Annals ofTropical Medicine &Parasitology ,Vol. 97,Supplement No. 1,S29–S45 (2003)

HIV and thetransmission of Leishmania

R.MOLINA*,L. GRADONI † andJ. ALVAR* *WHOCollaboratingCentre for Leishmaniasis, Serviciode Parasitolog ´õ a,Centro Nacional deMicrobiolog ´õ a,Instituto deSaludCarlos III, CarreteraMajadahonda– Pozuelo Km 2, 28220Majadahonda, Madrid, Spain †Laboratoriodi Parassitologia,Istituto Superiore diSanita`,VialeRegina Elena 299,00161, Rome, Italy Received andaccepted 30 May 2003

Inmany countries, Leishmania/HIVco-infectionis now changingthe epidemiology ofvisceral leishmaniasis. The levelsof transmission ofthe parasitescausing such leishmaniasis werepreviously dependenton the conventional zoonoticcycle, in which sandies transmitted the parasitesfrom infected canids to othercanids or humans. The co-infection,however, has lednot only to marked increasesin the sandy transmission ofthe parasitesfrom immuno- depressedindividuals directlyto otherhumans but also, probably, to artiŽcial transmission betweenimmunodepressed intravenous-drugusers, as the resultof needle sharing.

Theepidemiology of humanleishmaniases and The zoonotic form of VLiscaused by the biologyof the phlebotominesand ies that Leishmaniainfantum (=Leishmaniachagasi ), transmitthe causative parasitesare naturally with infectionpredominan tly inwild and closelylinked (Killick-Kendrick, 1990). There domesticcanids and occasionally in humans. islittle evidenceto indicatethat immuno- Thisform, which is mainlyendemic, extends competenthumans can develop leishmaniasis fromthe Mediterraneanthrough Central Asia exceptas aconsequenceof beingbitten intoChina and also exists in Latin America. by infectedsand ies, although the parasites Severalspecies of the genera Phlebotomus may occasionallybe transmittedas the result (OldWorld) and Lutzomyia (New World) of bloodtransfusions (Cohen et al., 1991; have beenincriminated in the transmission Grogl et al.,1993; Singh et al.,1996; Kubar of Le. infantum . et al., 1997; Luz et al.,1997; Le Fichoux Theanthropo– zoonotic form, caused by et al.,1999; Otero et al.,2000). Theresults Le.donovani or Le.infantum ,occursin East ofrecentxenodiagnostic tests with sandies Africaand the south–west of the Arabian indicatethat the amastigotesin samples of peninsula.It is normally endemic but can blood(taken fromcases of Leishmania/HIV causeextensive epidemics in humans. Trans- co-infection)remain viable forat least 8days missionof the parasitesthat causethis form after the bloodhas beencollected (R. Molina, of VLispredominantly human to humanvia unpubl.obs.). non-synanthropicvectors such as P. martini Froman epidemiolog icalstandpoint, and P.orientalis ,althoughthere may also threemain forms of humanvisceral leish- beother mammals acting as ‘reservoir’ maniasis(VL) have beenidentiŽ ed: zoonotic, hosts. anthropo–zoonotic and anthroponotic. Theanthroponotic form, caused by Le. donovani,occurson the Indiansub-continent. Reprint requeststo: R.Molina. Itisnormally endemic but severe epidemics E-mail: [email protected]; fax: + 3491 5097034. candevelop among humans. The parasite is

