Prevalence and genotype distribution of norovirus and rotavirus among children under five with diarrheal infection in lower-middle-income countries after Rotavirus vaccine introduction: A systematic review and meta-analysis

Firayad Ayele; Tesfaye Assebe; Miheret Mandefro; Desalegn Admassu Ayana; Nega Assefa

doi:10.1371/journal.pone.0359886

. 2026 Oct 13;21(10):e0359886. doi: 10.1371/journal.pone.0359886

Prevalence and genotype distribution of norovirus and rotavirus among children under five with diarrheal infection in lower-middle-income countries after Rotavirus vaccine introduction: A systematic review and meta-analysis

Firayad Ayele ^1,^*, Tesfaye Assebe ¹, Miheret Mandefro ¹, Desalegn Admassu Ayana ¹, Nega Assefa ¹

Editor: Ricardo Q Gurgel²

PMCID: PMC13660929 PMID: 42868165

Abstract

Background

Rotavirus and norovirus are the leading viral causes of acute diarrheal disease worldwide. Both viruses encompass multiple genotypes and subgenotypes, and this genetic diversity presents a significant challenge to rotavirus vaccine effectiveness and development. High-quality evidence on the prevalence and circulating genotypes of these enteric viruses among children under five years of age remains limited, particularly following the introduction of rotavirus vaccination in low and middle-income countries (LMICs). Therefore, this systematic review and meta-analysis aimed to synthesizes available evidence on the prevalence and genotype distribution of norovirus and rotavirus in this population.

Methods

A systematic literature search was conducted across five major databases PubMed, Scopus, ScienceDirect, Embase, and Web of Science between February 20 and 28, 2026. Duplicate removal, title and abstract screening, full-text review and data extraction were performed independently by two investigators using the Rayyan web-based platform. STATA version 17 software was used for statistical analysis. A random-effects model was applied to estimate pooled prevalence and effect sizes. Heterogeneity was evaluated using Cochran’s Q statistic, the I² statistic, forest plots, and Galbraith plots. Publication bias was assessed using both graphical (funnel plots) and statistical methods.

Results

A total of 35,704 children under five years with diarrheal infection from LMICs were included across eligible studies. The overall pooled prevalence of norovirus and rotavirus combined was 18% (95% CI: 15–21%). Rotavirus had a higher pooled prevalence than norovirus, at 24% (95% CI: 19.6–28.3%) and 13% (95% CI: 11–15%), respectively. Substantial heterogeneity was observed across studies (I² = 98.72%, p < 0.001). Among norovirus genotypes, GII was predominant (76%), with subgenotype GII.4 accounting for 53% of cases, followed by GI (23%). The most frequently identified rotavirus genotypes were G3[P8] (39%) and G1[P8] (17%).

Conclusions

Norovirus and rotavirus remain significant causes of diarrheal disease among children under five in LMICs, even after the introduction of rotavirus vaccination. Approximately one in five children with diarrheal illness were infected with at least one of these virusesGII, particularly subgenotype GII.4, was the dominant norovirus strain, while G3[P8] and G1[P8] were the most prevalent rotavirus genotypes. These findings underscore the need to strengthen surveillance systems, consider circulating genotypes in vaccine formulation, and continuously monitor genotype shifts within the population.

Introduction

Viral diarrheal infection, also referred to as viral gastroenteritis, is a condition characterized by the frequent passage of watery, loose stools caused by a pathogenic virus [1]. The primary viral agents responsible for this condition are rotavirus, which predominantly affects children, and norovirus, which can infect individuals of all age groups [2,3].

Rotavirus is characterized as a non-enveloped, double-stranded RNA virus containing 11 gene segments [4]. These segments encode six structural proteins, designated VP1-VP4 and VP6-VP7 [5,6]. Additionally, the rotavirus genome encodes six non-structural proteins (NSP1 to NSP6) [7] and there are nine antigenically distinct groups (A through J) [8]. Geno-typically, rotavirus is classified into 36 G types and 51 P types based on the VP7 glycoprotein and VP4 protease-sensitive protein. The most prevalent strains causing severe diarrhea in humans are G1P, G2P, G3P, G4P, and G9P, with G1-G4, G9, and G12 being the most common G genotypes and P[4], P[6], and P[8] the most frequently observed P genotypes worldwide [9]. However, with the introduction of four rotaviruses A vaccine, the incidence of disease caused by rotavirus has decreased significantly, although norovirus remains a leading cause of gastroenteritis [4,10,11].

Norovirus (NoV) belongs to the Caliciviridae family and is a single stranded, non- enveloped, positive sense virus [12,13]. The genome of this virus categorized into six Geno groups, with Geno groups I and II (GI and GII) accounting for most human infections [14]. Each Geno group can be subdivided into various genotypes, with GI consisting of at least 9 genotypes and GII containing around 27(GII.1–GII.27) [15]. The majority of human NoV infections are attributed to GII strains, particularly the GII.4 genotype, which is responsible for approximately 55–85% of clinical cases globally [16].

Diarrheal disease is the second most prevalent illness and the third leading cause of death among children under five, resulting in approximately 443,832 deaths and 1.7 billion cases annually [17]. In lower- and middle-income countries, diarrhea is responsible for about 25% to 30% of deaths in children, leading to high mortality rates, morbidity, and economic crises. Notably, over 70% of diarrhea-related fatalities in children under five occur in Africa [18], with the majority of morbidity and mortality concentrated in middle income countries, where 88% of these cases are related to unsafe water, inadequate sanitation, and poor hygiene [19,20].

