=49) alignments(Table1).Asetoffullgenomereference sequences(Genbank:AF004885,Genbank:AB253421, Genbank:AB253429,Genbank:AF286241,Genbank: AF286237,Genbank:K03455,Genbank:AY423387,Genbank:AY173951,Genbank:AY331295,Genbank: DQ853463,Genbank:U52953,Genbank:U46016,Genbank:AF067155,Genbank:AY772699,Genbank:K03454, Genbank:AY371157,Genbank:AY253311,Genbank: U88824,Genbank:AF077336,Genbank:AF005494,Genbank:AF075703,Genbank:AJ249238,Genbank: AY371158,Genbank:AJ249236,Genbank:AJ249237, Genbank:AF377956,Genbank:AF084936,Genbank: AF061641,Genbank:U88826,Genbank:AF190127,Genbank:AF190128,Genbank:AF005496,Genbank: EF614151,Genbank:AF082394,Genbank:AF082395, Genbank:AJ249235,Genbank:AJ249239,Genbank: AY271690)weredownloadedfromtheLosAlamosHIV database.AnadditionalsetofsequencesofPR/RTfor subtypesHIV-1CRF02-AGandsubtypeBweredownloadedfromtheLosAlamosdatabase.Thecriterionfor inclusioninthisdatasetincludedthefollowing: sequenceswerepublished,werenotamplifiedinculture andhadaknownlocationandyearofsampling.The HIV-1Bdatasetwasreducedforcomputationalpracticalityandincludedrepresentativesequencesonlyfrom majorgeographicareas.Mul tiple-sequencealignments wereobtainedbycodon-alignmentwiththeCLUSTAL algorithm,andsubsequentlymanuallyeditedforoptimization(Alignmentsareavailablefromtheauthorsupon request).PhylogeneticanalysisMaximumlikelihood(ML)t reeswerefirstinferred usingtheMoroccansequencesandfullgenomereferencessequences.Analyseswereperformedassuming theGTR+Gammamodelofnucleotideevolution.Statisticalsupportwasassessedbynon-parametricbootstrapping(numberofreplicates=500)usingPHYML version3.0[16].Sequencesthatclusteredwithapure subtypewithabootstrapvalueof>80wereclassified assuch.SequencesthatclusteredwiththeCRF02_AG withabootstrapvalue>50wereclassifiedasHIV-1Akrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 Page2of8

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Table1DatabaseofHIVsequencesincludedinthestudySampleCodeNOrigin RiskGroup SexAgeSub-typePRSubtypeRT N1BBB BTetouan PatientwithTB M35 B B N1CBB CTetouan PatientswithSTI sM3 2BB N1AB ATetouan PatientwithTB M39 B N2NBB NRabat Maleprisoners M30 B B N238B 38Rabat FemaleSWinprison F19 B S4DBB DCasablancaPatientswithSTI sF4 1BB S4EBB ECasablancaPatientwithTB M39 B B S4Q1BB Q1CasablancaPregnantwomen F19 B B S3MBB MBeni-MellalMaleprisoners M25 B B S3LBB LSafi NA B B S2J1BB J1MarrakechMaleprisoners M28 B B S136BB 36Agadir PatientswithSTI sF3 5BB S1B1BB B1Agadir FemaleSWinMC F28 B B S1C1BB C1Agadir PatientswithSTI sF3 0BB S1D1BB D1Agadir Marines M28 B B S1E1BB E1Agadir Pregnantwomen F38 B B S1F1BB F1Agadir FemaleSWinprison F21 B B S1G1BB G1Agadir FemaleSWinprison F21 B B S1RBB RAgadir PatientwithTB M20 B B S1VBB VAgadir Maleprisoners M24 B B S1ZBB ZAgadir PatientswithSTI sF1 8BB S1V1BB V1TaroudantPregnantwomen F38 B B S235BB 35MarrakechPatientswithSTI sM3 9BB SX2K1O1BB MarrakechNA B B S2I1BB I1MarrakechPatientswithSTI sF2 3BB S2L1BB L1MarrakechPregnantwomen F27 B B S2M1BB M1MarrakechMaleprisoners M25 B B S2N1BB N1MarrakechMaleprisoners M22 B B S1WBB WAgadir FemaleSWinprison F25 B B S115DB 15Agadir Maleprisoners M25 B B S1A1DB A1Agadir Maleprisoners M27 B B S114DB 14OuladTaimaFemaleSWinMC F31 B B S118DB 18OuladTaimaPregnantwomen F30 B B S3JBB JSafi Maleprisoners M28 B B S29BB 9MarrakechPatientswithSTI sF2 2BB S216BB 16MarrakechPatientswithSTI sM4 0BB SX1K1O1BB Agadir NA 2929 B B S1H1BB H1Agadir Pregnantwomen F24 B B S225B 25MarrakechMaleprisoners M29 B S240B 40ChichaouaPatientswithSTI s F34 B S28B 8ChichaouaPatientswithSTI s F44 B S311B 11Safi Pregnantwomen F26 B S3KB KSafi PatientswithSTI s F41 B S139B 39Agadir PatientswithSTI s F25 B S13B 3OuladTaimaFemaleSWinMC F29 B S113B 13OuladTaimaFemaleSWinMC F39 B N319CC 19Meknes PatientswithSTI sF2 1CC Akrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 Page3of8

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CFR02_AG.AllMoroccansequenceswithaconfirmed subtypewereassembled,andsequencesfromthesame subjectwithconcordantsubtypesinPRandRTwere concatenated.MLtreeswereinferredusingthefinal alignmentsforeachsubtypeusingconcatenatedPR/RT sequences.Analyseswereperformedassumingthe GTR+Gammamodelofnucleotideevolution.Statisticalsupportwasassessedbynon-parametricbootstrapping(numberofreplicates=500)usingPHYML.MolecularclockanalysisTheevolutionaryrate(nucleotidesubstitutionspersite peryear)andthetimeofthemostrecentcommon ancestor(TMRCA,years)ofHIV-1BinMoroccowere inferredusingsequencessampledatdifferenttime pointsbytheMCMCapproachimplementedin BEAST[17].Theanalyseswereperformedwiththe samenucleotidesubstitutionmodeldescribedinthe previoussection,anddifferentcoalescentpriors(constant,exponentialandBayes ianSkylinePlot),assuming astrictorarelaxedmolecularclock[18].AnMCMC wasrunfor100,000,000generationswithsampling every10,000thgeneration.Theresultswerevisualized inTracer.Theeffectivesamplesize(ESS)valuefor eachparameterwas>500indi catingsufficientmixing oftheMarkovchain.ResultsSixtyHIV-1positiveseraweregenotyped:41fromthe south(68.3%),14(23.3%)fromthecenterand5(8.3%) fromtheNorthofMorocco.Sexdistributionwas62% femalesand38%males,withanaverageageof29years. Theriskgroupsrepresentedinthestudysample includedpersonsattendingSTIclinics(38.6%),male