Feco-oral transmitted disease (FOTD) refers to the risk of contracting a viral, bacterial or parasitic infection carried by excrements of sick or asymptomatic people or animals . Intestinal parasites and enteropathogenic bacteria are directly or indirectly transmitted through food, water and fingers . Whatever the infectious agent involved, the consequences are digestive disorders, particularly diarrheal episodes which can progress to dehydration or even malnutrition in children .
Enteropathogenic bacteria and intestinal parasites remain by far a public health problem in Sub-Saharan Africa (SSA). The incidence of Salmonella spp infection in developing countries is 540 cases per 100,000 inhabitants (versus 0.2 cases/100,000 in temperate countries). In the most affected regions, the peak incidence occurs among children and adolescents aged from 2 to 15 years . Among the Salmonella infections, S. Typhi is responsible for approximately 21 million new infections each year. Annual mortality of this infection has increased by 39% from 1990 to 2010 and is estimated to be more than 190,000 worldwide . Salmonella infections are endemic in developing countries and associated with high mortality . On the other hand, intestinal parasitic infection (IPI) is one of the leading causes of childhood morbidity and mortality worldwide . It is estimated that three and a half billion people are infected with 450 million people symptomatic; the majority being children . On the educational level, the impact of IPI is noticeable with the decline in the child’s physical and intellectual capacities .
The particularity of these infections is that there are healthy carriers for both Salmonella and intestinal parasites. In fact, after healing from typhoid fever 2% - 5% of individuals continue to harbor S. Typhi which are excreted episodically in the stool and which can, therefore, be the cause of secondary cases . This asymptomatic carriage is common among children of poor neighborhoods as demonstrated by Praharaj et al. in India . In Africa, Le Noc et al. found an asymptomatic carriage of Salmonella in 2.6% of primary school children while the prevalence of 39.7% was found in the city of Buea  . Concerning IPI, a prevalence of 11.6% was found in a children’s population in Cameroon . Asymptomatic carriers for Salmonella and IPI represent a risk for the community (as they contribute to the persistence of the endemic state in households) and risk for the carrier (particularly in immunocompromised patients, who are more exposed to invasive salmonellosis and malignant anguillulosis) . However, data on the asymptomatic carriage of Salmonella and IPI are rare in our setting. It was, therefore, important to determine their prevalence and identify associated risk factors. This process could reduce the burden of enteropathogens in a limited-resources setting.
Study Design and Setting: A cross-sectional descriptive study was carried out in two public primary schools in Yaoundé (Minkoa Meyos II for the rural part and Nkolbisson II-A for the urban part) from October 2017 to May 2018. These schools are located in the 7th precinct of Yaoundé, the capital city of Cameroon, with a catchment of two million inhabitants.
Study Population: The children attending both educational institutions were recruited after the obtention of parents’ and administrative authorizations. The precinct, school and educational level of pupils were chosen after a random sample drawn at each level. Detection of IPI was done using a direct examination between slide and cover slide under photonic microscopy while Salmonella spp diagnosis was performed using stool culture on Hektoen medium after enrichment on Mueller-Kaufman medium.
Variables and Measurements: All pupils, whose parents/legal guardians had signed an informed consent form, were included. All children whose parents refused their participation or whose consent was withdrawn during the study or were on antibiotics or who presented fever, vomiting or diarrhea were excluded from the study. The screening was conducted by trained medical personnel. All participants (and their parents) were subjected to a face-to-face interview. Data were collected (using a standardized questionnaire) on sociodemographic characteristics (age, gender, vaccination status, risk factors for enteropathogens), social characteristic (availability of water at home or canteens at school). Clinical data were also recorded (general status, weight, abdominal exam findings) as well as bacteriological and parasitological parameters (type of analysis performed, cell morphology).
Laboratory Testing: For each patient, two stool samples were collected, using rectal swabs. The swabs were transported in their packaging to the bacteriology laboratory of the Yaoundé University Teaching Hospital (YUTH) where the analysis was carried out immediately. One rectal swab was used for direct examination under optic microscopy, in order to detect parasites or yeast cells. The other rectal swab was used for performing stool culture. Enrichment was done on Mueller-Kauffmann medium then subculturing was carried out on Hektoen agar with a drop of the solution after a maximum of three to six hours of incubation at 37˚C. The research for Salmonella spp on this medium was orientated by the appearance of the colonies. The likely colonies were H2S-positive and lactose-negative. At least five likely isolated suspicious colonies were identified. We tested for urease on the urea-indole medium. This was carried out on each suspect colony and incubated at 37˚C for 24 hours. Urease-positive colonies (urea-indole medium in two hours) were eliminated. We inoculated a classic mini gallery with urease negative colonies from the indole urea medium. The results were reported on the lab bench and the identification form.