Viral gastroenteritis has a substantial effect on patients, especially on children under five. Severe diarrheal episodes can lead to dehydration and loss of water and electrolytes (sodium, chloride, potassium, bicarbonate), which may be fatal in young children. Additionally, children under five often experience concurrent malnutrition and weight loss [18]. These health consequences impose economic burdens on families, healthcare systems, and negatively affect national productivity, alongside increasing morbidity and mortality. In low- and middle-income countries, factors such as low maternal education, poor sanitation, contaminated water, short breastfeeding duration, inadequate handwashing, lack of rotavirus vaccination, improper feces disposal, young child age, and poor food hygiene further worsen the problem [17,19,21].

In LMICs, to the best of our knowledge there is no high-quality evidence on the pooled prevalence and genotypes distribution of rotavirus and norovirus among under five children, especially following the introduction of rotavirus vaccines. Filling this gap would improve understanding of the molecular epidemiology and pooled prevalence information that is crucial for outbreak preparedness and response and for guiding the development of vaccines targeting the most common genotypes. Therefore, the present study, aimed to systematically review and quantitatively synthesize evidence on prevalence and genotype distribution of rotavirus and norovirus among children under five with diarrheal infection in lower-middle-income countries following the introduction of rotavirus vaccination.

The findings of this study provide valuable evidence for low and middle-income countries, health professionals, NGOs, policymakers, and other stakeholders involved in childhood diarrheal disease. The findings also serve as a baseline for future research and vaccine development.

Methods and materials

Registration and reporting

This systematic review and meta-analysis was carried out according to the protocol registered in the International Prospective Register of Systematic Reviews (PROSPERO), registration number CRD420261 349950 (https://www.crd.york.ac.uk/PROSPERO/viewrecord.php?ID=CRD420261349950).

Data source and study search strategy

A comprehensive search for relevant literature was conducted across various online databases, including PubMed, Scopus, Science Direct, Embase, and Web of Science. The search was done from February 20–28/2026 using the searching terms included “Rotavirus, Norovirus, Calicivirus, “Norwalk virus”, “viral diarrhea”, “enteric viruses” Genotype, prevalence, “Under-Five Children”, “pediatric gastroenteritis”, diarrhea” without any filters applied for date. Boolean operators such as AND, OR were utilized to refine the search results (the detail S1 Table).

Inclusion and exclusion criteria for studies

Studies were eligible for inclusion if they met all of the following criteria: (1) conducted in low- and middle-income countries (LMICs; see S2 Tab le for the full list of eligible countries); (2) for rotavirus-focused articles, published after the national introduction of the rotavirus vaccine in the respective country; (3) enrolled children under five years of age as the study population; (4) used stool specimens for laboratory diagnosis; (5) included a minimum sample size of 50 participants; (6) published in the English language; (7) employed a case-control, cross-sectional or large scale surveillance design; (8) reported the prevalence or genotype distribution of rotavirus, norovirus, or both; (9) studies with full-text articles; and (10) achieved a quality assessment score greater than 50% (moderate quality or above) on the JBI critical appraisal checklist.

Studies were excluded if they were case reports, clinical trials, editorials, commentaries, meta-analyses, previously published systematic reviews, or conference abstracts lacking full-text availability.

Study selection

Upon completing the literature search across various databases, the search results were imported into Rayyan (AI-Powered Systematic Review Management Platform, https://www.rayyan.ai/). Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)(S3 Table) format and studies screening, duplicate removal, title/abstract screening, and full-text screening was conducted by two independent authors. Any conflict during screening discussed in detail to reach a consensus on whether to accept or reject the articles for inclusion in the report.

Data extraction

Eligible articles were extracted using a Microsoft Excel spread-sheet by two authors. The full texts were thoroughly reviewed to ensure that all relevant information required for the analysis was captured. Each study was assigned a unique identification code to prevent duplication and misclassification. After the full text review, the authors extracted information such as the first Authors name, year of publication, country, study design, detection methods, sample size, event/cases, prevalence, outcome (rotavirus or norovirus), and genogroup/genotype.

Study risk of bias and quality assessment

The overall quality of each eligible article was evaluated by two independent authors. A standardized checklist developed by the Joanna Briggs Institute (JBI) [22] specifically for assessing the quality of prevalence studies was used. This checklist included nine main criteria, each criterion could be answered Yes (Y), No (N), unclear (U) and not applicable (NA). An overall quality assessment score was assigned to each study based on the answers to these nine questions. The overall quality of the studies was categorized as ‘high’ (scores ≥8 points), ‘moderate’ (scores >5 to <8 points), or ‘low’ (scores ≤5 points).

Evidence synthesis and analysis

All the extracted data are qualitatively described and presented in textual description and also presented in tables and Figs. For quantitative analysis STATA version 17 software was used, and the pooled prevalence of norovirus, rotavirus and their genotype was estimated using a Restricted Maximum Likelihood (REML) random-effects model with corresponding 95% confidence intervals. Studies reporting zero prevalence were retained in the meta-analysis and were not excluded on the basis of a zero event. Heterogeneity across the included studies was assessed using a Q statistic, I² statistic, tau-squared (τ²), forest plot & Galbraith plot, where I² statistic values of 0%, < 25%, 25–75%, and >75% indicated no, low, moderate, and high heterogeneity, respectively. Publication bias was evaluated using funnel plots, egger’s test, begg test & contour-enhanced funnel plot. A leave-one-out sensitivity analysis was also performed to assess the robustness of the pooled estimates.