prisoners(17.5%),femalesexworkers(15.8%),pregnant women(14%),peoplewithTB(8.8%),drugusers(5.3%), marines(1.7%)andhotelworkers(1.7%)(Table1). ThePRandRTsequenceswerebothpositivefor47 (78.3%)samples.Ofthese,43(91.5%)hadconcordantsubtypeassignmentsincluding36(83.7%)subtypeB,6(14%) CRF02_AGand1(2.3%)subtypeC(Table1).Theremaining4(8.5%)samplesinwhichbothPRandRTregions werepositiverevealeddiscordantsubtypesthatrepresent intersubtypesand/orinter-CRFrecombinantviruses.They includeB/C,A/C,A/CRF01_AEandCRF02_AG/Bwhich arerepresentedbyonesampleeach.Finally,ofthe13specimenswithHIV-1subtypeassignmentforonlyoneviral region,8PRand2RTsequenceswereofsubtypeBand3 PRsequenceswereofsubtypeCRF02-AG. HIV-1subtypesappearedtobedifferentlydistributed inMoroccangeographicregions.SubtypeBstrains appearedtobewidelydistributedwithlittlegeographic compartmentalizationfromr egiontoregion,whereas thesinglesamplesofsubtypeC,A/C,B/CandA/ CRF01_AEwereallconcentratedinthenorthern regionsofMorocco. Figure1showsaMLtree,includingHIV-1subtypeB sequencesfromMoroccoaswellasworldwidereference sequencesdownloadedfromtheHIVdatabases(http:// www.hiv.lanl.gov/content/index).Overall,Moroccan strainsarehighlyintermixedwithreferencestrainsfrom differentgeographicregions,suggestingmultipleintroductionsofsubtypeBinMoroccooverarelativelylong periodoftime.Twohighlysupportedmonophyletic clades(100%and94.5%bootstrapsupport,respectively) ofMoroccanstrainsappeartoberelatedtoHIV-1B sequencesfromEurope,whereasathirdlargeclade clusteredtogetherwiths equencesfromtheUnited States,althoughthecladewasonlyweaklysupportedby bootstrapping(<50%).Thetimeofthemostrecent commonancestor(TMRCA)ofHIV-1BMoroccan strainscalculatedbymolecularclockanalysisdatedback to1983(95%highposteriordensityintervals:1975Table1DatabaseofHIVsequencesincludedinthestudy (Continued)N124AC 24Tanger Drugusers M27 A C N220BC 20Rabat PatientwithTB F33 B C N241AAE 41Rabat PatientswithSTI s F25 A AE N3HAGAG HMeknes Pregnantwomen F22 AG AG N229AGAG29Rabat PatientswithSTI s F26 AG AG N2QAGAGQRabat PatientswithSTI s F29 AG AG N2OAG ORabat PatientswithSTI s F29 AG N2PAG PRabat PatientswithSTI s M28 AG N446AG 46Oujda PatientswithSTI s F40 AG S1TAGAG TAgadir Hotelsworkers M35 AG AG S1SAGB SAgadir FemaleSWinMC F30 AG B S27AGAG 7ChichaouaConsultantforSTI F24 AG AG S317AGAG17Safi ConsultantforSTI F39 AG AG Akrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 Page4of8

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1987)accordingtotheconstantpopulationsizecoalescentpriorenforcingarelaxedmolecularclock.Different coalescencepriorsalsoproducedverysimilarestimates (datanotshown). Figure2showsaMLtreeofMoroccanandreference CRF02_AGstrainsavailableinHIVdatabases.Incontrast tothesubtypeBMLtree,theMoroccanstrainsarehighly localizedintwodistinctmonophyleticcladesrelatedto sequencesfromCameroonandSenegal.Althoughthe resultshouldbeinterpretedwithcaution,giventherelativelysmallnumberofavailablesequencesforphylogenetic comparison,thetreesuggeststwoseparateintroductions ofCRF02_AGinMoroccofromsub-SaharanAfrica,dated in1995and1998respectively,accordingtotheconstant populationsizecoalescentpriorenforcingarelaxedmolecularclock.Again,differentcoalescencepriorshadlittle effectontheestimates(datanotshown).DiscussionWepresentthefirstdataonthemolecularepidemiology ofHIV-1inMoroccofromthenationalHIVsentinel surveillancesurveydata.Asof2005,subtypeBisstill predominant(76.7%),yetfollowingsubtypeB,thereisa highdiversityofnon-Bsubtypes,especiallyCRF02_AG recombinant(15%).Geographicsubtyperepartitionsuggeststheco-evolutionofamoreancientdiffusionof EuropeansubtypeB,andofamorerecentspreadof sub-SaharanAfricanstrainsinsomeMoroccanregions. TheseresultsdemonstrateahighdiversityofHIV-1 strainsinMorocco.Thisisdifferentfromwhatwas reportedin1997wherethedistributionofsubtypeB,A andFstrainsinMoroccowere93.5%,1.0%,and0.5% respectively[12].However,theseresultsareconsistent withourpreviousresults[19]andthemorerecent resultsdescribedfromtheCasablancaregion[13]. Thesefindingsarealsoconsistentwithresultsdescribed inothercountriesoftheregion,includingtheneighbouringWestAfricancountries[14].Theincreaseof HIVnon-Bsubtypeswasalsorecentlyreportedinmany WesternEuropecountries.StudiesconductedinFrance, Spain,Switzerland,andPortugalhavefoundthatthe proportionofnon-Bsubtypesmayexceed20%[20,21]. Figure1 HIV-1BMaximumlikelihood(ML)trees .TheMLtreeincludesHIV-1BMoroccansequencesforwhichwehaveRTandPRsequences, aswellas46subtypeBreferencesequencesfromtheHIVdatabasethatwererandomlychosentorepresentmajorgeographicareasinthe world.ThetreewasgeneratedusingtheGTR+Gmodelofnucleotidesubstitutionusingtheconcatenated RT and PR genes.Branchesaredrawn inscale,accordingtothebaratthebottom,andcoloredtoreflectgeographicoriginaccordingtothelegendofthefigure.Thenumberalong abranchindicatessignificantbootstrapsupport(>65%).Sequenceswerenamedusingtheyearofsamplingprecededbythetwolettercounty codeoforigin,accordingtotheHIVdatabaseguidelines. Akrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 Page5of8

