Gastrointestinal infections are very common inpatients with HIV infection or AIDS1. Diarrhoea isa common clinical presentation of these infections.Reportsindicatethatdiarrhoeaoccursin30-60percentofAIDSpatientsindevelopedcountriesandinabout90percentofAIDSpatientsindevelopingcountries2.Theaetiologicspectrumofentericpathogenscausingdiarrhoea includes bacteria, parasites, fungi andviruses3. The presence of opportunistic parasites

OpportunisticparasiticinfectionsinHIV/AIDSpatientspresentingwithdiarrhoeabythelevelofimmunesuppression

S.V.Kulkarni,R.Kairon,S.S.Sane,P.S.Padmawar,V.A.Kale,M.R.ThakarS.M.Mehendale&A.R.Risbud

National AIDS Research Institute, (ICMR), Pune, India

ReceivedNovember28,2007

Background & objectives: Enteric parasites are major cause of diarrhoea in HIV infected individuals. The present study was undertaken to detect enteric parasites in HIV infected patients with diarrhoea at different levels of immunity.Methods: The study was carried out at National AIDS Research Institute, Pune, India, between March 2002 and March 2007 among consecutively enrolled 137 HIV infected patients presenting with diarrhoea. Stool samples were collected and examined for enteric parasites by microscopy and special staining methods. CD4 cell counts were estimated using the FACS count system. Results: Intestinal parasitic pathogens were detected in 35 per cent patients, and the major pathogens included Cryptosporidium parvum (12%) the most common followed by Isospora belli (8%), Entamoeba histolytica/Enatmoeba dispar (7%), Microsporidia (1%) and Cyclospora (0.7%). In HIV infected patients with CD4 count < 200 cells/l, C. parvum was the most commonly observed (54%) pathogen. Proportion of opportunistic pathogens in patients with CD4 count 200-499 and 500 cells/l (P=0.001, P=0.016) respectively.Interpretation & conclusions: Parasitic infections were detected in 35 per cent HIV infected patients and low CD4 count was significantly associated with opportunistic infection. Detection of aetiologic pathogens might help clinicians decide appropriate management strategies.

Key words Diarrhoea-HIV-opportunisticparasites

Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli and Microsporidia are documented inpatientswithAIDS4.NonopportunisticparasitessuchasEntamoebahistolytica,Giardia lamblia, Trichuris trichiura, Ascaris lumbricoides, Strongyloides stercoralis and Ancylostoma duodenalearefrequentlyencountered in developing countries but are notcurrently consideredopportunistic inAIDSpatients5. In immunocompromised patients, the intestinal

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opportunistic parasites probably play amajor role incausing chronic diarrhoea accompanied by weightloss6.The incidenceandprevalenceof infectionwitha particular enteric parasite inHIV/AIDS patients islikelytodependupontheendemicityofthatparticularparasiteinthecommunity7.C. parvum, I. belliand E. histolyticahavebeenreportedas themost frequentlyidentified organisms in HIV infected individuals with diarrhoeafromIndiaandotherpartsoftheworld8-15.

The present study was undertaken to study theprevalenceofentericparasitescausingdiarrhoeaandtheirassociationwith immunestatus inHIVinfectedpatientsinPune,India.

Material & Methods

ThestudywascarriedoutinsevenclinicssetupbytheNationalAIDSResearch Institute (NARI), India,in thecityofPune,Maharashtra.Twoof thesesevenclinicsarelocatedattwotertiarycarehospitals,twoinmunicipalcorporationdispensaries,twoareHIVreferralclinicsonNARIcampusandoneissetupin theredlightareaofPune.STDandHIVdiagnosticservices,careandsupportservicestoHIVinfectedpatientsareprovidedattheseclinics.DuringMarch2002toMarch2007consecutiveHIVinfectedpatientspresentingwithdiarrhoea at these seven clinics were considered forinclusioninthestudy.Studypatientswereinterviewedusing the structured questionnaire and informationwasobtainedondemographiccharacteristics,presentandpasthistoryofdiarrhoeaandantibiotictreatment.Diarrhoea was defined as two or more liquid or three or moresoftstoolsperday.Patientsalreadyonantibiotictreatmentwereexcludedfromthestudy.Atotalof137patientswereenrolledinthestudy.

Blood samples (plain&EDTA) 5ml eachwereobtainedfromenrolledpatients.SerumsampleswereusedforHIVtesting.HIVserostatusofthepatientswasdetermined by using commercially available ELISAantibody tests (Genetic system, Biorad Labs, USAandTridot,JMitra&Co.,NewDelhi)usingNationalAIDS Control Organisation (NACO) recommendedalgorithm16.