©2003The Liverpool Schoolof Tropical Medicine DOI:10.1179/ 000349803225002516 30 MOLINA ET AL. transmittedfrom human to humanby the andSpain give positive results in leishmanin bite of aspeciesof sandy that isstrictly skintests (Pampiglione et al.,1975, 1976; peridomestic, P.argentipes . Gramiccia et al.,1990; Meller-Melloul et al., Sincethe globalepidemic of HIVinfection 1991; Marty et al.,1992; Arbaji et al., 1993; began,there has beena steady risein the Acedo-Sa´nchez et al.,1996; Alvar et al., annualnumber of reportednew cases of VL 1996; Morillas et al.,1996), manydetected associatedwith the virus. Leishmania/HIVcasesof Leishmania/HIVco-infection in these co-infectionis emerging as anewand worry- countriesare probably the resultof latent ingdisease, particularly in south– western Leishmania infectionsthat have beenactivated Europe(Desjeux, 1998). InSpain — the orre-activated as the later HIVinfection countryreporting the highestincidence of causesimmunosuppression. Many now think the co-infection— 68% of the casesare that Leishmania spp.should be considered intravenous-drugusers (Desjeux and Alvar, as opportunisticpathogens in HIV-infected 2003). InFrance,Italy, Portugaland Spain, patients (Alvar et al.,1992). Evenin the the outbreakof HIVinfectionhas changedabsence of HIV, Le. infantum infections the epidemiologyof VL, froma diseasepre- may bere-activated followingimmunological dominantlyfound in children to onemore distress (Ma et al.,1979; Kubar et al., 1998). commonin adults (WHO, 1999). HIVco- Ifthe naturalhistory of VLinHIV- infectionmay re-activate latent leishmanialpositive individuals is to befullyunderstood, infections,increase the levelof transmission long-term,longitudinal, immunological and of Leishmania (particularlyhuman– human parasitological investigations on cohorts of transmission)by phlebotominesand ies, and individualsat riskof both HIVand Leishmania faciliate artiŽcial transmission of Leishmania,infectionwill have to beconducted. As the via the sharingof contaminatedsyringes and incidenceof leishmaniasisin the areasmost needles,from one intravenous -druguser a Vectedby the co-infectionis relatively low, (IVDU) to another(see below). the cohortsstudied will have to belarge. The relevantepidemiological data alreadyavailable may be suYcient,however, to estimatethe NEWLY ACQUIRED VERSUS probabilitiesthat clinicalVL ina HIV-positive RE-ACTIVATED VISCERAL individualis (1) the resultof anewlyacquired LEISHMANIASIS:THE ITALIAN leishmanialinfection or (2) the resultof EXAMPLE are-activated latent infection.In 1998, L.Gradoniand A. Scaloneattempted to Itis estimated that onlyone in every Ž ve to estimatethese two probabilitiesin Sicily, 10 immunocompetentindividuals who have by assimilatingand updating the relevant beeninfected with Le.donovani or Le.infantum epidemiological-surveillancedata andthen everdevelops clinical VL (Desjeux,1992; usingthe resultantdata-set inan epidemio- Badaro´ et al.,1996). Sincea T-lymphocyte- logicalmodel (unpubl. obs.). Their analysis mediatedimmune response is necessary to ofthe naturalhistory of the Leishmania/HIV control Leishmania infection,however, those co-infectionis described below, in extenso. residentsof endemicareas who have been madeimmunodeŽ cient as the resultof HIV infectionare far moreprone to VLthan their Surveillance Methodology immunocompetentneighbours .Strainsof In1989, aprogrammebased on the active Leishmania that areusually non-pathogenic detection of Leishmania/HIVco-infection was inhumans, and even lower trypanosomatids implemented,in collaboration with some148 (Chicharroand Alvar, 2003), may all causeinfectious-disease units sited in universities illnessin HIV-positive individuals. As many andhospitals throughout Italy (Gradoni et al., asymptomatic individualsin Italy, France1996). Theunits selected, from the 260 HIV AND Leishmania TRANSMISSION 31 clinicaldiagnostic centres which were then TheSituation in Italy recordingAIDS cases,were chosen because Between1985 and1997, 183 casesof they werenot only reporting relatively high Leishmania/HIVco-infectionwere recorded numbersof HIV-positivepatients butwere inItaly. Most(68%) of the co-infections alsoin regions where VL was endemic.were in patients whofulŽ lled the criteriafor Collaboratorswere asked to provideserum, AIDS.In areas of Italy whereleishmaniasis bone-marrow,peripheral-blood or skin-biopsy was endemic,the incidenceof VLamong samplesfrom any patient who,when sero- HIV-positiveresidents (1.6 cases/100) was positivefor HIV orsu Veringfrom AIDS, about 500 timeshigher than that amongtheir developedan infection that was clinically HIV-negativeneighbours. In four distinct suspectedto beVL. Oneor more of the ‘hotspots’, 12%–100% ofthe HIV-positives followingclinical and laboratory Žndingswere developedVL. Theannual number of co- consideredindicative of VL: feverof unknowninfection cases showed a sharpincrease in origin,splenomegaly, hepatomegaly, hyper- 1991 butthen stabilized, at about 21 cases/ gammaglobulinaemia,and pancytopenia. year (Fig.1). Thepresence of singleor multiple nodular/ ulcerativeskin lesions was consideredindi- cative of cutaneousleishmaniasis (CL). A EpidemiologicalModel retrospectivesurvey was conductedto identify Amodelwas constructedto test two alternative any casesof co-infectionthat hadpresented hypotheses: that the VL seenin HIV-positives at the collaboratingunits before 1989; con- represented(1) newlyacquired infections, Žrmatorydiagnosis was thenbased on the or(2) re-activation of latent leishmanial laboratory examinationof storedsera and infections. bone-marrowsmears.Possible Leishmania Totest the Žrstof thesehypotheses, the infectionsdiagnosed in AIDS patients were numberof expectedco-infections was derived routinelyreported, through the nationalby applyinga knownforce of leishmanial systemof AIDS-casenotiŽ cation, with other infection ( p;seebelow) to anestimated HIV- associatedpathologies ( i.e.those not indi- infected population exposed to Leishmania cative of leishmaniasis)to the National AIDS during each Leishmania-transmissionseason. Registry. It was assumedthat the leveland mode

