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Prevalence of enteric adenovirus and co-infection with rotavirus in children under 15 years of age with gastroenteritis in Qom, Iran

Aim: The current study is the first performed in Qom to determine the prevalence of adenovirus and co-infections with rotavirus in children aged <15 years with gastroenteritis symptoms. Background: Gastroenteritis-associated viral infections are a cause of death among young children worldwide, especially in developing countries. The Adenovirus species F (40 and 41) are responsible for a range of acute diarrhea cases among infants and children. Methods: Over a period of 9 months, a total of 130 children suffering from intestinal problems who referred to the infectious ward of Children's Hospital were enrolled in the current study. After clinical examination and collection of demographic information, fecal samples were obtained from the patients. Viral genomes were extracted with a commercial kit and amplified and typed by adenovirus-specific PCR assay. Adenovirus-positive samples were also evaluated for co-infection with rotavirus. Results: Patients had a mean±SD age of 2.66±2.72 years; 63.1% of patients were male and 36.9% were female. Adenovirus infection was identified in 23 cases (17.7%), 21 (91.0%) and 2 (9.0%) of which were type 41 and type 40, respectively. Fever was the most common clinical manifestation among adenovirus-positive patients. No significant difference was observed between adenovirus infection and clinical symptoms, seasonal pattern, or serum laboratory results. Co-infection was found in only 5 cases (21.7%). Conclusion: This study was the first to demonstrate adenovirus infection with a relatively high prevalence among children, especially infants, in Qom. The findings further revealed co-infection with rotavirus, indicating a health problem in this region.


Introduction
1 Acute gastrointestinal infections are the second leading cause of death in children, especially those the most important etiological agents responsible for about 70.0% of cases of gastroenteritis infection in children (4,5). Adenoviruses are relatively large, nonenveloped, ds-DNA viruses with >60 recognized serotypes that are grouped into 7 species, A to G (6).
Human adenoviruses (HAdVs) are the cause of common cold or flu-like symptoms and other ailments such as ophthalmic, gastrointestinal, conjunctivitis, hemorrhagic cystitis, exanthema, and urinary tract diseases in humans (7,8). Adenovirus species F (40 and 41) are responsible for 2.0-23.0% of cases of acute diarrhea among young children, especially infants, and has a higher prevalence in immunocompromised patients (9,10). These serotypes are mostly associated with gastroenteritis in pediatrics and can be transmitted predominantly via the fecal-oral route (11). Clinical features of enteric adenovirus types 40 and 41 in children include diarrhea, fever, vomiting, abdominal pain, and mild dehydration (12). Compared to other viral infections, adenovirus-induced disease has been reported with prolonged diarrhea (average 10.8 days), acute onset, less frequent vomiting, mild dehydration, and abdominal pain (13).
Infection with more than one microorganism simultaneously, including viruses, virus-bacteria, etc., is known as co-infection (14). Some studies have reported on co-infection of adenovirus with other viruses, including rotavirus, which can be associated with more severe symptoms (15)(16)(17). There are also reports on the prevalence and genotype of enteric adenovirus in some parts of Iran (17,18), but no information is yet available about this virus in Qom. Therefore, the present study aimed to determine the prevalence of adenovirus and coinfection with rotavirus in children aged <15 years experiencing gastroenteritis symptoms in this region. In addition, clinical symptoms, seasonal distribution, and laboratory data were compared in both adenoviruspositive and -negative groups.

