Prevalence and associated factors of intestinal parasite infection by HIV infection status among asymptomatic adults in rural Gabon

Introduction Intestinal parasites infections are endemic in Gabon. Nevertheless, they are rarely described in people living with HIV (PLHIV). Objective The frequency of intestinal parasite infection was estimated and compared between HIV-positive and HIV uninfected individuals in Gabon; factors associated with intestinal parasites were also analysed. Material and Methods Using a cross-sectional study design sociodemographic data, life style habits, antiretroviral therapy, cotrimoxazole use and CD4 cell count were recorded.. Stool samples from participants living in Koulamoutou and Oyem were analysed using microscopy. Chi-squared or fisher's exact tests and logistic regression were performed. Results Among participants (n=332), female gender was predominant (73.7%; n=135/183) and the median age was 45 [33–57] years old. Among 183 samples, 53.6% (n = 98/183) were infected by intestinal parasites. The proportion was higher (72.1%) in HIV negative participants compared to PLHIV (42.6%) (p <0.01). PLHIV were more frequently poly-infected. Infection was frequent in patients using external toilets and tap water (>70.0%). Conclusion Prevalence of intestinal parasites is higher in seronegative participants but polyparasitism is more frequent in PLHIV. Strategies are focused on HIV negative population, but this study shows the importance of sensitization for PLHIV to improve their quality of life.


Background
Intestinal parasite infections (IPIs) affect about half of the world's population, especially in developing countries where environmental conditions are favorable to their development. 1 In sub-Sahara Africa, 36.7 million people are living with HIV (PLHIV) and are also exposed to several infectious diseases including intestinal parasitic infections that threaten the health of these people. 2, 3 In absence of highly active antiretroviral therapy (HAART), the destruction of the immune system due to HIV leads to greater susceptibility to opportunistic Corresponding author: Denise Patricia Mawili-Mboumba, Faculty of Medecine, Department of Parasitology Mycology. Email: dpmawili@gmail.com and other intestinal parasites. 4 Other parasitic diseases such as blastocystosis of which prevalence varies between 20 to 50% in PLHIV, giardiasis and entamoebiasis are common, even in the absence of diarrhea. [5][6][7] HIV and parasitic infections interact with each other, potentially leading to activation of HIV replication and accelerating disease progression from HIV infection to AIDS. 8,9 The risk of co-infection of these diseases (helminths infection, intestinal protozoa)were shown to be higher in PLHIV with CD4 cell counts low 200cells/ mm 3,5 . Moreover, co-infections may worsen the progression of AIDS. [10][11][12][13] In Gabon, soil transmitted helminths (STH) and intestinal protozoa asymptomatic carriage is frequent. M'Bondoukwé et al found a high proportion of IPI carriers (75%) in shanty towns of Libreville; Blastocystis sp was the main pathogen detected. 14 In another study, protozoa and helminths were found in 61.2% of population in rural and urban areas. 15 These studies were performed in patients whose HIV status was not determined. Blastocystis sp is an emergent protozoan; a significant association was observed between immunosuppression among PLHIV and its occurrence. 16 The Parasitic Diseases National Control Program performs surveys to monitor parasitic diseases throughout Gabon. Data on IPIs in PLHIV are scarce although this population is also exposed to opportunistic and non-opportunistic parasitic diseases. 4,17 PLHIV, are approximately 53000 people, and represent between 2 to 13% of the Gabonese population and are followed up in care and treatment centre (CTC) for PLHIV in the whole country. 18 They are not targeted by IPIs preventive strategies although their living conditions did not differ, from the ones of the rest of the population. IPIs control strategies objectives may not be achieved without investigating the burden of IPIs in the whole population including PLHIV. The present study aimed to compare the prevalence of intestinal parasites in people according to their HIV infection status and CD4 cell counts in two rural areas, Oem and Koulamoutou where no data on intestinal parasites had been recorded.