FIG.1. Temporal distribution ofthe casesof Leishmania co-infectionamong those with AIDS ( f ) and other HIV-positiveindividuals ( e )recordedduring epidemiological surveillance in Italy. 32 MOLINA ET AL. of Leishmania transmissionto andamong the numberof HIV-positiveresidents of Sicily HIV-positiveindividuals were the sameas wholived in areas where leishmaniasis was thoseto andamong the HIV-negatives,that endemic. eachHIV-positive individual infected with Leishmania developedclinical VL, andthat p hadbeen constant during the 10-year study forceof infection ( p) period(1985– 1994). Theforce of infection(Lysenko and Beljaev, Totest the secondhypothesis, the number 1987) was estimated,as the meannumber of expectedco-infections was calculatedby of timesa residentof Sicilywas inoculated applyingan estimate of the annualincidence with Leishmania overa year, fromLST of HIVinfection to apopulationthat included data indicatingthe prevalenceof leishmanial manyindividuals — the numberbeing indi- infectionamong Sicilans aged 0– l5 years,who catedby the resultsof leishmaninskin tests wereassumed to benon-immune. Surveys (LST) —whowere asymptomatic carriers basedon LST have beencarried out in Sicily of Leishmania.Itwas assumedthat each since1975. Theirresults indicate that the individualfound positive in an LST hada forceof infectionin Sicily varies little from latent leishmanialinfection that woulddevelop year to year andis similar to thosein other intoclinical VL followingthat individual’s leishmaniasis-endemicareas of southernItaly infectionwith HIV,and that the age-related (Pampiglione et al.,1975; Gradoni et al., prevalenceof LSTpositivity was constant 1993; unpubl.obs.). overthe studyperiod. thepopulation potentially harbouring Leishmania Sourcesof Data Theresults of the LSTsurveysin Sicily prevalenceand incidence of HIV werealso used to estimatethe numberof infection individualson the islandwho were LST- Theprevalence and incidence of HIV positiveand therefore possibly carrying latent infectionin Italy in1994 wereestimated Leishmania infections.Particular attention usingmathematical models,‘ back-calculation’, was paidto the LSTresultsfor residents of the annualnumbers of AIDScases notiŽ ed, Sicilywho were aged 20– 30 years,this age- andthe resultsof pilotstudies on HIVsero- groupbeing considered the oneat greatest conversion(Pezzotti et al., 1995; Rezza,1998). riskof HIVinfection. Prevalencewas estimatedby subtractingthe numberof HIV-attributable deathsprior to 1994 fromthe accumulativeincidence of HIV reportedincidence ofthe infection.Annual incidence represented all Leishmania/HIV co-infection the newHIV infectionsthat occurredin Theannual numbers of cases Leishmania/HIV 1994. co-infectiondetected among Sicilian residents Gradoni et al.(1996) usedmaps of the duringepidemiological surveillance between distributionof leishmaniasisin Sicily (based 1985 and1994 werecompared with those onaccurate locality data forall casesof predictedusing each of the two mathematical humanand canine leishmaniasi sthat had models. beenrecorded over the previous18 years) to estimatethe sizeof the humanpopulation Evaluation oftheModels at risk of Leishmania infectionon the island. They thenused the samemaps and the hypothesis 1 (‘new infections ’) relevantAIDS-case notiŽ cation reports, stored In Sicily,the numberof HIV-positivesliving at the National AIDSRegistry, to estimate inareas where VL was endemicranged from HIV AND Leishmania TRANSMISSION 33 approximately800 in1985 to approximatelyin the period1986– 1989, to 16–24 cases/year, 4200 in1994 (Fig.2). Thecorresponding with atotal of 376 casesover the study numbersof AIDScases showed the sameperiod. trend(Fig. 2). Themean force of leishmanial infectionin these areas was calculatedto be 0.003 infection/person-year,indicating that observed HIV/VL cases the areasare only hypo-endemic for VL. By Theactual recordednumbers of HIV- applyingthis p-valueto the numberof HIV- positiveindividuals found to have VLin positivespresent in the areasof the island Sicilyincreased from nil in 1985 to ninein whereVL isendemic, the annualnumbers 1994, with atotal of 35 HIV/VLcasesover of casesof co-infectionwere predicted to the studyperiod. The trend in the numbers increasefrom two in1985 to 12 in1994, of reportedcases was similarto that pre- with atotal of74 casesover the studyperiod. dictedwhen the ‘new-infection’hypothesis was applied,and di VeredsigniŽ cantly from that predictedwhen the ‘re-activation’hypo- hypothesis 2 (‘re-activations ’) thesiswas assumedto becorrect ( P<0.01; Theestimated incidence of HIVinfection in Fig. 3). Sicilypeaked at about 600–700 cases/year between1986 and1989 andthen decreased to about 200–300 cases/year (Fig.2). Most Discussion of thesecases were aged 20– 30 years.A mean InItaly, the problemof HIV/VLco-infection of8.0% ofthe Sicilianresidents aged 20– 30 isno longeras alarmingat it appearedto be yearswho were checked in LST surveyswere inthe early 1990s, whenthe numberof cases foundLST-positive. By applyingthis pro-was showingdramatic increases from one portionto the sizeof the HIV-positivepopu- year to the next.Although, in some areas of lation,the numbersof casesof co-infectionthe country,the incidenceof VLdoesappear werepredicted to decreasefrom 48– 56/ year to have increasedrecently, this upwardtrend

FIG.2. AIDS incidence( f )and the estimatedtrends of HIV prevalence( e )and incidence( ^)in leishmaniasis- endemicareas of Sicily. 34 MOLINA ET AL.

