Rotavirus Infection Among Hospitalized Children With Gastroenteritis in A Teaching Hospital in Central Iran: A Cross-Sectional Study

Human Rotaviruses are important causative agent of acute gastroenteritis among young children globally. Therefore, the study of diarrheal samples obtained from children in order to determine the prevalence and dominant genotypes of Rotavirus is indispensable to prevent inappropriate prescription of antibiotics and to assess the necessity of vaccine immunization. We aimed to investigate the prevalence of Rotavirus infections among children with acute diarrhea and to study the association of the disease with paraclinical parameters in Qom, central Iran. Methods We collected stool samples from children admitted with acute diarrhea to a teaching hospital. Enzyme-Linked-Immunosorbent Assay (ELISA) was used to detect Rotavirus antigens. Blood samples were taken to measure CBC, ESR, and CRP levels. Rotavirus infections in and dryer with the peak rate happening in Winter(7, 17–19), the region countries including Iran are an exception showing no distinct peak(20). Our results showed a higher albeit non-signicant rate of rotavirus positivity in Winter (33%) among children hospitalized with acute diarrhea without any clear peaks. This lack of signicant variation may be due to the hot desert climate in Qom with low rate of rainfalls. The same factor may account for the lower rate found in this study compared with other regions of Iran. We found signicantly lower levels of CRP, ESR, and WBC in patients with rotavirus-related diarrhea compared to rotavirus-negative children. This nding may be due to the higher rate of bacterial infections in rotavirus-negative children hospitalized for diarrhea.


Introduction
Rotavirus, a double-stranded RNA virus from the Reoviridae family, is one of the major causes of gastroenteritis among children and teenagers globally. It can also cause encephalitis, exanthema subitum, otitis media, necrotizing enterocolitis and hepatic abscesses (1)(2)(3)(4).
Symptomatic infection is most common in children aged 6 to 24 months, however, asymptomatic infection can occur at younger ages too (when the maternal antibodies are still in circulation) (5). Antirotavirus antibodies are highly prevalent among adults, probably due to asymptomatic re-infection. The virus is transmitted through the oral-fecal route, as well as iatrogenically(6).
Studies show that 500 to 600 thousand children die annually due to Rotavirus infection, 85 percent of which are from low-income countries (1,7,8). An effective vaccination program can reduce hospitalization rate and expenses(8-10). Due to its high mortality and case-fatality rates (8, 11), the World Health Organization(WHO) has recommended hospital monitoring to identify common rotavirus genotypes in order to develop vaccines. Typing is based on two capsid proteins VP4 (P) and VP7 (G) (12). Rotavirus detection in stool samples is not a routine laboratory work-up and can be achieved using a variety of methods with different levels of diagnostic accuracy including enzyme-linked immunosorbent assay (ELISA), latex agglutination (LA), electron microscopy (EM), and molecular methods (13). Due to the lack of information about the prevalence of Rotavirus infection in Qom province, physicians usually treat diarrhea empirically. This approach leads to antibiotic over-prescription which in turn can increase antibiotic resistance where conservative treatment would su ce. This study aimed to investigate the prevalence of Rotavirus infection and its association with clinical and laboratory markers among hospitalized children with diarrhea.

Patients and design
We conducted a cross-sectional study of rotavirus prevalence among 199 patients hospitalized with diarrhea at Hazrat-e-Masoumeh pediatric hospital during a period of one year. We included children more than 1 month of age who were hospitalized with acute diarrhea. Individuals with bloody diarrhea were excluded from the study. All patients underwent laboratory testing including CBC, ESR, and CRP as well as stool examination for rotavirus infection using antigen(Ag) detection method. The study protocol was approved at ethics committee of Qom university of medical sciences under the ethics code (IR.MUQ.REC.1394.47).

Specimen collection
All patients were enrolled after consenting for participation. Stool samples (1-2 gr) were collected inside a clean, dry and waterproof container with no detergent. Peripheral blood samples were taken for laboratory evaluation including CBC, ESR and CRP at the time of enrollment. All data were recorded in uni ed data collection forms.

