Opportunistic and other intestinal parasites infections among HIV-positive patients in the era of combination antiretroviral therapy and preventive treatment in Ouagadougou, Burkina Faso

Background: One of the particularities of human immunodefi ciency virus (HIV) infection in the tropics is its frequent association with parasitic diseases so frequent in this area. Aim: The aim of this study was to update the epidemiological and clinical profi le of intestinal parasites among HIV-positive patients in the era of combination antiretroviral therapy (ART) and preventive treatment in Ouagadougou, Burkina Faso. Materials and Methods: A cross-sectional study was conducted in a single health care system, Yalgado Ouédraogo University hospital, between January 2013 and December 2014. A total of 131 HIVinfected patients were included in the study. Blood and Stool samples were collected for CD4 counts and for intestinal parasitic examination using direct, formol-ether concentration, a concentration method using sodium chloride solution, modifi ed Ziehl-Neelsen and Weber modifi ed trichrome. Odds ratio was calculated to estimate the risk attributable to different factors with confi dence intervals. Chi-square (X2) or Fisher’s Exact Test statistical analysis was used to test level of signifi cance at p<0.05 using SPSS 20.0.0. Results: A prevalence of 73.3% of intestinal parasites was recorded. Most of the parasitic infections were protozoa (97.9%) with few helminths (2.1%). Cryptosporidium spp. (29.2%), was the most commonly found parasite followed by Entamoeba coli (23.9%) and Isospora belli (21.9%). There were 76 cases (78.5%) of single infection and 20 cases (21.5%) of mixed infection. Paradoxically, patients who use preventive treatment were more infected with opportunistic parasites (p= 0.05, OR=2.16, 95% CI= 1.06-4.40). In multivariate analysis, patients with diarrhea (OR=4.04, 95% CI=1.94-8.41), and illiterate (OR=3.70, 95% CI=1.68-8.14) had higher risk of opportunistic parasites. The diarrheic patients were 0.29 times more likely to be infected with opportunistic parasites. Conclusion: Despite the advent of combination ART and preventive treatment of opportunistic infections, intestinal parasites remain very prevalent and associated with diarrhea among patients with HIV in Ouagadougou. Research Article Opportunistic and other intestinal parasites infections among HIVpositive patients in the era of combination antiretroviral therapy and preventive treatment in Ouagadougou, Burkina Faso Adama Zida1,2, Abdourahamane Yacouba1*, Marcel P Sawadogo1, Ismael Diallo3, Ibrahim Sangare4,5, Sanata Bamba4,5, Bassory Ouattara1,6 and Tinga R Guiguemde2,4 1Service de parasitologie-mycologie, centre hospitalier universitaire Yalgado Ouédraogo, 03 BP 7022, Ouaga 03, Ouagadougou, Burkina Faso 2Département des sciences biologiques appliquées, unité de formation et de recherche en sciences de la santé, université Ouaga I Joseph Ki-Zerbo, Ouagadougou, Burkina Faso 3Service de Médecine interne, centre hospitalier universitaire Yalgado Ouédraogo, 03 BP 7022, Ouaga 03, Ouagadougou, Burkina Faso 4Département des sciences fondamentales et mixtes, Institut supérieur des sciences de la santé, université Polytechnique de Bobo-Dioulasso, Bobo-Dioulasso, Burkina Faso 5Service de parasitologie-mycologie, centre hospitalier universitaire Souro Sanou, BoboDioulasso, Burkina Faso 6Ecole privée de santé sciences nouvelles (ESSN), Ouagadougou, Burkina Faso Dates: Received: 28 July, 2017; Accepted: 28 August, 2017; Published: 29 August, 2017 *Corresponding author: Abdourahamane Yacouba, MD, Medical Biologist, Service de Parasitologiemycologie, Centre Hospitalier Universitaire Yalgado Ouédraogo, 03 BP 7022, Ouaga 03, Ouagadougou, Burkina Faso, Tel: +226 75904270; E-mail:

The spectrums of opportunistic parasitic infection vary in different regions and usually refl ect the infections prevalent in these regions [10].
In Burkina Faso, in 2016, the national HIV prevalence was estimated at 0.8% (0.7-1.0) with women and youths being predominantly infected. The overall antiretroviral therapy (ART) coverage among Burkinabe people living with HIV/ AIDS was estimated at 60% [11]. The fi rst study (hospital based cross-sectional study, conducted in 1997) on intestinal parasites in patients with HIV showed that the country pays a heavy toll to the infections by these parasites (16 (19.3%) patients died, among them 11 (68.7%) had opportunistic parasites) [12]. Seventeen years later, supported measures of the intestinal parasites, knew several novelties such as: introduction of a mass treatment with Albendazole/Ivermectine in 2001, Praziquantel from 2005, respectively by the National Program to Eliminate Lymphatic Filariasis (NPELF) and the National Control Program on Schistosomiasis (NCPS); last but not least the introduction of Trimethoprim-sulfamethoxazole (SXT) preventive treatments for patients with HIV and a CD4 count <200 cells /μL. Added to these preventive treatments, improved diagnostic techniques for parasites screening are still unknown.
The aim of this study was to update the epidemiological and clinical profi le of intestinal parasites among HIV-positive patients in the era of combination ART and preventive treatment, in Ouagadougou, Burkina Faso.