FIG.3. Thenumbers ofcases of visceral leishmaniasis observedamong the HIV-positiveresidents of Sicily between1985 and 1994( f ),and the numbers expectedif hypothesis 1( ^;‘newlyacquired leishmanial infections’) orhypothesis 2( e ;‘re-activationsof latent, leishmanial infections’) werecorrect. appearsto beunrelated to HIV(Gradoniwho have frequentlytaken theirvacations in et al.,1996). It seemsthat the predicted regionsof the Mediterraneanlittoral where oodof HIV/VLcaseshas beenstemmed the diseaseis endemic. bythe largelysuccessful introduction of anti- Theepidemiological models have to be retroviraltreatment for those found sero- treated with somecaution as they may betoo positivefor HIV. The quarterly incidence of simplistic.Although the two hypotheseswere AIDSinItaly peakedat 10 cases/100,000 assumedto bemutually exclusive, both new inhabitants in1995, fallingto sixcases/ infectionsand re-activations may contribute 100,000 inthe Žrstquarter of 1998. to the actual numberof clinicalHIV/ VLcases. Theresults of the epidemiologicalmodelling Althoughthe predictednumber of casesfor discussedabove indicatethat, inSicily at eachyear was comparedwith the actual least, mostcases of HIV/VLco-infectionare numberof casesdiagnosed in the sameyear, the resultof individualswho are already HIV- an incubation period — the lengthof which positivebeing newly infected with Leishmania. is diYcultto estimate— willhave separated Hadsuch co-infection been predominantly co-infection from diagnosis. Some of the the resultof thosewith (latent)leishmanial assumptionsand estimates made appear more infectionbeing infected with HIV,then many reasonable than others.The estimates of p morecases should have beendetected in the andof the proportionof the populationwho Žrstfew yearsof the presentstudy period, may have latent Leishmania infections,for as HIVswept throughSicily. Such large example,were both basedon LST data numbersof caseswere not observed at that showedconsistency over a longperiod. this timein Sicily, in other areas of the Theseestimates were supported by astable Mediterraneanbasin where VL isendemic, incidence of recordedVL amongthe immuno- orin the youngpeople of northernEurope competentpopulation of Sicilyduring the HIV AND Leishmania TRANSMISSION 35 studyperiod, in the absenceof measuresto increasesthe riskof VLby 100- to 1000- controlthe caninereservoir or phlebotomine foldin areas where VL isendemic (WHO, vectorsand of dramaticchanges in local 1999). InwesternEurope, IVDU who share environmental conditions(Cascio et al., syringesform the mainpopulation at riskof 1997). Thepossibility that HIVinfection HIV infectionand account for 44% of the has ledto, oris associated with, unusual HIV/AIDScases (Desjeux, 1998). Inajoint routesof leishmanialinfection (see below) consultative meeting on the Leishmania/HIV was notconsidered in the epidemiological co-infection,held in September 1998, itwas models. establishedthat IVDUhad a 2.5- to 3.6-fold higherrisk of VL/AIDSthan otherAIDS cases(WHO, 1998). InSpain and Italy, ALTERNATIVEANTHROPONOTIC biochemicalvariants of Le. infantum that CYCLES FOR Leishmaniainfantum rarelyif evercause leishmaniasis in immuno- competentpatients have beenrecovered Inthe predominant,natural cycle of trans- fromcases of the HIV/ Leishmania co-infection mission,an individual, whether HIV-positive (Gramiccia et al.,1995; Jime´nez et al., 1995; orHIV-negative, becomes infected with Le. Pratlong et al.,1995; Agostoni et al., 1998). infantum whenhe or she is bitten (usually Amastigote-infectedmacrophages can be inrural or peri-urban settings) by asandy foundin approximately 50% ofbloodsmears that has alreadybeen infected when taking fromco-infected patients (Mart ´õ nez et al., abloodmealfrom an infected canid. The 1993; Medrano et al.,1993), andpro- possibilityof otherroutes of transmission, mastigotescan be found in 67% of NNN suchas needle-mediatedinfection, should culturesof bu Vycoats fromsuch patients not,however, be ruled out (Alvar et al., (Lo´pez-Ve´lez et al., 1995). 1992). Directhuman– sand y– human trans- TheAIDS-case surveillance system that missionmay alsooccur, particularly from coversthe Madridregion of Spainhas also HIV-positives,who carry particularly high provideddata indicatingthat needle-sharing numbersof leishmanialamastigotes in their carriesa riskof HIV/ Leishmania co-infection peripheralblood. (Amela et al.,1996). Asthis systemonly recordsAIDS cases,VL diagnosesin any HIV-infectedindividuals who fail to meet An ArtiŽcial Cycleof Transmission the diagnosticcriteria for AIDS are not Itseems likely that notonly HIV butalso included. In total, 6652 casesof AIDSwere Leishmania may betransmitted, on and in diagnosedbetween 1982 and1993 and sharedneedles and syringes, among IVDU. recorded by the Madridsystem. Only 166 Theobservation that mammalsmay be (2.5%) of thesecases developed VL, and