Patients and sample collection
The current work was reviewed and approved by the Medical Ethics Committee of Qom University of Medical Sciences (Code No. IR.MUQ.REC.1394.087). The participants were enrolled in the study after providing the written consent of either the patients or their parents. During a 9-month period, 130 children from the total number of patients who referred to the infectious ward of Hazrat Masoumeh Children's Hospital in Qom, Iran, and had suspected signs/symptoms of viral gastroenteritis were included in the study. The sample size was calculated based on the prevalence estimation formula, according to the prevalence of 20.0% in the Shokrollahi study (19), 5.0% of type I error, and 7.0% precision.
After collecting demographic data, fresh fecal samples were obtained from the patients and immediately transferred to the laboratory. For molecular assessment, a part of each sample was stored at -80 °C until adenovirus/rotavirus polymerase chain reaction (PCR) assay. To evaluate intestinal pathogenic bacteria, the samples were cultured on blood agar, Hektoen enteric agar, xylose lysine deoxycholate agar, and thiosulfate citrate bile-salt sucrose agar, and all were incubated for 48 hours at 37 °C. Other laboratory tests such as white blood cell count (WBC), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) in serum and also stool examination for WBC, red blood cells (RBC), and ova/parasite were performed. Signs/symptoms from each patient were also documented.

Preparation
of stool samples, genome extraction, and PCR The stool samples were homogenized and treated with chloroform and phosphate buffer saline (PBS) (e.g., ~200 μl of stool, 9 ml of PBS, and 1 ml of chloroform). The mixture was vigorously vortexed for ~20 min. Finally, samples were centrifuged at 2100 g for 20 min at 4 °C (20). The supernatant was directly used for genome extraction using a commercial kit (CinnaPure Viral kit, SinaClon, Iran) according to the manufacturer's protocol. To detect infection and typing of HAdVs, a partial fiber gene of the virus was amplified by PCR. The primers used were AdF1 (5′-ACTTAATGCTGACACGGGCAC-3′) and AdF2 (5′-TAATGTTTGTGTTACTCCGCTC-3′) (21) to produce 541 bp and 586 bp fragments from Ad40 and Ad41, respectively. Adenovirus-positive samples were also evaluated by a conventional PCR using VP6 primers (F: 5′-GACGGVGCRACTACATGGT-3′ and R: 5′-GTCCAATTCATNCCTGGTG-3′) for co-infection with rotavirus. The expected fragment length for detecting rotavirus was 380 bp (22).
Gastroenterol Hepatol Bed Bench 2022;15(3):256-262 PCR reactions were performed in a final volume of 25 μL containing 10 μL of 1X Master Mix (Ampliqon, Denmark), 3 µl of extracted genome, 1 μL of each primer (10 pmol/μL), and 10 μL of sterile deionized water in a thermocycler (Eppendorf, Hamburg, Germany). PCR amplification thermal cycling conditions were as follows: an initial genome denaturation step at 95 °C for 3 min (one cycle), followed by 33 cycles of denaturation at 95 °C for 30 s, primer annealing (adenovirus: 54 °C for 30 s; and rotavirus: 55 °C for 60 s), extension at 72 °C for 30 s, and then a final extension step at 72 °C for 10 min. Finally, the products were electrophoresed on 1.0% agarose gel.

Statistical analysis
Student's t and chi-square tests were used for statistical analysis of the data. The age of patients was calculated as the mean±standard deviation (SD). A pvalue <0.05 was considered statistically significant. Statistics were analyzed using SPSS statistics software version 22 (IBM, NY, USA).
Adenovirus infection was detected among 23 cases (17.7%), of which 21 cases (91.0%) and 2 cases (9.0%) were determined as type 41 and type 40, respectively. The rate of adenovirus infection was higher in males (18 cases, 22.0%) than females (5 cases, 10.4%) (p=0.096). The age range in positive patients was from 2 months to 9 years (mean±SD age of 2.0±2.37 years). The prevalence was higher for patients <2 years of age (15 cases, 19.5% vs. 8 cases, 15.1%), but this difference was not statistically significant (p=0.520). No significant relationship was observed between the clinical symptoms and adenovirus infection (p>0.05).
After 48 hours, no growth of intestinal bacteria such as Salmonella, Shigella, or Vibrio spp. was observed. All stool samples were negative for the presence of parasites or their larvae. No relationship was found between infection and serum laboratory findings such as WBC, PMN, ESR, and CRP. Compared to adenovirus-negative cases, no RBC in the stool was observed in positive patients. The seasonal prevalence of adenovirus infection was mostly detected in spring and winter seasons, but no significant relationship was observed (p=0.285). More information is summarized in Table 1.
Co-infection of adenovirus and rotavirus was found in only 5 cases (21.7%) of patients. All positive cases were females (100.0%), and their mean±SD age was 4.23±3.52 years (range: from 5 months to 9 years). In the five cases of co-infection, fever (in 80.0% of cases) was the most common symptom followed by vomiting and abdominal pain, each accounting for 60.0% of cases. Most cases of co-infection (60.0%) were detected in the spring.

