Etiological surveillance of viral gastroenteritis in children aged less than 5 years in Tehran Province, Iran, 2023–2024

Abstract

Background

Acute gastroenteritis (AGE) is a primary cause of death in children less than five years old in low- and middle-income countries (LMIC), mostly caused by viral intestinal infections. While the disease poses a significant global burden, further investigation is needed to clarify the role of enteric viruses in disease pathogenesis. This work aimed to study the frequency of five major enteropathogenic viruses—Rotavirus (RV), Norovirus (NoV), Adenovirus (AdV), Sapovirus (SaV), and Astrovirus (AstV)— in stool samples of young children in Tehran, Iran, during 2023–2024.

Methods

A cross-sectional study was conducted on 203 stool samples collected from children under 5 years of age clinically diagnosed with AGE. Viral genomic RNA was extracted and amplified for the most common AGE viruses using the RT-qPCR assay.

Results

Out of 203 tested samples, 101 (49.8%) were positive for at least one of the viruses assessed. The most frequently detected enteropathogenic viral agents in children under 5 years were RV and AdV, while AstV was rarely detected. The positivity rate varies by age, gender, and season. Mixed infections were determined in 16 (7.9%) patients, with a predominance of RV and AdV. Infection with enteric viruses can occur throughout the year (peaking during mild-to-cold months), with fever, diarrhea, and nausea/vomiting being the most common symptoms among children.

Conclusions

Enteric viruses, particularly AdV and RV, contribute greatly to AGE among children in Iran. Continuous surveillance and long-term monitoring of enteropathogenic agents is essential to provide insight into the local circulation and characterization of AGE-causing viruses, guiding the implementation of targeted interventions and public health strategies to mitigate the risk of future outbreaks in the region.

Introduction

Acute gastroenteritis (AGE) is recognized as a common disorder involving infants and young children (aged ≤ 5 years) with diarrhea-related dehydration, posing a significant burden on public health and remaining a continuous threat to at-risk children [1, 2].

According to World Health Statistics, it is estimated that gastroenteritis is responsible for approximately 1.7 billion cases of diarrhea in childhood, with an annual rate of 525,000 deaths worldwide, standing out as the main cause of hospital admission in children living in industrialized nations and a major cause of death in children in developing countries [1, 3]. In the US, for instance, the annual incidence rate of AGE is estimated to be more than 10 million among outpatient visits and more than a million cases of severe infections [4]. Although the disease is often benign with self-limiting short-term clinical symptoms of diarrhea, fever, malaise, nausea, and abdominal discomfort, in low-and middle-income countries, the lack of basic sanitation, along with nutritional deficiencies, exacerbates the disease severity, increasing the risk of death in childhood, which typically requires medical attention [5]. In children aged ≤ 5 years, the disease is the second leading cause of death, with approximately 1,000,000 diarrheal cases and 500,000 deaths each year, with inadequate access to clean water and sanitation services making the disease a neglected threat [6]. Recent advances and the widespread application of molecular diagnostic tools with the aim of identifying etiological causes of AGE have revolutionized our understanding of epidemiological characteristics of viral gastroenteritis. It is now recognized that, in addition to toxic chemicals, enteric infectious agents are etiologically linked to AGE, which include viruses [∼70%], bacteria [10–20%], and parasites [< 10%], with Rotavirus (RV) being the most frequently detected viral agent and Norovirus (NoV) representing the second most frequent AGE-causing virus often associated with outbreaks in schools and childcare centers [7–9]. In addition to RV and NoV, a few additional viruses have been identified as etiologic agents of viral gastroenteritis, causing epithelial destruction and fluid absorption, with Astroviruses (AstVs), Adenoviruses (AdVs), and Sapoviruses (SaVs) showing variation in incidence rates and geographical distributions [10]. Worldwide, Rotavirus is responsible for 19-78.2% of cases of acute gastroenteritis. Norovirus accounts for approximately 16–18%, and AstV, AdV, and SaV together are also considered local etiological agents in causing the disease, with a prevalence rate of about 2–10% [11–15].

