Pathogenicity and non-opportunistic character of Blastocystis spp.: a hospital-based survey in Central Cameroon.

INTRODUCTION
Blastocystis spp. is a protist found in humans. Although usually the most frequent protozoa found in stool samples of both symptomatic and healthy subjects, its pathogenic or rather opportunistic role is yet to be clearly elucidated. To attempt to fill this gap, a cross-sectional study was conducted to compare the frequency of Blastocystis spp. in HIV positive (HIV+) versus HIV negative (HIV-) individuals in four health facilities of the Center Region of Cameroon.


METHODOLOGY
Stool samples were collected from 283 HIV positive and 245 HIV negative subjects and analyzed using direct diagnostic tests.


RESULTS
A total of 46 (8.7%) individuals were found infected with Blastocystis spp., including 6.7% HIV positive and 11.0% HIV negative. This species was more frequent in urban and semi-urban areas than in rural areas, but evenly distributed among genders and age groups as well as among all sectors of activity. The prevalence of Blastocystis spp. (11.3%) was higher in HIV+ patients with a CD4 count ≥ 500 cells / mm3, but no significant difference was found among HIV clinical stages. Likewise prevalence, the mean number of cysts per gram of stool was similar between HIV positive and HIV negative individuals. People infected with Blastocystis spp. showed diverse clinical signs, but only flatulence was significantly more prevalent. The frequencies of these clinical signs were not related to HIV status.


CONCLUSION
No clear relationship links the infection with Blastocystis spp. to HIV, although its presence was associated with digestive disorder, suggesting that this parasite might not be opportunist.


Introduction
Blastocystis spp. is a protist found in humans and some animals [1][2][3][4][5][6].Previous studies have shown a wider distribution in underdeveloped countries compared to developed ones [7][8][9].Within the affected communities, this protozoan is highly prevalent amongst the poorest and most deprived individuals, exhibiting poor hygiene [6].Although it is the most frequent protozoan reported in stool samples of both symptomatic and healthy individuals, its pathogenicity or clinical significance remains uncertain and controversial [4][5][6][7][10][11]. Indeed, Blastocystis spp. was considered as a commensal organism living in the human and animal intestine.The possibility that the immunosuppression influences the presence or severity of symptoms was proposed [12], though the presence of Blastocystis spp. in Human Immunodeficiency Virus (HIV) immunosuppressed individuals could not be confirmed [13].
The present study was an attempt to fill this gap by investigating the pathogenicity and the opportunistic character of Blastocystis spp.To this end, a crosssectional study was conducted in the Central Cameroon in order to compare the frequency of this species between HIV positive (HIV + ) and HIV negative (HIV -) individuals.

Ethical consideration
The study was approved by the Institutional Review Board of the Faculty of Medicine and Biomedical Sciences of the University of Yaoundé I. Prior sampling, authorizations were granted by the Directors of the health facilities (Yaoundé University Teaching Hospital, Yaoundé Central Hospital, Ntui District Hospital and Akonolinga District Hospital) where the participants were enrolled.Verbal agreements were obtained from those who agree to participate, under the discretion of the leaders of health facilities.All participants were attributed a unique identifier and their data thus analyzed anonymously.

Study areas
This study was conducted in three divisions (Mfoundi, Mbam & Kim and Nyong & Mfoumou) of the Center Region of Cameroon.These divisions were chosen to represent different levels of urbanization.Indeed, the Mfoundi division is generally considered as an urban area (distances between buildings is less than 200m), although some of its peripheral quarters are rural settings.Ntui and Akonolinga, capitals of Mbam & Kim and Nyong & Mfoumou respectively, are semiurban and semi-rural cities (intermediate zones), though the villages that are close to these cities are rural [14].

Study design
A hospital-based cross-sectional survey was carried out between July 2013 and September 2015 at the Yaoundé University Teaching Hospital (CHUY), the Yaoundé Central Hospital (HCY), the Ntui District Hospital and the Akonolinga District Hospital to compare Blastocystis spp prevalence between HIV infected and non-infected individuals.These health facilities were chosen since they have Units for management of HIV patients.People infected with HIV and visiting the management unit of each targeted health facility were eligible for the study and those who consented to participate in the study were enrolled as the reference group.HIV negative individuals were approached in the counselling services of the targeted health facilities after withdrawal of their HIV test results.Those HIV negative subjects who had already spent at least one year in the study area were eligible for the study and those who consented to participate were recruited and considered as the control group.Children under one year of age were excluded from the study due to the very low likelihood for exposure to contaminated water.All volunteers who agreed to participate in the study underwent a brief questionnaire and provided stool samples for Blastocystis spp.searching.