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CRF02_AG,whichpredominatesinWestAfrica,is increasinglymoreprevalentamongthenon-Bsubtypes intheseWesternEuropeancountries. TheoverallincidenceofHIV-1inMoroccohasbeen increasingatapproximately15%peryearsince2000. Theincreasingincidence ofHIVcombinedwiththe identificationofadditionalnon-Bsubtypesraisesconcernsregardingthecontrolofthecurrentepidemic. TheentryofnewHIVrecombinantvirusesislikelythe consequenceofactiveexchangebetweendifferentpopulations,suchasMoroccangr oupsatriskandpersons migratingthroughMoroccofromsub-SaharanAfrica. Before1997,thepresenceofSub-SaharanAfricanindividualsinMoroccowasmostlylimitedtostudentsand tourists.However,migrationofpeoplefromsubSaharanAfricatoandthroughMoroccohasbeen increasingsincethelate1990s.In2007,theMoroccan MinistryoftheInteriorest imatedthatapproximately 15,000irregularmigrantsflowthroughMoroccoeach year[22].Inresponse,theEuropeanUnionhastighteneditsboardercontrolandimmigrationpolicies.Asa result,manyofthemigrants settleinMorocco,waiting foranopportunitytocrossintoEurope.Inaddition, regularandirregularmigrantsfacemanyeconomicand socialissuesthatmayincreasetheirriskforHIVtransmission.Forexample,issues suchashumantrafficking andprostitutioncouldcontributetothecirculationof non-BHIVsubtypessuchasCRF02_AGthroughoutthe country. ThefactthatsubtypeBwasmoredistributedthroughoutthecountry,especiallyinthebig-touristiccities (Agadir,MarrakechandCasablanca),suggestthis Figure2 HIV-1CRF02_AGMaximumlikelihood(ML)trees .TheMLtreeincludesHIV-1MoroccanCRF02_AGsequences,forwhichwehave RTandPRsequences,togetherwith28CRF02_AGstrainsdownloadedfromtheHIVdatabasesforwhichthefullgenomesequenceswere available.ThetreewasgeneratedusingtheGTR+Gmodelofnucleotidesubstitutionusingtheconcatenated RT and PR genes.Branchesare drawninscale,accordingtothebaratthebottom,andcoloredtoreflectgeographicoriginaccordingtothelegendofthefigure.Thenumber alongabranchindicatessignificantbootstrapsupport(>65%).Sequenceswerenamedusingtheyearofsamplingprecededbythetwoletter countycodeoforigin,accordingtotheHIVdatabaseguidelines. Akrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 Page6of8

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subtypemayreflectanolderinfection.Personswith subtypeCRF02_AGwerealsowidelydistributedgeographically;however,thissubtypewasnotdetectedinMoroccobefore1997,suggestingamorerecentepidemic. Thesefindingsarealsosupportedbyourmolecular clockanalysis.Theothernon-BsubtypesandrecombinantsrepresentedmorelocalisedtransmissionduetoC, A/C,B/CandA/CRF01_AEstrainsinthenorthernpart ofMorocco. AsthefirstcaseofHIV/AIDSinMoroccowas reportedin1986,thereisanexcellentagreementwith theTMRCAofHIV-1Bof1983estimatedbymolecular clockanalysis.Sincethatdate,HIVhasbeenspreading throughoutthecountry,mainlybyheterosexualtransmission[11].Accordingtothesentinelsurveillancesystem,theoverallHIVprevalenceislessthan1%in Morocco.However,eventhoughMoroccoisalowprevalenceepidemic,HIV/AIDScasesaresteadilyrising, chieflyinthesouthernMoroccoregionofAgadirand neighbouringareasthatmayrepresenttheepicentreof theepidemicwithinMorocco[23]. Ourfindingsshouldbeinterpretedinlightofstudy limitations.WhileouranalysisincludedallHIVpositive specimensfromthe2004-2005survey,thesamplesize wasrelativelysmall.Therefore,itisnotpossibletogeneralisetheresultsasanationaltrendinMorocco.In addition,routesoftransmissionandclinicalandimmunologicstatusoftheHIV-in fectedindividualswerenot availableforthisstudy,sincetheyarenotrequiredin thesurveillanceprocess.However,thepresentdata shouldpromptustocontinuetotrackthemolecular epidemiologyoftheHIVvirusinMoroccoatthe nationallevel.Inthiscontext,reinforcementofpreventivemeasurestolimitthespreadoftheepidemiciscrucial.Lastly,bylimitingourphylogeneticanalysistoonly the pol generegion,wemayhavemissedsomerecombinantsandthereforeunderestimatedtheirdistribution. However,ourmainfindingthatCRF02_AGisincreasing inMorocco,signifyingashiftfromanepidemicpreviouslydominatedbyserogroupB,remainstrue.Inconclusion,theresultsofthisstudydisplayedthatHIV diversityismoredynamicinMoroccoanditspatternis shiftingfromtheEuropeantosub-Saharanone,i.e.with moresubtypesnon-B,namelytheCRF02_AG.However, morestudiestoconfirmthetrendobservedduringthis studyandtobettercharacterizethemolecularHIVepidemicinMoroccowillbeofgreatimportance.When takentogether,thesedatademonstrateadynamicevolutionintheHIVdiversityinMorocco.Theemergenceof newHIVsubtypesarecharacterisedbyanimportant presenceofnon-Bsubtypesthatappeartobelinkedto sub-Saharanpopulations.Moredataareneededtobetterunderstandthefactorsresponsibleforthe introductionandspreadofnewHIV-1subtypeepidemicsintoregionswheretheydidnotexistpreviously.Abbreviations AIDS :AcquiredImmuneDeficiencySyndrome; CRF :circulatingrecombinant factor; ESS :effectivesamplesize; HIV-1 :HumanImmunodeficiencyVirus1; IDU:injectiondrugusers; MENA: MiddleEastandNorthAfrica; ML : MaximumLikelihood; PCR :polymerasechainreaction; PR :Protease; RT : ReverseTranscriptase; STI:SexuallyTransmittedInfections; TB :tuberculosis; TMRCA :TimetoMostRecentAncestor; WHO :WorldHealthOrganization Acknowledgements TheauthorsacknowledgethestaffoftheNationalprogramtofightHIV/ AIDS,inthedepartmentofepidemiologyanddiseasescontrol,Moroccan ministryofhealth;andallthestaffworkingonHIVsentinelsurveillance