The study was conducted from November 2018 to March 2020 in three care centers in the Abidjan district, namely the Urban Community Health Training Center (FSUCOM) of Anonkoua-Kouté, the Research and Training Care Center (CePReF) of Yopougon Attié, and the Pneumo-Physiology Department (PPH) of the University Hospital Center (CHU) of Cocody. This was a cross-sectional, prospective, and descriptive study that involved adult patients managed in each of the 3 study sites who gave informed consent. The three study sites were chosen to be representative of the Abidjan district. Microscopic and molecular analyses of this study were performed at the Parasitology Unit of the Institut Pasteur of Côte d’Ivoire. The immunological analyses were performed in each of the management centers of our study.

Before participation in the study, the objectives of the study and the procedures to be followed were explained in simpler and more accessible terms to the patients. Therefore, each patient enrolled in this project gave informed consent by signing a consent form in French according to the patient’s preference. Sociodemographic data (age, gender, place of residence and occupation), clinical data (medical history, HIV status, weight loss, vomiting, abdominal pain and fever) were collected. Some biological data (HIV/AIDS status, CD4/mm³) were also collected from all patients included in the study, using a questionnaire administered by the treating physician.

For patients who reported diarrhea, additional questions were asked to determine the cause of the diarrhea. The physical examination was performed by the attending physician at each management center. At the time of enrollment, each patient was informed about the parasitological analysis of the stool and the assessment of the CD4+ T-cell count to be performed on each purple tube blood sample. A 125-mL sterile stool jar was distributed to each volunteer for collection of their stool samples.

- Inclusion criteria

Participants of the study included HIV-infected adults over 18 years of age old, with or without diarrhea. All the participants were monitored in a health care center in Abidjan and their approval written informed consent was obtained.

- Exclusion criteria

Persons excluded from the study were those under 18 years of age, patients that were not followed in a treatment center, and those who refused to give their written informed consent.

Patient stools collected at each site were sent to the Parasitology Unit of the Institut Pasteur de Côte d’Ivoire (IPCI) and analyzed within two hours of release to identify vegetative forms of intestinal parasites. The collected stool samples were separated into three samples: the first sample was preserved in potassium dichromate (K₂CR₂O₇), the second sample was preserved in sodium chloride (NaCl) 9‰, while the third sample was used for microscopic examinations.

Once the stool arrives at the IPCI Parasitology Unit laboratory, it is first examined macroscopically by triturating it with a clean rod to note its consistency (appearance) and the presence of blood or mucus. Once the macroscopic examination is completed, 1 g of stool is taken from different places (3 to 4 places depending on the stool appearance) with a thin clean rod. This sample is diluted in a drop of physiological water (9‰) placed on a slide and covered with a coverslip. This preparation is observed under a light microscope with a 10× and 40× objective respectively (this preparation must not be thick and must be read completely). To better identify some parasites, a drop of Lugol’s was finally placed on the edges of the cover slip and the observation was done at the 40× objective. Finally, the staining by the Zielh Neelsen method was performed in order to highlight the parasite oocysts under the light microscope, first at the ×40 objective and then at the ×100 magnification.

This study was approved by the National Ethics Committee for Life Sciences and Health (CNESVS) of Côte d’Ivoire. All patients were informed of the objectives of the study protocol and an informed consent form was presented to them to obtain their agreement before the collection of stool and blood samples.

R software (version R x64 3.6.0 and R i386 3.6.0) was used for the analyses. Prevalence of infection is the proportion of positive samples out of all tested samples. Prevalence of infection was compared with sex and age group using the chi-square test or Ficher’s exact test (when at least one cell was less than 5).

The sensitivity of a diagnostic technique is the ability of the method to correctly identify the presence of a parasitic infection.

The specificity of a diagnostic technique is a measure of the performance of the test when used on negative individuals.