Results

Study selection process

A total of 13,031 articles were identified from various electronic databases. Of these, 3,606 were removed as duplicates, leaving 9425 articles for title and abstract screening. After screening, 265 articles were selected for further assessment; full text could not be retrieved for 5 articles. Therefore, 260 articles go through full-text screening, and 215 were excluded for not meeting the predetermined eligibility criteria. Finally, 45 articles were included in the data extraction process (Fig 1).

Risk of bias and quality assessment

The risk of bias and methodological quality of the included studies were assessed using the standardized Joanna Briggs Institute (JBI) Critical Appraisal Checklist for Studies Reporting Prevalence Data [22].. All 45 articles were assessed; quality scores ranged from 55.5% (moderate) to 100% (high), with the majority of studies attaining high quality scores. Based on this assessment, all articles were considered valid and were included in the review (S4 Table).

Characteristics of included studies

A total of 45 eligible studies were included in evidence synthesis. All studies are reported from lower and middle-income countries. Among 45 studies, 43 studies were considered for genotype distribution. Across these studies, the total number of study participant (children under five years of age) found to be 35,704. For articles reporting norovirus, no publication-year restrictions were applied. For studies reporting rotavirus, we restricted inclusion to those conducted from the rotavirus vaccine introduction year onward; accordingly, studies from 2006–2025 were included. Across the included studies, norovirus prevalence ranged from 3.12% to 30.50%, while rotavirus prevalence ranged from 17.90% to 46.50%%.

India contributed the largest number of included studies, with a total of 20 studies [23–41] followed by 4 studies each from Bangladesh [42–45] and Ethiopia [46–49]; 3 studies from Nigeria [50–52]; two studies from Honduras [53] and Morocco [54]; and one study each from Angola [55], Bhutan [56], Cameroon [57], Gabon [58], Kenya [59], Lebanon [60], Nepal [61], Pakistan [62], Senegal [63], and Zambia [64].

Of the 45 studies included, most (34) were cross-sectional while 11 were large-scale surveillance studies. In terms of detection methods, 23 studies used RT‑PCR 19 studies used both RT‑PCR and ELISA 3 studies used ELISA and multiplex RT‑PCR. Sample sizes for detection ranged from 55 to 5,408. Regarding outcome variable, 24 studies reported norovirus as an outcome variable, 21 reporting rotavirus outcomes and two studies reported both viruses (Table 1).

Table 1. Summary characteristics of the 45 study reports for the systematic review and meta-analysis on prevalence and genotype distribution of norovirus and rotavirus among children under five with acute diarrhea in lower-middle-income countries after rotavirus vaccine introduction, 2026.