network,inMorocco.WethankalsoDrOumzilHicham,MsImnaeBelbacha, DrMengadRajae,MsBennaniOuafaeandMrAminaSiwanifromthe NationalReferenceLaboratoryforHIV,NationalInstituteofHygiene.Finally, theauthorsthankallmembersofthemolecularbiologylaboratorygroup whohelpedwiththisproject.ThisstudyhasafundingsupportfromESTHER Initiative,CNRST-INSERMCollaboration,andfromtheEmergingPathogens InstituteandtheCenterforAIDSResearchattheUniversityofFlorida. Authordetails1MolecularBiologyUnit,NationalReferenceLaboratoryforHIV,National InstituteofHygiene,Rabat,Morocco.2HIVDiagnosisandImmunologic FollowupUnit,NationalReferenceLaboratoryforHIV,NationalInstituteof Hygiene,Rabat,Morocco.3DepartmentofZoology,UniversityofOxford, Oxford,UK.4LaboratoiredeVirologie,HpitaldelaCroixRousse,Lyon, France.5DepartmentofEpidemiologyandEmergingPathogensInstitute, UniversityofFlorida,Gainesville,USA.6DepartmentofPathology, ImmunologyandLaboratoryMedicine,SchoolofMedicineandEmerging PathogensInstitute,UniversityofFlorida,Gainesville,USA. Authors contributions MA:Conductionofthestudy,samplescollection,sequencingandanalysis, preparationofthemanuscript;SL:samplepreparation,sequencingand analysis;EE:samplediagnosisandcollection,HIVscreeningand confirmation,preparationofthemanuscript;RG:phylogeneticanalysisof samplesandconstructionofphylogenetictrees;JCT:sequencingand analysis,preparationofthemanuscript;RLC:interpretationof epidemiologicaldata,preparationofthemanuscript;MS:phylogenetic analysisofsamplesandconstructionofphylogenetictrees,preparationof themanuscript;PA:sequencingandanalysis,preparationofthemanuscript; TA:preparationofthemanuscript;REA:conductionofthestudy,preparation ofthemanuscript.Allauthorshavereadandapprovedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:22September2011Accepted:14February2012 Published:14February2012 References1.LeitnerT,KorberB,DanielsM,CalefC,FoleyB: HIV-1subtypeand circulatingrecombinantform(CRF)referencesequences. HIVsequence compendium 2005 :41-48. 2.LosAlamosNationalLaboratory: HIVSequenceDatabase. ,AvailableatURL http://www.hiv.lanl.gov,accessedJanuary,2012. 3.RambautA,PosasaD,CrandallKA,HolmesEC: Thecausesand consequencesofHIVevolution. NatureReviewsGenetics 2004, 5 :52-61. 4.SalemiM: TowardarobustmonitoringofHIVsubtypesdistribution worldwide. AIDS 2011, 25 :713-714. 5.RobertsonDL,AndersonJP,BradacJA,CarrJK,FoleyB,FunkhouserRK, GaoF,HahnBH,KalishML,KuikenC,LearnGH,LeitnerT,McCutchanF, OsmanovS,PeetersM,PieniazekD,SalminenM,SharpPM,WolinskyS, KorberB: HIV-1nomenclatureproposal. Science 2000, 288 :55-56. 6.RobertsonDL,HahnBH,SharpPM: RecombinationinAidsviruses. Jmol evolution 1995, 11 :1423-1425.Akrim etal AIDSResearchandTherapy 2012, 9 :5 http://www.aidsrestherapy.com/content/9/1/5 Page7of8

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!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui 1742-6405-9-5ji 1742-6405fm
dochead Research
bibl
title
p HIV-1 Subtype distribution in morocco based on national sentinel surveillance data 2004-2005
aug
au id A1 snm Akrimfnm Mohammedinsr iid I1 email mohammedakrim@yahoo.fr
A2 LemrabetSanaesanae.lem@gmail.com
A3 ElhartiElmirI2 elhartie@yahoo.fr
A4 Graymi RRebeccaI3 grayr@pathology.ufl.edu
A5 Tardymnm ClaudeJeanI4 jean-claude.tardy@chu-lyon.fr
A6 ca yes CookLRobertI5 cookrl@ufl.edu
A7 SalemiMarcoI6 salemi@pathology.ufl.edu
A8 AndrePatricepatrice.andre@chu-lyon.fr
A9 AzarianTajtaj.azarian@epi.ufl.edu
A10 AouadElRajaerajaeelaouad@yahoo.fr
insg
ins Molecular Biology Unit, National Reference Laboratory for HIV, National Institute of Hygiene, Rabat, Morocco
HIV Diagnosis and Immunologic Follow up Unit, National Reference Laboratory for HIV, National Institute of Hygiene, Rabat, Morocco
Department of Zoology, University of Oxford, Oxford, UK
Laboratoire de Virologie, Hôpital de la Croix Rousse, Lyon, France
Department of Epidemiology and Emerging Pathogens Institute, University of Florida, Gainesville, USA
Department of Pathology, Immunology and Laboratory Medicine, School of Medicine and Emerging Pathogens Institute, University of Florida, Gainesville, USA
source AIDS Research and Therapy
issn 1742-6405
pubdate 2012
volume 9
issue 1
fpage 5
url http://www.aidsrestherapy.com/content/9/1/5
xrefbib pubidlist pubid idtype pmpid 22333070doi 10.1186/1742-6405-9-5
history rec date day 22month 9year 2011acc 1422012pub 1422012
cpyrt 2012collab Akrim et al; licensee BioMed Central Ltd.note 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.
kwdg
kwd HIV-1
subtypes
phylogeny
Morocco
abs
sec
st
Abstract
Background
Little is known about HIV-1 subtype distribution in Morocco. Some data suggest an emergence of new HIV subtypes. We conducted phylogenetic analysis on a nationally representative sample of 60 HIV-1 viral specimens collected during 2004-2005 through the Morocco national HIV sentinel surveillance survey.
Results
While subtype B is still the most prevalent, 23.3% of samples represented non-B subtypes, the majority of which were classified as CRF02_AG (15%). Molecular clock analysis confirmed that the initial introduction of HIV-1B in Morocco probably came from Europe in the early 1980s. In contrast, the CRF02_AG strain appeared to be introduced from sub-Saharan Africa in two separate events in the 1990s.
