Molecular Characterization of Enteric Viruses Causing Acute Gastroenteritis among Children under 5 Years Old in Distrito Central, Honduras

Diarrheal diseases constitute a significant public health problem in terms of mortality and morbidity. The World Health Organization (WHO) estimates that this disease causes approximately 2 million deaths annually, mostly in children under 5 years of age in developing countries [1]. In Honduras, as well as worldwide, rotaviruses (RVs) have consistently emerged as the single most important etiologic agent in infants and young children [2]. However, other viruses such as noroviruses (NoVs) [3, 4] and astroviruses (HAstVs) [5, 6] have also been shown to be responsible for acute gastroenteritis among children under 5 years old.

RVs are classified in a binary system of genotypes based upon the main capsid genes, namely, the VP4 gene (P genotype) that encodes for the spike protein and the VP7 gene (G genotype) that encodes for the major outer capsid glycoprotein. Although 42 G and 58 P genotypes have been described in humans and animals worldwide [7], only a few combinations of G and P genotypes are predominantly detected in humans [8]. The most frequently detected human rotavirus genotypes are G1P[8], G2P[4], G3P[8], G4P[8], G8P[8], G9P[8], and G12P[8] [9, 10]. Regarding NoVs, they are now classified into ten genogroups (GI-GX) and 48 genotypes [11, 12], of which GI, GII, and GIV genogroups cause disease in humans [13]; GI and GII noroviruses are further divided into 9 and 22 different genotypes, respectively [14]. HAstVs are classified into eight classic genotypes (i.e., HAstV-1–8) known to cause mild to severe gastroenteritis, usually in young children [6]. The monovalent rotavirus vaccine (Rotarix™ GlaxoSmithKline Biologicals, Belgium) is currently being applied in Honduras and several Latin American countries; however, no broadly effective vaccines are developed for NoVs and HAstVs [15].

Unfortunately, there is limited information in the country concerning the etiologic role of these agents in acute gastroenteritis. This study aimed to investigate the frequency, determine the genotypes, and determine the epidemiological characteristics of three of the major enteropathogenic viruses, rotavirus group A (RV-A), NoVs, and HAstVs, in preschool children in Distrito Central, Honduras.

The study population consisted of children under 5 years old who visited selected healthcare centers for medical attention and experienced an episode of acute gastroenteritis between October 2010 and July 2011. An acute gastroenteritis episode was defined as three or more depositions in the last 24 h of liquid or semi-liquid consistency, with or without fever and vomiting, and less than 14 days of duration. We selected three healthcare centers: Maternal-Child Block of the Hospital Escuela Universitario; Alonso Suazo Healthcare Center; and Specialties Hospital of the Instituto Hondureño de Seguridad Social (IHSS), in the cities of Tegucigalpa and Comayagüela of the Distrito Central, Honduras (Fig. 1). Parents or legal tutors of children who fulfilled the case definition of acute gastroenteritis were invited to participate. Only children from whom written informed consent, an epidemiological questionnaire, and stool samples were provided were included in the study.

Stool samples were collected from all participants and immediately transported on ice to the laboratory of the Centro de Investigación en Cáncer y Patogenos Asociados (CICPA) at the Universidad Nacional Autónoma de Honduras (UNAH) in Tegucigalpa, Honduras. RV-A and HAstVs detection was carried out in fresh samples. Then, a 10% fecal suspension was prepared in Tris-HCl 0.01 m with 0.0015 m CaCl₂ pH 7.2 and stored at −20°C for molecular characterization, including molecular detection and genotyping of RV-A, HAstVs, and NoVs.

Stool samples were screened for RV-A using a commercial immunochromatographic assay (Rota-Strip, Bio-Concept, Belgium). An enzyme-linked immunosorbent assay (RIDASCREEN Astrovirus, R-Biopharm, Germany) was also applied to screen stool samples for HAstVs. The tests were performed according to the manufacturer’s instructions.