Questionnaire
All relevant information regarding HIV status, socio-demographic factors and clinical signs were gathered both by interview and consultation of medical notebooks of participants.The Cluster Differentiation 4 (CD4) cell counts were recorded from the medical registers of HIV positive patients.Indeed, CD4 counts were done every three months, according to the recommendations of the World Health Organization.

Sample collection and processing
A plastic vial was provided to each enrollee for stool sample collection.These vials were returned back with stools early in the morning the next day for analyses.Two approaches were used for stool sample processing:

Direct microscopy (wet mount)
The direct wet mounts of stools were performed using normal saline (0.9% NaCl solution), stained with Lugol's iodine (Art Pharmaceutical Ltd, Brossard, QC, Canada), and examined under light microscope (magnification 400x).Blastocystis spp.are spherical bodies of various sizes (4 -15 µm of diameter) that seemed optically empty, with a large vacuole pushing to the periphery the cytoplasm that contains small refractive nuclei [4,[15][16].

Formalin -Ether sedimentation
Stool samples provided by participants were weighted and 500 mg placed in a clean conical centrifuge tube containing 10 mL formalin (10%).In each conical centrifuge tube, the sample was dissolved and mixed thoroughly using a stick.The resulting suspension was filtered through a sieve into a beaker and the filtrate poured back into the same tube.The debris trapped by the sieve were discarded.Four milliliters of ether were then added to the mixture and homogenized by vigorous hand shaking.The content was therefore centrifuged at 2,000 rpm for 3 minutes.The supernatant was poured away and the sediments were stained with iodine.The preparation was then spread on a slide, covered with cover slip and examined using light microscope (magnification 400×) [17][18][19].

Statistical Analyses
All data relevant for this study were recorded into a purpose-built Microsoft Excel dataset and subsequently exported into PASW Statistics Version 18 (SPSS Inc., Chicago, IL, USA) for statistical analysis.Prevalence were expressed as the percentage of infected individuals among the total number of individuals examined.The mean number of Blastocystis spp.cysts was used to express the intensity of infection.The sampling fluctuation of intensity of infection was estimated using standard deviation (SD).Chi-square (χ 2 ), Fisher exact (f) and Mann-Whitney (U) tests were used to compare prevalence and mean intensity of infection between different groups (HIV status, sociodemographic classes and clinical status).For all statistical analyses, the threshold for significance was set to 5%.

Socio-demographic characteristics of the enrollees
Table 1 displays socio-demographic parameters of individuals enrolled in the study, both HIV negative and positive.A total of 528 individuals were enrolled, of which 283 (54%) were HIV positive and 245 (46%) were HIV negative.Among these enrollees, 294 were originated from urban settings, 162 from intermediate or semi-rural/semi-urban, and 72 from rural areas.Regarding gender, 172 (32.6%) males and 356 (67.4%) females were recorded.The sex ratio was female biased both among HIV positive patients (2.45) and seronegative subjects (1.72).According to the occupation of enrollees, 84% of participants were in the informal sector (small businesses and farmers), unemployed and inactive (children, pupils and students).The mean age of positive subjects was 43.3 (SD: 10.5) years, with a minimum of 19 and a maximum of 68 years old, whereas the mean age in HIV negative participants was 30.3 (SD: 17.4) years old, varying between 1 and 80 years of age.

Prevalence and intensity of Blastocystis spp. infection
A total of 46 (8.7%) individuals were found infected with Blastocystis spp., including 19 (6.7%) HIV positive and 27 (11.0%)HIV negative subjects (Table 1).Blastocystis spp. was more frequent in urban (10.9%) and intermediate (7.4%) settings than in rural areas (2.8%).The difference observed between the prevalence of Blastocystis spp. was significantly higher among HIV -compared to HIV + subjects in urban areas (P = 0.0156).Males (9.9%) and females (8.1%) were similarly infected with Blastocystis spp.Most of the age groups were infected, the frequencies being generally higher in seronegative than in HIV positive individuals.Blastocystis spp. was significantly more prevalent among HIV negative individuals aged 29 -45 years (P = 0.0119).This parasite was found in all sectors of activity, HIV negative individuals involved in the formal sector being most affected than their HIV positive counterparts (P = 0.0116).As regard to intestinal antiparasitic treatment, the prevalence of Blastocystis spp. was significantly higher among HIV - than in HIV + individuals who had taken an antiparasitic drug less than one month ago (P = 0.0333).When taking into account the immunity and clinical status of HIV positive individuals, the prevalence of Blastocystis spp. was higher in the patients presenting with a CD4 count higher or equal to 500 cells / mm 3 (P = 0.0453) (Table 2).No significant difference was found in Blastocystis spp.prevalence as regard to HIV clinical stages and antiretroviral treatment.