Conclusions
Subtype CRF02_AG has been emerging in Morocco since the 1990s. More information about the factors introducing HIV subtype-specific transmission will inform the prevention strategy in the region.
bdy
Introduction
HIV-1 variability remains a formidable challenge for designing a protective vaccine or an effective cure. The HIV-1 is divided into 4 groups: M, N, O and P. Group M is responsible for the current pandemic and includes more than 49 circulating recombinant forms (CRFs), 9 subtypes, 5 sub-subtypes, and unique recombinant forms (URFs) abbrgrp
abbr bid B1 1
B2 2
. HIV genetic diversity is generated by the high rate of virus mutation, rapid viral turnover and frequent recombination events between subtypes
B3 3
. Furthermore, there is an unequal geographic distribution of HIV-1 subtypes and CRFs around the world characterized by different epidemic behaviours and growth rates
B4 4
. For instance, in western Europe and North America, subtype B is the most prevalent whereas in sub-Saharan Africa subtypes A, C, D and CRF02_AG predominate
B5 5
B6 6
B7 7
. This geographic distribution of HIV-1 subtypes could result from migration, travel, or geographic accessibility. These factors may contribute to the transmission of these clades outside the regions where they are most prevalent
B8 8
B9 9
. The increasing diversity of HIV-1 underscores the need for diagnostics, patient monitoring tools, and treatment options that are effective across the full spectrum of known groups, subtypes, and recombinant forms.
The first reported case of HIV/AIDS in Morocco occurred in 1986. Up to December 2010, a cumulative total of 2,914 persons have been diagnosed with AIDS in Morocco, and estimates suggest approximately 26,000 persons are living with HIV in the country
B10 10
. Among them, 58% were identified during the 6 last years. Furthermore, more than half of cases are from 3 regions: the Agadir region (22%), the Marrakech region (16%) and the Casablanca region (14%). Young adults (15-39 years) represent 64% of all the cases, and the proportion of HIV infections in women has increased from 18% (1986-1990) to more than 40% (2004-2008). HIV-1 transmission is reportedly attributed to heterosexual transmission in more than 80% of individuals.
A national HIV sentinel surveillance network has been implemented in Morocco since 1993
B11 11
. This surveillance is based on an anonymous, unlinked study and is approved by the WHO Ethical Committee. Studied groups include pregnant women, patients consulting healthcare centres with STIs, persons with tuberculosis, prisoners, injecting drug users (IDUs) and sex workers.
A study of HIV subtypes in Morocco from 1997 showed a predominance of HIV-1 subtype B (93.5%), a pattern more similar to Europe than sub-Saharan Africa
B12 12
. More recently, an analysis of HIV subtypes from a single region of Morocco suggests an increase in persons with HIV CRF02-AG, which has typically been associated with infections from sub-Saharan Africa
B13 13
. Since the mid-1990s, Morocco has been experiencing a significant immigration of persons from sub-Saharan Africa, many of which are attempting to enter Europe. More recently, Spain and the European Union have intensified their border and coastal surveillance. Consequently, Morocco has shifted from being a transit country to the final station for many migrants. Other countries in the MENA region are also demonstrating a more diverse HIV epidemic in terms of HIV subtype distribution
B14 14
.
The study of HIV subtype distribution may reveal epidemiological patterns of transmission or distinct networks associated with specific risk behaviours. Phylogenetic analysis can further expand the identification of specific epidemiological clusters of HIV infection from a common origin
9
. We investigated the pattern of HIV-1 subtype diversity and high-resolution phylogenetic analysis of a representative sample of 60 HIV-infected persons identified through the Morocco national HIV sentinel surveillance program.
Materials and methods
Sample collection
Samples were collected as part of the sentinel surveillance system, a national HIV epidemic AIDS surveillance survey carried out each spring by the Moroccan Ministry of Health to assess trends in the HIV epidemic. The sera were collected from different regions of Morocco during 2004 and 2005. All of the 60 HIV-positive samples during the 2004-2005 survey were included in this study. Samples were screened HIV-1 positive by Elisa (HIV1/2 Genscreen plus, Bio-Rad, France) and confirmed by Western blot test (Genlabs, USA). These samples were representative of the different regions of Morocco and were consistent with the age and sex distribution of reported HIV cases within the country (Table tblr tid T1 1).
tbl Table 1caption Database of HIV sequences included in the studytblbdy cols 8
r
c left
b Sample Code

Origin
Risk Group
center
Sex
Age
Sub-type PR
Subtype RT
cspan
hr
N1BBB
B
Tetouan
Patient with TB
M
35
B
B
N1CBB
C
Tetouan
Patients with STI's
M
32
B
B
N1A B
A
Tetouan
Patient with TB
M
39
B
N2NBB
N
Rabat
Male prisoners
M
30
B
B
N238B
38
Rabat
Female SW in prison
F
19
B
S4DBB
D
Casablanca
Patients with STI's
F
41
B
B
S4EBB
E
Casablanca
Patient with TB
M
39
B
B
S4Q1BB
Q1
Casablanca
Pregnant women
F
19
B
B
S3MBB
M
Beni-Mellal
Male prisoners
M
25
B
B
S3LBB
L
Safi
NA
B
B
S2J1BB
J1
Marrakech
Male prisoners
M
28
B
B
S136BB
36
Agadir
Patients with STI's
F
35
B
B
S1B1BB
B1
Agadir
Female SW in MC
F
28
B
B
S1C1BB
C1
Agadir
Patients with STI's
F
30
B
B
S1D1BB
D1
Agadir
Marines
M
28
B
B
S1E1BB
E1
Agadir
Pregnant women
F
38
B
B
S1F1BB
F1
Agadir
Female SW in prison
F
21
B
B
S1G1BB
G1
Agadir
Female SW in prison
F
21
B
B
S1RBB
R
Agadir
Patient with TB
M
20
B
B
S1VBB
V
Agadir
Male prisoners
M
24
B
B
S1ZBB
Z
Agadir
Patients with STI's
F
18
B
B
S1V1BB
V1
Taroudant
Pregnant women
F
38
B
B
S235BB
35
Marrakech
Patients with STI's
M
39
B
B
SX2K1O1BB