For molecular characterization, the total RNA extraction of 140 μL fecal suspensions of all specimens was conducted using a commercial kit (QIAamp viral RNA mini kit, Qiagen, Germany) following the manufacturer’s instructions. A complementary DNA (cDNA) reaction was carried out using a random initiator, as described by Ferreira et al. [16]. Briefly, 10 μL of total RNA were mixed with 2 μL of DMSO, heated at 97°C for 7 min, and rapidly cooled on ice for 2 min. Then, 4 μL dNTPs 2.5 mm (Promega, USA), 10 μL 5X PCR buffer (Promega, USA), 5 μL MgCl₂ 25 mm (Promega, USA), 1 μL SuperScript III reverse transcriptase 200 U/µL (Invitrogen, USA), 2 μL random initiator 50 ng/μL (Invitrogen, USA), and 16 μL nuclease-free water (Promega, USA) were added to each tube for a final volume of 50 μL. The mixed reaction was centrifuged at 16,000 g for 20 s and then incubated at 25°C for 5 min, 50°C for 1 h, and 70°C for 20 min. The synthesized cDNA was stored at −20°C until use.

RV-A capsid genes were detected by RT-PCR with a pair of consensus primers for VP7 (9con1/9con2) or VP4 (4con3/4con2) genes (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000540253), as previously described [17‒20]. These products were genotyped by heminested multiplex-PCR with sets of specific primers for the G1–G5, G9, and P[4], P[6], P[8], and P[9] types (online suppl. Table 1), as previously described [17‒20].

All positive and 10% negative samples in the HAstV immunoassay were subsequently confirmed by RT-PCR using specific primers Mon269/Mon270 directed to a region of ORF2 of HAstV (online suppl. Table 1), as previously described [21]. The 449-bp RT-PCR products were analyzed by agarose gel electrophoresis and further genotyped by sequencing. For genotype assignment, isolated sequences were submitted to BLAST and compared for homology with reference sequences available in GenBank.

All fecal samples were tested by RT-PCR using a generic NoV PCR system based on the degenerate primers Mon431/Mon433 and Mon432/Mon434 for region B within the 3′-end of ORF1 (RNA polymerase) as previously described [22, 23] (online suppl. Table 1). Positive samples were further genotyped by sequencing the 213-bp RT-PCR products. The Norovirus Typing Tool (https://www.rivm.nl/mpf/typingtool/norovirus/) was used for the assignment of NoV genogroup, genotype, and variant of the GII.4 genotype [24].

RT-PCR products corresponding to NoV and HAstV were quantified by Nanodrop 2000 (Thermo Scientific, USA) and adjusted to a final concentration of 30 ng/μL with nuclease-free water (Promega, USA). PCR products were shipped to Macrogen (New York, USA) for purification and sequencing. Sequences were visualized with Sequencher version 5.0 (Gene Codes Corporation, USA) and aligned with BioEdit version 7.1.3 (Informer Technologies, USA). The evolutionary history was inferred using the neighbor-joining method. The bootstrap consensus tree inferred from 1,000 replicates represents the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees where the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in units of the number of base substitutions per site. The analysis involved 32 and 38 nucleotide sequences for HAstV and NoV isolates, respectively. Nucleotide sequences included those isolated in this study, as well as a set of representative reference sequences and sequences isolated by authors from Latin America and available in GenBank. Codon positions included were 1st, 2nd, 3rd, and noncoding. All positions containing gaps and missing data were eliminated. There were a total of 334 and 192 positions in the final dataset for HAstV and NoV isolates, respectively. Evolutionary analyses were conducted in Mega 11 [25].

All participants’ data were anonymized and managed confidentially. An electronic database was created in Microsoft Excel (Microsoft, USA). Data was analyzed with EpiInfo version 7.2.6.0 (Centers for Disease Control and Prevention, USA) [26]. Measurements of central tendency for discrete variables and frequencies for qualitative variables were calculated. The χ² or Fisher test was used to evaluate and compare proportions between variables. A p value <0.05 was considered statistically significant.

A total of 100 stool samples from children under 5 years of age with acute gastroenteritis were tested for RV-A, NoVs, and HAstVs. Of all the samples analyzed, 31.0% were positive for at least one of the three viral agents under study. Table 1 shows each viral agent’s individual frequencies: RV-A 14.0%, NoVs 13.0%, and HAstVs 5.0%. Of the total positive samples, RV-A was detected in 45.2% (14/31), NoVs in 41.9% (13/31), and HAstVs in 16.1% (5/31). Mixed infections between RV-A and the other viral agents were not detected. However, a coinfection between NoVs and HAstVs was identified in one of the samples, representing 20.0% (1/5) of the samples positive for HAstVs.