Materiel and methods Study area
The study was carried out in June 2016 in two regions of Gabon where data on IPIs are scarce: Koulamoutou, South Eastern part of Gabon with a population estimated at 16222 inhabitants and Oyem in the north with a population of near 60,000 inhabitants. The climate is equatorial with two rainy and two dry seasons, temperatures ranges of 23-33 °C and 80% humidity on average. In the rural areas of Gabon, water is mainly obtained from public water pumps or rivers. The dwellings are made of terracotta and the streets are not always tarred. The main activity in these areas is agriculture.

Study population Study design
This was a prospective comparative cross-sectional study. Asymptomatic PLHIV attending their routine follow-up visits in two HIV care and treatment centers (CTCs) were invited to participate in the study. For the comparison of intestinal parasites prevalence, data from volunteer adults, accompanying patients, were also collected in the Regional Hospital of Koulamoutou which is located near CTC.

Data collection
Participants were included after being given informa-tion about the study. Briefly, data on demography (age, sex and education level), self-medication with anthelminthic drugs, type of drinking and housekeeping water, presence of toilets in/or outside the house were recorded for both groups using a structured questionnaire. Among PLHIV, antiretroviral treatment (ART) use and cotrimoxazole (CTX) prophylaxis, CD4 cells counts were recorded from patients HIV clinic registers.

Inclusion criteria for seronegative participants and PLHIV:
Only individuals, including the ones accompanying the PLHIV, who accepted to be tested for HIV, who had been living at the site for at least six (6) months before the start of the study and who gave their written informed consent were recruited. Exclusion criteria for seronegative participants and PLHIV: Participants who had diarrhoea or abdominal pain and anti-parasitic intake in the 2 weeks prior to collection were excluded.

HIV infection diagnosis
HIV test was performed for all volunteers not followed up at the HIV CTC according to the Gabonese national HIV testing algorithm. None were found positive.

Study procedures: specimen collection and processing
Clean well-labelled stool collection vials were provided to the participants with clear instructions to ensure that stool samples would be correctly collected. Each vial was identified by a code number specific to each participant. They were instructed to use the spatula provided to transfer a thumb-sized faecal sample to the container making sure that the sample was not contaminated with urine. Stool specimens were processed and examined microscopically for parasite detection.

Direct microscopic examination
A small amount of stool was mixed with a drop of saline and was examined under a light microscope for the observation of motile parasites. STH including Schistosoma (S.) intercalatum eggs, cysts and vegetative forms of Entamoeba sp, Blastocystis sp, G. duodenalis can be detected with this method.

MIF (Merthiolate)-Iodine-Formaldehyde) concentration
The MIF concentration (MIFc) method was carried out as described by Sapero and Lawless. 15,19 MIFc is a simple and rapid staining method in which faecal specimen is concentrated by crushing in thiomersal-formaldehyde solution and stained with 5% Lugol's solution. Eggs of A. lumbricoides, T. trichiura, S. intercalatum, cysts and vegetative forms of amoeba and G. duodenalis, cysts of Blastocystis spare stained. The use of a combination of MIF and Lugol's solution for staining is sensitive for the detection, fixation and storage of vegetative forms and cysts of protozoa. Parasite species observed after direct examination are detected equally after MIFc. Samples were considered positive upon the detection of a single parasite in the sample.

Parasite culture
Parasites culture was performed as previously described for N. Americanus and S. Stercoralis larvae detection. 15,20,21 The parasite culture was considered as positive if at least one larva was present.

Modified Ziehl Neelsen staining method
Modified Ziehl Neelsen method was used for the identification of Cryptosporidium sp and Isospora belli 22 . Slides were examined under a microscope at a magnification of ×100. Samples were considered as positive upon the detection of at least one parasite in the sample. The samples that tested negative using direct microscopic examination were confirmed after staining and the results were returned to the participants.

ART use
According to the national guidelines, the drugs administered as first line antiretroviral therapy ( ART) were Zidovudine (AZT) or Tenofovir (TDF) plus Lamivudine (3TC) plus Efavirenz (EFV) or Nevirapine (NVP); for the second line treatment it was Abacavir (ABC) or Zidovudine (AZT) plus Didanosine (DDL) plus Lopinavir/r (Lopi/rito).

