Campylobacter and Rotavirus co-infection in diarrheal children in a referral children hospital in Nepal

Diarrhea, although easily curable, is a global cause of death for a million children every year. Rotavirus and Campylobacter are the most common etiological agents of diarrhea in children under 5 years of age. However, in Nepal, these causative agents are not routinely examined for the diagnosis and treatment. The objective of this study was to determine Campylobacter co-infection associated with Rotaviral diarrhea in children less than 5 years of age. A cross-sectional study was conducted at Kanti Children's Hospital (KCH), Kathmandu, Nepal from November 2017 to April 2018. A total of 303 stool specimens from diarrheal children were processed to detect Rotavirus using rapid Rotavirus Ag test kit, and Campylobacter by microscopy, culture and biochemical tests. Antibiotic susceptibility test of Campylobacter isolates was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines 2015. Of 303 samples, 91 (30.0%) were positive for co-infection with Rotavirus and Campylobacter ; Rotavirus monoinfection was detected in 61 (20.1%), and Campylobacter monoinfection was detected in 81 (26.7%). Patient’s age, month of infection, untreated water and frequent soil contact were the major risk factors for infections. Clinical features such as >9 loose motions per day, fever, vomiting, mild to moderate dehydration, diarrhea persisting 6-9 days and presence of mucus in stool were significant (p<0.05) clinical features and were more severe in coinfection compared to monoinfections in multivariate analysis. The study shows a high rate of Rotavirus and Campylobacter coinfection in children with diarrhea. Diagnostic based management of diarrheal cases can guide the specific treatment. In addition, the associated factors identified in this study can guide clinicians for clinical

Studies revealed that co-infection does exist between enteric bacteria and viruses (16)(17). This evidence collectively demonstrates that co-infection by bacterial and viral pathogens play a critical role in disease progression. Infectious diseases cause most of the child deaths in developing countries (18)

Results
Three hundred and three diarrhoeal patients less than 5 years of age were included in the study with age from 10 days to 59 months. The highest number of patients (n = 118) belonged to age group 7-12 months. In total, there were 207 (68.3%) samples from males and 96 (31.7%) from females.

Detection rate of pathogens
The study was focused on detection of Rotavirus and Campylobacter spp., and at least one of these pathogen was detected in 233 (76.9%) samples. Among 303 children with acute watery diarrhea, Rotavirus monoinfection was detected in 61 (20.1%), Campylobacter monoinfection was detected in 81 (26.7%), co-infection was detected in 91 (30.0%) of the cases (Figure 1).

Age wise distribution of different infections in children
The highest number of Rotavirus monoinfection was detected in 7-12 months age group category which accounted 29 (47.5%) of total Rotavirus monoinfection.
Similarly, highest number of Campylobacter monoinfection 34 (42.0%) was found in the <6 months age group. The co-infection was observed highest (35; 38.5%) on 7-12 months of age ( Table 1). Distribution of different infections in age groups was statistically not significant (p>0.05).

Risk factors for different infections in children
In multivariate analysis, infection in February was associated with a decreased risk of Rotavirus monoinfection (adjusted odds ratio AOR = 0.26, 95% CI = 0.07-0.98, P = 0.047) than in November. Except 25-36 months age, all age were significantly associated with decreased risk of Campylobacter monoinfection compared to <6 were significantly associated with a higher risk of co-infections compared to November (Table 2).
Abdominal pain and presence of pus cells in stool were less common features, which were significantly associated with Rotavirus monoinfection in multivariate analysis.
Pus cells in stool was common clinical feature, while fever and vomiting were less prevalent but significantly associated with Campylobacter monoinfection in multivariate analysis. More than 9 loose motions per day, fever, vomiting, presence of mucus in stool were most striking clinical features, while mild-moderate dehydration was less common compared with no-minimal dehydration but significantly associated with co-infections in multivariate analysis (Table 3).

Discussion
This hospital based cross sectional study explored the association between Campylobacter and Rotavirus, possible risk factors and specific clinical features. In this study, most cases of acute gastroenteritis were infants. Studies also revealed that diarrhea incidence peaks at age 6-11 months and then decreases with age (19)(20)(21).
Our study indicated that a higher frequency of diarrhea was seen in males consistent with past studies (9, 12, 22, 23). Males' preponderance to develop diarrhea can be explained by their increased susceptibility to outdoor physical activities thus exposure to unhygienic surroundings and flood-water during rainy seasons. In addition, it could be due to simply increased presentation of male patients at the hospital more than females (24). ). It appears that infants less than 6 months of age were initially protected against severe diarrhea, to some extent, by maternal antibodies and they seem to have acquired adequate immunity between 12 and 16 months of age.
Infants and young children aged 6-12 months appear to be at greater risk because of declined levels of maternal antibodies to rotavirus infection (12).
Age, sex, household income, breast feeding, meat consumption, food habit, water source, drinking water type, hygiene and education level of parent were not predictors of Rotavirus monoinfection in these children. While, the month of diarrheal illness does possess a risk for Rotavirus monoinfection, a higher infection during January-February contradicts with our study (15). In our study, age group was the significant predictor of Campylobacter monoinfection. No soil contact was associated with a reduced risk of Campylobacteriosis as compared to frequent soil contact. In Nepal, Campylobacter is not included in routine microbiological testing in patients with diarrhea for identification of cases of Campylobacteriosis. We did not find effects of age, sex, occupation, household income, breast feeding, meat consumption, food habit, water source, drinking water, contact with soil, hygiene and parent's education level as risk factor for co-infection in children.
In multivariate analysis, clinical picture of children with co-infection was more     Antibiotic resistance patterns in isolated Campylobacter spp.