As shown in Table 2, at least one viral agent was detected in all age groups. In the group of 49–60 months, only RV-A was identified. The frequency of enteric viruses was highest (46.9%) in the 13–24 months group. RV-A mainly affected the population aged ≤12 months (35.7%), while NoVs and HAstVs affected the group aged 13–24 months in a more significant proportion (61.5% and 60.0%, respectively). On average, 75.0% of viral gastroenteritis cases were identified in the population younger than 24 months; however, this result was not statistically significant (p = 0.3051).

In this study, only a statistically significant association related to gender was determined in rotavirus infection (p = 0.0421); Table 3 shows that RV-A was detected in 85.7% of boys and 14.3% of girls. In NoVs infection, it was observed that boys were more infected than girls (76.9% and 23.1%, respectively), while in HAstVs infection, it was observed that girls were infected more frequently than boys (60.0% and 40.0%, respectively); however, these results were not statistically significant (p = 0.1905 and p = 0.2793, respectively).

Table 4 shows that the highest percentage (64.3%) of rotavirus infection was observed during February 2011. On the other hand, NoVs were more frequent during November 2010 and May-June 2011 (23.1%, respectively), while HAstVs were detected intermittently throughout the study. Our results show that 92.8% of RV-A cases and 40.0% of HAstV cases were detected in the year’s first quarter, while 61.6% of NoV cases were detected in the second quarter (Fig. 2).

As shown in Table 5, the symptoms most associated with acute gastroenteritis of viral etiology were: diarrhea (100.0%) with an average of 7 depositions in the last 24 h; vomiting (83.9%); fever (74.2%); and dehydration (48.4%). In general, the symptoms are common among each etiological agent, but RV-A infection seems more severe since it showed a higher percentage (42.9%) of hospitalizations secondary to dehydration. As shown in Table 6, a total of 82 outpatient children and 18 hospitalized children were included in the study. A viral agent was detected in 12 of the 18 (66.7%) hospitalized children, corresponding to 50.0% RV-A, 41.7% NoVs, and 8.33% HAstVs.

All one hundred samples were initially screened for HAstV using an enzyme-linked immunosorbent assay (RIDASCREEN Astrovirus, R-Biopharm), and 5 positive samples (5.0%) were detected. These positive samples, along with 10% of the negative samples, were then subjected to molecular analysis, which demonstrated a remarkable 100% concordance in the RT-PCR with the previously positive samples in the immunoassay. A fragment of 449 bp was considered positive for HAstVs (Fig. 3a). The genotype of the five isolates detected in preschool children in Distrito Central, Honduras, was determined by sequencing. Table 7 shows the circulation of three of the 8 classic HAstV genotypes in this population, with genotypes HAstV-1 and HAstV-8 being the most frequent. Honduran HAstVs isolate nucleotide sequences were deposited in GenBank under accession numbers KF156804 – KF156808.

The HAstV isolates reported in this study were grouped into three distinct genotypes: HAstV-1, HAstV-2, and HAstV-3 (Fig. 4). In the case of isolates corresponding to HAstv-1, it was observed that they were grouped in the same cluster with isolates from Argentina and Brazil (Fig. 4) with a maximum divergence in the nucleotide sequence of 2.4%; this group presents a high homology with the reference sequence (96.6%). In the HAstV-8 genotype, a more homogeneous distribution was observed since all the strains were grouped in the same group with reference sequence (Fig. 4); Honduran isolates of HAstV-8 presented an average nucleotide sequence divergence of 2.6% with the reference sequence and 1.05% with a Brazilian isolate. Interestingly, the Honduran isolate of HAstV-2 formed an independent cluster (Fig. 4), exhibiting 6.6% nucleotide sequence divergence from the HAstV-2 reference sequence.