Study site and study design
A cross-sectional study was conducted between January 2013 and December 2014, at the Yalgado Ouédraogo University Hospital (CHU-YO), 03 BP 7022, Ouaga 03, Ouagadougou, Burkina Faso. Socio-demographic, clinical, and laboratory data were obtained from the study subjects using a structured questionnaire and laboratory analysis of stool and blood samples.

Study population
Patients admitted in the CHU-YO were the study population. The inclusion criteria were: Patients with confi rmed HIV/ AIDS, with or without signs of diarrhea. The exclusion criteria were: those on specifi c anthelminthic, anti-protozoan, or who had treatment for intestinal parasitism in the last two weeks preceding specimen collection and those who had antacid in the last two weeks preceding specimen collection. The use of antacids can distort protozoan morphology contributing to the diffi culty in identifying the organism.

Sample collection and processing
Fresh stool samples were collected from these patients. In the fi rst time, macroscopic analysis of stool sample was done to determine the consistency, color of the fecal material and element present in the feces such as mucus, blood, and eventually worms (Taenia segment). In second time, microscopic analysis was done including direct and complementary special staining methods; direct microscopy by mixing a small amount of the specimen in 0.9% sodium chloride solution was done to detect trophozoites of Entamoeba spp. Trichomonas intestinalis and Giardia lamblia; formol-ether concentration was used to detect cyst of protozoa (Entamoeba spp., Blastocystis hominis, Giardia intestinalis) and eggs of helminths (Schistosoma spp., Taenia spp.); a concentration method using sodium chloride solution (Willis method) was used to identify hookworm (Ancylostoma duodenale), tapeworm (Hymenolopis nana) and Ascaris lumbricoides. Weber modifi ed trichrome was used for detection of microsporidia spores and modifi ed Ziehl-Neelsen technique was used to detect oocysts of Cryptosporidium spp. and Isospora belli.
Intestinal parasitic infection-positive patients were defi ned as those who had their stool analysis positive for one or more parasites in many forms: cyst, trophozoite, eggs and/or adult.
The CD4 cells count determination was done at the laboratory of Bacteriology-Virology, largest referral center for HIV/AIDS in Burkina Faso. We categorized CD4 cell counts into 2 groups: <200 cells/μL and >200cells/μL.

Statistical analysis
The statistical analysis was done using SPSS version 20.0.0. Prevalence was calculated. Chi-square test or Fisher's Exact Test was used to determine the association between different variables in the structured questionnaire and intestinal parasitic infection. Odds ratio was calculated to estimate the risk attributable to different factors with confi dence intervals. Multivariate analysis was performed to assess crude and adjusted ratio. Observed differences in data were considered signifi cant if p < 0.05.

Ethical considerations
An informed consent was obtained from all study participants, after explaining the importance of the study.

Results
A total of 131 subjects were included in the study. The mean age of the study population was 40.24 years (extreme: 16 and 64 years). The sex ratio was 0.79 (Table 1).

