LJM 1993-2820 1819-6357 Libyan Journal of Medicine, Vol. 5, No. 0, October 2010, pp. 1–6 sici xxx PUBMED Abbreviation Frederick Olusegun Akinbo

*Frederick O. Akinbo, Pathology Department University of Benin Teaching Hospital, Department of Animal and Environmental Biology, University of Benin PMB 1111 Benin City Nigeria +2348033796874

Christopher E. Okaka Richard Omoregie Department of Pathology University of Benin Teaching Hospital

Benin City Nigeria

Department of Animal and Environmental Biology University of Benin

Benin City Nigeria

School of Medical Laboratory Sciences University of Benin Teaching Hospital

Benin City Nigeria

Libyan Journal of Medicine ORIGINAL ARTICLE Intestinal parasitic infections among HIV-positive patients This study was carried out to determine the presence of intestinal parasites and their correlation with CD4⁺ T-cell counts and demographics among human immunodeficiency virus (HIV)-positive patients in Benin City, Nigeria. Stool specimens from 2,000 HIV-positive patients and 500 controls (HIV-negative individuals) were examined for ova, cysts, or parasites, using standard procedures. In addition, patient's blood samples were analyzed for CD4 counts by flow cytometry. An overall prevalence rate of 15.3% was observed among HIV-positive patients while 6.2% was noted among non-HIV subjects. HIV status was a significant (P<0.0001) risk factor for acquiring intestinal parasitic infections. Male gender, CD4 count <200cell/µl, and diarrhea were significantly associated with an increased prevalence of intestinal parasitic infections among HIV-positive patients. The level of education, occupation, and source of water among HIV patients significantly (P<0.0001) affected the prevalence of intestinal parasitic infections. Ascaris lumbricoides was the most predominant parasite in both HIV-positive patients and controls. A CD4 count <200 cells/µl was significantly associated with only Isospora belli and Cryptosporidium infections. The presence of pathogenic intestinal parasites such as A. lumbricoides, hookworm, Giardia intestinalis, Entamoeba histolytica, Trichuris trichiura, and Taenia species among HIV-infected persons should not be neglected. Cryptosporidium species and I. belli were the opportunistic parasites observed in this study. Routine screening for intestinal parasites in HIV-positive patients is advocated. intestinal parasites HIV CD4 count Demographics Benin City Parasitic infections are among the most widespread of all chronic human infections worldwide. The rate of infection is remarkably high in sub-Saharan Africa, where the majority of human immunodeficiency virus (HIV) and AIDS cases are concentrated 1 2. Individuals with HIV/AIDS are threatened by a great number of diseases including those caused by different kinds of biological agents. The progressive decline and ultimate collapse of immune system functions, which are characteristic for AIDS, usually result in morbidity and ultimately death due to opportunistic bacterial, viral, and parasitic infections 3. Helminths and HIV infection have major effects on the host immune response and co-infection is widespread 4. With the progressive development of AIDS, especially once CD4⁺ T-lymphocyte counts have fallen below 200 cells/µl, patients often become co-infected by bacteria, parasites, or viruses 5. Such co-infections, generally, are the proximate cause of death of AIDS patients 6 7 8 . Several parasites have been implicated as major contributors to morbidity in HIV-infected persons living in developing countries, and the parasites frequently encountered include: Cryptosporidium spp., Isospora belli, Microsporidia spp., Entamoeba histolytica, Giardia intestinalis, Trichuris trichiura, Ascaris