infectedwith Leishmania,eitheraccidentally the datesof diagnosisof both the AIDS (Owens et al.,2001) orexperimen tally andVL wereavailable foronly 137 of the (Palatnik deSousa et al.,1996), by trans-co-infection cases. The VL was diagnosed fusionsof bloodfrom infected hosts under- beforethe AIDSin 33 cases,after the AIDS linesthe possibilityof this modeof trans-in 65 cases,and at the sametime as the mission.There has alsobeen at least one AIDSin 39 cases.The prevalence of VL caseof laboratory-acquired Le. donovani amongthe AIDScases who were IVDU was infectionas the resultof aneedlestickinjury signiŽcantly higherthan that inany of the (Freedman et al.,1987). Althoughthere is, other exposure-groups (relative risk =2.57; as yet, nodirect evidence of the spreadof 95% conŽdence interval =1.64–4.01). Leishmania throughthe sharingof syringesIn Italy, the unexpectedoccurrence of amongIVDU, there is much indirect evi- smallhotspots of HIV/ Leishmania co-infection, denceindicating that this happens.AIDS inwhich every detected case of HIVinfection 36 MOLINA ET AL. may have VL, alsoindicates that transmissionEurope between 1990 and1998, the pre- isnot restricted to the normal,canid– dominanceof IVDU,who were identiŽ ed sandy– human route. The observations that as the mainpopulation at risk,was clear IVDUrepresent 84% of the Italian casesof (WHO,1999). HIV/Leishmania co-infectionbut only 64% of ItalianAIDS patients without leishmaniasis Natural Human–sand y– human (64%) andthat the zymodemespectrum of Transmission the Le.infantum fromthe co-infectedcases Mosthumans are infected with Le. infantum diVersfrom that of the parasitic isolates whenthey arebitten by sandies that have fromHIV-negative adults, and the distances alreadybeen infected when they fedon dogs betweenthe hotspots,also support the view orother canids harbouring the parasite. that leishmanialparasites can be transmitted Asuninfected sand ies are not frequently onneedles and syringes (Gradoni et al., infectedwith Le.infantum as they feedon 1996). InnorthernItaly, 17 (77%) of the 22 immunocompenthumans carrying the para- co-infectedpatients investigatedby Agostoni site,human– human transmissio nof Le. et al.(1998) wereIVDU. infantum,via asandy, israre. There is, Pineda et al.(1998) investigatedthe however,considerable evidence indicating factorsassociated with VLinindividuals that uninfectedsand ies feeding on indi- infectedwith HIV-1 wholived in southern vidualsco-infected with Le. infantum and Spain.When Giemsa-stained smears of bone- HIVarequite likely to becomeinfected with marrowaspirates from 291 HIV-1 carriers the parasite. Transmissionof leishmanial wereexamined, 45 werefound positive for parasitesfrom a HIV-positivehuman is there- amastigotes.Thirty-two of the amastigote- foremore likely than transmissionfrom a positivecarriers of HIV-1 hadsymptomatic HIV-negativeindividual. Most of the data VL, the other13 havingsubclinical infections indicatingthat sandy-mediated transmission with Leishmania.SymptomaticVL again of Le.infantum fromco-infected HIV-positive appearedparticularly common among the individualsoccurs have beencollected during HIV-positiveIVDU included in the study. xenodiagnostictests with sandies. Althoughthis association was notquite found to bestatistically signiŽcant, whenadjust- mentswere made for clinical category and indirectxenodiagnosis genderin a multivariateanalysis, Pineda TheconŽ rmation of asuspecteddiagnosis of et al.(1998) thoughtthat Leishmania trans-leishmaniasis in immunodepressed patients missionthrough the sharingof needleswas frequentlyrequires the useof severaltech- very probable.The isolations of the MON-18 niques.Although the examinationof bone- zymodeme of Le.donovani froma Portuguese marrowaspirates for amastigotes has been drugaddict with clinicalVL andAIDS proposed as the besttechnique, it requires (Campino et al.,1994), andof the MON- aninvasive and painful procedure and is not 253 zymodemeof Le.infantum froma clusterhighly sensitive. In the searchfor a dia- of threeco-infected patients, all IVDU, gnostictest that isless invasive and less pain- livingin the sametown in north– eastern ful, for patients whomay alreadybe seriously Spain(Chicharro et al.,1999), alsosupport ill,the useof indirectxenodiagnosis (IXD) — the view that leishmanialamastigotes are that is,the feedingof uninfected(usually beingtransmitted on shared needles. When laboratory-bred)sand ies, through a mem- Cruz et al.(2002) checkedsyringes discarded brane, on a sampleof venousblood from by IVDUin Madrid, they found34%– 52% the suspectedcase of leishmaniasis— has to bePCR-positive for leishmanial DNA. beenconsidered. Molina et al.(1992) found Ina retrospectiveanalysis of 965 cases IXD with Phlebotomusperniciosus ,animportant of co-infectionreported in south– western vector of Le. infantum inthe south–western HIV AND Leishmania TRANSMISSION 37

