Intestinal protozoan infections among HIV positive persons with and without Antiretroviral Treatment (ART) in selected ART centers in Adama, Afar and Dire-Dawa, Ethiopia

Background: In developing countries, gastroenteritis caused by intestinal parasites may be complicated and is a major cause of morbidity, in general, and kills millions of AIDS patients annually. Thus, the consequences of parasitic diseases are among the major health problems in tropical developing countries. Methods: A total of 200 HIV positive patients on and without-ART aged from 18 to 65 years, of both sexes participated in the study. Each study participant was provided with a faecal collection vial containing 10% formalin for microscopic examination of ova, larvae, and cysts. For detection of Cryptosporidium spp, lsospora belli and Cyclospora spp, the modified Zeihl-Neelsen staining method was used. Results: Most (60%) of the study participants were on antiretroviral therapy (ART). Out of those, only two (1.5%) were diagnosed with an opportunistic parasite, and 96 (48%) of the non-ART study participants were infected with at least one other intestinal parasite species. The prevalence was 16% for Giardia lamblia, 13% for Entamoeba histolytica/ E. dispar, 8% for Cryptosporidium spp, 5% for Isospora belli, 1.5% for Blastocystis hominis, 2.5% for Ascaris lumbricoides and 2% for Hymnolepis nana. Diarrhoea was significantly associated with cryptosporidiosis, giardiasis, and isosporiasis. Significant association was observed between lower CD4 T cell count (<200 cells/μL) and the prevalence of Cryptosporidium spp, Isospora belli, and Blastocystis hominis. The three parasites were significantly prevalent in HIV sero-positive patients not on -ART. Conclusion: The finding showed that patients under ART had lower prevalence of diarrheogenic protozoan parasites suggesting that ART through improvement of the immune status of patients may have contributed to controlling parasites in HIV/AIDS patients. [Ethiop. J. Health Dev. 2009;23(2):133-140] Introduction Human immunodeficiency virus (HIV) infection, a worldwide phenomenon, is a serious public health problem (1). HIV infection has globally claimed over 20 million lives, and currently over 40 million people carry the infection (2). Even though AIDS remains a global pandemic, Ethiopia is one of the highly affected SubSaharan countries. It is estimated that as much as 60% of the World’s population is infected with intestinal parasites, which may play a significant role in morbidity due to intestinal infections (3). Intestinal parasitic infections are among the most common infections world-wide. It is estimated that some 3.5 billion people are affected, and 450 million are ill as a result of these infections (4). The rate of infection is remarkably high in Sub-Saharan Africa, where the majority of HIV and AIDS cases are concentrated (5). The incidence of intestinal parasitic infections is 50% in developed countries, whereas it reaches up to 95% in developing countries. These infections are caused both by protozoa and helminths and the main clinical manifestation of the disease caused by them is diarrhoea (6). In developing countries, acute gastroenteritis caused by intestinal parasites is complicated and it is a major cause of illness and kills millions of AIDS patients annually (7). Reports indicate that diarrhoea occurs in 30-60% of AIDS patients in developed countries, whereas it reaches up to 90% in developing countries (8). The progressive decline of mucosal immunologic defence mechanisms predisposes patients to precocious, intermediate, or late gastrointestinal manifestations such as diarrhoea (9). In the late stages of AIDS, the protective effects of nonspecific defence mechanisms, production of IgA antibodies and local cellular immune responses are diminished, thus increasing susceptibility to various intestinal opportunistic agents, such as Cryptosporidium parvum, Isospora belli, and Microsporidiium species (10). After the emergence of AIDS, opportunistic parasites, known until then solely in veterinary medicine, were no longer considered commensal organisms, and they currently are recognized as common opportunist pathogens affecting HIV infected patients, constituting a major secondary aggravating factor of the disease. These enteric infections frequently cause severe diarrhoea, which often is responsible for the grievousness of the disease, and may sometimes lead to death. 134 Ethiop. J. Health Dev. Ethiop. J. Health Dev. 2009;23(2) In general, diarrhoeal diseases are significant causes of morbidity and mortality in all age groups, but immunocompromised and pediatric patients experience more frequent and severe illnesses (11). Moreover, nowadays diarrhoeal illnesses are becoming one of the most common clinically observable gastrointestinal manifestations in AIDS patients, occurring at late stages of HIV infection; usually due to opportunistic infections (12). Intestinal parasites are highly prevalent in Ethiopia due to shortage of clean water, lack of sewage system and other unhygienic factors that increase the probability of infection. The purpose of this study was to test the hypothesis that ART will reduce the prevalence of diarrheogenic protozoan parasites in HIV/AIDS patients. Methods The study area: The study was carried out in selected Health Centers