Enteric Opportunistic Parasites among HIV Infected Individuals: Associated Risk Factors and Immune Status

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1 Jpn. J. Infect. Dis., 60, 76-81, 2007 Original Article Enteric Opportunistic Parasites among HIV Infected Individuals: Associated Risk Factors and Immune Status Kaushal Kumar Dwivedi 1 *, Ganga Prasad 2, Sanjeev Saini 3, Surbhi Mahajan 1, Shiv Lal 1 and Usha Krishan Baveja 1,4 1 Centre for AIDS and Related Diseases, National Institute of Communicable Diseases, Delhi; 2 Department of Microbiology, Gurukul Kangari University, Haridwar; 3 Department of Microbiology, Maulana Azad Medical College; and 4 William J. Clinton Foundation, New Delhi, India (Received September 11, Accepted December 20, 2006) SUMMARY: Data on various etiologic agents causing diarrhea in human immunodeficiency virus type-1 (HIV- 1) infected individuals are sparse in Delhi, India. The present study was undertaken to identify various causative agents, the role of associated risk factors and immune status. A case-control study was conducted among 75 HIV- 1 infected individuals, 50 with and 25 without diarrheal infection. Fecal samples were screened for coccidian parasites, enteric protozoa, and helminthes by using various staining techniques. The CD4 + T-lymphocyte count was estimated. Enteric parasites were identified among 62.7% individuals, of which Cryptosporidium emerged as the single largest pathogen predominant among 33% of the individuals (P < 0.025). Other parasites diagnosed that were significantly associated with diarrhea were Giardia lamblia (13.3%), microsporidia (6.7%), and Isospora belli (2.7%). Chronic infected diarrheal cases were found to have polyparasitic infections. The mean CD4 + cell count was found to be lower among the diarrheal cases when compared with the non-diarrheal cases (mean, 141 cells/mm 3 versus 390 cells/mm 3 ). Similarly, among diarrheal individuals, the chronic diarrheal cases had a comparatively lower CD4 + cell count than the acute cases (mean, 123 cells/mm 3 versus 265 cells/mm 3 ). Risk factors found significant during multivariate analysis were: residence in a slum, exposure to pets and animals, use of public toilets, and practice of unsafe homosexual activity. Enteric coccidian parasites were identified as significant agents associated with diarrhea, especially among those with improper hygiene, multiple infections and a lower CD4 + cell count. Thus, this study emphasizes the need for routine screening of enteric parasites as well as education about practicing personal hygiene and taking timely and appropriate prophylactic measures. *Corresponding author: Mailing address: Centre for AIDS and Related Diseases, National Institute of Communicable Diseases, 22- Shamnath Marg, Delhi , India. Tel & Fax: , dwivedikaushal@yahoo.com INTRODUCTION Human immunodeficiency virus (HIV)/AIDS ranks among the most dreaded diseases afflicting mankind, causing dysfunction of both limbs of the immune system, resulting in overwhelming and fatal opportunistic infections. Gastrointestinal involvement, primarily in the form of diarrhea, is a universal problem affecting almost 90% of HIV infected patients in developing countries (1). Because of the increase of HIV in India (an estimated 5.2 million HIV infected persons with 111,000 AIDS cases among them) (2) and the high incidence of diarrhea among persons with HIV/AIDS, there is a strong need for regional and parameter-specific studies of these infections. In particular, Cryptosporidium parvum, Isospora belli, microsporidia, Cyclospora cayetanensis, Giardia lamblia, and bacterial and viral pathogens have been reported to be associated, as validated by various studies in this region (3,4). These pathogens are recognized as an important cause of gastroenteritis in the community as well as in immunocompromised individuals. Various risk factors like using contaminated drinking water, animal exposure, etc. have been studied in other countries, but data related to such studies is sparse in India, particularly in Delhi. Therefore, the present study was undertaken with the objective of accounting for the prevalence of enteric parasites in HIV infected individuals with and without diarrhea, and to measure associated risk factors and their correlation with the CD4 + T-lymphocyte count. MATERIALS AND METHODS This case-control study was conducted between January 2004 to February 2005 in the Centre for AIDS and Related Diseases, National Institute of Communicable Diseases, Delhi, after the approval of the institutional ethical committee. This center is one of the premier centers for HIV/AIDS in India and provides services to all walk-in patients. Therefore, the enrolled patients included those referred by