Mediterraneanregion, very usefulin con- Pakistan have ledto the hypothesisthat skin Žrmingthat anAIDS patient hadVL. parasites,rather than parasitescirculating in Subsequently,Molina et al. (1994) investi- bloodmonocytes, might be the principal gatedthe potentialusefulness of IXDinthe sourceof infectionfor sand ies in areas routinedetection of leishmaniasisin those whereanthroponotic leishmaniasis is found co-infectedwith Le. infantum and HIV, (Rab et al., 1992).] andattempted to establisha standardpro- UsingIXD, parasitescan be clearly tocol.The study, designed to investigatethe observed48 hafter sandy feeding;parasites infectivity to P.perniciosus of 10 HIV-positive incultures of aspiratesand blood usually individualswho had symptoms indicative of take longerto multiplysu Ycientlyfor them VL (N=9) orhad duodenal amastigotes to bedetected.Although IXD isvery useful detectedduring routine endoscopy ( N=1), fordetecting Le. infantum inHIV-positive alsoserved to comparethe IXDwith the patients, its applicationis, for the moment, moreconventional methods of diagnosis. largelyrestricted to thosecases who have Theother methods, tested in parallel with givennegative results in other diagnostic the IXD, wereIFAT for the detectionof tests eventhough there is strong clinical evi- anti-Leishmania antibodies,the microscopical denceindicating that they have leishmaniasis. examinationof Giemsa-stainedsmears of IXDisa highlysensitive technique when bone-marrowaspirates and peripheral blood, usedon immunodepressed patients. Molina andNNN culturesof bone-marrowaspirates et al.(1998), forexample, used IXD to andperipheral mononuclear cells. Records demonstratethe presenceof viable parasites weremade, for each patient, of associated in21 (95%) of 22 bloodsamples from cases infections,fever (for 2weeks),spleno- > of Le.infantum /HIVco-infection and in blood megaly,treatment for the leishmaniasisand samplesfrom two otherpatients who, though the responseobserved, the sandy-feeding HIV-negative,were immunodepressed as the procedure,and other information (Table 1). resultof acutelymphoblastic leukaemia. The P.perniciosus usedcame from a local Theobservation that bloodfrom cases laboratory colonykept at 27 ±1°C and of Leishmania/HIVco-infectionremains 90%–100% relative humidity,with a17-h- light:7-h-darkphotoperiod (Molina, 1991). infectiveto the sandies used in IXD even if usedafter storagefor at least 8days Batchesof 30–150 female P.perniciosus collected4– 21 days post-eclosionwere fed, (unpubl.obs.) adds support to the ideathat inthe presenceof somemales, through a leishmanialinfections can be transmitted by membraneon anticoagulated peripheral blood bloodtransfusion. fromeach patient. Thebloodfed sand ies IXD has usesin the Želdof epidemiology weredissected 2– 7 days after takingtheir otherthan the demonstrationof leishmanial bloodmealsso that theirguts could be checked co-infectionin those who are HIV-positive. forpromastigotes. All 10 ofthe patients were It has beenused, for example, to showthe foundpositive by the IXDbutonly eight, importanceof haematogenicdissemination seven,Ž ve andŽ ve, respectively,had positive inthe developmentof mucosalleishmaniasis culturesof bone-marrowaspirates, positive andother metastatic manifestationsof Le. culturesof peripheralblood monocytes, braziliensis infection(Mart ´õ nez et al., 1992). amastigotesdetected in their bloodsmears, Da-Cruz et al.(1992) usedIXD to studythe orwere found seropositive in the IFAT failureof T-cell-mediatedimmune responses (Table 1). Evenin the absenceof cutaneous to preventdi Vusecutaneous leishmaniasis macrophagescarrying amastigotes, most developingin HIV-positives infected with (upto 93%) of the sandies taking a blood- Le. braziliensis .Asthe resultof using mealwere infected. [The results of studies P.argentipes inIXD, Addyand Nandy conductedon Le. infantum -relatedVL in(1992) concludedthat the skinparasites in 38 MOLINA ET AL. , 0 U 0 P 3 P s s s 4 0 o 4 . : 0 7 0 D 4 e e e . C 7 0 D C 3 1 M + + + 1 3 9 2 N 7 Y Y Y N 1 P 5 V P 5 I < ; n o i t c e s e U 0 f 0 8 s s 8 1 p o 5 o o . 8 0 2 n e e 4 . a 9 4 3 D i : 3 l M ­ ­ + 2 9 7 N 6 N N Y Y 1 1 1 e 1 V 1 s I R u r i v o l e a s 0 g U 0 7 s s 9 p o o o 2 e . 8 0 0 0 8 e e a 8 7 D 3 0 + + l + M 2 6 2 1 4 N N N : m Y Y 1 e 1 V 1 1 o I t R y c , V e s s i 0 B U 0 s 1 s s 4 M p . o o 6 0 o 2 0 3 4 T e e . a 7 D 4 0 1 M ­ + l + C n 2 4 7 2 N N P : 8 Y Y 1 e g 1 V 2 ; 1 I E a s R i i s d o a i n d e i x 0 d U 0 3 s s s s 4 t . : n 9 C 0 1 8 e e e e c 6 9 0 6 D 8 a e 1 M + + + 2 6 5 9 Y Y Y Y O r c 4 V i 3 I < d l a n e i r s o y a b , C C C d 0 E e 0 9 s s s s 4 t O S 4 . : , 0 8 8 5 6 e e e e . a 5 0 2 1 M + ­ + 3 3 4 7 3 ; B g H 8 Y Y Y Y s i 9 4 i t T < s s e P o l v u n i c r n e 0 o b i U 0 7 s s s s 4 t 2 . : u 5 0 6 4 C . e e e e c . 4 D 0 6 F t 2 1 e + + + e 2 5 5 8 7 E Y Y Y Y f 6 V n 2 y n I < i r o - a d o n c t o o V n m U I 0 2 l P s s s o . , A A A 5 8 4 e e e 8 u 3 0 H D C : 4 M + + + / 3 6 2 N p D Y Y N N N Y 1 1 V P I , m N u B ; t e T l n a b P f 0 a U V 0 ; l n 5 s s 4 4 l i i o o 3 . : 3 0 0 e e . a a 2 6 0 6 D 7 M 1 M + + + 3 4 1 a N N 8 v Y Y u 1 i 3 V 2 a C x I n < e t a s o o m n r h e , s t i C A e e 0 h U O 0 8 s s N L 4 o o 6 . 