All one hundred samples were assessed for NoV by RT-PCR, and 13 positive samples (13.0%) were detected. A fragment of 213 bp was considered positive for HAstV (Fig. 3b). All the NoV isolates detected in this study were sequenced and then genotyped with the genotyping tool NoroNet. In this study, assigning the genogroup to 100% of isolates was possible, while the genotype could only be assigned to 84.6% of them. 92.0% of the samples positive for NoV belong to genogroup II (GII) and 8.0% to genogroup I (GI); in this study, the circulation of genogroup IV was not detected. Table 8 summarizes the genogroups, genotypes, and variants of GII.4 found in preschool children with acute gastroenteritis in Distrito Central, Honduras. Among the samples for NoV, the most prevalent genotype was GII.4 (61.5%), followed by GII.16 (23.1%); 15.4% of isolates could not be genotyped based on the RNA-dependent RNA polymerase (RdRp) gene region. Honduran NoV isolates nucleotide sequences were deposited in GenBank under accession numbers KF177224–KF177231.

Nine of the thirteen sequenced isolates were chosen to build a phylogenetic tree to demonstrate the evolutionary relationships of Honduras NoV isolates. This selection was made based on a complete characterization of the isolate up to genotype and an equal sequence’s length than other isolates used in the alignment. Our results show that Honduran NoV isolates were clustered into only two genotypes within the GII genogroup (Fig. 5). In relation to the isolates belonging to the GII.4 genotype, most of them were grouped in the same cluster with Brazilian isolates of NoVs (Fig. 5). However, within this cluster, two lineages are distinguished: one that clustered isolates 08, 09, 10, and 11 with a maximum divergence of 1% in their nucleotide sequence, and another that clustered the Honduran isolate 06 and most of the Brazilian isolates with a maximum divergence between them of 3.1% in their nucleotide sequence. Honduran NoVs isolate 01 was found to cluster together with a GII.4 reference isolate, with which it shares 94.3% homology. Honduran NoV isolates 03 and 04 presented a 4.7% divergence in the nucleotide sequence with the GII.16 genotype reference sequence. This study demonstrates that the Honduran NoVs isolates belonging to the GII.4 genotype were clustered into two variants: 2006b and 2010, of which 88% are of the 2006b variant (Fig. 6).

RV-A was detected in 14 of the 100 samples (14.0%) studied by the immunochromatographic test. All positive samples were analyzed by RT-PCR for the VP7 and VP4 genes (Fig. 3c). In this study, we achieved 100% concordance between the immunochromatographic test and the RT-PCR. Table 9 shows that genotype G2P[4] was identified in 92% of RV-A positive samples, in addition to the circulating G3P[4] and G3P[8] genotypes. Interestingly, in vaccinated children who presented with symptomatic rotavirus infection, only the G2P[4] genotype was detected.

RV-A was detected in 28.6% of vaccinated children and 71.4% of unvaccinated children (Table 10). Interestingly, the rotavirus-vaccinated children who presented with RV-A infection were all less than 24 months old and had the completed vaccination schedule (two doses) at the time of entering the study (Table 9). Our study’s findings, estimating the protective role of rotavirus vaccination against RV-A infection, have direct implications for public health. We calculate the relative risk (RR), a ratio of the proportion of disease (RV-A infection) among the exposed (children vaccinated) relative to the proportion of disease among the unexposed (children unvaccinated) [27]. The RR value obtained in this study, less than 1.0, suggests that the risk of the outcome (RV-A infection) is decreased by the rotavirus vaccination (exposure), which is a “protective factor” against RV-A infection (RR = 0.2667, 95% CI = 0.0898–0.7918, p = 0.011) [26, 27].

In Honduras, childhood diarrhea has been a persistent health issue. This study, however, provides a significant contribution by shedding light on the etiology of acute diarrhea in preschool children in Distrito Central, Honduras. The results underscore that RV-A remains a major cause of acute gastroenteritis in children, albeit with a lower prevalence than that previously reported [28, 29], and similar to that observed in other countries of the Central American region [3, 30, 31]. Importantly, this research is a game-changer, being the first molecular study of the presence of human NoVs and HAstVs as diarrheal agents in Honduras, potentially paving the way for more targeted interventions.