There is little information about enteric viruses circulating in Iran, with the roles of several medically important viruses—i.e., RV, NoV, AdV, AstV, and SaV—in community-acquired severe gastroenteritis having rarely been studied, and the burden of these viruses remains poorly characterized. Therefore, in designing the current hospital-based study, we aim to investigate the prevalence of five common enteric viruses among pediatric patients in Iran, analyze the clinical features, and explore the association between infection and age, sex, and the seasons in which the cases presented between 2023 and 2024.

Materials and methods

Study population

From October 2023 to October 2024, 300 samples were collected from children aged ≤ 5 years old, clinically diagnosed with AGE, complaining of ≥ 3 loose watery stools per day. These children attended the referral Children’s Hospital of the Tehran University of Medical Sciences, from whom 97 patients were excluded owing to incomplete demographic or medical data; thus, 203 patients were included for testing. This study was approved by the Ethics Committee of Tehran University Medical Science with approval ID (IR.TUMS.SPH.REC.1402.218). Informed consent was obtained from the parents or guardians of the children enrolled for the collection of samples and data.

Sample collection and preparation

For the present study, the sample size was calculated using the Exact binomial test (two-tailed), which yielded a minimum sample size of 172 participants. Studying a larger sample size than the calculation, a total of 203 fresh stool samples from children seeking medical care for AGE were collected mostly within the first few days after the onset of the disease, following the National Surveillance Protocol for Viral Diarrhea. These children mostly exhibited symptoms of fever, diarrhea, nausea/vomiting, and abdominal pain in combination with other rarely reported clinical manifestations. Children with underlying chronic diseases, malnutrition, positive bacterial cultures, or recent antibiotic therapy were excluded. After being collected using sterile, leak-proof containers without preservatives, all specimens were transported on ice to the Department of Virology at the Pasteur Institute of Iran in Tehran for enteric viral agent detection and stored at -70 °C until further processing. Demographic and clinical data (including sex, age, and symptoms) were gathered through a standard structured questionnaire and review of patient medical records. Details of the sample preparation procedure are provided in a recent publication [16]. Briefly, stool samples (1 g, pea-sized) were suspended in 10% (wt/vol) phosphate-buffered saline (PBS) (pH 7.4) (w/v) and centrifuged at 1500 ×g for 20 min after 10-second mixing. All supernatants prepared after centrifugation were divided into aliquots and stored at -70 °C before viral nucleic acid extraction and PCR analysis.

Viral nucleic acid extraction

Viral RNA extractions were carried out using the Nucleic Acid Extraction Kit (Zybio, China), in accordance with the manufacturer’s instructions. The eluted RNA was assessed for quality and quantity using the NanoDrop™ One spectrophotometer (Thermo Fisher Scientific, USA) and was kept at -70 °C until use.

Molecular detection of viral pathogens

The extracted RNAs were first analyzed using the GA Gastro5G OneStep RT-PCR Kit (GeneovA, Tehran, Iran) with an internal amplification control, which enables simultaneous screening for five gastroenteritis-causing viruses, including RV, NoV, AdV, AstV, and SaV in the same tubes. The assay, consisting of three fluorescent TaqMan probes, including FAM (for Norovirus, Sapovirus, Astrovirus), HEX (for Rotavirus), and ROX (for Adenovirus), was used for the experiment in the QIAquant Real-Time PCR cycler (Qiagen, Germany). Each reaction contains the same volume of GA Gastro5G Mastermix, DNase/RNase-free water, and DNA template (7µL) to make a 21 µl reaction. Thermal cycling conditions were 53 °C for 15 min, initial denaturation at 95 °C for 3 min, 46 cycles of two steps—5 s at 94 °C (denaturation) and 30 s at 60 °C (annealing, extension, and fluorescence measurement). The cutoff CT value for the tested viruses was considered 40. To ensure the reliability of the results, a negative, a positive, and an internal control were used in each test round.