No	Authors name, year	Country	Study Design	Region	Detection Methods	Sample size	Event/Cases	Total prevalence	Outcome
1.	Abubakar et al,2023	Nigeria	Cross-sectional	West Africa	RT‐PCR	200	42	21%	Norovirus
2.	Alam et al, 2015	Pakistan	Cross-sectional	South Asia	RT‐PCR	255	41	16.10%	Norovirus
3.	Arowolo, K.O, et al, 2023	Nigeria	Cross-sectional	West Africa	RT‐PCR	175	13	7.40%	Norovirus
4.	S.K. Dey et al, 2007	Bangladesh	Cross-sectional	South Asia	RT‐PCR	917	41	4.50%	Norovirus
5.	El Qazoui et a, 2024	Morocco	Cross-sectional	North Africa	ELISA, multiplex PCR	335	89	26.60%	Rotavirus
6.	El Qazoui et a, 2024	Morocco	Cross-sectional	North Africa	ELISA, multiplex PCR	335	54	16.10%	Norovirus
7.	A. Esteves et al, 2018	Angola	Cross-sectional	Southern Africa	RT‐PCR	343	58	17.40%	Norovirus
8.	A. Gelaw et al, 2019	Ethiopia	Cross-sectional	East Africa	RT‐PCR	450	60	13.30%	Norovirus
9.	T.N. Hoa-Tran et al,2015	Nepal	Cross-sectional	South Asia	RT‐PCR	4437	356	8%	Norovirus
10.	Gopalkrishna et al,2021	India	Cross-sectional	South Asia	ELISA, RT‐PCR	308	44	14.30%	Norovirus
11.	Gopalkrishna et al,2021	India	Cross-sectional	South Asia	ELISA, RT‐PCR	308	94	30.50%	Rotavirus
12.	Japhet et al, 2012	Nigeria	Cross-sectional	West Africa	RT‐PCR	55	14	25.50%	Norovirus
13.	Kebe et al, 2021	Senegal	Surveillance	West Africa	RT‐PCR	599	79	13.20%	Norovirus
14.	R. Kulkarni et al, 2016	India	Cross-sectional	South Asia	RT‐PCR	1712	126	7.40%	Norovirus
15.	S.E. Lekana-Douki et al, 2015	Gabon	Cross-sectional	Central Africa	RT‐PCR	317	73	23.40%	Norovirus
16.	M. Lo et al,2021	India	Cross-sectional	South Asia	RT‐PCR	2812	170	6.04%	Norovirus
17.	Melhem NM et al., 2016	Lebanon	Cross-sectional	Middle East	RT‐PCR	739	83	11.20%	Norovirus
18.	Menon et al, 2013	India	Cross-sectional	South Asia	RT‐PCR	282	28	9.90%	Norovirus
19.	Mugyia et al., 2019	Cameroon	Cross-sectional	Central Africa	RT‐PCR	902	76	8.40%	Norovirus
20.	Nataraju et al, 2010	India	Surveillance	South Asia	RT‐PCR	2495	78	3.12%	Norovirus
21.	Ortiz-Quintero et al,2024	Honduras	Cross sectional	Central America	RT‐PCR	100	13	13%	Norovirus
22.	Satter et al 2022	Bangladesh	surveillance	South Asia	RT‐PCR	1008	137	14%	Norovirus
23.	N. Sharif et al, 2023	Bangladesh	Cross sectional	South Asia	RT‐PCR	404	27	6.70%	Norovirus
24.	Wangchu et al, 2017	Bhutan	Cross sectional	South Asia	RT‐PCR	623	147	23.60%	Norovirus
25.	A. Abebe et a, 2018	Ethiopia	Surveillance	East Africa	ELISA, RT‐PCR	815	161	20.00%	Rotavirus
26.	Kumar et al, 2022	India	Cross sectional	South Asia	ELISA, RT‐PCR	240	35	14.58%	Rotavirus
27.	Namboodiripad1, 2025	India	Cross sectional	South Asia	ELISA, RT‐PCR	643	115	17.90%	Rotavirus
28.	Kumar, et al, 2025	India	Cross-sectional	South Asia	ELISA, RT‐PCR	510	237	46.50%	Rotavirus
29.	Nyamanga et al, 2024	Kenya	Cross sectional	East Africa	ELISA, multiplex PCR	200	23	11.50%	Rotavirus
30.	Singh et al, 2025	India	Cross-sectional	South Asia	ELISA, RT‐PCR	461	165	35.80%	Rotavirus
31.	Gurung, et al, 2025	India	Cross sectional	South Asia	ELISA, RT‐PCR	200	40	20.00%	Rotavirus
32.	jafet et al, 2024	Honduras	Cross sectional	Central America	ELISA, RT‐PCR	100	14	14.00%	Rotavirus
33.	jain, et al, 2021	India	Surveillance	South Asia	ELISA, RT‐PCR	734	95	12.94%	Rotavirus
34.	Simwaka et al., 2021	Zambia	Surveillance	Southern Africa	ELISA, RT‐PCR	424	153	36.00%	Rotavirus
35.	Engade et al, 2025	India	Surveillance	South Asia	ELISA, RT‐PCR	534	173	32.70%	Rotavirus
36.	Mohanty et al, 2025	India	Surveillance	South Asia	ELISA, RT‐PCR	5408	1870	34.60%	Rotavirus
37.	Mohanty et alb, 2025	India	Surveillance	South Asia	ELISA, RT‐PCR	288	67	23.30%	Rotavirus
38.	Rajani et al, 2025	India	Cross-sectional	South Asia	ELISA, RT‐PCR	405	97	23.90%	Rotavirus
39.	Rawal et al, 2021	India	Surveillance	South Asia	ELISA, RT‐PCR	444	107	24.10%	Rotavirus
40.	Pradhan, et al, 2021	India	Cross-sectional	South Asia	ELISA, RT‐PCR	1963	715	36.40%	Rotavirus
41.	Vyas et al, 2025	India	Surveillance	South Asia	ELISA, RT‐PCR	1156	206	17.90%	Rotavirus
42.	India et al 2025	India	Cross-sectional	South Asia	ELISA, RT‐PCR	114	13	11.40%	Rotavirus
43.	Redda et al. 2025	Ethiopia	Cross sectional	East Africa	RT‐PCR	247	30	12.14%	Rotavirus
44.	Rahman et at.,2016	Bangladesh	surveillance	South Asia	RT‐PCR	547	149	27%	Norovirus
45.	Tegegne D, et al,2024	Ethiopia	Cross-sectional	East Africa	RT‐PCR	160	20	12.5%	Norovirus

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ELISA=Enzyme-Linked Immunosorbent Assay, RT‐PCR =Reverse Transcription Polymerase Chain Reaction

Pooled prevalence of Norovirus and Rotavirus

The pooled prevalence of norovirus and rotavirus among children under five with diarrheal infection in LMICs was 18% (95% CI: 15–21%), with substantial heterogeneity (I² = 98.85%, p < 0.001) (Fig 2).

Subgroup analysis, sensitivity analysis, and publication bias

In this systematic review and meta-analysis, the results of Cochran’s Q test (3832.35, df = 44, p < 0.001), tau-squared (τ² = 0.0096), and particularly the I² statistic (98.72%) revealed substantial and statistically significant heterogeneity, which was further illustrated by the Galbraith plot (Fig 3) and the forest plot (Fig 4). Accordingly, subgroup analyses were performed based on outcome (norovirus vs rotavirus), region, study design, and detection methods. In addition, a meta-regression analysis was conducted to examine the effect of sample size.