various clinicians for CD4 + T-cell estimation and parasitic identification in fecal samples. Patients with confirmed HIV seroreactivity who had not received any antiprotozoal or antiretroviral treatment in the past 3 weeks were enrolled. Initially, 100 patients were enrolled, but 25 of them were later excluded from the study: 12 patients who did not submit fecal samples, six who had taken medications in the past 3 weeks, five who did not provide consent, one patient who was HIV seronegative and one patient in whom the CD4 + cell count could not be performed. The final study was conducted among 75 HIV seropositive patients, 50 who presented with diarrhea and were thus considered case-subjects, and 25 without diarrhea who were considered case-controls. In addition, the controls had to be non-diarrheals for at least 14 days prior to enrollment in the study. Background information, including the sociodemography, type of illness, clinical symptoms and risk factors (for the last 3 months) were 76

2 collected from each patient. In the study, diarrhea was defined as having three or more loose bowel movements within a 24-h period. These cases were further subdivided into acute and chronic groups. Acute cases were defined as those in whom diarrhea had lasted for 14 days, while those having diarrhea for more than 2 weeks were identified as chronic. The HIV-1 serostatus of each participant was confirmed by adopting standard guidelines formulated by the National AIDS Control Organization (NACO), Government of India (5). A single fecal sample was collected in a leak proof, labeled and sterile plastic container with 10% buffered formalin as a preservative. The samples were concentrated by the formalin-ether sedimentation technique and were examined as a wet saline mount and iodine preparation for the detection of ova, larvae and cysts (both direct and concentrated specimens). The samples were subjected to the modified Ziehl-Neelsen technique for intestinal coccidian infection (6). Positive or doubtful cryptosporidial samples were further confirmed by direct immunofluorescence assay (IFA) (Cellabs, Brookvale, Australia). A modified trichome stain was used for confirmation of microsporidia (7). No attempt was made to isolate bacteria and viruses. Three mililiters of whole blood was collected in a vacutainer containing K 3 EDTA (Becton Dickinson, Paramus, N.J., USA) for enumeration of the CD4 + T-lymphocytes by flow cytometer on an FACSCount system (Becton Dickinson). Statistical analysis was performed to test the significance of association between enteric parasites and risk factors by using the chi-square ( 2 ) test, bivariate tabulation and calculations of odd ratios (OR) with a 95% confidence interval (CI) limit. Factors found significant in univariate analysis were entered in a multivariate model. A P value of <0.05 was considered statistically significant. RESULTS Of the 75 enrolled cases, 57 (76%) were males and 18 (24%) females. The males were older than the females (median age, 34 versus 31.5 years; range, years). The majority of the study participants were heterosexual (45, 60%) followed by homosexuals (18, 24%) and transfusion recipients (12, 16%). Most of the participants resided in slums (27, 36%), followed by rural (25, 33.3%) and urban (23, 30.7%) localities. Many of the enrolled participants (10.7%) had taken prophylactic antibiotics in the past few months (although not within the 3 weeks prior to enrollment), especially for Pneumocystis carinii pneumonia (PCP), and 18 (24%) had experienced antiretroviral treatment but had discontinued it because of side effects or because of financial hardship. Twenty-seven (36%) and 16 (21.3%) individuals took up treatment for tuberculosis and oral ulcers, respectively. The overall prevalence of enteric parasites was 62.7% (47 participants). Among diarrheal cases, 72% (36 participants) were found positive for intestinal parasites, of whom Cryptosporidium alone was predominant in 23 (63.9%) along with other mixed infections. Furthermore, this infection was significantly higher in diarrheal cases when compared with non-diarrheal cases (P < 0.01). A significantly higher proportion of diarrheal cases were found to harbor enteric parasites when compared with nondiarrheal cases (72 versus 44%; P < 0.05). Among the infected diarrheal cases, enteric parasites were more frequent in chronically infected individuals as compared to those having acute infection (66.7 versus 33.3%). Overall, Cryptosporidium and Ascaris were the parasites identified more frequently. In addition, after comparing the parasites found in diarrheal cases with non-diarrheal cases on the basis of the CD4 + count, the former were found to be infected more with Cryptosporidium, Giardia, Ascaris, and