6 0 0 1 , e e , . f 1 2 6 ; D 1 2 M + ­ + 7 3 4 7 s 8 N N : S o B Y Y i 2 V 2 1 s s I T H e o s P l ; a r u c e c s r 0 e u 1 ) ? b h g e r u t ( o t u f a r s r y i i d a r s - t p a s o s a n u a n d o g o l d a w n e a i m t l c e o i d c r v u e ) g r o e a r s p o f n a r l n y a u t o n ) o e s t r i a i t l m i l n t t x i - i ) d s m s x c u d / l e ( o , o a e e c s n m n t r f ? s n / e ) , i r u U e o a n t s l g r s t i l a B o p y b a l n o a D y f i e / i c i e f T c d s r V d V o s c n t o I m o I P n ) p o e c a e s i s n a ) l r e n E ; H u ( + d e  e r o a m l C e e e ; t n r o r 4 l l e ° d i a u h x ) t a i o m a ( o t o i c s 4 l l n i l l i f D - t i t / n m t t a i p t e n b s u c m g d r c s C l i p n ( a c e e l ( T o ( e a ? f r n n u f u r t c i d o i i y C e t A l n l o , o o w n e n d i g s i f r n f s s F a b c i o ) F u a n b I t - g e e g u r d s t . i r l s t t ( d o e o r ; r e o o n 1 l a t o y e f e c o o a e a c r t y e c r t o y g l g g o m e l s t e o E a i i g c a u o m i y ? o e t t t m b h s + o - o l l L r t m c s s d ( l n m e f o p c e M e 4 a a a u u o o i B o e t e e n o s p e v l u e s r x r , o a a D r A g s m m e x e o l e l e n e p e T S A E A F S L C H P A A S B P T R B % M P HIV AND Leishmania TRANSMISSION 39 apatient with nodulo-ulcerativepost-kala-azar HIV-positiveindividual who is co-infected dermalleishmaniasis represented a sourceof with Le. infantum ,raisesthe possibilitythat infectionin a VLfocusin West Bengal. the casesof co-infection(and any other Afterseveral years of usingIXD to immunocompromisedcases of VL)may act test bloodsamples from Spanish cases of as secondaryreservoirs of VL, ina naturalbut Le.infantum /HIVco-infection,Molina et al. anthroponoticcycle of the disease.Direct (1996) developeda standardprotocol. xenodiagnosis (DXD) —inwhich uninfected Samplesof peripheralblood are collected in sandies are allowed to feeddirectly on tubeswith anticoagulant(usually heparin, humans — has recentlybeen used to explore althoughEDTA or sodium citrate areequally this possibility(Molina et al.,1999). Each satisfactory) andbrought to the laboratory ofsixco-infected patients was askedto place refrigerated,at 4 °C.Each istested by o Ver- oneof hisor her hands into a smallcage ing50, 7-day-old,laboratory-bred, female containing 25, 7-day-old,laboratory-bred, P.perniciosus a1.5-ml sampleof blood,held female P.perniciosus (anda similarnumber at 37°C,for1 hina sterilemembrane- of male ies)for 15 min(Fig. 4; Table2). feedingapparatus, usingthe skinof a3-day- Theunfed  ieswere carefully removed with oldchicken as the membrane.Any unfed an electrical aspirator and killed, while the iesare then removed with anelectrical blood-fedfemales were kept inthe cagefor aspiratorand killed with chloroformor CO .at least 72 hbeforebeing dissected (Molina 2 Thefed  iesare maintained on 30% fructose et al.,1996). Allsix patients werefound to solution,replaced daily, for48– 72 hbeforebe infective to the sandies, their infectivity the dissectionsbegin. Each yto bedis- beingnegatively correlated with theirCD4 + sectedis anaesthetized with CO ,placedin cell counts (Fig. 5). Countsof CD4 cells 2 + adropof sterilephosphate-bu Vered saline may thereforebe a usefulindicator of the (PBS)on a sterilemicroscope slide, and infectivity of aco-infectedpatient. decapitatedwith sterileneedles. The midgut Tosummarize, the resultsof xeno- isthen drawn out, transferred to anotherdiagnoses have indicatedthat: (1) vector dropof sterilePBS, coveredwith asterile sandies are readily infected by feedingon coverslipand examined under the micro-immunodepressed cases of Le.infantum /HIV scopefor promastigotes. If a gutis found to be co-infection,whereasimmunocom petent heavily infectedit isgently ruptured, by press-individuals infected with Le. infantum are ingon the coverslip,and used to inoculate poorlyinfective to suchinsects; (2) CD4 + NNN mediumso that the Leishmania straincell counts are useful indicators of the canbe isolated and typed by iso-enzyme infectivity of co-infectedpatients; and(3) at analysis.If a gutis found to containonly a least someof the Le. infantum in the blood few promastigotes48 hafter the infective of thoseco-infected with HIVremain infective feed,the surviving iesthat hadfed on the to sandies for at least 8days after the blood samesample are maintained until at least has beencollected. day 7post-bloodmeal,before they tooare dissectedin the hopethat, by then,some will beheavily infectedand of usefor parasite EpidemiologicalImplications isolation. Theease with whichsand ies may be infectedwhen fed, directly or indirectly, on the bloodof thosewith Le. infantum /HIV directxenodiagnosis co-infectionhas considerableepidemiological Theease with whichsand ies can be infected implications, particularly as the casesof co- with leishmanialparasites by feedingthem, infectionmay remainasymptomatic, the VL througha membrane,on blood from a inHIV-positive individuals often responds 40 MOLINA ET AL.