Subgroup analysis based on outcome showed that the pooled prevalence of norovirus and rotavirus was 13% (95% CI: 11–15) and 24% (95% CI: 19.6–28.3), respectively (Fig 5). Regarding subgroup analysis by region, the prevalence of norovirus and rotavirus was highest in Southern Africa at 26% (95% CI: 8–45), followed by North Africa at 21% (95% CI: 11–31), South Asia at 19% (95% CI: 15–24), Central Africa at 16% (95% CI: 1–30), West Africa at 16% (95% CI: 8–23), East Africa at 14% (95% CI: 11–17), Central America at 13% (95% CI: 9–18), and the Middle East at 11% (95% CI: 9–14) (Fig 6). The pooled prevalence of norovirus and rotavirus according to study design was 17% (95% CI: 14–20) for cross-sectional studies and 21% (95% CI: 16–27) for surveillance studies (Fig 7). Regarding subgroup analysis by detection method, the pooled prevalence was 25% (95% CI: 20–29) for ELISA combined with RT-PCR, 18% (95% CI: 9–27) for ELISA combined with multiplex PCR, and 13% (95% CI: 10–16) for RT-PCR alone. Meta-regression analysis indicated that sample size was not significantly associated with the effect size (β = −1.98 × 10⁻⁶, p = 0.887), despite substantial heterogeneity (I² = 98.72%, Q-res p < 0.001).

The leave-one-out meta-analysis was conducted to evaluate the influence of individual studies on the overall effect size (Fig 8). The results showed that no single study had a significant impact on the pooled estimate, as the overall effect remained stable when each study was sequentially omitted, with the p-values remaining significant. Egger’s regression test indicated significant small-study effects (β = 3.25, p = 0.035). Similarly, Begg’s rank correlation test provided evidence of publication bias (p = 0.039). These findings suggest the presence of potential publication bias among the included studies (Fig 9). Additionally, a trim-and-fill analysis was performed, which showed that the overall pooled estimate remained unchanged despite evidence of possible publication bias.

Genotype distribution Norovirus and Rotavirus

A total of 43 studies were included to synthesize evidence on the genotype distribution of norovirus and rotavirus. A total of 35,704 samples were collected from under five children, 9,889 were sequenced to determine genotype, yielding 1,840 identified genotypes. Most norovirus detections belonged to Genogroup II (GII), 665 of GII-positive cases were reported across Nigeria, Pakistan, Morocco, Senegal, Gabon, Lebanon, Cameroon, India, Honduras, Bangladesh, and Ethiopia [25,40,43,45,49,50,53,54,56–58,60,62,63]. Genogroup I (GI) accounted for 129 cases reported from Nigeria, Morocco, Angola, India, Senegal, Gabon, Lebanon, Cameroon, Honduras, and Bhutan [23,25,41,50,53–58,60,63]. Sub genotype analysis showed that GII.4 was the most common GII subtype, with 563 cases reported from Bangladesh, Angola, Ethiopia, Nepal, Senegal, Lebanon, India, Cameroon, and Bhutan [23,24,40–44,46,55–57,60,61,63]. The next most frequent subtype was GII.3, with 145 cases reported from several LMICs including Angola, Nepal, Lebanon, India, Bangladesh, and Bhutan [24,40,41,43,55,56,60,61](Fig 10).

Across low- and middle-income countries (LMICs) after rotavirus vaccine introduction, the most frequently observed genotype was G3[P8], with 1,437 detections reported primarily from India, and also from Ethiopia and Kenya [26–28,30–35,37,39,47,48,59]. The second most common genotype was G1[P8], with 501 reported detections from Morocco, Ethiopia, and India [23,26–31,33,35,36,39,47,54,64]. The third most frequent was G2[P4], accounting for 489 detections reported in India, Ethiopia, Honduras, and Zambia [23,26–36,47,52,64]. A total of 384 mixed-type rotavirus detections were reported, mainly from studies in India and Zambia, with the majority coming from India [26,27,30,31,33,35,38,39,64]. The review also recorded five partially typable detections from India [30] and 16 non-typable detections from Ethiopia(Fig 11).

Pooled prevalence of predominant genotype Norovirus and Rotavirus

The predominant norovirus genotype was genogroup II (GII), with a pooled prevalence of 76% (95% CI: 61–91%), while the GI genogroup had a pooled prevalence of 23% (95% CI: 9–37%). Within GII, the sub-genotype GII.4 accounted for 53% (95% CI: 39–68%) of cases. Regarding rotavirus genotypes, G3P[8] was the most prevalent, with an overall pooled prevalence of 39% (95% CI: 28–51%), followed by G1P[8] with a pooled prevalence of 17% (95% CI: 10–25%). The pooled prevalence estimates for GII, GII.4, GI, G3P[8], and G1P[8] are presented in Figs 12–16, respectively.

Discussion

Despite technological advances, improved treatments, widespread vaccine use, and overall better sanitation, viral diarrhea remains a leading cause of childhood mortality in low- and middle-income countries. Transmission often arises periodically due to scarce resources, high population density, and low public awareness of how the infection spread in these counties [65–67]. Therefore, knowing the burden, prevalence of norovirus and rotavirus, it also equally important to knowing genotype distribution for vaccine targeting and development [68]. Therefore, this systematic review and meta-analysis ultimately included 45 studies from low- and middle-income countries (LMICs) reporting the prevalence of norovirus and rotavirus, and 43 studies reporting genotype distribution, among children under five years of age with diarrheal infection following the introduction of the rotavirus vaccine.