lumbricoides, Strongyloides stercoralis, and hookworm, which are associated with diarrhea and iron deficiency anemia 9. Parasitic infections in HIV-infected patients are common in many regions and populations across Nigeria and represent a lasting public health challenge. It has long been recognized that interactions between HIV and other infective agents, including parasites, influence the health status of people living with HIV/AIDS. There are a number of studies on parasites among HIV/AIDS patients in Nigeria, but with conflicting prevalence. Some authors reported a low prevalence of 28.4% in Abeokuta, Nigeria 10, while others observed a high prevalence of 79.3% in Osun state, Nigeria 11. There is a paucity of information on the prevalence of parasites among HIV-positive patients in Benin City. Against this background, this study focuses on these intestinal parasitic infections in relation to clinical manifestations, demographics, and immune status. The study was carried out between August 2007 and August 2009 at the University of Benin Teaching Hospital, Benin City – a teaching hospital with a referral status and center for HIV/AIDS management under the President's Emergency Plan for AIDS Relief (PEPFAR). A total of 2,500 subjects consisting of 2,000 (668 males and 1,332 females) HIV-positive patients attending HIV clinics and 500 (209 males and 291 females) apparently healthy HIV-negative individuals, who were contacted through HIV outreach programs in their homes and offices and served as controls, were included in this study. The age of the study subjects ranged from 21 to 70 years (mean±standard deviation=37.06±9.19 years). Verbal informed consent was obtained from all subjects prior to specimen collection. The study was approved by the Ethics Committee of the University of Benin Teaching Hospital, Benin City, Nigeria. A pre-designed structural questionnaire was utilized to collect the socio-demographic characteristics of the subjects. Blood and stool specimens were collected from each subject. The blood specimens were placed in ethylene diamine tetraacetic acid containers and the stool specimens were collected in clean wide-mouthed containers. Stool specimens were examined microscopically for ova, cysts, or parasites, using saline and iodine mounts on grease-free slides. Following this, each fresh stool sample was preserved in 10% formol saline. The fecal sample preserved in 10% formol saline was concentrated and used to detect oocysts of Cryptosporidium species, I. belli, and Cyclospora cayetanensis using the modified Ziehl-Neelsen staining technique described by Akinbo et al. 12. Giemsa solution stain was used in detecting the spores of Microsporidium species using an earlier described method 13. Blood samples were analyzed for CD4⁺ T lymphocyte cell estimation using flow cytometry (Partec, GmbH, Germany). Briefly, 20 µl of CD4 PE antibody was placed into a Partec test tube and 20 µl of well-mixed whole EDTA blood was added, mixed gently and incubated in the dark for 15 min at room temperature. The mixture was agitated during incubation every 5 mins. Eight hundred microliters of CD4 buffer was added to the mixture of antibody and sample and mixed gently. This was then plugged to the counter for counting. The data were analyzed using the Chi square (&khgr;²) test and odds ratio (OR) analysis using the statistical software INSTAT^®. The prevalence of intestinal parasitic infection among HIV-infected patients and non-HIV subjects was 15.3 and 6.2%, respectively. HIV status was a significant (P<0.0001) risk factor for acquiring an intestinal parasitic infection (HIV-positive vs HIV-negative: 15.3 vs 6.2% OR=2.722, 95% CI=1.855, 3.995). In a similar vein, the prevalence of a parasitic