Definitions: IPI was defined by the identification of parasites at the direct stool test (either protozoa or helminths). Salmonella infection was defined by the identification of any Salmonella colony on the culture medium.
Sample Size and Statistical Analysis: The sample size was calculated using Lorenz’s formula (StatCalc of Epi Info software). Using a previous prevalence of 39.7% of Salmonella infection in Buea (Cameroon) , with an 80% power to detect associations or differences and a 5% accepted margin of error, the minimal sample size estimated was 368 participants. Data were analyzed using EXCEL 2016 and EPI-INFO v.3.5 software. Discrete variables were presented as counts and percentages, and continuous variables as mean (standard deviation). The Chi-square test was used, and also the Student t-test where appropriate.
Characteristics of the Study Population
We included 368 (192 boys) pupils from both schools (184 in each) with a mean age of 8.99 ± 2.21 years. The most frequent age group was 8 - 10 years with 154 (41.85%) participants (Figure 1). They included pupils who were respectively recruited in the following classes: class 1 (n = 31), class 2 (n = 32), class 3 (n = 30), class 4 (n = 31), class 5 (n = 32) and class 6 (n = 28).
School and Clinical Characteristics
Schools had no running water or toilets with wet excreta disposal. Latrines were present at the urban school but not in the rural one. There was no canteen
Figure 1. Distribution of the study population by age groups and gender.
in both schools, children have to buy food from itinerant traders. None of the children lived near a farm. All children had been vaccinated as recommended by the national vaccination planning. They all said that they followed basic hygiene rules. The abdominal exam was normal for all children (no tenderness, no mass).
Bacteriological and Parasitological Profile
None of the stool samples was positive for Salmonella after stool culture.
The overall prevalence of IPI was 9.80% (6.52% in the urban school and 13.04% in the rural school, p = 0.026). This prevalence tended to be higher in females (11.98%) than males (7.39%) however, no evidence of statistical significance was found (p = 0.095). Monoinfection was found in 33 (91.66% of all infected children) children while coinfection was found in 3 (8.34%).
Among the parasites, we found 8.42% of protozoa (5.43% in the urban school and 11.41% in the rural one) and 1.35% of helminths (1.08% in the urban school and 1.63% in the rural one). This protozoa prevalence was significantly higher in the rural than the urban one (p = 0.028), unlike helminths whose prevalence was almost similar in the two groups. In monovariate logistic regression analysis, the 5 - 7 years (p = 0.008) and 11 - 13 years (p = 0.023) age groups were associated with IPI. However, the 5 - 7 years age group tended to be protective of IPI. Table 1 summarizes the factors associated with IPI.
The pathogenic protozoan species were represented by Entamoebahistolytica and Giardia intestinalis representing respectively 11.11% and 25% of positive stool exams. However, Entamoebacoli was the most common protozoa found in stool exams. The helminths were represented by Ascarislumbricoides and Enterobiusvermicularis respectively 5.56% and 8.33% of the positive stool exams.
Table 1. IPI associated risk factors.
Interestingly, the prevalence of Blastocytishominis, whose pathogenicity is controversial, is similar to that of Entamoeba. The prevalence of all parasites is presented in Figure 2.
We conducted this cross-sectional descriptive study for assessing the prevalence and associated factors of asymptomatic carriage of Salmonella and IPI in a pupil population from two primary schools in a sub-Saharan African setting. This study revealed no asymptomatic carriage of Salmonella, a prevalence of IPI of 9.80% (with protozoa as the main intestinal parasites) and an association with 8 - 10 years age group and rural school.
We did not find any asymptomatic carriage of Salmonella in our study. This result differs from that of Devi et al. and Le Noc et al., who respectively found a Salmonella carriage in 1% of pupils in India, and 2.6% of pupils in Cameroon and Ivory Coast  . This difference could be explained either by the intermittent excretion of Salmonella in the stool . Similarly, this result is lower than the 39.7% of carriage found in Buea (Cameroon) . The difference in age group could also explain this discrepancy, as the study in Buea was carried out on the global population and knowing that the risk for chronic carriage of
salmonella increases with age .
Figure 2. Prevalence of identified species of intestinal parasites.