Discussion
After rotavirus and noroviruses, adenovirus serotypes 40 and 41 are the most commonly recognized viral agents in infants and young children and are the cause of diarrhea with fever lasting usually for 2 weeks. These serotypes have been estimated to account for 5% to 20% of hospital admissions, depending on economic status or geographic location (23)(24)(25). Therefore, infection caused by these viruses is very important in health systems.
To evaluate human adenoviruses directly in clinical samples, species-specific PCRs are better techniques because of their speed, sensitivity, and reliability compared to other methods, such as cell culture and enzyme-linked-immunosorbent assay (ELISA) (26). Therefore, the present study, analyzed samples by PCR to identify adenoviruses.
The isolation rate of Ad40 and Ad41 in the current study was 17.7% and ranged from 2 months to 9 years, indicating a relatively high prevalence of the disease. reported HAdV DNA in 2.0%, 4.3%, and 2.6% of patients, respectively (29)(30)(31). However, the current results are similar to the results described by Shokrollahi et al. (2014) in a report on HAdVs (20.0%) (19).
The prevalence of the viruses among patients with enteric infections in industrialized countries such as Japan, Australia, and France was 5.0%, 3.1%, and 3.1%, respectively (32)(33)(34). In developing countries, however, enteric adenovirus has been reported to be highly variable. The reported rates from Vietnam, Bangladesh, and Thailand were 2.8%, 3.4%, and 3.1% (i.e., 4.4% in children with gastroenteritis and 1.8% in children without gastroenteritis), respectively (34-36), while higher rates were reported from Nigeria (22.3%) (12) and Iran (20.0%) (19). In Iraq, the prevalence of adenovirus is reported at 14.6% (37). Qom is a religious city, and many people from neighboring countries travel to this city; therefore a higher rate of infection is not unexpected.  (38).
According to the age distribution of children in the present work, the highest incidence of adenovirus was found in children under 2 years of age (15, 19.5%). Other studies have also reported higher prevalence rates of adenovirus in younger children and infants. Sanaei Dashti observed a significantly higher prevalence in children <12 months of age (18 (40).
Although no significant difference was observed, we noted a high frequency of adenovirus infection in spring and winter. The seasonal prevalence was also reported by Motamedifar et al., who found adenovirus to be predominant in July and October (17). In some earlier reports, however, adenoviruses were identified throughout the year with no seasonal pattern or peak of virus infection frequency throughout the year (41).
In the current study, HAdV-41 and -40 contributed to 91% (21 of 23) and 9% (2 of 23) of positive samples, respectively. These findings are similar to those of Kotloff et al. (42), who indicated that Ad41 was more prevalent (68%) than Ad40 (32%  (41). It seems that viral co-infections have significantly increased (15). Unlike some previous studies that reported more co-infection in males (17,45), the current study found co-infection in females to be predominant. Moreover, the clinical manifestations of the disease such as fever, vomiting, and abdominal pain were higher in patients with coinfection than in others. The prevalence of co-infection was more significant in the spring and was similar to reports by Kamal Allayeh et al. in Cairo, Egypt, which stated that spring was the peak season for rotavirus and enteric adenovirus (40).

Conclusion
In summary, the present study is the first of its kind to investigate adenovirus infection in Qom, Iran, and data suggests that a relatively high prevalence of HAdVs could play an important role in diarrheal infections among children, especially infants. In addition, the results showed that co-infections of adenovirus and rotavirus are frequent, indicating a serious problem in our area. This may be due to poor hygiene in some parts of the province, so identifying gastrointestinal viruses in the sewage systems and drinking water sources can be valuable in preventing infections caused by them in the future.