infection among HIV-infected patients in relation to gender was 18.3 and 13.7% (male and female, respectively) (P=0.0096), while 4.8 and 7.2% was observed among non-HIV subjects (male and female, respectively) (P=0.3476). The prevalence of an intestinal parasitic infection among HIV-infected patients with a CD4 count <200 cell/µl and ≥200 cell/µl was 24.7 and 13.9%, respectively (P<0.0001). Diarrhea was significantly associated with an increased prevalence of intestinal parasitic infections among HIV-positive patients (Table 1). Characteristics No. tested No. with infection OR 95% CI P-value HIV status  HIV patients 2000 305 (15.3) 2.722 1.855, 3.995  Non-HIV subjects 500 31 (6.2) 0.367 0.250, 0.539 <0.0001 Gender (HIV patients)  Male 668 122 (18.3) 1.403 1.092, 1.803  Female 1332 183 (13.7) 0.713 0.555, 0.916 0.0096 Non-HIV  Male 209 10 (4.8) 0.646 0.298, 1.403  Female 291 21 (7.2) 1.548 0.713, 3.360 0.3476 CD4 count (cells/µl) of HIV patients  <200 255 63 (24.71) 2.038 1.487, 2.794  ≥200 1745 242 (13.87) 0.491 0.358, 0.673 <0.0001 Clinical manifestation of HIV patients  Diarrhea 374 82 (21.93) 1.767 1.332, 2.344 <0.0001 The effect of demographics on the prevalence of intestinal parasitic infections among HIV-positive patients is shown in Table 2. The level of education among HIV-positive patients significantly (P<0.0001) affected the prevalence of intestinal parasitic infections, and patients with no formal education had the highest prevalence of the infection. Artisans, farmers, and security officers were significantly affected (41, 40, and 37.5%, respectively) while traders (2.9%) were (P<0.0001) least affected with intestinal parasitic infections among HIV-positive patients. HIV-infected patients whose source of water was from a stream/river (54.6%) and the municipal water supply (50%) were significantly affected with these infections (P<0.0001). Defecating in nearby bushes resulted in a significantly increased prevalence of intestinal parasitic infections among HIV-positive patients. The rate of infection among those who had contact with animals ranged from 15.4% (dog) to 25% (cats and goats) while for those who had no contact with animals, the rate was 14.7%. Demographics No. tested No. infected (%) P-value Level of education  No formal 57 27 (47.37)  Primary 287 42 (14.63)  Secondary 1281 174 (13.58)  Tertiary 334 63 (18.86) <0.0001 Occupation  Civil servant 221 41 (18.6)  Business man/woman 169 30 (17.8)  Security officer 16 6 (37.5)  Artisan 427 175 (41.0)  Traders 1055 31 (2.9)  Farmers 15 6 (40.0)  Housewife 62 12 (19.4)  Student 35 4 (11.4) <0.0001 Source of water  Municipal water 34 17 (50.0)  Borehole 1811 252 (13.9)  Well/rain 133 34 (25.6)  Stream/river 22 12 (54.6) <0.0001 Type of toilet  Water cistern 1077 121 (11.24)  Pit latrine 917 181 (19.24)  Bush 6 3 (50.00) <0.0001 Animal contact  Cat 16 4 (25.0)  Dog 26 4 (15.39)  Birds 125 22 (17.6)  Goat 44 11 (25.0)  Cattle 2 2 (100.0)  None 1787 262 (14.66) 0.0055 A. lumbricoides was the most predominant parasite, followed by Cryptosporidium species and Gardia intestinalis was the least prevalent. G. intestinalis was recovered only from females (Table 3). Among HIV-negative subjects, A. lumbricoides and hookworm were the only intestinal parasites detected and the ratio was 3:1. Males had more A. lumbricoides while females had more hookworm infections (Table 3). HIV-infected HIV-negative Parasite Male (%) Female (%) Total (%) Male (%) Female (%) Total (%) Entamoeba histolytica 5 (3.5) 7 (3.3) 12 (3.3) – – Giardia intestinalis 0 (0.0) 2 (0.9) 2 (0.6) – – Isospora belli 8 (5.5) 20 (9.3) 28 (7.8) – – Cryptosporidium spp. 31 (21.4) 49 (22.8) 80 (22.2) – – Ascaris lumbricoides 43 (29.7) 76 (35.4) 119 (33.1) 9 (90.0) 12 (66.7) 