1. Introduction

Diarrhea is a major cause of childhood morbidity and mortality in socio-economically developing countries. More than one million episodes of diarrhea occur every year among children under five years of age causing approximately 2.5million deaths ^{[1, 2]} Various microbial agents have been incriminated in diarrhea among which are enteric bacteria, parasites and viruses ^{[3, 4, 5]}. Candida, Trichosporon and Geotrichum are fungi that have been reported to cause diarrhea ^{[6, 7]}. Different groups of viruses have been showed to be responsible for the high incidence of acute viral diarrhea among children during their first years of life ^[8]. These viruses include rotavirus, norovirus, adenovirus and astrovirus ^{[9, 10, 11]}. The contribution of the various pathogens to diarrhea may differ substantially between regions depending on local meteorological, geographic and socio-economic conditions. Underlying reasons for the spread of diarrheal diseases are found in poor hygiene and sanitation, limited access to safe drinking water as well as inadequate education of health care providers and recipients ^{[12, 13]}.

In many developing countries including Nigeria, research works have mainly focused on bacterial and parasitic agents of diarrheal diseases, with little information on viral and fungal diarrhea. This research work tends to look into the aforementioned areas of need in this locality. It may be interesting to note that in many Nigerian hospitals, routine viral and fungal analysis of stool specimens are not carried out on patients with diarrhea infections. This may be due to the poor attention given to health issues in the country. Against this background, this research work was carried out to determine the prevalence of viral agents of diarrhea with particular emphasis on rotavirus, adenovirus and norovirus as causative agents as well as fungal agents with emphasis on Candida species. This work was carried out among children under 5 years of age with diarrhea in a tertiary health institution in Edo State, Nigeria.

2. Methodology

Study Area and Population: This study was carried out at the University of Benin Teaching Hospital, Benin City, Edo State and it is located in the Southern part of Nigeria. A total of two hundred and seven (207) stool specimens comprising 153 diarrheic stools and 54 non-diarrheic stool specimens were collected from children under 5 years of age.. This study was carried out between February 2011 and August 2012. Verbal informed consent was obtained from patients or guardian of the children prior to sample collection. Children who had taken antibiotics and/ or antifungal drugs in the last one month were excluded from this study. Ethical approval was obtained from the hospital ethics committee.

Specimen collection and processing: The samples were collected into sterile wide mouth specimen containers and were processed immediately for viral and fungal pathogens..

Detection of viral enteropathogens: Rotavirus, adenovirus and norovirus were detected by the immunochromatographic method. Rotavirus and adenovirus antigens were detected in the stool specimens of patients with clinical symptoms of diarrhea using VIKIA Rota-Adeno rapid test device (BioMerieux, France). The results were interpreted according to manufacturer’s instructions. Similarly, norovirus was detected using RIDA Quick Norovirus (N1403) rapid test device (R-Biopharm AG, Germany). The results were interpreted according to the manufacturer’s instructions.

Isolation and identification of fungal agents: This was carried out following standard microbiological technique. Yeast counts were determined by colony count and counts of >10⁴ cfu/ml were considered as overgrowth and therefore significant for diarrheal infection ^[7].

Statistical Analysis: This was carried out using odd ratio and chi-square(X²) test. A P-value of less than 0.05 was taken as statistically significant.

3. Results

A total of 153 children with diarrhea were tested for three viral agents (rotavirus, adenovirus and norovirus) and Candida species associated diarrhea. 56(36.6%) were pathogenic for at least one viral or fungal agent while none of the 54 children without diarrhea was pathogenic for viral or fungal agent. Fungal pathogenicity was based on fungal colony count greater than. 10⁴ cfu/ml which was considered as overgrowth and significant for diarrheal infection. Among patients with diarrheal infection, Candida growth were observed in 38 cases, but only 14 cases were found with Candida overgrowth (i.e >10⁴ cfu/ml). The control group had nine of Candida growth, but none had overgrowth.