The overall pooled prevalence of norovirus and rotavirus was 18% (95% CI: 15%–21%). This result highlight that both viruses remain significant causes of acute gastroenteritis In LMICs and nearly one in five diarrheal cases may be associated with enteric viral infections (Norovirus or Rotavirus). Subgroup analysis based on outcome revealed that the pooled prevalence of norovirus was 13% (95% CI: 11%–15%). This finding is consistent with previously reported pooled estimates from China (11.54) [69], Latin America 15% [70], and Africa 15% [70]. However, the present estimate is lower than findings from global systematic reviews and meta-analyses, which reported prevalence of 17.7% [71], and 18% [72], respectively. In contrast, the current finding is higher than the pooled prevalence reported in sub-Saharan Africa (6.6%) [73]. Additionally, previous studies in Africa have reported pooled norovirus prevalence of 15% (95% CI: 15%–18%) in the pre rotavirus vaccination period and 13% (95% CI: 9%–17%) in the post-vaccination period [74]. The observed discrepancies may be due to differences in the sensitivity of detection methods [75], variations in sanitation, hygiene practices [76], and access to healthcare, as well as differences in sample size, including hospital-based cross-sectional studies versus larger surveillance studies [77]. Temporal factors, including the introduction and scale-up of rotavirus vaccination programs, may also influence the observed burden of norovirus by altering pathogen dynamics.

In the current systematic review and meta-analysis, the pooled prevalence rotavirus found to be 24% (95% CI: 19.6%–28.3%). This means out of 4 children with diarrheal infection one may be due to rotavirus infection. This estimate is comparable to findings reported from Burkina Faso (27.7%) [78], South Africa (24%) [79], and Ethiopia (23%) [80], supporting the evidence that rotavirus remains a common cause of acute diarrheal disease in sub-Saharan Africa. However, the current estimate is lower than pooled prevalence reports from Africa overall (31.0%) [81], Bangladesh (30.1%) [82], and sub-Saharan Africa (29.2%) [73]. In contrast, the present finding is higher than estimates reported from Latin America (16.1%) following the introduction of rotavirus vaccination [83]. Notably, studies from this region have documented a decline in rotavirus prevalence from 24.3% to 16.1% after vaccine implementation, highlighting the substantial impact of vaccination programs in reducing disease burden. These variations may be explained by differences in rotavirus vaccine introduction year, vaccine coverage across regions [84], seasonal variation, geographic differences, and health system strength are also contributing factors to the observed differences in detection reports.

In the current systematic review and meta-analysis, the most frequently detected norovirus genotype was GII, with a pooled prevalence of 76% (95% CI: 61%–91%). This estimate is comparable to the global pooled prevalence of 84.6% [81].This genotype is considered the globally dominant strain in norovirus infections. However, the estimate is slightly lower than the systematic review and meta-analysis conducted in Africa (91.2%) [74]. At the sub-genotype level, GII.4 was the most prevalent, with a pooled estimate of 53% (95% CI: 39%–68%). This finding is consistent with reports from China (45.18%) and global estimates (59.3%) [71], which confirm GII.4 as the most common norovirus genotype circulating in the population. However, the observed prevalence is lower than the systematic review and meta-analysis reported from Africa (79.3%) [81]. In addition to GII and GII.4, other genotypes such as GII.3 were also reported, with prevalence estimates of 11.92% in China [69] and 14.9% globally [71], suggesting moderate but consistent circulation across regions.

In the current systematic review and meta-analysis, the most common G/P genotype combination of rotavirus was G3P[8], with a pooled prevalence of 39% (95% CI: 28%–51%). This estimate is higher than reports from Asia (24%) [10], where G3P[8] has also been identified as a predominant circulating genotype. In contrast, lower prevalence estimates have been reported from Africa (11.7%) [81], Ethiopia (12.9%) [80], and South Africa (7.02%) [79], suggesting considerable regional variation in genotype distribution. Similarly, G1P[8] was identified as the second most common genotype in the current systematic review and meta-analysis, with a pooled prevalence of 17% (95% CI: 10%–25%). This estimate is comparable to findings from South Africa and Nicaragua (20.3%) [83]. However, it is higher than estimates reported from Asia (8%) [13], Africa (7.1%) [81], and Chile (6.8%) [83], while remaining lower than reports from several Latin American countries, including Brazil (54.2%), Argentina (46.6%), Ecuador (50.0%), and Colombia (57.3%) [83]. Therefore, rotavirus remains the major public health problem in LMICs even after the introduction of rotavirus vaccine. It is very important to strengthen immunization programs, improve vaccine accessibility and enhance surveillance systems to monitor disease trends and vaccine impact.

Limitations of the study

The findings of this study may inform evidence-based decision-making; however, several limitations should be considered. First, the search was restricted to studies published in English, which may have introduced language bias by excluding potentially relevant studies published in other languages. This restriction may have reduced the comprehensiveness and geographical representativeness of the evidence included in the review. Second, the majority of the included studies were cross-sectional, which limits the ability to establish temporal associations. Third, substantial heterogeneity was observed across studies, which may limit comparability and should be considered when interpreting the pooled estimates. Fourth, the possibility of publication bias may have affected the pooled estimates, as studies with statistically significant or positive findings may be more likely to be published than studies with null or negative findings. Finally, the absence or limited availability of studies from some LMIC regions reduces the generalizability of the findings, while the large proportion of studies from India may have disproportionately influenced the overall pooled estimates and limited comparability across geographical regions.