21 (75.0) Hookworm 33 (22.8) 41 (19.07) 74 (20.6) 1 (10.0) 6 (33.3) 7 (25.0) Strongyloides stercoralis 10 (6.9) 13 (6.05) 23 (6.4) – – Trichuris trichiura 12 (8.3) 6 (2.8) 18 (5.0) – – Taenia sp. 3 (2.07) 1 (0.5) 4 (1.1) – – Total 145 (40.28) 215 (59.72) 360 A CD4 count <200 cells/µl was significantly associated with only I. belli and Cryptosporidium infections (P<0.0001) (Table 4). CD4 count (cells/µl) Parasitic agents <200 ≥200 OR 95% CI P-value Entamoeba histolytica 0 12 0.283 0.017, 4.794 >0.05 Giardia intestinalis 0 2 1.422 0.068, 29.727 >0.05 Isospora belli 11 16 5.085 2.331, 11.090 <0.0001 Cryptosporiduim spp. 59 21 26.037 15.472, 43.817 <0.0001 Ascaris lumbricoides 16 103 1.115 0.647, 1.922 >0.05 Hookworm 9 65 0.987 0.485, 2.008 >0.05 Strongyloides stercoralis 3 20 1.070 0.316, 3.630 >0.05 Trichuris trichiura 4 15 1.916 0.631, 5.823 >0.05 Taenia sp. 1 3 2.382 0.247, 23.006 >0.05 Total 103 257 Parasitic infections remain an important cause of morbidity and mortality in developing countries especially among HIV-infected persons 14. In the present study, HIV infection was a significant risk factor for acquiring an intestinal parasitic infection. This is in agreement with previous findings 15 16 17 . It is important to note that the prevalence (15.3%) observed among HIV-infected patients in this study is within the range of 11.4 and 18.4% reported by some authors in Iran and Ethiopia 18 19. However, other investigators reported 27.9% in Apulia (Italy), 28.1 and 42.9% in (Abuja and Abeokuta, respectively) Nigeria, 50% in Thailand, 63.9% in Rio de Janeiro (Brazil), 69.2% in Ethiopia, and 84.3% in South Africa 20 21 22 23 24 25 . The difference could be due to sample size as our study presented a larger sample size than that reported by other authors. Gender significantly affected the prevalence of intestinal parasitic infections among HIV-infected patients while it was not a risk factor for acquiring these infections among non-HIV subjects. This finding is inconsistent with Mohammad et al. 18. The reason for this association between gender and intestinal parasites may be adduced to more males being exposed than females based on occupational grounds. The CD4 count of HIV-infected patients with intestinal parasitic infections was significantly lower than those of HIV-seronegative patients (383.57±136.15, 1029.84±117.42, respectively). Cellular immunity is the major defense against intestinal parasitic infections 13. Therefore, the reduction in CD4 count by the HIV virus predisposes HIV-infected patients to opportunistic intestinal parasitic infections 22 26. It is generally accepted that a CD4 count <200 cells/µl predisposes HIV-infected persons to opportunistic infections 27. In this study, a CD4 count <200 cells/µl resulted in a significantly higher prevalence of intestinal parasitic infections (P<0.0001). In HIV infection, diarrhea is a major sign of progression to AIDS, which results from opportunistic infections, and this may explain the findings in this study. We found diarrhea significantly associated with intestinal parasitic infections among HIV-positive patients. Others reported similar findings 28 29 30 31 32 33 . The occupation of the HIV-infected persons significantly affected the prevalence of intestinal parasitic infections, with artisans having the highest prevalence (47.98%). Artisans are more likely to eat food and drink water from questionable sources as they carry out their work. They are also likely to have a poor educational background and to a large extent, poor hygiene standards. This may be the reason for the high prevalence in this group. The source of water significantly affected the prevalence of intestinal parasitic infections with HIV-infected patients