Table 1. Overall prevalence of viral and fungal enteropathogens of diarrhea

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Table 2. Relationship between Candida species overgrowth and diarrhea

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Table 3. Sex distribution of infection

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Single viral pathogen had 39(25.5%), single fungal pathogen had 12 (7.8%) and mixed viral and fungal pathogens had 2(1.3%). (Table 1). There was an association between Candida species and diarrhea (X²= 4.72; P=0.030) (Table 2). The sex distribution of infection showed that males had 31 (36.9%) while females had 25(36.2%), but this was not statistically significant (P=0.92) (Table 3). Age group of the children did not show statistical significant (P=0.96) with infection (Table 4). The frequency distribution of viral and fungal enteropathogens of diarrhea showed rotavirus as the most predominant with 19.6% (Table 5). The distribution of patients with single and mixed infections is showed in Table 6.

Table 4. Age group distribution of enteropathogens

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Table 5. Frequency distribution of viral and fungal enteropathogens

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Table 6. Distribution of patients with single and mixed infections

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4. Discussion

The prevalence of three viral agents namely rotavirus, adenovirus and norovirus as well as fungal agents (Candida species associated diarrhea were investigated among children with diarrhea who were under 5 years of age. The overall prevalence of infection was 56(36.6%). A breakdown of infection rate showed that viral infection was 42(27.5%), fungal infection was 12(7.8%) and mixed viral and fungal infection was 2 (1.3%). The results of this survey showed the contribution of viral and fungal agents to diarrheal infections in this locality which are usually missed out due to non-performance of such investigations. However, it has been reported that 30 to 40% of all acute episodes of gastroenteritis are caused by viruses ^[14]. Similarly, fungal agents have been reported to cause diarrhea ^{[15, 16, 17]}..

In this study an association was observed between Candida species overgrowth and diarrhea (X²=4.72; P=0.030). This is consistent with the report of Nkuo-Akenji et al_. ( 17), who also found an association between Candida and diarrhea. Moreover, in a group of Nigerian children with diarrhea, 77% had fungi isolated from the stools; Candida was the most predominant isolates and was strongly associated with diarrhea ^[18]. A subsequent study in India reported Candida as the sole pathogen in 15% of diarrheal stool specimens ^[19]. Thus, these aforementioned studies further support the association between Candida and diarrhea.

There was no association between sex and infection (P=0.92), This could be due to the fact that risk factors associated with diarrhea are environmental and socio-demographic, rather than biological ^[20]. Age group of children also did not show statistical significance (P=0.96) as infection was found in all age group. However, 34 out of the 56 infected children were between 0 and 24 months. This is consistent with the reports of Patwari et al ^[21] and Orlandi et al ^[22] who stated that diarrhea is highly prevalent in children in the first two years of life. This could be attributed to the fact that children within this age bracket are exposed to a lot of playful activities which make them come in contact with potential pathogens.

The distribution of enteropathogens showed that rotavirus was the most prevalent with 16.3%, and was also found in both single and mixed infections. This is consistent with other reports ^{[23, 24]}.

5. Conclusion

A prevalence of 36.6% viral and fungal associated diarrhea was found in this study. This goes to show that viral and fungal agents played significant role in childhood diarrhea in this locality and they should not be undermined when investigating diarrhea. The non-inclusion of viral and fungal tests as part of routine analysis of stool specimens for diarrheal patients means that such important pathogens of interest are missed out (i.e unidentified), thereby compounding data collation as regards diarrheal infection. Thus, there is great need for health practitioners to look inward and see viral and fungal agents as possible causes of diarrheal infections and thus provide the means for carrying out such tests on routine bases and this will go a long way in checking the indiscriminate use of antibiotics which can give rise to bacterial resistance as well as antibiotic-associated diarrhea.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

We thank the Management of the hospital for permission to use the facility. We also thank the clinicians, medical laboratory scientists and other health staff for their support.

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