Conclusions and recommendations

Conclusions

This systematic review and meta-analysis synthesizes evidence. on the prevalence and genotype distribution of norovirus and rotavirus among children under five years of age in low- and middle-income countries following rotavirus vaccine introduction. Both viruses remain major causes of diarrheal disease in this population. Approximately one in five children with diarrheal illness may be attributable to at least one of these viruses. Specifically, norovirus was associated with approximately one in eight diarrheal cases, while rotavirus was associated with approximately one in four. Among norovirus genotypes, GII particularly the GII.4 subgenotype was predominant, whereas G3[P8] and G1[P8] were the most commonly identified rotavirus genotypes. However, given the considerable heterogeneity in reported prevalence and genotype distribution across studies, these findings should not be considered conclusive.

Recommendations

Policymakers, programmers and researchers should work closely together to regular monitoring and strengthening surveillance systems to track the prevalence, genotype shifts, and genotype distribution within the population to control and reduce the infection. Vaccine manufacturers should consider the full range of circulating genotypes prior to vaccine manufacture, and the vaccine should include at least the predominant rotavirus genotypes associated with infection in low- and middle-income countries. Health interventions, as well as water, sanitation, and hygiene (WASH) practices, should be strengthened. Future research should focus on longitudinal and large-scale surveillance studies using molecular methods.

Supporting information

S1 Table. Full search strategy for a systematic review and meta-analysis for prevalence and genotype distribution of norovirus and rotavirus among children under five with acute diarrhea in lower-middle-income countries after rotavirus vaccine introduction from February 20–28/2026.

(DOCX)

pone.0359886.s001.docx^{(22KB, docx)}

S2 Table. List of LMICs with the year of rotavirus vaccine introduction for prevalence and genotype distribution of norovirus and rotavirus among children under five with acute diarrhea in lower-middle-income countries after rotavirus vaccine introduction, 2026.

(DOCX)

pone.0359886.s002.docx^{(22.6KB, docx)}

S3 Table. PRISMA 2020 checklist.

(DOCX)

pone.0359886.s003.docx^{(267.7KB, docx)}

S4 Table. Risk of bias and quality assessment results of the 45 studies included in the systematic review for prevalence and genotype distribution of norovirus and rotavirus among children under five with acute diarrhea in lower-middle-income countries after rotavirus vaccine introduction, 2026.

(DOCX)

pone.0359886.s004.docx^{(28.8KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0359886.r001

Decision Letter 0

Ricardo Q Gurgel

18 Aug 2026

Dear Dr. Ayele,

Please submit your revised manuscript by Oct 17 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Ricardo Q. Gurgel, PhD

Academic Editor

PLOS One

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

**********

Reviewer #1: Overall, this manuscript addresses a highly relevant public health topic, particularly given the increasing importance of norovirus following the introduction of rotavirus vaccination. The research question is pertinent, and the study employs an appropriate systematic review and meta-analysis methodology. However, several methodological, statistical, and writing issues reduce the overall strength of the conclusions.

The manuscript would benefit from careful language editing by a native English speaker or a professional scientific editing service. There are numerous grammatical errors and awkward sentence constructions throughout the text. For example, the sentence "Approximately one in five children presenting with diarrheal illness infected..." is grammatically incorrect and should be revised. In addition, several expressions, particularly "This systematic review", are repeated excessively, making the text unnecessarily repetitive.

Some methodological aspects also require clarification. The authors do not describe how studies reporting zero prevalence were handled in the meta-analysis, which is an important issue when pooling prevalence estimates. Restricting the search to English-language publications may have introduced language bias and should be acknowledged more explicitly. Furthermore, combining studies that used different diagnostic methods, such as RT-PCR and ELISA, is likely to have contributed to the substantial heterogeneity observed. A meta-regression including diagnostic method as a covariate would help determine whether the laboratory technique explains part of the between-study variability.

Reviewer #2: Attached is the full report,

Herein, the authors present a systematic review and meta-analysis of the pooled prevalence and genotype distribution of norovirus and rotavirus among children under five with diarrheal infection across 45 studies (35,704 children) from low- and middle-income countries (LMICs), restricted to the period following national rotavirus vaccine introduction. Using a random-effects (REML) model, the overall pooled prevalence was 18% (95% CI: 15–21%), with rotavirus (24%) more prevalent than norovirus (13%). Heterogeneity was substantial throughout (I² ≈ 98–99%) and was explored via subgroup and meta-regression analyses. GII, particularly GII.4, was the dominant norovirus genotype, while G3P[8] and G1[P8] led among rotavirus genotypes. The authors conclude that both viruses remain a significant burden in LMICs post-vaccination and call for continued genomic surveillance.

**********

what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: CATHARINA CORREA COSTA

Reviewer #2: No

**********

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Attachment

Submitted filename: Peer_Review_Norovirus_Rotavirus_Manuscript.pdf

pone.0359886.s005.pdf^{(278KB, pdf)}

PLoS One. 2026 Oct 13;21(10):e0359886. doi: 10.1371/journal.pone.0359886.r002

Author response to Decision Letter 1

1 Sep 2026

Response to Academic Editor

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We sincerely thank the Academic Editor for your time, effort, and valuable guidance. We have carefully revised the manuscript in accordance with the journal’s requirements and have addressed the necessary corrections. We kindly request that you consider the revised manuscript for further evaluation.