Sensibility ( % ) = Numberoftruepositivecases Numberoftruepositivecases + Numberoffalsenegativecases × 100

Specificity ( % ) = Numberoftruenegative   cases Numberoftruenegativecases + Numberoffalsepositivecases × 100

Cohen’s kappa coefficient (ƙ) assesses the degree of agreement between diagnostic techniques and uses the 2 × 2 contingency table. The statistical coefficient (ƙ) was used to determine the strength of agreement based on the following criteria:

—<0 = no agreement;

—0 - 0.20 = poor agreement;

—0.21 - 0.40 = average agreement;

—0.41 - 0.60 = moderate agreement;

—0.61 - 0.80 = considerable agreement;

—0.81 - 1 = near perfect agreement.

P-values less than 0.05% and 95% confidence intervals were considered statistically significant associations.

Logistic regression was used to determine factors associated with Cryptosporidium spp. using R software (versions Ri3864.0.0.0). For this purpose, the association between Cryptosporidium spp. infections was defined as the independent variable.

The dependent variables were analyzed using univariate analysis and the strength of each association was measured by an Odds Ratio (OR). All variables with a minimum p-value criterion (p ≤ 0.2) were specified and included in the multivariate analysis at a 95% Confidence Interval (CI). A p-value ≤ 0.05 was considered statistically significant.

The parasitological survey was conducted on 363 HIV-infected patients of whom 114 (31.40%) were men [95% CI 37 - 47] and 249 (68.59%) were women [95% CI 52 - 62].

The mean age of the participants was 44.87 years with extremes ranging from 18 years to 72 years (Table 1). The age groups of 40 - 50 and 51 - 61 years were the most represented with respectively 37.74% and 27.82% of the studied population. Patients of 40 - 50 years (36.52%) old were the most numerous to participate in the study. However, the lowest participation was observed in adults over

Note: ND: Not defined.

61 years old (2.20%).

Several types of HIV were identified in this study in which HIV Type 1 represented 86.22% of the study population.

The liberal profession (67.21%) was predominant among all the professions in the study population. Professional occupation (67.21%) was predominant among all occupations in the study population (Table 1). The majority (92.83%) of the PLHIV in the study were on ARV treatment (Table 1).

Direct examination of samples by Zielh Nelseen staining revealed 14 samples positive for Cryptosporidium spp. (Figure 1) out of 363 samples, representing

3.86%. Only the genus of the parasite has been identified.

A total of 14 of 363 stool samples examined microscopically contained Cryptosporidium spp. oocysts, for a prevalence rate of 3.86%. Males (4.38%) were more parasitized than females (3.61%) (Figure 2). No statistically significant difference was observed (p-value = 0.962). The 29 - 39 age group had the highest prevalence rate (8.97%) (Figure 3).

The COWP gene targeted by the two primer pairs by Nested PCR revealed fragments of 640 base pairs corresponding to the genus Cryptosporidium. Analysis of the Nested PCR (molecular biology) results identified 18 samples positive for Cryptosporidium spp. (Figure 4) out of 363 samples analyzed, a proportion of 4.95%. This analysis technique was used to identify 18 egg-infested samples of the genus Cryptosporidium.

Figure 5 shows that the prevalence of Cryptosporidiosis infection in females (5.22%) is higher than in males (4.38%). However, this difference is not significant (χ² = 1.3415, p = 0.247). In terms of age, the 29 - 39 age group had the highest prevalence rate (10.25%) (Figure 6). However, statistical analysis showed that there was significant variation between age groups (χ² = 43.0411, p = 0.00481).

The observed sensitivity rate showed that PCR is more sensitive than microscopy,

but no statistically significant difference was observed (p-value = 0.24). A Cohen’s kappa of 0.17 was obtained between the diagnostic techniques, reflecting poor agreement between these two diagnostic techniques for the identification of Cryptosporidium spp. (Table 2).

Concerning the main variables identified, namely gender, age, occupation, only CD4 count was the factor associated with Cryptosporidium spp. infection for both microscopy (OR = 0.887, p = 0.001) and PCR (OR = 0.896, p = 0.001) (Table 3).

Note: NA: Not Applicable; P: p-value; OR: Odds Ratio.