with ART service at Adama 100km from the capital Addis Ababa, Afar (“Awash Sebat Kilo”), 225km from Addis Ababa and Dire-Dawa which is 525 km from Addis Ababa, Ethiopia from December 2007 to January 2008. The study sites were selected considering the location, cost and time constraints. The study population: The study participants were people living with HIV (PLHIV) of both sexes 18 to 65 years of age on and not on ART selected randomly. Socio-demographic and clinical information including diarrhoea and medication histories were obtained from the study participants by interview and their CD4 T-cell counts were obtained from their medical records at the Health Centers. CD4 T-cell counts were considered only when it was done at the time of or very close to stool sample collection. Stool collection and processing: A single fresh stool sample was collected from consenting study participants (n=200). The questionnaires concerning the prevalence were filled by the principal investigator who interviewed the study participants during sample collection. A portion of the stool was preserved in 10% formalin in a proportion of 10g of stool in 3ml of formalin (13). Direct Microscopy (Wet mount): A direct wet mount of stool in normal saline (0.85% NaCl solution) was prepared at Adama, Afar (“Awash Sebat Kilo”) and DireDawa ART Centers; and examined for the presence of motile intestinal parasites and trophozoites under light microscope (400X magnification). Lugol’s iodine staining was used to detect cysts of intestinal parasites. Formalin-Ether concentration: Using an applicator stick, about 5g of preserved stool sample was placed in a clean 15 ml conical centrifuge tube containing 7 ml of 10% formalin. The sample was dissolved and mixed thoroughly with applicator stick. The resulting suspension was filtered through a sieve (cotton gauze) into a beaker and the filtrate poured back into the same tube. The debris trapped on the sieve was discarded. After adding 3 ml of 99.5% diethyl ether to the mixture and hand shaking, the content was centrifuged at 2000 rpm for 3 minutes. The supernatant was poured away and the tube was placed in its rack. The sediments were stained with Iodine and put on slide and covered with cover slip. The entire area under the cover slip was examined using 400X objective magnification (14). Microscopic examinations were done independently by experienced clinical laboratory technicians; the determination and verification was finally made by the investigator. Modified Zeihl-Neelsen Method: Modified ZeihlNeelsen staining; based on direct and concentration methods, for detection of oocysts of opportunistic coccidial intestinal parasite-Cryptosporidium spp, Isospora belli, Cyclospora cayetanensis was done. Fresh faecal samples were collected and thin smears were prepared, air-dried, fixed with methanol for 5 minutes in the field and stained by Zeihl-Neelsen technique at the Biomedical Sciences Laboratory of the Department Biology, Addis Ababa University. The same procedures were used for smears prepared after concentration. Smears were prepared from the concentrated stool samples and were stained as described by Adegbola et al. (15) with some modifications. In this technique, the slides were stained with carbol-fuchsine for 30 minutes and then were washed with tap water. The slides were decolorized in 1% HCl acid-alcohol for 1 minute and were counter stained with 1% ethylene blue for another 1 minute. Finally, the stained smears were microscopically examined using 1000x magnification (16). Data analysis: Statistical analysis was done by using SPSS version 13.0. Data were summarized using frequency tables and bar charts. Categorical risk factors for diarrhoea analyzed; and the strength of association measured by using the chi-square and its associated pvalue. Values were considered to be statistically significant when the p-value obtained was less or equal to 0.05. Ethical Consideration: This study was conducted with the approval of the Ethical Review Committee on Health Research, Faculty of Science, Department of Biology, Addis Ababa University. Informed written consent was obtained from each study participant. Participants were also informed that they are free to withdraw consent any time and their medical records and specimens were examined by qualified persons. More over, all personal information of the participants was treated strictly confidential. Study participants positive for intestinal parasites were treated for free using standard drugs. The drugs were administered by clinicians working at the study sites. HIV/AIDS, ART, parasites, diarrhoea, HIV positive, Ethiopia 135 Ethiop. J. Health Dev. 2009;23(2) Results A total of 200 HIV sero-positive individuals participated in this study. Among those, 110 (55%) were males and 90 (45%) were females. The mean age of the study participants was 41.5 years (range: 18 to 65). The majority, 143 (71.5%), were from the rural vicinities of the towns. They had direct and indirect contact with domestic animals. Of the total study participants, 62 (31%) were from Adama and its surroundings, while 70 (35%) were from Afar (“Awash Sebat Kilo”) and 68 (34%) were residents of Dire-Dawa and its surroundings (Table 1). Table 1: Age, Sex and Area distribution of study participants (n=200) in Adama, Afar and Dire-Dawa, Ethiopia (2008).