Ancyclostoma and to have a statistically significant lower mean CD4 + count (P < 0.001, < 0.01, < and < 0.05, respectively) as compared to the latter. Similarly, when all protozoa and helminths were compared between the two groups, a significantly lower mean CD4 + count was observed in the diarrheals than the non-diarrheals (P < and < 0.005) (Table 1). We compared certain characteristics of the participants of both groups, out of which gender and age were found to be non-significant factors responsible for enteric parasitic infestations. A significant trend of infection was detected among diarrheal individuals residing in slums (86.4%), followed by Table 1. Prevalence of different enteric parasites among HIV/AIDS patients with comparison to CD4 + T-lymphocyte count Diarrheal (n = 50) Non-diarrheal (n = 25) Parasite 1) % Mean CD4 count % Mean CD4 count P value 3) (cells/mm 3 ) (cells/mm 3 ) Cryptosporidium parvum 2) <0.001 Giardia lamblia <0.01 Microsporidium Isospora belli Entamoeba coli NS Cyclospora cayetanensis 0 0 Ascaris lumbricoide <0.001 Ancyclostoma duodenale <0.05 Strongyloides stercoralis 0 0 All protozoa <0.001 All helminths <0.005 Total infected <0.05 1) : The number of parasites may exceed the number of patients, as more than one parasite was found in some patients. 2) : P-value < (chi-square test). 3) : Student t test to compare CD4 count between diarrheal and non-diarrheal cases. 77

3 Table 2. Prevalence of enteric parasites in HIV/AIDS patients with and without diarrhea on the basis of different characteristics Characteristic Diarrheal Non-diarrheal Odd ratio P value Odd ratio P value n = 50 n = 25 (95% CI) ( 2 test) (95% CI) ( 2 test) n (%) n (%) Male 29/40 (72.5) 1.1 ( ) <0.9 8/17 (47.0) 1.5 ( ) <0.95 Female 7/10 (70.0) 1 3/8 (37.5) 1 Age (yrs) /6 (50.0) 1 1/3 (33.3) /35 (74.2) 2.8 ( ) <0.1 10/20 (50.0) 2 ( ) < /9 (77.8) 3.5 ( ) 0/2 (0) Residence Slum 19/22 (86.4) 7.9 ( ) 4/5 (80.0) 1.4 ( ) Rural 13/19 (68.4) 2.7 (2-10.6) < /6 (50.0) 0.9 ( ) <0.75 Urban 4/9 (44.4) 1 for trend 4/14 (35.7) 1 for trend Diarrheal status Acute 12/23 (52.2) 1 0 (0) Chronic 24/27 (88.9) 1.7 ( ) < (0) No 0 (0.0) 11/25 (44.0) * Pets and animal contact Yes 27/31 (87.1) 7.5 ( ) <0.01 8/11 (72.7) 9.8 ( ) <0.05 No 9/19 (47.4) 1 3/14 (21.4) 1 Toilet facility In-house 2/9 (22.2) 1 < /8 (12.5) 1 <0.05 Open field 11/15 (73.3) 9.6 ( ) for trend 4/8 (50.0) 7 ( ) for trend Public 23/26 (88.5) 26.8 ( ) 6/9 (66.7) 3.5 ( ) Source of drinking water Hand pump 11/15 (73.3) 1.7 ( ) 3/9 (33.3) 2.5 ( ) Water tanker 17/22 (77.3) 2.1 ( ) <0.25 5/9 (55.6) 1.4 ( ) <0.75 Tap water 8/13 (61.5) 1 for trend 3/7 (42.9) 1 for trend Risk group Heterosexual 23/31 (74.2) 5.8 ( ) < /14 (42.9) 1.5 ( ) <0.75 Homosexual 11/13 (84.6) 11.0 ( ) 3/5 (60.0) 3.0 ( ) Transfusion recipient 2/6 (33.3) 1 2/6 (33.3) 1 *Odd ratio cannot be estimated. CI, confidence interval. those living in rural (68.4%) and urban areas (44.4%), (Pvalue for trend <0.025). Similarly, in the non-diarrheal cases, inhabitants of slums were also found to be harboring more parasitic infestation (80%) as compared to those from rural and urban settings. In the diarrheal cases, chronic diarrhea was observed in 27 (54%) and acute diarrhea in 23 (46%) cases, of whom chronic cases had more enteric parasites than acute ones (88.9 versus 52.2%). Contact with pets and animals emerged as a significant factor with higher odds, in both the diarrheal (P < 0.01) as well as the non-diarrheal (P < 0.05) groups harboring enteric parasites. As far as toilet facilities were concerned, public toilet users in diarrheal cases were at a higher risk of harboring enteric parasites compared to open field and in-house toilet users (P-value for trend <0.001). A similar trend was also noticed in non-diarrheal cases (P < 0.05). Regarding the source of drinking water, no significant difference was noticed in both groups, perhaps because water tanker users were found to be slightly more infected than hand pump or tap water consumers. When comparing the impact of risk groups among infected individuals, homosexual males were found to be at a significantly higher risk (P < 0.025) in diarrheal cases (Table 2). In addition, diarrheal cases also reported various symptoms like vomiting (6%), nausea (13%), flatulence (17%), fever (20%), anorexia (30%), weight loss (32%), and abdominal pain (33%). However, again in chronically infected cases, weight loss (67%) and anorexia (65%) were found to be statistically significant (P < and P < 0.05, respectively) as compared to acutely infected ones. Furthermore, 14 (51.8%) out of 27 chronically infected individuals and 6 (26.1%) out of 23 acutely infected individuals presented with clinical symptoms suggestive of AIDS (data not shown). Of the total infected (n = 47), multiple parasitic infestations were identified in 17 (36.2%) individuals, among whom 16 (94.1%) were from the diarrheal group and only one (5.9%) from the non-diarrheal group (P < 0.001). In addition, of multiple infected diarrheal individuals, 14 (87.5%) and 2 (12.5%) were diagnosed with double and triple parasitic infections, respectively, though in the non-diarrheal cases, only one case of double parasitic infection was detected. The immune status of the enrolled participants was assessed by measuring the CD4 + T-cell count. The mean CD4 + count of the study population was 224 ± 160 cells/mm 3. Among diarrheal cases it was 142 ± 97 cells/mm 3 (range, 2-399), whereas in non-diarrheal cases it was 390 ± 129 cells/mm 3 Table 3. Single and multiple parasitic infections in HIV/AIDS patients Diarrheal (n = 36) Non-diarrheal (n = 11) Infection % Mean CD4 count % Mean CD4 count (cells/mm 3 ) (cells/mm 3 ) Single Dual Triple

4 Table 4. Pattern of enteric parasitic infection in different immune categories on the basis of status of diarrhea CD4 count 0 (n = 14) 1 (n = 20) 2 (n = 14) 3 (n = 2) Total (n = 50) (cells/mm 3 ) Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Table 5. Multivariate logistic regression analysis showing association of different risk factors with and without diarrhea Risk factors Odd ratio 95% CI P-value ( 2 test) Residence in slum <0.001 Pets and animal contact <0.001 Public toilets <0.005 Homosexual risk activity <0.001 CI, confidence interval. (range, ). The mean CD4 + count was found to be significantly lower in the diarrheal cases with triple parasitic infection (33 cells/mm 3 ) as compared to those with double (105 cells/mm 3 ) and single infections (161 cells/mm 3 ). Both individuals with a triple infection were found to have CD4 + counts 50 cells/mm 3. Only one individual was found to be infected with a double parasitic infection in the non-diarrheal group with a CD4 + count of 354 cells/mm 3 as compared to 10 individuals infected with a single parasite (mean, 412 cells/ mm 3 ). However, diarrheal individuals with either single or multiple infections had a lower mean CD4 + count than the non-diarrheal cases (Table 3). The pattern of enteric parasitic infections at various immunity levels on the basis of diarrheal status showed that the chronically infected diarrheal individuals had a significantly lower mean CD4 + count than the acute infected individuals (123 cells/mm 3 versus 265 cells/mm 3 ). Furthermore, enteric parasites were more prevalent in the chronic group, of whom the maximum number of individuals (11, 30.6%) belonged to the immune category with a CD4 + count of 50 cells/mm 3, followed by those in an immune category with a CD4 + count of cells/mm 3 (7, 19.4%) (Table 4). Similarly, in cases of acute infection, 8 (66.7%) individuals were in the range of cells/mm 3 followed by 2 (16.7%) in cells/mm 3. Risk factors found significant in univariate analysis, namely residence, exposure to pets and animals, and using public toilet were entered into the multivariate model, where residence in a slum (P < 0.001), exposure to pets and animals (P < 0.001), using public toilet (P < 0.005) and practicing homosexual risk activity (P < 0.001) emerged as significant factors related to enteric parasitic infection (Table 5). DISCUSSION Due to deterioration of the immune system because of HIV infection, numerous opportunistic infections occur, of which gastrointestinal parasitic infection is a universally recognized problem. This largely presents with diarrheal symptoms leading to life threatening complications (8). In the present study, the overall prevalence of intestinal parasites was found to be considerably high (62.7%); of which diarrheal and non-diarrheal cases accounted for 76.6 and 23.4%, respectively. Studies carried out by other workers have documented a relatively lower prevalence ( %) in diarrheal cases compared to that found in our study. However, in non-diarrheal cases, workers have reported the prevalence of intestinal parasites ranging from % compared to 23.7% found in our study (1,9,10). A high prevalence of Cryptosporidium was noticed in this study (33.3%), as compared to other studies that reported a prevalence ranging between % in India (1,3,4,11) and % from other countries (9,10,12). However, our results are in accordance with the study carried out in Tanzania, where an up to 36% prevalence was reported (9). To the best of our knowledge, such a high prevalence of Cryptosporidium has been reported for the first time in Delhi, India. This high prevalence may be due to overcrowding and a low level of sanitation, as the majority of infected individuals were residing in slums and practicing personal unhygiene. In addition to this, use of a direct