Discussion
The female predominance and the younger age of our patients are consistent with the epidemiological data of HIV infection in Burkina Faso [13]. This might be due to men having less recourse to health services in Burkina Faso comparatively to women and also due to HIV epidemic mainly affecting the young and sexually active population [14]. Like in the present study, it has been reported that a high proportion of intestinal parasitic diseases occurs in the low education level, and among housewife [12,15,16]. This could result from little information on the importance of sanitation.
Seventeen years after the fi rst study which showed a prevalence of 61.4%, our study found that intestinal parasitism has not decreased (73.3%) [12]. However, this fi nding may be biased through the fact that we used screening techniques (Willis, Ritchie, chromotrope 2R) which were absent in the 1997 survey. This prevalence is also higher than that reported in Nepal (30.1%) [17]. Among amoeba, the prevalence of Entamoeba coli (23.9%) was higher to the prevalence found in Equatorial Guinea (15.8%) [15].The higher prevalence of Entamoeba coli among amoeba was also recently found in prisoners in Ouagadougou, Burkina Faso [23]. The prevalence of Entamoeba histolytica (8.3%) was in concordance with the fi nding in Nigeria and Ethiopia respectively 9.2% and 6.1%, but lower than those found in India 24.4% [5,18,22]. The prevalence rate of Giardia intestinalis (9.3%) was higher than an earlier study and that found in India, 4.1% [12,24]. In general, such a prevalence of these non-opportunistic parasites could have been due to water and food contamination, or with to poor personal hygiene [24].
The high prevalence of non-opportunistic parasites with HIV infected patients also draws attention to the need to include routine stool examination in HIV/AIDS management.
To our knowledge, this study for the fi rst time showed the presence of intestinal microsporidia in Burkina Faso. But we were unable to differentiate Enterocytozoon bieneusi of  Encephalitozoon intestinalis. For this differentiation, molecular method which is not used in this survey is needed. We can nevertheless notice with many authors that these two species exist in African countries and have been estimated to vary from 1.5% to 50.0%, depending on the geographic region concerned and the diagnostic methods applied [25][26][27]. Since the administration of antiretroviral therapy has enabled HIVinfected patients to restore their immune status, the number of cases of microsporidia infections has decreased dramatically in developed countries [28]. This high frequency of microsporidia in our study allows us to say that, unlike some countries like the Congo where infection with those protozoa is emerging, the microsporidia are a reality in Burkina Faso [25]. But so far, they have attracted little attention from clinicians and have not been included in the SXT preventive therapy. Indeed, none of the two parasite species, Enterocytozoon bieneusi and Encephalitozoon intestinalis, is reported to be sensitive to this molecule [10].
Like in the present study, it has been reported the rarity of helminths in the recent surveys on intestinal parasites in Burkina Faso [23,29]. This fi nding was also in agreement with other studies done in African countries [5,25,30]. This low prevalence of helminths among our population is directly on the one hand, due to the treatments of mass with Albendazole/ Ivermectine but also with Praziquantel led respectively by the NPELF and the NCPS, on the other hand, to extensive antiretroviral treatment administration, secondary to a stabilization of immune response. In contrast to our fi nding, the dominance of helminths was found in Ethiopia [24]. Whether the particular absence of Strongyloides stercoralis, helminth opportunistic in HIV / AIDS, can be related to its sensitivity to Albendazole, it is reported in many similar studies in the same context of treatment of mass in African countries like Nigeria, Lao People's Democratic Republic and Ethiopia [24,[31][32][33]. This absence in our series can also be explained by the reason why we did not use a special research technique (Baermann and Lee, Koga Agar Plate, PCR) [34]. The presence of Schistosoma intercalatum among the few (n=2) helminths also raises a comment. Indeed, to the best of our knowledge, this study is the second which indicates the presence of this parasite species in Burkina Faso. The fi rst detection was made in 1969 [35]. This extreme rarity is due to the fact that the West African region is not one of the usual foyers of this parasite. The usual foyers concern the rain forest areas of Central Africa [36].
In our study, diverse modalities of parasitism are observed with a monoparasitism rate (78.5%) higher than the polyparasitism (21.5%). The presence of polyparasitism was also found in other studies done in Burkina Faso, Nigeria and India where they were respectively 3.6%, 23.8% and 21.5% [5,12,37]. The presence of parasitic associations indicates one very faecal and sanitary food low level of hygiene and the unfavorable living conditions [38]. The predominance of association of protozoa is explained by the fact that these parasites often have a similar mode of infestation.
In the present study, 62.5% and 37.5% were the isolation rate for parasites in patients with and without diarrhea respectively (p= 0.00). This association was in agreement with other studies which have shown that parasites are important etiological agents of diarrhea with HIV-positive patients [18,21,24].
There was a signifi cant association between the opportunistic parasites and CD4 count <200 cells/μL. The presence of opportunistic parasites with the HIV-positive patients was inversely proportional to the CD4+ T-cell counts with these patients, i.e. the lower the CD4 counts the more the chances of isolation of parasites with these patients were. This fi nding is in concordance with other studies where decreasing opportunistic parasite positivity has been reported as CD4 count of HIV/AIDS patient increases [17,21,39,40]. This raises the question of the role of cell-mediated immunity in host defense against the parasite.
Paradoxically, in our study, patients who use preventive treatment (SXT and/or albendazole) were more infected with opportunistic parasites (p=0.05). However, there was no signifi cant association between opportunistic parasites and the use of preventive treatment (p=0.05). The high prevalence of opportunistic parasites can be explained by the fact of inadequate immune reconstitution or poor adherence and preventive treatment interruption, among HIV+ subjects on ART.
This study gives preliminary elements to investigations for more elaborate via methods of diagnosis more successful, in particular PCR, the detection of antigen, and the iso-enzymatic analysis. In addition, a longitudinally designed study with a stronger power, incorporating additional confounding factors, would be needed to further understand the possible immunological and epidemiological mechanisms underlying HIV infection with intestinal parasite interactions.
There are some limitations of our study. This study do not precise for how long have the patients been on SXT therapy or on ART. HIV negative control groups were not included. These limitations may introduce bias. Therefore, the fi ndings of this study, conducted in a single health care system, may not be generalizable to other health care systems and should be interpreted in light of the study limitations.

Conclusion
Despite the advent of combination antiretroviral therapy and preventive treatment of opportunistic infections, intestinal protozoa remain very prevalent and strongly associated with diarrhea among patients with HIV in Ouagadougou. Those results posit the need for considering early detection and treatment of intestinal parasites in HIV infected individuals in order to reduce their morbidity. In addition, this fi nding calls for establishment of specifi c diagnostic tests in all health laboratories and train health professionals with special attention to opportunistic intestinal parasites in HIV-positive patients.