Marrakech
NA
B
B
S2I1BB
I1
Marrakech
Patients with STI's
F
23
B
B
S2L1BB
L1
Marrakech
Pregnant women
F
27
B
B
S2M1BB
M1
Marrakech
Male prisoners
M
25
B
B
S2N1BB
N1
Marrakech
Male prisoners
M
22
B
B
S1WBB
W
Agadir
Female SW in prison
F
25
B
B
S115DB
15
Agadir
Male prisoners
M
25
B
B
S1A1DB
A1
Agadir
Male prisoners
M
27
B
B
S114DB
14
Oulad Taima
Female SW in MC
F
31
B
B
S118DB
18
Oulad Taima
Pregnant women
F
30
B
B
S3JBB
J
Safi
Male prisoners
M
28
B
B
S29BB
9
Marrakech
Patients with STI's
F
22
B
B
S216BB
16
Marrakech
Patients with STI's
M
40
B
B
SX1K1O1BB
Agadir
NA
29
29
B
B
S1H1BB
H1
Agadir
Pregnant women
F
24
B
B
S225B
25
Marrakech
Male prisoners
M
29
B
S240B
40
Chichaoua
Patients with STI's
F
34
B
S28B
8
Chichaoua
Patients with STI's
F
44
B
S311B
11
Safi
Pregnant women
F
26
B
S3KB
K
Safi
Patients with STI's
F
41
B
S139B
39
Agadir
Patients with STI's
F
25
B
S13B
3
Oulad Taima
Female SW in MC
F
29
B
S113 B
13
Oulad Taima
Female SW in MC
F
39
B
N319CC
19
Meknes
Patients with STI's
F
21
C
C
N124AC
24
Tanger
Drug users
M
27
A
C
N220BC
20
Rabat
Patient with TB
F
33
B
C
N241AAE
41
Rabat
Patients with STI's
F
25
A
AE
N3HAGAG
H
Meknes
Pregnant women
F
22
AG
AG
N229AGAG
29
Rabat
Patients with STI's
F
26
AG
AG
N2QAGAG
Q
Rabat
Patients with STI's
F
29
AG
AG
N2OAG
O
Rabat
Patients with STI's
F
29
AG
N2PAG
P
Rabat
Patients with STI's
M
28
AG
N446AG
46
Oujda
Patients with STI's
F
40
AG
S1TAGAG
T
Agadir
Hotels workers
M
35
AG
AG
S1SAGB
S
Agadir
Female SW in MC
F
30
AG
B
S27AGAG
7
Chichaoua
Consultant for STI
F
24
AG
AG
S317AGAG
17
Safi
Consultant for STI
F
39
AG
AG
PCR and DNA sequencing
To study diversity, samples were sequenced in it pol gene region [protease gene (PR) and 2/3 5' region of the reverse transcriptase gene (RT)]. The viral RNA was extracted and PR and RT genes were RT-PCR amplified as previously described
B15 15
. The fragments obtained were sequenced on both strands using an automated sequencer Beckman CEQ 2000 DNA Analysis System, and subtyped by using the Rega HIV-1 Subtyping tool version 2.0 (http://dbpartners.stanford.edu/RegaSubtyping).
GenBank accession numbers for the sequences reported in this study are ext-link ext-link-id JQ316543 ext-link-type gen JQ316543 to JQ316600 JQ316600 and JQ344156 JQ344156 to JQ344204 JQ344204 for PR and RT sequences respectively.
Dataset assembling
All sequences from Morocco were divided into protease (PR, N = 58) and reverse transcriptase (RT, N = 49) alignments (Table 1). A set of full genome reference sequences (Genbank:AF004885 AF004885, Genbank:AB253421 AB253421, Genbank:AB253429 AB253429, Genbank:AF286241 AF286241, Genbank:AF286237 AF286237, Genbank:K03455, Genbank:AY423387 AY423387, Genbank:AY173951 AY173951, Genbank:AY331295 AY331295, Genbank:DQ853463 DQ853463, Genbank:U52953, Genbank:U46016, Genbank:AF067155 AF067155, Genbank:AY772699 AY772699, Genbank:K03454, Genbank:AY371157 AY371157, Genbank:AY253311 AY253311, Genbank:U88824, Genbank:AF077336 AF077336, Genbank:AF005494 AF005494, Genbank:AF075703 AF075703, Genbank:AJ249238 AJ249238, Genbank:AY371158 AY371158, Genbank:AJ249236 AJ249236, Genbank:AJ249237 AJ249237, Genbank:AF377956 AF377956, Genbank:AF084936 AF084936, Genbank:AF061641 AF061641, Genbank:U88826, Genbank:AF190127 AF190127, Genbank:AF190128 AF190128, Genbank:AF005496 AF005496, Genbank:EF614151 EF614151, Genbank:AF082394 AF082394, Genbank:AF082395 AF082395, Genbank:AJ249235 AJ249235, Genbank:AJ249239 AJ249239, Genbank:AY271690 AY271690) were downloaded from the Los Alamos HIV database. An additional set of sequences of PR/RT for subtypes HIV-1 CRF02-AG and subtype B were downloaded from the Los Alamos database. The criterion for inclusion in this dataset included the following: sequences were published, were not amplified in culture and had a known location and year of sampling. The HIV-1B dataset was reduced for computational practicality and included representative sequences only from major geographic areas. Multiple-sequence alignments were obtained by codon-alignment with the CLUSTAL algorithm, and subsequently manually edited for optimization (Alignments are available from the authors upon request).
Phylogenetic analysis
Maximum likelihood (ML) trees were first inferred using the Moroccan sequences and full genome references sequences. Analyses were performed assuming the GTR + Gamma model of nucleotide evolution. Statistical support was assessed by non-parametric bootstrapping (number of replicates = 500) using PHYML version 3.0
B16 16
. Sequences that clustered with a pure subtype with a bootstrap value of > 80 were classified as such. Sequences that clustered with the CRF02_AG with a bootstrap value > 50 were classified as HIV-1 CFR02_AG. All Moroccan sequences with a confirmed subtype were assembled, and sequences from the same subject with concordant subtypes in PR and RT were concatenated. MLtrees were inferred using the final alignments for each subtype using concatenated PR/RT sequences. Analyses were performed assuming the GTR + Gamma model of nucleotide evolution. Statistical support was assessed by non-parametric bootstrapping (number of replicates = 500) using PHYML.
Molecular clock analysis
The evolutionary rate (nucleotide substitutions per site per year) and the time of the most recent common ancestor (Tsub MRCA, years) of HIV-1B in Morocco were inferred using sequences sampled at different time points by the MCMC approach implemented in BEAST
B17 17
. The analyses were performed with the same nucleotide substitution model described in the previous section, and different coalescent priors (constant, exponential and Bayesian Skyline Plot), assuming a strict or a relaxed molecular clock
B18 18
. An MCMC was run for 100,000,000 generations with sampling every 10,000sup th generation. The results were visualized in Tracer. The effective sample size (ESS) value for each parameter was > 500 indicating sufficient mixing of the Markov chain.