Definitions of variables of interest •
Patients who had a positive MIF concentration or stool culture or modified Ziehl Neelsen staining are classified as having intestinal parasites.
• Presence of at least two different intestinal parasite species was considered as "multiple parasite infections" or "polyparasitism".

•
Person was considered to be educated if they completed/attended primary school.
• type of drinking and housekeeping water indicates the source of the water (tap, well, river and mix) • presence of toilets in/or outside the house indicates the location of the toilets Data analysis StatView 5.0 (SAS Institute Inc) was used for data analysis. Age and CD4 count were presented in medians with interquartile (IQR) ranges (non-Gaussian distribution). Differences of the frequency between groups (PLHIV and seronegative patient) and associated factors were assessed using chi-squared or Fisher's exact tests. P values below 0.05 was considered significant, if p values were between 0.05 and 0.10, it was considered as a trend. Crude odds ratios (cORs) and 95% confidence intervals (CIs) were used to assess the association between intestinal parasite groups, and age, education level, type of water, use of external toilet, cotrimoxazole use and CD4 level count. For all these tests, the difference was considered significant if P < 0.05. All the reported P-values are for two-tailed tests. Logistic regression was performed to estimate the adjusted odds ratio (aOR) between intestinal parasite and all variables with a p<0.2, a multivariate mixed logistic regression model using likelihood ratio estimation for discrete choice modelling of small datasets was performed. The aORs and their 95% CIs were calculated. Associations were found significant if P values were below 0.05 and a trend was indicated if P -values were between 0.05 and 0.10.

Ethical considerations
The study protocol was authorized by the Ministry of Public Health and approval of the National Ethics Committee was obtained in February 2016 (PROT N°003/2016/SG/CNE). Informed consent was obtained. Biological testing was offered to participants, appropriate treatment according to national recommendations was administered by the specialist physician for any participant infected with pathogenic protozoa or STH.

Sociodemographic characteristics of the study population
During the study period, 332 individuals accepted to participate in the study; among them 233 were PLHIV. A total of 183 (55%) participants brought their stool for analysis; they were asymptomatic and not actively detected. The majority were female (73.7%; n=135/183) and their median age was 45 [33-57] years old. Among these participants, more than half of them used tap water as source of drinking water (77.7%; n=133/171) and for housekeeping (66.4%; n=101/152) (Table 1).