Differential methods for Norovirus, Sapovirus, Astrovirus

Considering the inability of the study’s multiplex Real-Time PCR to differentiate NoV, SaV, and AstV in the FAM channel within a single tube, an in-house TaqMan RT-qPCR assay was carried out for NoV detection. Briefly, thermal cycling conditions were 37 °C for 30 min, initial denaturation at 95 °C for 5 min, followed by 45 cycles of 15 s at 94 °C and 30 s at 58 °C, using REVERTA-L RT reagents and PCR kit (AmpliSens, Russia) in the QIAquant Real-Time PCR cycler (Qiagen, Germany).

RT-PCR was then performed for AstV on the remaining samples that tested negative for NoV in the previous step, using the following in-house protocol: primary denaturation at 94 °C for 10 min; 40 cycles of 30 s at 94 °C for secondary denaturation, 30 s at 52 °C for annealing, and 45 s at 72 °C for extension, conducted in the SensoQuest Labcycler (SensoQuest GmbH, Germany).

In cases where both NoV and AstV were negative, a positive result with the GA Gastro Kit was considered indicative of SaV infection.

Statistical analysis

Statistical analysis of data was performed using chi-square and Fisher’s exact test in the SPSS v.27 program, and graphs were drawn using Prism v 8.0.3 software. In order to compare the groups of our study, the data in the 2 × 2 table were calculated based on the frequencies calculated in the crosstabulation table. OR was calculated according to the following formula: No. of infected cases in the exposed group × No. of non-infected cases in the non-exposed group and No. of non-infected cases in the exposed group × No. of infected cases in the non-exposed group.

Results

Among the 203 children with AGE included in the study, there were 116 males (57.1%) and 87 females (42.9%) with a sex ratio of 1.3 and a median age of 12 months, divided into three age groups: under 12 months (72 subjects, 40.9%), 12–24 months (58 subjects, 33%), and over 24 months (46 subjects, 26.1%) Table 1.

Table 1.

Distribution of study population by age and sex

	Total	Male		Female
		n	%	n	%
Total	203	116	57.1	87	42.9
Infected	101	57	56.4	44	43.6
< 12 Months*	37 (36.6)	24	64.9	13	35.1
12–24 Months*	30 (29.8)	16	53.3	14	46.7
> 24 Months*	27 (26.7)	12	44.4	15	55.6
Rotavirus	39	20	51.3	19	48.7
Adenovirus	40	18	45	22	55
Norovirus	24	14	58.3	10	41.7
Sappovirus	11	7	63.6	4	36.4
Astrovirus	4	4	100	0	0

* Indicates a P value > 0.05

Among all participants, 102 (50.2%) tested negative for the studied viruses which were RV, NoV, AdV, AstV, and SaV, while 101 (49.8%) were infected with one or more of these included viral pathogens of which 85 samples (41.9%) had mono-infections, and 16 samples (4.9%) had mixed infections, including 15 dual infections (7.4%) and a single triple infection (0.5%), showing typical seasonal patterns. AdV (40 cases, 19.7%) was the main cause of AGE, followed by RV (39 cases, 19.2%), NoV (24 cases, 11.8%), SaV (11 cases, 5.4%), and AstV (4 cases, 2%) (Fig.1). The prevalence of these pathogens varied by age and gender: under 12 months, with 64.9% males and 35.1% females, showed RV and AdV each at 33.3%, followed by NoV at 26.2%, SaV at 7.1%, and AstV at 0%; 12–24 months, with 53.3% females and 46.7% males, showed RV at 34.2%, AdV at 31.6%, NoV at 18.4%, SaV at 10.6%, and AstV at 5.3%; and over 24 months, with 44.4% males and 55.6% females, showed AdV at 45.2%, RV at 25.8%, SaV at 12.9%, NoV at 9.7%, and AstV at 6.5% (Fig.3). The most common dual co-infection involved RV and AdV (20.5%), followed by AdV and SaV (10%), RV and SaV (5.1%), AdV and NoV (5%), RV and NoV (2.6%), and AdV and AstV (2.5%). A triple co-infection (RV + AdV + SaV) was identified in 2.6% of samples (Fig.4 ). The patients showed various clinical symptoms, with diarrhea (48.2%), fever (35.1%), and vomiting/nausea (35.1%) being the most common (Fig.2).