We found an IPI prevalence of 9.80% in this pupil population (these subjects harbor one or more intestinal parasites). This prevalence rate is lower than that of Ahmed Salem et al. (33.4%), in 2012, among school children of the rural area of Mauritania and that of Adou-Bryn et al. (38.9%) among school children in Ivory Coast in 1997  . Our prevalence is comparable to that found by Saotoing et al. (10.74%) in schoolchildren of the Far-North region, in Cameroon . This difference could be explained by the systematic deworming program initiated in schools’ settings in Cameroon since 2004 and the laboratory methods (they used concentration and enrichment techniques for improving their results).
Children from the 11 - 13 years age group seem to be more infested than the other age groups. This finding is comparable to that of Adou-Bryn et al., who described that the 11 - 13 years age group was most affected by IPI . Ahmed Salem et al., however, found that children who are aged 10 years or less were the most infested . These results could be explained by the fact that between 11 - 13 years, it is the beginning of adolescence and this age group has less awareness of disease transmission methods and less adherence to hygiene measures.
IPI prevalence was statistically higher in rural than in urban areas (p = 0.026). This observation was similar to that made by Lehman et al. who found a prevalence of 14.67% in Douala and 39.22% in Njombé (p < 0.001) . This result could be explained in our study by the absence of latrines in rural areas, the absence of water and the presence of itinerant traders.
IPIs among children in Yaoundé were mainly protozoan (protozoan prevalence 8.4%). This finding is similar to that of Saotoing et al. (8.52%) and Adou-Bryn et al. (69.8% of the parasites encountered)  . This high rate of intestinal protozoan diseases indicates the high level of contamination of water and food with feces and the lack of hygiene and sanitation measures in these regions .
The pathogenic protozoan species were represented by Entamoebahistolytica and Giardia intestinalis. A similar observation was made by Ahmed Salem et al. who found a prevalence of 3.75% for Entamoebahistolytica and 9% for Giardia intestinalis . The same protozoa in different proportions (30.7% for Entamoebahistolytica and 5.5% for Giardia intestinalis) were found byBamba et al. . The prevalence of helminths in our study was 1.35% represented by Ascarislumbicoides and Enterobiusvermicularis (pinworm). This result is different from that of Adou-Bryn et al. (10.4% for helminths represented by Trichuristrichiura, Hymenolepisnana) and of that from Kyambikwa et al. (73.8% for helminths, represented by Strongyloidsstercoralis, Ascarislumbricoides, Trichuristrichiura, and Taeniasaginata)  . It is surprising to have cases of E. vermicularis. This should be because we used the swabs method which is close to the scotch test, the recommended technique for the search of E. vermicularis.
This study should be interpreted in light of some limitations. The stools were collected by swabbing which did not allow us to have a sufficient quantity of stool to carry out the stool concentration methods. These limitations could lead to an underestimation of the prevalence of intestinal parasites.
This study suggests that among pupils in Yaoundé, Salmonella spp infection is zero prevalent, while intestinal parasites are frequent. This parasitism is essentially protozoan and associated with the rural area and the 11 - 13 years age group. Regular screening followed by adequate treatment associated with water supply, toilet facilities and environmental hygiene will ensure the prevention of these parasitic infestations.
Availability of Data and Materials
The dataset analyzed during this study is available from the corresponding author on a reasonable request.
Conception and design: RNM, MT, HG. Data collection: RNM. Administrative support: MT, HG. Data analysis and interpretation: RNM, SRSN, NCNA. Drafting of the manuscript: SRSN, RNM, NCNA. Reviewing manuscript: MT, ELM, HG. All the authors read and approved the final draft for publication.
This work was approved by the institutional review board of the Faculty of Medicine and Biomedical Sciences, Yaoundé, Cameroon (registration number 0333/UY1/FMSB/VDRC/CSD). Administrative authorization from the Yaounde University Teaching Hospital, the Ministry of Basic Education, the Regional delegate for basic education and the Inspector of Basic Education of the Yaoundé 7th precinct and parents’ consent were obtained before collection. This work was carried out in accordance with the declarations of Helsinki . Patients were free to attend the study without any outside constraint. Consent forms were obtained, informed and signed by each participant’s parent. This work is reported in compliance with the STROBE checklist.
We thank all the staff of the laboratory units of the Yaounde University Teaching Hospital.
List of Abbreviations
FOTD: Faeco-oro transmitted disease
IPI: Intestinal parasite infection
SSA: Sub-Saharan Africa
SPSS: Statistical Package for Social Sciences
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