FIG.4. Directxenodiagnosis of a suspectedcase of Leishmaniainfantum /HIVco-infection.

TABLE 2. Clinical andparasitological datafor six cases of Leishmania infantum /HIVco-infection investigated bydirect xenodiagnosis

Case

1 2 3 4 5 6*

Age ( years) 26 27 29 38 27 32 Associatedinfection(s) No PCP OC T, MAI OC No Fever? Yes Yes Yes No Yes Yes Splenomegaly? Yes Yes Yes Yes No No Leucocytecount ( leucocytes/ ml) 22003910 2400 4450 980 720 CD4+count (CD4+cells/ml) 120 4 45 12 39 28 Haemoglobin (g/l) 8.6 8.7 10.1 9.6 7.9 8.7 Plateletcount ( platelets/nl) 125 300 72 56 125 70 Serology(IFAT titre) 1:640 <1:40 ND ND 1:320 <1:40 Bone-marrowculture ND ND + + + ­ Peripheral-blood-monocyte culture + + ND ND ND ­ Resultof direct xenodiagnosis + + + + + + %ofbloodfed sandies found infected9.1 85.7 37.5 88.9 38.5 18.2

*Relapseafter treatment for leishmaniasis. PCP, Pneumocystis carinii ;OC, oralcandidiasis; T, toxoplasmosis; MAI, Mycobacterium avium infection;ND, not done. poorlyto treatment,and VL may developat 1992; Arbaji et al.,1993; Acedo-Sa´nchez any stageof the HIVinfection(Pineda et al., et al.,1996; Morillas et al.,1996). Le Fichoux 1998). Theresults of leishmaninskin tests et al.(1999) detectedleishmanial DNA in andlymphoproliferative tests in vitro indicate nineblood samples (out of 76 foundsero- that, inareas where VL isendemic, many positive for Leishmania)fromthe Monaco individuals,though asymptomatic, may be bloodbank, indicatingthat Le. infantum carryingleishmanial infections (Pampiglione circulates, albeit perhapsonly intermittently et al.,1975, 1976; Gramiccia et al., 1990; andat alowdensity, in the bloodof asymp- Meller-Melloul et al.,1991; Marty et al.,tomatic blooddonors. HIV AND Leishmania TRANSMISSION 41

FIG.5. Therelationship betweenthe infectivityof untreated cases of Leishmaniainfantum /HIVco-infectionto laboratory-bred Phlebotomus perniciosus (e ) and the CD4+cellcounts of the cases( f ).

Itseems that someiso-enzymatic pheno- the strictly anthroponoticVL, causedby Le. types of Le. infantum ,whichhave been donovani,seenin India, where even HIV- detectedin HIV-positive individuals but negative cases of VLarehighly infective to notin immunocompetent humans or dogs, sandies (Theodor, 1964). Asmost(91.5%) areparticularly associated with immuno-cases of Le. infantum /HIVco-infection depression(Gramiccia et al.,1995; Jime´nez detectedin south– western Europe have low et al.,1995; Gradoni et al.,1996; PratlongCD4 +counts, of <200 cells/ml (WHO, et al., 1995; Rosenthal et al., 1995; Harrat et al., 1999), manyare probably infectiveto their 1996; Agostoni et al.,1998; Chicharro et al.,localsand ies. The overall prevalence of 1999). Suchphenotypes occur among the humanco-infection in the VL-endemicareas manyzymodemes to befoundin wild sand y of Europeis very lowwhen compared with populations(Rioux et al., 1986; Gradoni et al., that of canineleishmaniasis. There may 1991; Mart´õ n-Sa´nchez et al.,1994, 1995, befoci, however, in which human– human 1996) andat least someof themcan infect transmissionvia sandies is playing an laboratory-bred P.perniciosus (J.M.Lohseimportant part inthe localepidemiology of andR. Molina,unpubl. obs.). VL. Cases of Leishmania/HIVco-infection Inthe presenceof HIV, Le. infantum may beclustered together, for example, in adaptedto the peridomesticenvironment institutionsspecializing in the careof AIDS couldevolve without the interventionof any patients orthe detoxiŽcation of IVDU. othervertebrate host apart fromhumans, to Insome countries, including Spain, such atrueanthroponosis (Tesh, 1995). Insomeinstitutions are usually in peri-urban or iso- aspects,this situationwould emulate that of lated,rural settings in which sand ies are 42 MOLINA ET AL. oftencommon. 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