Fig. 1.

Prevalence (%) of detected enteric viral pathogens (Rotavirus, Adenovirus, Norovirus, Sapovirus, and Astrovirus)

Fig. 3.

The prevalence of Rotavirus (RV), Norovirus (NoV), Adenovirus (AdV), Sapovirus (SaV), and Astrovirus (AstV) based on the identified pathogens in each age group. Each pie chart corresponds to an age group, and each slice of the chart represents a virus defined by the legend

Fig. 2.

The prevalence rate of clinical manifestations of the studied populations

Fig. 4.

Comparison of gender, prevalence, and infection rates based on age groups and type of pathogen among participants. The blue and pink bar charts represent the distribution of gender among the study participants, the prevalence of infection in the total population, in different age groups, and based on the investigated pathogens. The red bar charts indicate the rate of Co-infection based on the dominant pathogen in the study. The dot points represent the (OR) odds ratio of infection for the pathogens between men and women and co-infection based on the dominant pathogen, plotted with a 95% CI and a reference line at OR = 1. For cases where the OR (95% CI) was not calculated but a p-value is reported, it is due to the non-observation of that pathogen in one group

Seasonally, viral detection rates fluctuated across the four studied seasons, with the highest rates during mild to cold weather. AdV was mostly detected in fall (42.9%), spring (41.7%), and summer (40.6%), with only 28.6% in winter. Most RV cases occurred in winter (50%), but were also present in fall (42.9%), spring (39.6%), and summer (31.3%). NoV was primarily found in winter (35.7%), summer (25%), and spring (20.8%). SaV was detected year-round with similar prevalence in fall (14.3%) and winter (14.3%), followed by spring (10.4%) and summer (9.4%). AstV was rarely detected, with all positive samples found during summer (3 cases, 10.4%) and fall (1 case, 14.3%) (Fig.5).

Fig. 5.

Seasonal pattern of Rotavirus (RV), Norovirus (NoV), Adenovirus (AdV), Sapovirus (SaV), and Astrovirus (AstV) prevalence among infected patients in each season

Discussion

Acute gastroenteritis (AGE) is one of the leading causes of death among children living in developing countries. Factors increasing the risk of AGE among children are enteric pathogens, the age and sex of patients, the pattern of seasons, the level of parental information, and living in poor hygienic conditions with limited access to clean water [17]. Therefore, it underscores the need for implementing population-based active surveillance and regular risk assessments in regions with limited medical resources and poor healthcare systems to develop effective evidence-driven strategies aimed at minimizing future risks. In developing countries, including Iran, understanding the etiological causes of childhood diarrhea allows policymakers to plan disease control strategies, including implementing vaccination of vulnerable populations. In this study, we examined the prevalence of Rotavirus (RV), Norovirus (NoV), Adenovirus (AdV), Sapovirus (SaV), and Astrovirus (AstV) in stool samples from children diagnosed with nonbacterial gastroenteritis at a pediatric hospital in Tehran, Iran. Over the past few decades, advances in sequencing, omics studies, and new diagnostic techniques have revolutionized the investigation of gastrointestinal viruses. The recent improvement and widespread application of molecular diagnostic techniques have provided clinicians and scientists with a better understanding of the epidemiological characteristics of AGE-causing viral infections. Using multiplex PCR and differential tests, we analyzed 203 stool samples from children under age five and found that 85 samples (41.9%) tested positive for at least one virus causing gastroenteritis. Recent studies have suggested that about 70% of childhood gastroenteritis is viral, involving RV, NoV, AdV, SaV, and AstV [18]. Our data indicated that RV (39 cases, 19.2%) and AdV (40 cases, 19.7%) had the highest positivity rates across all age groups. AdV and RV were most prevalent in children under 12 months. The prevalence of AdV varies across regions of Iran; Saeed Shams et al. reported 17.7%, Motamedifar et al. found 9%, and Arashkia et al. reported 4.3% [18–20]. Our results align more with Shokrollahi et al.‘s study, which reported an AdV rate of 20% [21]. RV vaccination, the primary cause of acute gastroenteritis in children worldwide, began in 2008 and has reportedly reduced infection rates, mortality, and hospitalizations by around 40% among children under 5 years old [22]. In Iran, RV vaccination has just recently been incorporated into the national immunization program; therefore, our samples were collected from children who had not yet received the vaccine. Reports from Iran reveal RV prevalence rates of 61.1% in Babol in 2008, 24.3% in southern Iran in 2013, and 32% in Tehran in 2024 [16, 23, 24]. The nearly equal prevalence of RV and AdV infections in children under 5 in our study, along with discrepancies in rates, highlights differences in diagnostic methods, surveillance data, population sizes, and socioeconomic development levels. Consequently, more attention should be given to the rising number of positive cases, particularly AdV, in this age group.