Discussion
Blastocystis spp. is a protozoan known to dwell in the colon of its human host where it reproduces by binary division [1,[20][21][22][23][24][25].The objective of this study was to evaluate the pathogenicity and the opportunistic character of Blastocystis spp.among HIV negative and HIV positive individuals.Overall, 8.7% of individuals tested were infected with Blastocystis spp.The prevalence of Blastocystis spp. was not significantly different between HIV positive and HIV negative individuals (P = 0.0802).These results corroborate those of Trabelsi et al. [6] who reported a prevalence of 7.3% in Tunis, and those of Haileeyesus et al. [26] who observed a prevalence of 6.3% in HIV positive patients in Fitche in Ethiopia.Although Blastocystis spp.prevalence reported by Tian et al. [13] were relatively higher (13.9% in HIV + patients and 21.7% in HIV - subjects) in Fuyang in China than in our study, the difference was not significant between the two groups (P = 0.1225).
Blastocystis spp. was more frequently observed in urban and intermediate areas than in rural areas.This parasite was evenly distributed among gender, age groups and was also found in people of all activity sectors, likely due to the cosmopolitan character of Blastocystis spp.[1,[21][22].
Regarding HIV positive individuals, the prevalence of Blastocystis spp. was higher in patients presenting with a CD4 count ≥ 500 cells / mm 3 .A significant difference in prevalence of infection with Blastocystis spp. was observed between CD4 slices (P = 0.0453) as previously reported by Tian et al. [13].Indeed, these authors found an average CD4 count of 668 cells / mm 3 among HIV + subjects infected with Blastocystis spp.Furthermore, this parasite was recorded only in patients at the clinical stage B, and in people receiving antiretroviral, although no significant difference was found in prevalence of Blastocystis spp. between different HIV clinical stages, as well as when individuals were receiving antiretroviral therapy or not.These results, together with the fact that no significant difference was found in cysts' loads and HIV status, suggest that this organism has a non-opportunistic character [13].
People infected with Blastocystis spp.present, for most of them, clinical symptoms associated with digestive disorder.The frequencies of clinical signs were not associated with HIV status.The clinical signs observed were abdominal pain, flatulence, asthenia, constipation, presence of blood in stool, diarrhea, but only flatulence was found to be associated with infection by this species.Trabelsi et al. [6] found similar results as they had observed symptoms in 72.1% of individuals infected with Blastocystis spp.These results are slightly different from those reported by Bourré [27] who found symptomatic infection only in 38.7% of cases.Individual susceptibility likely associated with parasite strains having higher pathogenic characteristics may explain these differences [5,28].Also, Tan and Suresh [25] suggested that the symptoms may be due to the accumulation of amoeboid forms of Blastocystis spp.strongly adhesive to the intestinal wall of the host.Lastly, co-infections were observed with yeasts, Entamoeba histolytica, E. dispar, Pentatrichomonas intestinalis, Iodamoeba buetschlii in the framework of the present study, the presence of these pathogens being likely amenable to induce potential bias related to the clinical disorder observed [6,[20][21][22][23][24]29,30].

Limitations of the study
In the present study, Blastocyctis spp. was detected using only direct diagnostic tests of stool samples.This approach might have led to the underestimation of the prevalence of the disease since only microscopy was used to diagnose this parasitic species.Indeed, in vitro culture of Blastocystis spp. as well as nested Polymerase Chain Reaction (PCR) using DNA extracted from human stool samples have been described to generally display more positive cases than direct microscopy or standard PCR [31][32].Also, the controversial results observed among different studies (including the present study) might be due to the presence of pathogenic and non-pathogenic genotypes.Unfortunately, the samples collected in the present study were not genotyped and this hypothesis could not be tested.

Conclusion
Blastocystis spp. is one of the most common human intestinal parasite.It was found in urban, semi-rural and rural settings in the Center Region of Cameroon.The presence of Blastocystis spp. was neither associated with HIV infection, nor with the clinical stage of HIV infection, suggesting that Blastocystis spp.might be a non-opportunistic parasitic species.Although the presence of this parasite was associated with digestive disorder, flatulence was the most important sign observed.

Table 1 .
Prevalence of Blastocystis spp.infection among HIV positive and HIV negative individuals according to socio-demographic parameters.

Table 2 .
Prevalence of Blastocystis spp.among HIV positive patients according to the immunity, treatment and clinical status.

Table 3 .
Frequencies of clinical signs presented by Blastocystis spp.infected individuals during the last month prior sampling.
N: number of individuals infected with Blastocyctis spp; NW: number with.

Table 4
Frequencies of clinical signs presented by all individuals during the last month prior sampling.
N: number of persons examined; NW: Number with; *: Significant difference.