Discussion
Little is known about the epidemiology of intestinal parasites infections in people living with HIV in Gabon, especially in rural areas where intestinal infections are the most frequent 15 . Thus, this study shows that IPIs prevalence is high in this asymptomatic population of adults, although it was higher among the HIV seronegative ones. However, polyparasitism was highly detected among PLHIV. Protozoans were the most frequent parasites detected in both type of participants. The main species found was Blastocystis sp associated to E.coli. Presence of parasites was associated with the quality of the water used, the use of external toilet, but not with CTX and low CD4 count.
The study provides local data on IPIs prevalence and human reservoir among seronegatives individuals and PLHIV and on associated risk factors. These data are important for the design or the readjustment of control strategies for the prevention of these infections. Nevertheless, this study has some limitations. Stool samples were collected only once from the subjects, potentially accounting for the lower prevalence of intestinal helminths than of intestinal protozoa. The intensity of IPIs was not determined. The overall prevalence of intestinal parasites infections found in the present study was 53.6% in the whole study population. This proportion was significantly higher among HIV uninfected patients compared to PLHIV (72.1% vs 42.6%) presumably due to antiretroviral treatment. In a study carried out in Ethiopia, HIV positive patients on ART had significantly lower prevalence of intestinal parasitic infection compared to HIV negative individuals. 23 In this study, the majority of PLHIV was under ARTs. However, the impact of ART on IPIs is controversial. 24,25 Considering the parasites infection distribution, the prevalence found among HIV negative patients (72.1%) is comparable to those obtained in previous studies performed in different areas of Gabon. 14,15 In PLHIV, the frequency of IPIs (42.6%) is higher than that reported in other African countries such as in Ethiopia (35.9%) and Brazil (28.8%). 26,27 Such difference may be explained by the study site location. These studies were conducted in both rural and urban areas, while the present study was exclusively carried out in rural areas. Indeed, in rural areas IPIs are more frequently encountered related to environmental conditions and inhabitants lifestyle.
Moreover, in the present study where the participants are all asymptomatic, the prevalence of IPIs in PLHIV is lower compared to that found in PLHIV with diarrhoea (59.8%) by Assefa et al. 10 In infected participants, protozoa were seven fold more frequent than helminths whatever HIV status. The same was true in other countries such as Malaysia (18.5% vs 7.5%) or in Ethiopia (76% vs 22.1%) where intestinal protozoa were more frequently detected. 4,10 This decrease of helminths frequency has also been observed in previous studies in Gabon. 14, 15 It may be due to self-medication by anthelminthic drugs in the populations in cases of diarrhoea or abdominal pain but also as a preventive measure. Despite the low prevalence of helminths, T. trichiura was found in 5.5% of individuals. It has been shown that this helminth was not fully sensitive to albendazole or mebendazole treatment. 28 Nevertheless, the predominant species identified was Blastocystis sp in both populations as reported by others authors. 7,10,14 This species was almost two-fold less frequent in PLHIV compared to HIV negative participants (22.6% vs 47.1%). Such prevalence is similar to that found by Paboriboune et al (26%) in Lao People's Democratic Republic. 5 Blastocystis sp usually considered as a commensal (non-pathogenic) protozoa living in the human intestine, is now described as a pathogen. 29 It has been isolated in PLHIV having diarrhea. 16,30 One study has shown that cotrimoxazole administered to PLHIV, with diarrhea and infected by Blastocystis sp, contributes to the recovery of these patients. 31 In a study performed in Lambaréné, in the centre of Gabon, authors found a lower prevalence of intestinal helminths among patients taking cotrimoxazole compared to those who did not. 32 In the present study the frequency of the infection did not vary according to the CTX intake presumably due to the sample size. This may hampered the clinical significance of CTX use in PLHIV use in the present study.
I. belli and Cryptosporidium sp (<1.0%) were specifically found among people living with HIV confirming their opportunistic character. This low prevalence was similar to that found in PLHIV (3.8%) in Brazil. 27 Furthermore, the consumption of tap water and the use of external toilets were factors associated to the high prevalence of intestinal parasites (near 90%) as reported elsewhere. 33,34 Protozoa such as G. duodenalis and Cryptosporidium sp resist to the wastewater treatment process. 35 Participants immunodepressed (<200 cell/mm 3 ) had more frequently IPIs than PLHIV with higher level of CD4 count. This result was presumably not statistically significant due to the small sample size. In Nigeria, Ethiopia and other studies, the prevalence of IPIs was higher in subjects with low CD4 levels (<200cell/ mm 3 ). [36][37][38][39][40] Other authors reported a higher frequency of IPIs in PLHIV when CD4 rate was below 350cell/ mm 3.31 Intestinal polyparasitism was found in 20.4% of PLHIV. Globally, the majority of them carried two parasites, four had three parasites and only one individual had four parasites. Most of the participants who had two or four parasites were people living with HIV (n = 10; 24.4%). In other studies polyparasitism occurred more commonly among HIV/AIDS individuals, and is frequently associated with low CD4+ T cell. 39 A similar rate of infections was found in Algeria (15.4%) but this was higher in Malaysia (71.4%). 40,41 This high rate could be due to the environmental conditions of rural areas, difficulty of access to drinking water and poor hygiene.

Conclusion
The overall prevalence of intestinal parasite infection is high with a predominance of protozoa in PLHIV living in rural areas in Gabon. Polyparasitism is not negligible in this population and infection with pathogenic intestinal parasites is also common even in the absence of clinical signs. Routine diagnosis of stools should be advocated for PLHIV better case management. As strategies are focused on HIV negative population, who carries the highest load of intestinal infections, sensitization on the preventive measures must be integrated for PLHIV. This study provides data that will contribute to a better management of PLHIV care by identifying the parasites to which these participants were exposed.