The RV-A prevalence obtained in this study (14%) was lower than that reported in previous studies, as shown by a case-control study that included 521 children under 5 years of age between June 2000 and June 2002, which showed that RV-A was the most frequent causal agent with a 42.8% prevalence [28]. Similarly, the Surveillance System for rotavirus gastroenteritis of the Honduran Ministry of Health reported between 2006 and 2008 percentages of rotavirus positivity between 36 and 46% [29]. It is important to mention that the prevalences reported in these studies correspond to the period prior to the introduction of the rotavirus vaccine, which was in 2009 in Honduras. However, the prevalence documented in this study, despite the small sample size, suggests a positive trend that introduction of the rotavirus vaccine has indeed helped reduce the number of cases of acute childhood gastroenteritis. This finding is consistent with the promising results of post-vaccination studies in Latin America [2, 32].

In that sense, a study conducted in Brazil by Gurgel et al. [33], reported that after the introduction of the vaccine in 2006, there was a reduction in the overall number of consultations and hospitalizations for diarrhea in the northeast region of that South American country, especially in young children. The authors attributed this decline to vaccination and the implementation of sanitation measures. Richardson et al. [34] reported that after introducing the rotavirus vaccine, a significant decrease in the number of deaths related to diarrhea in Mexican children was observed, and they suggest that it may be due to the rotavirus vaccination. In Central America, de Palma et al. [35] conducted a study between January 2007 and June 2009 to evaluate the effectiveness of the monovalent rotavirus vaccine against severe cases of gastroenteritis in El Salvador and concluded that the monovalent vaccine (Rotarix) against rotavirus is highly effective in preventing the number of rotavirus diarrhea admissions in children under 2 years of age.

Importantly, this study shows that rotavirus was detected mainly in unvaccinated children, and it was determined that being vaccinated against rotavirus appears to have a protective effect (RR = 0.2667, 95% CI = 0.0898–0.7918, p = 0.011) against infection by this agent and the occurrence of severe symptoms. These findings suggest that widespread rotavirus vaccination could significantly reduce the burden of rotavirus-related morbidity and mortality. However, a broad study involving a more significant number of samples and representative of the national child population should be required to consistently evaluate the role of rotavirus vaccination in reducing the morbidity and mortality associated with this agent in the country. In that sense, passive surveillance has proven to be a powerful tool to evaluate the population-level impact of RV vaccination. Shioda et al. [2] using data from standardized sentinel RV surveillance, observed declines in the proportion of RV-positive acute diarrhea samples, a reduction in the number of RV hospitalizations, and changes to RV seasonality; they identified that the strength of the seasonal peak in RV incidence became smaller after vaccine introduction.

Several authors have established the importance of determining the circulating RV genotypes in the post-vaccination stage in order to identify changes in the distribution patterns of these viruses and to be able to correlate, at the same time, whether the vaccine is providing effective protection to the child population or whether the genotypes that infect a certain population at a given time are evading the immune response induced by the vaccine. In this sense, the results of this study show a predominance of the G2P[4] genotype, which was detected in 92% of the positive cases of rotavirus. This finding is consistent with that reported by other researchers in Latin America. For example, in Brazil in 2009, 96% of rotavirus cases of the G2P[4] genotype were reported after implementing rotavirus vaccination [36]. In general, the increase of the G2P[4] genotype in the post-vaccination stage has been reported by different research groups in recent years, reaching the conclusion that the monovalent rotavirus vaccine seems to be less effective in preventing diarrhea caused by RV-A of the G2P[4] genotype, which does not share any of the surface antigens with the vaccine strain [37]. The increase in the prevalence of this genotype and the reduced protection that the vaccine has against it may represent an imminent health problem since, in recent years, this type of rotavirus has been associated with the occurrence of outbreaks. In Honduras, in 2006, a major outbreak of rotavirus gastroenteritis occurred due to this genotype [31]. Additionally, unusual G-P combinations have been reported in Honduras; among these are the rare G9P[4] and G10P[14] genotypes that are infrequently found in Latin America [38, 39]. This highlights the importance of rotavirus gastroenteritis surveillance and detection of circulating genotypes and the need to develop efficient preventive measures against these viruses.