Following RV and AdV, NoV was the third most frequently detected virus in our study (11.8%), followed by SaV (5.4%) and AstV (2%). Unlike RV, NoV is the leading cause of gastroenteritis across all age groups, accounting for about 17.7% of cases in children [25]. In agreement with previous findings, vomiting and abdominal pain were reported to be the most common symptoms in children with NoV infection [26]. Additionally, a significant correlation between NoV infection and dyspnea was noted in 14.3% of cases (P-value = 0.02). AGE caused by NoV is usually mild and self-limiting, but can be fatal in immunocompromised individuals. Most research on symptoms beyond fever, vomiting, and diarrhea focuses on case reports or immunocompromised patients, and our study did not include such cases [27, 28]. We also found a significant association between NoV infection and the age of patients, with the highest incidence recorded in children under 12 months (p-value < 0.05). A meta-analysis by Mohammad Farahmand et al. supported our findings, showing higher NoV prevalence in males and children under 1 year [25]. Another study by Weihuazou et al. indicated that 87% of children positive for NoV were under 3 years old, suggesting a strong connection between age and infection risk [29].

The clinical symptoms of the children in this study are shown in Fig. 2. Consistent with our findings, a recent systematic review from the Middle East reported that acute viral gastroenteritis in children is primarily characterized by diarrhea, vomiting, fever, and dehydration, with Rotavirus and Norovirus remaining the predominant pathogens. In addition, AstV, enteric AdV, and SaV infections have been detected in pediatric AGE cases across the region, with generally similar clinical presentations and variable severity depending on age and hydration status [11].

The most common symptoms among our patients were diarrhea (48.2%), fever and vomiting (35.1%), abdominal pain (13.1%), and bloody stools (7.7%). SaV-infected children showed vomiting in 5.4% of cases, mainly those over 24 months. Similar to our results, El-Heneidy et al. found that 72% of symptomatic SaV cases had vomiting and 48% had diarrhea [30]. There is no effective vaccine or specific antiviral treatment for SaV, and the infection clinically resembles NoV with a high rate of coinfections [31, 32]. As in our study, co-infections of SaV with RV (5.1%) and AdV (10%) were observed. There was no significant difference in virus detection between genders, although AstV infections were only found in male samples (100%) and in children over 12 months. The global prevalence of AstV ranges from 1.3% to 40.4% among children [33, 34]. Our findings are consistent with studies from Spain and Brazil, reporting lower AstV detection rates in children under 1 year compared to older children [29, 30]. Similarly, Barari Savadkoohi et al. reported a small number of cases confirmed for AstV in the north of Iran (5/208), while Najafi et al. detected a 20.4% infection rate among children with gastroenteritis in southern Iran [24, 35]. Conversely, S.D. Mousavi Nasab et al. observed a high frequency of AstV in males and children under 12 months [36]. In our study, co-infections were detected in 7.4% of cases (dual) and 0.5% (triple). The most common combinations were RV-AdV (20.5%) and SaV-AdV (10%). A similar study reported mixed infections in 4.4% to 29% of cases [37].