Rotavirus Detection
Fifty mgs of a solid sample or two drops of liquid sample (equivalent to 50 mg) were transferred into the test tube. The tube was capped and shaken for two minutes to ensure dissolution into buffer liquid. Then two drops (equivalent to 80 cc) of the sample was loaded on the diagnostic kit making sure no bubbles were formed. Finally, the Rotavirus antigen was added to the kit and the test result was read after ve minutes. The results were reported within 10 minutes and were considered invalid if not read within 20 minutes of loading. The test was considered as positive for Rotavirus if both C and R lines were illuminated. The test result was considered invalid if the C line is not there.

Statistical Analysis
The data were analyzed using SPSS software (version 22). The mean and standards deviation were calculated for continuous variables. Absolute and relative abundance were counted for the numerical variables. The chi-squared, Fisher's exact test and t-test were used to assess the relationship between clinical and laboratory features with rotavirus positivity. A p-value less than 0.05 was considered statistically signi cant.

Results
One hundred and ninety-nine children were recruited to the study including 118 boys (59.3%) and 81 (40.7%) girls. The mean age of children was 3.3 year (1-15 year). Sixty-nine (31.7%) of patients received antibiotic treatment. The prevalence of Rotavirus-associated diarrhea was 20.1% among children hospitalized with diarrhea.
The prevalence of the infection was 22.8% and 16% among boys and girls respectively; this difference was not statistically signi cant (p = 0.23).
One-hundred-and-nineteen (59.8%) of 199 participants were hospitalized for diarrhea in spring being in prevalence followed by 47(23.6%) cases in summer, and 33(16.6%) cases in winter. The seasonal distribution of the Rotavirus-associated diarrhea was 15.9% in spring, 21.2% in summer, and 33.3% in winter; with no signi cant difference across seasons (p = 0.086).

Discussion
We found that 0.20 (95% CI 0.14-0.26) of collected stool samples from hospitalized children with diarrhea were positive for Rotavirus. This is considerably lower than previous studies from Iran. A 2014 systematic review on prevalence of Rotavirus infection in Iran showed that Rotavirus infection rates for hospitalized children were slightly higher than outpatients (5). They reported an overall proportion of 0.35 (95% CI, 0.28-0.41) among Iranian children with gastroenteritis with a wide variation between 11.36-79%(5). A 2017 meta-analysis showed a higher overall estimate of about 39% (14). One reason for the difference in our results compared to the overall rates in Iran may be due to the regional epidemiologic Also, our study population had a wider age range (1-15 years) than most of previous studies that only included patients younger than 5 years. This factor as well as the decreased chance of RV-related diarrhea as children get older, may account for the apparently lower rate of Rotavirus infection in the current study population.
In spite of the wide age variation in our population, the mean age of patients in the rotavirus-positive group was 2.3 years which was lower than the mean age of the sample. Based on this nding which is consistent with previous ndings, we postulate that the likelihood of rotavirus infection is most common in children 2-3 years of age (5,15,16).
While most studies have shown a higher rate of Rotavirus infections in colder and dryer seasons with the peak rate happening in Winter (7,(17)(18)(19), the Gulf region countries including Iran are an exception showing no distinct peak (20). Our results showed a higher albeit non-signi cant rate of rotavirus positivity in Winter (33%) among children hospitalized with acute diarrhea without any clear peaks. This lack of signi cant variation may be due to the hot desert climate in Qom with low rate of rainfalls. The same factor may account for the lower rate found in this study compared with other regions of Iran.
We found signi cantly lower levels of CRP, ESR, and WBC in patients with rotavirus-related diarrhea compared to rotavirus-negative children. This nding may be due to the higher rate of bacterial infections in rotavirus-negative children hospitalized for diarrhea.
Our study was limited by several factors: we used antigen detection method which has sub-optimal accuracy and did not assess for the genotypic variation of Rotavirus in our population. Also, we did not assess for the etiology of diarrhea in non-rotavirus cases making judgement about the difference in laboratory markers di cult.

Declarations
Ethics approval and consent to participate: All patients were enrolled into the study after taking an informed consent. The study protocol was approved at ethics committee of Qom university of medical sciences under the ethics code (IR.MUQ.REC.1394.47).
Consent for publication: All authors have read and agreed to publish the present manuscript.
Availability of data: The data obtained in the present study are fully presented in the manuscript.
Competing interests: The authors declare that there is no con ict of interest.