having streams/rivers and the municipal water supply as their sources of domestic water (P<0.0001). Streams/rivers are not very hygienic sources of water for domestic use as a number of activities, such as bathing, defecating, and washing, are known to occur in these bodies of water. Similarly, the municipal water may be contaminated as it is usually an untreated source of water. Thus, these sources of water are likely sources of intestinal parasitic infections. This may explain, in part, the findings of the present study. However, contrary to our findings, Ikeh et al. 32 reported that the source of water did not affect the prevalence of intestinal parasitic infections among HIV-positive patients. Type of toilet significantly only significantly affected the prevalence of intestinal parasitic infections with patient's defecating in bushes having the highest prevalence of 50%. Contact with animals was not a significant risk factor for acquiring intestinal parasitic infections in this study (P=0.0055). This is discordant with previous reports 34 35 36 . Among HIV-infected individuals, Cryptosporidium species has been reported as the major zoonotic parasites 35 36. A total of 360 intestinal parasites were detected in HIV-infected individuals, with A. lumbricoides being the most prevalent (33.1%). Other workers 16 22 25 also reported A. lumbricoides as the most prevalent intestinal parasite in HIV-infected patients. The presence of pathogenic intestinal parasites such as A. lumbricoides, hookworm, G. intestinalis, E. histolytica, T. trichiura, and Taenia species among HIV-infected persons should not be neglected. Cryptosporidium, Microsporidium, Cyclospora, and I. belli are opportunistic infections that have been reported among HIV-infected persons 19. In the present investigation, Cryptosporidium species and I. belli were the opportunistic infections observed. We found Cryptosporidium species only among HIV-infected persons (22.2%). Similar findings have been reported by a number of authors 13 16 24 25 30 31. The prevalence of I. belli among HIV-infected patients in this study was 7.8%. However, some authors reported similar prevalence 24 31 32 33 34 35 36 . The prevalence of hookworm (20.6%) observed in this study was higher than that previously reported 10 25 30. Zelalem et al. 24 observed a higher prevalence of S. stercoralis than was observed in our study. In a similar vein, the prevalence of T. trichiura was lower than that observed by other authors 16 24. Furthermore, a higher prevalence of E. histolytica than that observed in this study (3.3%) has been reported 10 24 30. The prevalence of Taenia species (1.1%) and Giardia intestinalis (0.6%) observed in this study was lower than that previously reported 39 37 38 39 40 41 . Out of 500 stool samples examined from HIV-negative patients, A. lumbricoides (4.2%) was the commonest parasite followed by hookworm (1.4%). There were no opportunistic coccidian parasites encountered from the specimens of the non-HIV subjects. Generally, females had a higher prevalence (59.72%) of Cryptosporidium than their male counterparts (40.28%) among HIV-infected persons, though this was not statistically significant. This finding is consistent with previous work of Okodua et al. 10 but disagreed with that of Mohandas et al. 37. The reason for this may have been that more females were tested in this study than previous works. Females have a higher prevalence of I. belli than males in this study (9.3 and 5.5%, respectively). This is not in agreement with Akinbo et al. 36, who found approximately the same prevalence between males and females. Females also have a higher prevalence of A. lumbricoides. Males have a higher prevalence of hookworm, T. trichiura, and