Even though the results of the prevalence of rotavirus and the observed effectiveness of the vaccine in the child population are very encouraging, it is important to return to the problem of acute gastroenteritis since, in recent years, an increase in the number of other viral agents associated with this important health problem has been observed [3‒6].

Different authors have evaluated the dynamics of the etiology of viral gastroenteritis in the post-rotavirus vaccination stage, defined norovirus as an emerging and important agent in the occurrence of sporadic and epidemic cases, and highlighted the importance of investigating or establishing a surveillance system for these viruses [40‒42]. In a study carried out in Nicaragua between 2007 and 2008 in order to determine whether the RV5 pentavalent vaccine reduced the number of cases of rotavirus infection, Bucardo et al. [3] concluded that the introduction of rotavirus vaccination reduces rotavirus transmission in the community and recommended that NoV has to be monitored in the post-vaccination stage by determining a high prevalence of NoV in wastewater compared to rotavirus.

In this study, a norovirus prevalence of 13% was determined in sporadic cases in children under 5 years of age. This rate is comparable to that reported in Nicaragua (12%) [43] and Venezuela (13%) [44], indicating a similar burden of NoV in these regions. However, it is lower than that reported in Peru (17.4%) [42], suggesting a potential difference in the epidemiology of NoV in this country. It is worth noting that although gastroenteritis due to NoV is usually referred to as moderate and recovers quickly, the high percentage (38.5%) of positive cases due to NoV that required hospitalization is of special interest, unlike that was reported in Nicaragua (15%) in 2008 [43]. This study’s findings are not only significant locally but also globally. 92.3% of NoV-positive cases were grouped in genogroup II (GII), a trend consistent with that has been reported in other countries such as Venezuela [42] and worldwide. While the cause of this marked predominance of genogroup II remains unclear, it has been shown that viral excretion for GII is 100 times greater than for GI, a factor that could facilitates its dissemination in the environment [45]. This underscores the need for international collaboration in understanding and managing NoVs.

The literature establishes that NoVs are viruses with high genetic variability. Interestingly, the strains circulating among children from Distrito Central, Honduras, correspond only to the GII.4 and GII.16 genotypes included in the GII, which are the most frequent in the region and worldwide [43, 46]. Unlike other studies in Latin America that reported the circulation of a greater number of genotypes [40], the phylogenetic analysis of the Honduran NoV strains allowed them to be grouped with the Brazilian and reference strains, observing a low divergence in the nucleotide sequence. It is important to note the difficulty in obtaining NoV sequences from other Latin American countries; this difficulty lies mainly in the use of different protocols for the molecular characterization of NoV based on different regions of the virus genome and suggests the need for harmonization in these methodologies to facilitate comparison between the results obtained in each country. A single genotype, GII.4, has been associated with all NoV gastroenteritis epidemics worldwide. This genotype uses different mechanisms of variation which results in the emergence of new antigen variants. Different epidemic variants of NoV have emerged in the last few years: in 1995–1996, 2002, 2004, 2006, 2007–2008, and 2009 [47]. Within the GII.4 genotype, the circulation of two variants was detected in Distrito Central, Honduras the 2006b and 2010 variants. The 2006b variant is a descendant of the NoV strain named Farmington Hills 2002. This variant circulated between 2006 and 2010 and became predominant during 2007. NoV variants may vary according to the geographic region; in Brazil, for example, it was recently reported the circulation of variants 2004, 2006a, and 2006b of the GII.4 genotype [40].

On the other hand, the frequency obtained from human astroviruses (5%) is comparable to that reported in other Latin American countries [48‒50]; however, according to the literature, these frequencies can fluctuate according to the geographical region and time of year in which the detection is carried out [51]. In this study, the circulation of three HAstVs was detected: HAstV-1, HAstV-2, and HAstV-8; HAstV-1 and HAstV-8 were the most frequent types, but a clear predominance of any of the three genotypes was not demonstrated, unlike what was reported in the literature of countries in the region [48, 50, 51]. Phylogenetic analysis of Honduran strains of HAstV demonstrates homology with strains of diverse types of astroviruses detected in Latin American countries.