IFA may have also led to an improved diagnosis, otherwise, samples with low number of oocysts may get overlooked by conventional acid fast staining (13). In addition, in this study we also noticed a low prevalence of I. belli (2.6%) with no cases of Cyclospora, though prevalence has been mentioned in some reports from different regions of this country (1,3,4). These observations may be justified by the explanation that most of these patients had taken trimethoprim-sulphamethoxazole as prophylactic measures for PCP, which is quite effective against I. belli and Cyclospora. This inexpensive drug may therefore have additional beneficial effects as far as parasitic infections are concerned. In addition, our findings for microsporidia compared well with the studies carried out elsewhere (12). We have found that the extent of deterioration of immunity, as measured by the CD4 + count, could predict the status of diarrhea, i.e., acute or chronic and the presence of coccidian parasites with or without multiple infections. It has been observed that patients with Cryptosporidium, microsporidia or multiple infections who presented with chronic diarrhea had a lower CD4 + count. These findings are consistent with other studies (12). It is relevant to mention here that CD4 + T- lymphocytes are necessary for the resolution of both acute and chronic cryptosporidiosis. The results of experimental infection studies with Cryptosporidium in mice and calves have shown that immunity is dependent on the number of CD4 + T cells. CD4 + cells help in increasing the intraepithelial lymphocyte population and generating gamma interferon. Interleukin-12 may play a role, possibly through its ability to induce gamma interferon production. Antigen-driven interleukin-12 production in macrophages requires interaction between CD40 on antigen presenting cells and CD40 ligands on CD4 + T-lymphocytes (14). Furthermore, the presence of Cryptosporidium in two individuals from the nondiarrheal group (though they may be acting as carriers shedding oocysts in feces without any clinical manifestation) may be explained by the fact that they were not co-infected with any other parasites and had a higher CD4 + count. In the present study, three helminths (Ascaris, Ancyclostoma, and Strongyloides) were taken into consideration. A very high 79

5 prevalence of Ascaris (16%) and Ancyclostoma (6.7%) was observed with no case of Strongyloides seen as compared to other studies reported from this country. Surprisingly, Strongyloides is very low in the general population (0.4%) as well as in HIV infected individuals ( %) in India except in Manipur, one of the northeastern states in which a high prevalence of this helminth (27.3%) has been reported (1,4,11,15-17). It is clear that there is no specific prevalence pattern of occurrence of parasites among HIV/AIDS patients. Distribution of parasites may depend on geographical/ecological variation and behavioral activities (18). Thus, this difference can be seen between country-to-country and even within different regions of the same country. A low CD4 + count was observed in diarrheal cases with helminth infestation as compared to non-diarrheal cases. This may be due to the fact that diarrheal cases with helminths presented with other enteric parasitic infections, which may lead to severe suppression of the immune system. Therefore, screening of helminths must be included in routine parasitic examination, as studies showed that helminthic infection in HIV infected individuals may accelerate HIV disease progression. It has also been shown that the treatment and successful elimination of intestinal worms was associated with decreased HIV plasma viral load (15,18,19). Interestingly, multiple infections were reported in 32% of diarrheal individuals as compared to other studies carried out in different region of India, which showed a comparatively lower prevalence (4,11). However, we are not able to explain this finding. As far as risk factors are concerned, we have taken eight variables into consideration, out of which four variables, residence, exposure to pets and animals, type of toilet used, and the practice of risky behaviors were found significant for acquiring parasitic infections. Poor sanitation emerged as a crucial factor in our study, as highlighted from the variables found significant in the multivariate model. The odds of acquiring various diarrheal parasites were found to be higher for slum residents compared to urban and rural inhabitants (OR, 10.8; 95% CI, ). However, in a study carried out in Africa, it was found that persons who had spent time in rural areas were at risk of having parasites in their stool (18). A higher prevalence of intestinal parasitic infection was not found in