Taenia species, while G. intestinalis was only detected in females. Prompt diagnosis of parasitic infections, especially intestinal parasitic infections, among HIV-infected persons is advocated in order to improve the management and quality of life of HIV-infected individuals. The authors have not received any funding or benefits from industry or elsewhere to conduct this study. We acknowledge with thanks the Management of University of Benin Teaching Hospital for permission to carry out this study. Chan MS Medley GF Jamison D Bundy DAB. The evaluation of potential global morbidity due to intestinal nematode infections Parasitology 1994 109 372 87 WHO. Communicable diseases chister. Severe falciparum malaria. Trans R Soc Trop Med Hyg 2002; 94: 551–90. Durack DI. Opportunistic infections and kaposis sarcoma in homosexual men New Engl J Med. 1981 305 1465 67 Bundy D Sher A Michael E. Good worms or bad worms: do worm infections affect the epidemiological patterns of other diseases Parasitol Today. 2000 16 273 74 Zheng XY. Acquired immunodeficiency syndrome prevention and control in China Clin J Epidemiol. 1999 20 131 4 Morris A Lundgren JD Masur H Walzer PD Hanson DL Frederick T Current epidemiology of Pneumocystis pneumonia Emerg Infect Dis. 2004 10 1713 20 Ramakrishnan K Shenbagarathai R Uma A Karitha K Rajendran R Thirumalaikolundusbramanian P. Prevalence of intestinal parasitic infections in HIV/AIDS patients with diarrhea in Madurai city, South India Jpn J Infect Dis. 2007 60 209 10 Nielsen NO Friis H Magnussen P Krarup H Magesa S Simansen P. Co-infection with subclinical HIV and Wuchereria bancrofti, and the role of malaria and hookworm in adult Tanzanians: infection intensities, CD4/CD8 counts and cytokine responses Trans R Soc Trop Med Hyg. 2007 101 602 12 Lucas SB. Missing infections in AIDS Trans R Soc Trop Med Hyg. 1990 84 34 8 Okodua M Adeyeba OA Talfeng YM Okpala HO. Age and sex distribution of intestinal parasitic infections among HIV infected subjects in Abeokuta, Nigeria Online J Health Allied Sci. 2003 4 3 5 Adesiji YO Lawal RO Taiwo SS Fayemiwo SA Adeyeba OA. Cryptosporidiosis in HIV infected patients with diarrhea in Osun State, Southwestern Nigeria Eur J Gen Med. 2007 4 119 22 Akinbo FO Okaka CE Machado RLD Omoregie R Onunu AN. Cryptosporidiosis among HIV-infected patients with diarrhea in Edo State, Midwestern Nigeria Malaysian J Microbiol. 2010 6 99 101 Omalu ICJ Yako AB Duhlinska DD Anyanwu GI Pam VA Inyama PU. First detection of intestinal microsporidia in Nigeria Online J Health All Sci. 2005 3 4 WHO. Intestinal protozoan and helminthic infections. WHO Technical Report Series 666. Geneva : WHO; 1981 . Büyükbaba BO Uysal H Alan S Nazlican O. Investigation of intestinal parasites in AIDS patients Mikrobiyol Bul. 2004 38 121 8 Oguntibeju OO. Prevalence of intestinal parasites in HIV-positive/AIDS patients Malaysian J Med Sci. 2006 13 68 73 Kuppamattus R Rajaiah S Alagappan U Karuppusamu K Rathinasainy R Ponniah T. Prevalence of intestinal parasitic infestation in HIV/AIDS patients with diarrhea in Madurai city, South India Jpn J Infect Dis. 2007 60 209 10 Mohammad RZ Ah JM Mostafa R Ahmed RM Siavash V Minoo M. Prevalence of intestinal parasitic pathogens among HIV-positive individuals in Iran Jpn J Infect Dis. 2004 57 268 70 Awole M Gebre-Selassie S Kassa T Kibru G. Prevalence of intestinal parasites in HIV-infected adult patients in southwestern Ethiopia Ethiopian J Health Dev. 2003 17 71 8 Moura H Fernandes O Viola JPB Silva SP Passes RH Lima DB. Enteric parasites and HIV infection: occurrence in AIDS patients in Rio de Janeiro, Brazil Mem Inst Oswaldo Cruz. 1989 84 527 33 Brandonisio O Maggi MA Lisi A Anriola A Acquafredda A Angarano G. Intestinal protozoa in HIV-infected patients in Apulia, South Italy Epidemiol Infect. 