Various studies establish that the association of rotavirus with other enteric pathogens is frequent, mainly other viral agents such as astrovirus [49, 51]; however, this study did not reveal mixed infections between these two viruses, except for coinfection between NoV and HAstV. The association between age and viral agents revealed in this study is consistent with what has been reported in previous studies in the country and in the global literature. RV-A and NoV appeared in practically all age groups, mainly in children under 2 years of age. Similarly, HAstVs were detected in children between 9 and 36 months of age, with 80% of them under 2 years of age. This coincides with reports that establish that the prevalence of HAstV is related to age [5, 51, 52]. It is known that infancy is a time of immunity to many pathogens due to maternal antibodies obtained from the mother. Once this stage is over, the infant begins the process of reacting to pathogenic agents without much protective success, which increases the possibility of infection by HAstV, as shown by the results of our research described in Tables 2 and 5.

Consequently, the low levels of frequency in children older than 2 years can possibly be attributed to the acquisition of protective immunity against these enteric viruses, resulting from exposure to these viruses from an early age [5]. It has been documented that the immune system plays an important role in protecting against human rotavirus and HAstV infection; the importance of the humoral immune response in protection against HAstV infection is highlighted [53].

Among the various factors associated with susceptibility to enteric virus infection, gender has been a topic of interest. In the case of RV-A, a significant difference has been observed, with boys showing higher susceptibility than girls. However, our study, due to the limited number of samples collected, could not establish this relationship. Interestingly, some authors have suggested that HAstV infection is not influenced by the gender of infected individuals [50, 54]. This finding, which is in line with our study’s results, is intriguing and warrants further investigation. Our study found no significant difference between gender and HAstV infection, which could have important implications for future research in this field.

Concerning other variables such as socioeconomic level, it was observed that the majority of cases came from lower-middle-class urban areas, which could suggest that enteric virus infection is more prevalent in children living in poor areas, as mentioned by Cruz et al. [54]. However, to establish that socioeconomic status is related to infection by HAstV or other enteric pathogens, comparative studies are needed that include participants from different sectors of the city. On the other hand, it is mentioned that socioeconomic variables are not determinant in the occurrence or not of enteric virus infections because they show similar prevalence in developed and developing countries, as is the case with NoV [55].

The fact that acute gastroenteritis continues to be a public health problem in the country makes it imperative to monitor and investigate its etiology to establish effective preventive measures. The information obtained in this study allows us to understand the diversity of viral etiological agents responsible for acute gastroenteritis in children. Based on this information, we are inspired to advocate for the need to develop research studies that cover a longer period of time and involve more healthcare centers. This will not only provide a better representation of the importance of these three viral agents in the occurrence of acute gastroenteritis but also significantly contribute to the field of viral etiology.

We thank Dr. José Paulo G. Leite for kindly providing training to J. Ortiz-Quintero in molecular characterization of enteric viruses at the Laboratório de Virologia Comparada e Ambiental of the Instituto Oswaldo Cruz (IOC/Fiocruz). We thank Mrs. Zoila Velasquez for her valuable technical assistance during the study. We also thank all medical staff, parents, and children who participated in this study.

This study protocol was reviewed and approved on October 8, 2010 by the Research and Bioethics Committee of the Teaching and Research Management Department of the Instituto Hondureño de Seguridad Social (IHSS). A written informed consent was obtained from the parents or legal guardians of all participants prior to participating in the study.

The authors have no conflicts of interest to declare.

We thank the support of the Honduras-Canada Teasdale-Corti Project 2007–2012 supported by the Program of the Global Health Research Initiative (GHRI), the Network for Research and Training in Tropical Diseases in Central America (NeTropica), and the Dirección de Investigación Científica, Humanística y Tecnológica (DICIHT) of the Universidad Nacional Autónoma de Honduras (UNAH).

J. Ortiz-Quintero and A. Ferrera designed the research. A. Ferrera supervised the project. J. Ortiz-Quintero and Y. Cabrera performed the research. J. Ortiz-Quintero, L. Bourdett-Stanziola, and A. Ferrera analyzed the data, thoroughly discussed it, and wrote the manuscript. All the authors approved the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.