We also analyzed the seasonal occurrence of the studied infections. Seasonal trends show that diarrhea outbreaks often peak during colder months, with some reports indicating that RV, AdV, and NoV also surge during the cold seasons. In our study, viral infections varied by season, with the highest number of cases reported in spring (48 cases), followed by summer, winter, and autumn, although these differences were not statistically significant. Viral gastroenteritis transmission is complex and multifactorial. Behavioral habits, such as eating certain foods at specific times or increased water consumption during hot months, combined with reduced hygiene and climatic factors like humidity and temperature changes, can impact infection rates and epidemiological trends [38]. For instance, heavy rainfall alters the transmission dynamics of waterborne viral diseases [38]. Recent droughts and climate changes in Iran may also influence these patterns [39]. Since many gastroenteritis symptoms overlap with bacterial infections and may sometimes lead to unnecessary antibiotic administration, accurate diagnosis and a clear understanding of the true burden of these viruses are essential. This can reduce health impacts, economic costs, and enhance surveillance efforts nationwide, especially for vulnerable individuals like young children. In our study, all collected samples were first examined microscopically and cultured for bacterial contamination after collection. They were also checked microscopically for parasitic contamination. Those samples with no such contamination were considered candidates for diagnosing viral infections.

Limitations and conclusion

Regarding the current investigation, there are several limitations that need to be acknowledged. First, the studied samples were a small number collected from a single center in Tehran over just one year, limiting the generalizability of the results to other areas and time periods. Second, assessing the samples for only a small number of viruses may limit our understanding of other etiological causes of AGE in the region, representing not the confidential picture of circulating viral strains in Iran. Third, the study failed to type the studied pathogens, making it impossible to determine which genotypes were more prevalent during the time of study. Fourth, there are some limitations in accessing complete demographic information, particularly the age of some participants. Fifth, this study did not analyze the frequency of other recognized enteric viruses (e.g., bocaviruses and aichiviruses) in collected samples, which may falsely suggest that the disease may be caused by a non-viral agent when there is a strong possibility for another viral pathogen. Sixth, clinical symptoms were recorded for only 168 participants, and reports were based on the number of symptoms. Seventh, the SaV differential test was not performed, so co-infections involving this pathogen could not be assessed. Therefore, more comprehensive, multi-center, and longitudinal studies with a wider range of pathogens and a longer time span are needed in the future to provide more robust conclusions. The last limitation of our study was sampling time. It is important to collect samples within the first three days after symptoms onset, however, in some cases there was a delay in referring to the target medical center which may affect the positivity rate of viral infections.

The results of the present study support the circulation of enteric viruses among Iranian children presenting with AGE, with RV and AdV as leading causes in the region. However, more well-designed studies are required to provide a better insight into the role of enteric viruses in acute gastroenteritis. With continuous molecular detection through a robust surveillance system, the contribution of enteric viruses and their emerging strain to AGE is well-established, guiding policymakers and encouraging public health authorities to prioritize resources and implement targeted interventions for controlling future AGE-causing viral infections [40].

Abbreviations

AGE

Acute gastroenteritis

LMIC

Low- and middle-income countries

RV

Rotavirus

NoV

Norovirus

AdV

Adenovirus

SaV

Sapovirus

AstV

Astrovirus

RNA

Ribonucleic acid

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

Author contributions

Conceptualization and supervision were performed by Z.S. and A.M. Project administration was carried out by S.S.H.F., Z.A., and Z.F. M.SH. was responsible for formal analysis. Investigation and data curation were conducted by S.S.H.F. and H.K. The original draft was written by S.S.H.F. and H.K. The manuscript was reviewed and edited by A.M. and S.J.

Funding

Not applicable.

Data availability

The data are available from the corresponding author on reasonable request, subject to ethical approval.

Declarations

Ethics approval and consent to participate

All procedures performed in this study involving human participants followed the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments orcomparable ethical standards. Ethical approval for this study was obtained from TUMS Medical Ethics [IR.TUMS.SPH.REC.1402.218]. Informed consent was obtained from the parents of all patients.

Consent for publication

All authors agree to publish the research data.

Competing interests

The authors declare no competing interests.

Declaration of generative AI use

The authors used Grammarly solely for the check of grammar, spelling, and punctuation marks with the aim of improving the text readability.