Results
Sixty HIV-1 positive sera were genotyped: 41 from the south (68.3%), 14 (23.3%) from the center and 5 (8.3%) from the North of Morocco. Sex distribution was 62% females and 38% males, with an average age of 29 years. The risk groups represented in the study sample included persons attending STI clinics (38.6%), male prisoners (17.5%), female sex workers (15.8%), pregnant women (14%), people with TB (8.8%), drug users (5.3%), marines (1.7%) and hotel workers (1.7%) (Table 1).
The PR and RT sequences were both positive for 47 (78.3%) samples. Of these, 43 (91.5%) had concordant subtype assignments including 36 (83.7%) subtype B, 6 (14%) CRF02_AG and 1 (2.3%) subtype C (Table 1). The remaining 4 (8.5%) samples in which both PR and RT regions were positive revealed discordant subtypes that represent intersubtypes and/or inter-CRF recombinant viruses. They include B/C, A/C, A/CRF01_AE and CRF02_AG/B which are represented by one sample each. Finally, of the 13 specimens with HIV-1 subtype assignment for only one viral region, 8 PR and 2 RT sequences were of subtype B and 3 PR sequences were of subtype CRF02-AG.
HIV-1 subtypes appeared to be differently distributed in Moroccan geographic regions. Subtype B strains appeared to be widely distributed with little geographic compartmentalization from region to region, whereas the single samples of subtype C, A/C, B/C and A/CRF01_AE were all concentrated in the northern regions of Morocco.
Figure figr fid F1 1 shows a ML tree, including HIV-1 subtype B sequences from Morocco as well as worldwide reference sequences downloaded from the HIV databases (http://www.hiv.lanl.gov/content/index). Overall, Moroccan strains are highly intermixed with reference strains from different geographic regions, suggesting multiple introductions of subtype B in Morocco over a relatively long period of time. Two highly supported monophyletic clades (100% and 94.5% bootstrap support, respectively) of Moroccan strains appear to be related to HIV-1B sequences from Europe, whereas a third large clade clustered together with sequences from the United States, although the clade was only weakly supported by bootstrapping (< 50%). The time of the most recent common ancestor (TMRCA) of HIV-1B Moroccan strains calculated by molecular clock analysis dated back to 1983 (95% high posterior density intervals: 1975-1987) according to the constant population size coalescent prior enforcing a relaxed molecular clock. Different coalescence priors also produced very similar estimates (data not shown).
fig Figure 1HIV-1B Maximum likelihood (ML) treestext
HIV-1B Maximum likelihood (ML) trees. The ML tree includes HIV-1B Moroccan sequences for which we have RT and PR sequences, as well as 46 subtype B reference sequences from the HIV database that were randomly chosen to represent major geographic areas in the world. The tree was generated using the GTR+G model of nucleotide substitution using the concatenated RT and PR genes. Branches are drawn in scale, according to the bar at the bottom, and colored to reflect geographic origin according to the legend of the figure. The number along a branch indicates significant bootstrap support ( 65%). Sequences were named using the year of sampling preceded by the two letter county code of origin, according to the HIV database guidelines./p
/textgraphic file="1742-6405-9-5-1"//fig
pFigure figr fid="F2"2/figr shows a ML tree of Moroccan and reference CRF02_AG strains available in HIV databases. In contrast to the subtype B ML tree, the Moroccan strains are highly localized in two distinct monophyletic clades related to sequences from Cameroon and Senegal. Although the result should be interpreted with caution, given the relatively small number of available sequences for phylogenetic comparison, the tree suggests two separate introductions of CRF02_AG in Morocco from sub-Saharan Africa, dated in 1995 and 1998 respectively, according to the constant population size coalescent prior enforcing a relaxed molecular clock. Again, different coalescence priors had little effect on the estimates (data not shown)./p
fig id="F2"titlepFigure 2/p/titlecaptionpHIV-1 CRF02_AG Maximum likelihood (ML) trees/p/captiontext
pbHIV-1 CRF02_AG Maximum likelihood (ML) trees/b. The ML tree includes HIV-1 Moroccan CRF02_AG sequences, for which we have RT and PR sequences, together with 28 CRF02_AG strains downloaded from the HIV databases for which the full genome sequences were available. The tree was generated using the GTR+G model of nucleotide substitution using the concatenated itRT /itand itPR /itgenes. Branches are drawn in scale, according to the bar at the bottom, and colored to reflect geographic origin according to the legend of the figure. The number along a branch indicates significant bootstrap support ( 65%). Sequences were named using the year of sampling preceded by the two letter county code of origin, according to the HIV database guidelines.p
textgraphic file="1742-6405-9-5-2"fig
sec
sec
st
pDiscussionp
st
pWe present the first data on the molecular epidemiology of HIV-1 in Morocco from the national HIV sentinel surveillance survey data. As of 2005, subtype B is still predominant (76.7%), yet following subtype B, there is a high diversity of non-B subtypes, especially CRF02_AG recombinant (15%). Geographic subtype repartition suggests the co-evolution of a more ancient diffusion of European subtype B, and of a more recent spread of sub-Saharan African strains in some Moroccan regions.p
pThese results demonstrate a high diversity of HIV-1 strains in Morocco. This is different from what was reported in 1997 where the distribution of subtype B, A and F strains in Morocco were 93.5%, 1.0%, and 0.5% respectively abbrgrp
abbr bid="B12"12abbr
abbrgrp. However, these results are consistent with our previous results abbrgrp
abbr bid="B19"19abbr
abbrgrp and the more recent results described from the Casablanca region abbrgrp
abbr bid="B13"13abbr
abbrgrp. These findings are also consistent with results described in other countries of the region, including the neighbouring West African countries abbrgrp
abbr bid="B14"14abbr
abbrgrp. The increase of HIV non-B subtypes was also recently reported in many Western Europe countries. Studies conducted in France, Spain, Switzerland, and Portugal have found that the proportion of non-B subtypes may exceed 20% abbrgrp
abbr bid="B20"20abbr
abbr bid="B21"21abbr
abbrgrp. CRF02_AG, which predominates in West Africa, is increasingly more prevalent among the non-B subtypes in these Western European countries.p
pThe overall incidence of HIV-1 in Morocco has been increasing at approximately 15% per year since 2000. The increasing incidence of HIV combined with the identification of additional non-B subtypes raises concerns regarding the control of the current epidemic. The entry of new HIV recombinant viruses is likely the consequence of active exchange between different populations, such as Moroccan groups at risk and persons migrating through Morocco from sub-Saharan Africa. Before 1997, the presence of Sub-Saharan African individuals in Morocco was mostly limited to students and tourists. However, migration of people from sub-Saharan Africa to and through Morocco has been increasing since the late 1990s. In 2007, the Moroccan Ministry of the Interior estimated that approximately 15,000 irregular migrants flow through Morocco each year abbrgrp