Delhi villages, which contradicts the finding reported in the African study. This may be due to the highly developed civil structure in Delhi villages. Exposure to pets and animals also accounted for a significant parasitic burden, as was obeserved in our study (OR, 8.8; 95% CI, ). After asking participants about their contact with animals, the majority of them (56%) reported a positive history of contact with pets and animals, mainly dogs, cows, buffalloes, pigs, sheep, goats, and other domestic animals within at last 3-4 months. They had generally kept these animals as sources of milk and meat within their house premises, and also had frequent contact with them for cleaning, milking, etc. Glaser et al. (20) in their study have shown that HIV positive patients who own dogs were at increased risk of Cryptosporidium infection (OR, 2.19; 95% CI, ; P = 0.05). Moreover, according to one other study, contact with farm animals, including cleaning up after them, milking them, etc., has also been shown to be associated with a risk of diarrhea (OR, 2.4; 95% CI, ) (21). After getting information regarding the type of toilet facility used, the majority of the participants using public toilets were found to be infected with various diarrheagenic parasites (OR, 22.6; 95% CI, ). Although, the mode of transmission cannot be identified, some possible modes may be through touching door handles or taps, or using the cups which are kept in public toilets for cleaning, as water is often used for cleaning in India rather than toilet paper. Being a homosexual was also found to be a significant factor for harboring enteric parasites (OR, 7.0; 95% CI, ). Eisenberg et al. have found that the odds of cryptosporidiosis were increased among those who had insertive anal sex (21). Hallard et al. have also supported the same finding. Moreover, they also found that men who had had more than one sexual partner in the past month were nearly 7 times more likely to have had Cryptosporidium diarrhea (OR, 6.67; 95% CI, ; P = 0.034) (22). There were some limitations to this study. First, the study was performed using a small sample size. Second, due to resource constraints we were not be able to isolate bacteria or viruses, or to perform sensitive tests like PCR for confirmation of parasites such as microsporidia. In addition, the participants were unwilling to participate in a follow up, and thus no attempt was made to obtain follow up information regarding diarrheal episodes, effectiveness of prophylactic regimens, etc. In conclusion, the present study highlights the magnitude of enteric parasites in HIV infected individuals with and without diarrhea. Cryptosporidium was found to be an important parasite in HIV positive patients with diarrhea (P < 0.01). Polyparasitic infection was further observed in diarrheal patients (chronic), particularly in those with a lower CD4 + count. Therefore, when assessing a patient s immune status and enteric pathogenic profile, it is recommended that a routine screening for fecal specimens is performed and that the CD4 + count is obtained. Pathogenic burden accelerates HIV disease progression and contributes towards early morbidity. Coccidian and other intestinal parasites were largely diagnosed in those practicing poor sanitation and improper personal hygiene practices. This emphasizes the need to educate HIV infected individuals about practicing safer sexual practices, better sanitation and personal hygiene, and in using boiled or properly filtered water and avoiding exposure to domestic pets. Furthermore, routine anti-enteric parasitic prophylactic measures should be provided in clinical settings on a regular basis to persons with a significantly lower CD4 + count. Finally, the findings in this study are limited due to the small sample size. A large, prospective cohort study is needed to obtain conclusive results so as to formulate further appropriate intervention strategies. ACKNOWLEDGMENTS We would like to thank Mr. Pritam Singh (Opportunistic Infection Unit, Centre for AIDS & Related Diseases, NICD) for his continuous support for various tests. REFERENCES 1. Mukhopadhya, A., Ramakrishna, B.S., Kang, G., et al. (1999): Enteric pathogens in southern Indian HIV-infected patients with and without diarrhoea. Indian J. Med. Res., 109, National AIDS Control Organization: Facts and Figures. Online at < Accessed 16 May Mohandas, K., Sehgal, R., Sud, A., et al. (2002): Prevalence of intestinal parasitic pathogens in HIV-seropositive individuals in Northern India. Jpn. J. Infect. Dis., 55, Kumar, S.S., Ananthan, S. and Saravanan, P. (2002): Role of coccidian parasites in causation of diarrhoea in HIV infected patients in Chennal. Indian J. Med. 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