CD4cellcountsweremeasuredbyusingaFACScount system (Becton Dickinson, Singapore BD).Patients were categorized by their immune statusaccording to the 1993 revised classification system fortheHIVinfectionbyCD4T-cellcategories17.

Stool examination: Stool specimens were collectedaccordingtotheWHOstandardprocedureandexamined

microscopically following direct and formalin-etherconcentrationmethods18.Stoolsampleswerecollectedat home in labelled, leak proof, clean sterile plasticcontainers and then submitted at NARI clinics andwere transported to the laboratorywithin threehoursof collection. The stool samples were fixed in 10 per centformalinsaline,concentratedusingformyl/ethylacetate and examined through direct observation insaline(0.85%NaClsolution).Lugolsiodinewasusedforthedetectionofova,larvae,trophozoitesandcystsofintestinalparasites.E. histolyticaandE. disparwasstudiedbylightmicroscopy(CarlZeissInc,USA)bytrainedobservers. Smearsofdirectandconcentratedspecimens were examined by modified acid fast staining for C. parvum, I. belli and Cyclospora18,19.Modified trichrome stain (Hi-media laboratories, India,QualigensFineChemicals,India)wasusedfordetectingMicrosporidia20.Thestudywasapprovedbyinstitutionalethicscommittee.

Statistical analysis: Data were analysed using SPSSsoftwareversion14.0(SPSSInc,USA).TheproportionsofopportunisticpathogenswerecomparedbetweentheCD4groupsbyusingZtest.

Results & Discussion

Ofthe137patients,100(73%)weremalesand37(27%)females.Themeanageofthemaleandfemalepatientswas34.67.51and33.29.95yrrespectively.The study population consisted of 19 patients withCD4count>500cells/l,53patientswithCD4count200-499cells/land65patientswithCD4count500 cells/l, of which H. nana wasdetectedin2,andE. histolytica/E. dispar, A. duodenaleandA. lumbricoidesweredetected in1patient each.Interestingly, I. belliwasdetectedinonepatient.TheproportionofopportunisticpathogensinpatientswithCD4count200cells/l(P=0.001andP=0.016). Numerous opportunistic infections occur inHIV infected patients, due to downregulation of theimmune system. Gastrointestinal parasitic infectionisauniversallyrecognizedprobleminthesepatients.Theseinfectionslargelypresentwithdiarrhoealeadingto life threatening complications20. In the presentstudy,entericparasitesweredetectedin35percentofHIVinfectedpatientswithdiarrhoea.Variousstudiesfrom India and other countries have reported a highprevalence of intestinal parasite, ranging from 30 to60 per cent10,14,21-23.Almost half of the patients withCD4count less than200cells/lwerefoundtohavegastrointestinal parasitic infections and amajority ofwhich were opportunistic parasites (37%). Amongtheopportunisticparasites,C. parvum (54%)wasthepredominant pathogen. Several studies from Indiaand other parts of the world also have reported thesame12,24-26.The prevalence of opportunistic parasitesinpatientswithCD4count200-499cells/lwasfoundtobe9percent.

Thus, like many other studies, we also foundthat infectionswithopportunisticpathogenswere theleadingcauseofdiarrhoeainHIVinfectedindividuals,especially, in subjects with advanced disease. C. parvum and I.belli,werethemostcommonpathogens.AmongthenonopportunisticpathogensE. histolytica/E. dispar seemed to contribute significantly has shown earlier12,20,22,24,25.Similartootherreports,Microspordia and Cyclosporawere detected in a few patientsonly10,27-29.

The reported prevalence of non opportunisticparasites varied from 5-30 per cent in HIV infectedpatients5,9. In the present study, non opportunisticparasitesweredetectedin13percentpatientsacrossdifferentCD4groups, thus,highlighting theneedfor

earlydetectionandtreatmentofsuchinfectionsamongHIV-infectedpatientstoreducethemorbidity.

Therewassomelimitationsinourstudy.ThestudywasdoneonasmallsamplesizeasamajorityofthepatientswhocametoNARIclinicswerereferredfromthegeneralpractionersorfromprimaryorsecondarycarecenters.PatientswerealsoreferredfromVoluntaryCounseling & HIV Testing centers. Majority of thepatients seen at these centers had already receivedantibioticspriortotheirvisitandthereforethenumberofsymptomaticpatientswasless.

In conclusion, intestinal parasitic infectioncauseddiarrhoeain35percentofthestudysubjects.Most of the infections in patients with CD4 count

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Reprint requests:DrSangeetaV.Kulkarni,DepartmentofMicrobiology,NationalAIDSResearchInstitute(ICMR) Pune411026,India e-mail:svk51@hotmail.com,svk952000@yahoo.co.in

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