abbr bid="B22"22abbr
abbrgrp. In response, the European Union has tightened its boarder control and immigration policies. As a result, many of the migrants settle in Morocco, waiting for an opportunity to cross into Europe. In addition, regular and irregular migrants face many economic and social issues that may increase their risk for HIV transmission. For example, issues such as human trafficking and prostitution could contribute to the circulation of non-B HIV subtypes such as CRF02_AG throughout the country.p
pThe fact that subtype B was more distributed throughout the country, especially in the big-touristic cities (Agadir, Marrakech and Casablanca), suggest this subtype may reflect an older infection. Persons with subtype CRF02_AG were also widely distributed geographically; however, this subtype was not detected in Morocco before 1997, suggesting a more recent epidemic. These findings are also supported by our molecular clock analysis. The other non-B subtypes and recombinants represented more localised transmission due to C, AC, BC and ACRF01_AE strains in the northern part of Morocco.p
pAs the first case of HIVAIDS in Morocco was reported in 1986, there is an excellent agreement with the TMRCA of HIV-1B of 1983 estimated by molecular clock analysis. Since that date, HIV has been spreading throughout the country, mainly by heterosexual transmission abbrgrp
abbr bid="B11"11abbr
abbrgrp. According to the sentinel surveillance system, the overall HIV prevalence is less than 1% in Morocco. However, even though Morocco is a low prevalence epidemic, HIVAIDS cases are steadily rising, chiefly in the southern Morocco region of Agadir and neighbouring areas that may represent the epicentre of the epidemic within Morocco abbrgrp
abbr bid="B23"23abbr
abbrgrp.p
pOur findings should be interpreted in light of study limitations. While our analysis included all HIV positive specimens from the 2004-2005 survey, the sample size was relatively small. Therefore, it is not possible to generalise the results as a national trend in Morocco. In addition, routes of transmission and clinical and immunologic status of the HIV-infected individuals were not available for this study, since they are not required in the surveillance process. However, the present data should prompt us to continue to track the molecular epidemiology of the HIV virus in Morocco at the national level. In this context, reinforcement of preventive measures to limit the spread of the epidemic is crucial. Lastly, by limiting our phylogenetic analysis to only the itpol itgene region, we may have missed some recombinants and therefore underestimated their distribution. However, our main finding that CRF02_AG is increasing in Morocco, signifying a shift from an epidemic previously dominated by serogroup B, remains true. In conclusion, the results of this study displayed that HIV diversity is more dynamic in Morocco and its pattern is shifting from the European to sub-Saharan one, i.e. with more subtypes non-B, namely the CRF02_AG. However, more studies to confirm the trend observed during this study and to better characterize the molecular HIV epidemic in Morocco will be of great importance. When taken together, these data demonstrate a dynamic evolution in the HIV diversity in Morocco. The emergence of new HIV subtypes are characterised by an important presence of non-B subtypes that appear to be linked to sub-Saharan populations. More data are needed to better understand the factors responsible for the introduction and spread of new HIV-1 subtype epidemics into regions where they did not exist previously.p
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pAbbreviationsp
st
p
bAIDSb: Acquired Immune Deficiency Syndrome; bCRFb: circulating recombinant factor; bESSb: effective sample size; bHIV-1b: Human Immunodeficiency Virus 1; bIDUb: injection drug users; bMENA: bMiddle East and North Africa; bMLb: Maximum Likelihood; bPCRb: polymerase chain reaction; bPRb: Protease; bRTb: Reverse Transcriptase; bSTIb: Sexually Transmitted Infections; bTBb: tuberculosis; bTMRCAb: Time to Most Recent Ancestor; bWHOb: World Health Organizationp
sec
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st
pCompeting interestsp
st
pThe authors declare that they have no competing interests.p
sec
sec
st
pAuthors' contributionsp
st
pMA: Conduction of the study, samples collection, sequencing and analysis, preparation of the manuscript; SL: sample preparation, sequencing and analysis; EE: sample diagnosis and collection, HIV screening and confirmation, preparation of the manuscript; RG: phylogenetic analysis of samples and construction of phylogenetic trees; JCT: sequencing and analysis, preparation of the manuscript; RLC: interpretation of epidemiological data, preparation of the manuscript; MS: phylogenetic analysis of samples and construction of phylogenetic trees, preparation of the manuscript; PA: sequencing and analysis, preparation of the manuscript; TA: preparation of the manuscript; REA: conduction of the study, preparation of the manuscript. All authors have read and approved the final manuscript.p
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pAcknowledgementsp
st
pThe authors acknowledge the staff of the National program to fight HIVAIDS, in the department of epidemiology and diseases control, Moroccan ministry of health; and all the staff working on HIV sentinel surveillance network, in Morocco. We thank also Dr Oumzil Hicham, Ms Imnae Belbacha, Dr Mengad Rajae, Ms Bennani Ouafae and Mr Amina Siwani from the National Reference Laboratory for HIV, National Institute of Hygiene. Finally, the authors thank all members of the molecular biology laboratory group who helped with this project. This study has a funding support from ESTHER Initiative, CNRST-INSERM Collaboration, and from the Emerging Pathogens Institute and the Center for AIDS Research at the University of Florida.p
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Abstract
Background
Little is known about HIV-1 subtype distribution in Morocco. Some data suggest an emergence of new HIV subtypes. We conducted phylogenetic analysis on a nationally representative sample of 60 HIV-1 viral specimens collected during 2004-2005 through the Morocco national HIV sentinel surveillance survey.
Results
While subtype B is still the most prevalent, 23.3% of samples represented non-B subtypes, the majority of which were classified as CRF02_AG (15%). Molecular clock analysis confirmed that the initial introduction of HIV-1B in Morocco probably came from Europe in the early 1980s. In contrast, the CRF02_AG strain appeared to be introduced from sub-Saharan Africa in two separate events in the 1990s.
Conclusions
Subtype CRF02_AG has been emerging in Morocco since the 1990s. More information about the factors introducing HIV subtype-specific transmission will inform the prevention strategy in the region.
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Akrim, Mohammed
Lemrabet, Sanae
Elharti, Elmir
Gray, Rebecca R
Tardy, Jean Claude
Cook, Robert L
Salemi, Marco
Andre, Patrice
Azarian, Taj
El Aouad, Rajae
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Akrim et al.; licensee BioMed Central Ltd.
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AIDS Research and Therapy. 2012 Feb 14;9(1):5
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