1999 123 457 62 Wiwanitkit V. Intestinal parasitic infections in Thai HIV-infected patients with different immunity Biomed Centr Gastroenterol. 2001 1 1 3 Udeh EO Goselle ON D-Popova DD Abelau M Popov TV Jane N The prevalence of intestinal protozoans in HIV/AIDS patients in Abuja, Nigeria Sci World J. 2008 3 1 4 Zelalem MT Abebe G Mulu A. Opportunistic and other intestinal parasitic infections in AIDS patients, HIV sero-positive healthy carriers and HIV sero-negative individuals in Southwest, Ethiopia East Afr J Public Health. 2008 5 169 73 Oguntibeju OO Vanden-Heever WMJ Van Schalkwyk FE. Effect of liquid nutritional supplement on viral load and haematological parameters in HIV-positive/AIDS patients Br J Biomed Sci. 2006 63 134 9 Kurniawan A Karyadi T Dwintasari SW Sari IP Yunihastuti E Diauzi S Intestinal parasitic infections in HIV/AIDS patients presenting with diarrhea in Jakarta in Indonesia Trans R Soc Trop Med Hyg. 2009 103 892 8 Lee JK Song HJ Yu JR. Prevalence of diarrhea caused by Cryptosporidium parvum in non-HIV patients in Jeollanam-do, Korea Korean J Parasitol. 2005 43 111 4 Endeshaw T Mohammed H Woldemichael T. Cryptosporidium parvum and other intestinal parasites among diarrheal patients referred to EHNRI in Ethiopia Ethiopian Med J. 2004 42 195 8 Vignesh R Balakrishnan P Shankar EM Murugavel KG Hands S Cecelia AJ High proportion of isosporiasis among HIV-infected patients with diarrhea in Southern India Am J Trop Med Hyg. 2007 77 823 4 Guptal S Narang S Nunavath V Singh S. Chronic diarrhea in HIV patients: prevalence of coccidian parasites Indian J Med Microbiol. 2008 26 172 5 Kulkarni SV Kairon R Sane SS Padmawar PS Kale VA Thakar MR Opportunistic parasitic infections in HIV/AIDS patients presenting with diarrhea by the level of immunosuppression Indian J Med Res. 2009 130 63 6 Ikeh EI Obadofin MO Brindeiro B Baugher G Frost F Vanderjagt D Intestinal parasitism in rural and areas of north central Nigeria: an update Intern J Microbiol. 2006 2 1 12 Ajjampur SSR Asirvatham JR Muthusamy D Gladstone BP Abraham OC Mathai D Clinical features and risk factors associated with Cryptosporidiosis in HIV-infected adults in India Indian J Med Res. 2007 126 553 7 Petersen E Lebech M Hojlyng N Holten-Andersen W Molbak K Hogh B Parasitic diseases in patients with impaired immune response, clinical picture and diagnosis Ugeskr Laeger. 1992 154 2475 80 Kumar SS Ananthan S Saravanan P. Role of coccidian parasites in causation of diarrhea in infected patients in Chennai Indian J Med Res. 2002 116 85 9 Akinbo FO Okaka CE Machacho RLD. Isosporiasis in HIV/AIDS patients in Edo State, Nigeria Malaysian J Med Sci. 2009 16 43 6 Mohandas K Sehgal R Sud A Malla N. Prevalence of intestinal parasitic pathogens in HIV-seropositive individuals in northern India Jpn J Infect Dis. 2002 55 83 4 Meamar AR Rezaiam M Mohraz M Zahabium F Hadighi R Kia EB. A comparative analysis of intestinal parasitic infections between HIV/AIDS patients and non-HIV infected individuals Iranian J Parasitol. 2007 2 1 6 Tuli L Gulati AK Sundar S Mohapatra TM. Correlation between CD4 counts of HIV patients and enteric protozoan in different seasons – an experience of the tertiary care hospital in Varanasi (India) Biomed Centr Gastroenterol. 2008 8 36 Weber R Biyan RT Owen RL Wilcox CM Gorelkin LG Visversve R. Improved light-microscopical detection of microsporidial spores in a stool and duodenal aspirates. The enteric opportunistic infections working group N Engl J Med. 1992 326 161 6 Fisseha B Patros B Woldemichael T Mohammed H. Diarrhea-associated parasitic infections agents in AIDS patients within selected Addis Ababa Hospitals Ethiopian J Health Dev. 1999 13 169 74