2. In the online submission form you indicate that your data is not available for proprietary reasons and have provided a contact point for accessing this data. Please note that your current contact point is a co-author on this manuscript. According to our Data Policy, the contact point must not be an author on the manuscript and must be an institutional contact, ideally not an individual. Please revise your data statement to a non-author institutional point of contact, such as a data access or ethics committee, and send this to us via return email. Please also include contact information for the third-party organization, and please include the full citation of where the data can be found.

Response: We thank academic editor. We have revised the Data Availability Statement in accordance with the PLOS ONE Data Availability Policy. Since this study is a systematic review and meta-analysis based on data extracted from previously published studies, we have clarified the source and availability of the data and removed the author as the data-access contact.

3. Please ensure that you refer to Figures 9 and 12 to 16 in your text as, if accepted, production will need this reference to link the reader to the figure.

Response: Thank you for pointing this out. We have revised the manuscript to ensure that Figures 9 and 12–16 are appropriately referred to in the text.

Response: we have included all the caption at the end of the manuscript.

Response: Okay thank you.

Response Reviewer #1

1. Overall, this manuscript addresses a highly relevant public health topic, particularly given the increasing importance of norovirus following the introduction of rotavirus vaccination. The research question is pertinent, and the study employs an appropriate systematic review and meta-analysis methodology. However, several methodological, statistical, and writing issues reduce the overall strength of the conclusions.

� Response: Thank you so much reviewer for the constructive evaluation of our manuscript. We appreciate the positive assessment of the relevance of the research question and the appropriateness of this manuscript. We have carefully considered all comments and have revised the manuscript accordingly. Please look at the newly revised manuscript.

2. The manuscript would benefit from careful language editing by a native English speaker or a professional scientific editing service. There are numerous grammatical errors and awkward sentence constructions throughout the text. For example, the sentence “Approximately one in five children presenting with diarrheal illness infected...” is grammatically incorrect and should be revised. In addition, several expressions, particularly “This systematic review,” are repeated excessively, making the text unnecessarily repetitive.

� Response: We thank the reviewer for this important observation. We agree that the previous version contained grammatical errors, awkward sentence constructions, and some repetitive expressions. We have therefore carefully revised the manuscript to improve grammar, readability, clarity, and scientific style. Please look at the newly uploaded manuscript.

3. Some methodological aspects also require clarification. The authors do not describe how studies reporting zero prevalence were handled in the meta-analysis, which is an important issue when pooling prevalence estimates.

� Response: We thank the reviewer for highlighting this important methodological issue. We agree that the treatment of studies reporting zero prevalence should be explicitly described in a prevalence meta-analysis. We have clarified the statistical methods used to handle zero-prevalence studies in the revised manuscript.

4. Restricting the search to English-language publications may have introduced language bias and should be acknowledged more explicitly.

� Response: We agree with the reviewer. Restricting the search to English-language publications may have resulted in the exclusion of potentially relevant studies published in other languages and may therefore have introduced language bias. We have mentioned it in the limitation part please look at the newly uploaded manuscript.

5. Furthermore, combining studies that used different diagnostic methods, such as RT-PCR and ELISA, is likely to have contributed to the substantial heterogeneity observed. A meta-regression including diagnostic method as a covariate would help determine whether the laboratory technique explains part of the between-study variability.

� Response: We agree that differences in diagnostic methods, including RT-PCR and ELISA, may contribute to between-study heterogeneity because these methods can differ in diagnostic performance and analytical characteristics. We therefore re-evaluated the diagnostic methods used across the included studies and considered diagnostic method as a potential source of heterogeneity. Where the number of studies and the distribution of diagnostic methods were sufficient to support a meaningful analysis, we performed meta-regression with diagnostic method as a covariate.

Response Reviewer #2

We sincerely thank Reviewer 2 for the time, constructive comments, and valuable suggestions. Your feedback has helped us improve the quality and clarity of the manuscript. We have carefully addressed all of your comments and suggestions and revised the manuscript accordingly. We also appreciate your efforts in highlighting both the strengths and weaknesses of our manuscript. Please kindly review the newly uploaded revised manuscript.

1. General Introduction & Summary

Weaknesses. Several numerical inconsistencies recur across sections and should be reconciled: the overall I² is variously reported as 98.85%, 98.72%, and 98.67%; GII pooled prevalence appears as both 76% and 75%; the GI prevalence (23%) is paired with a CI (10–17%) that does not contain the point estimate (Figure 14 gives 10–37%); and the stated rotavirus prevalence range (“17.90–24.10%”) does not match several Table 1 entries as high as 46.50%. Figure citations in the sensitivity-analysis subsection also appear mismatched against the actual figure captions, and a citation string in the genotype section is garbled, likely from a page-break artifact.

Beyond these, a more substantive point: given I² near 98–99% throughout, a single pooled headline figure (18%) may understate how disparate the underlying data are, even with subgroup analysis performed. Notably, the manuscript’s claim that “one study reported

both viruses” appears incorrect Table 1 shows two (El Qazoui et al., 2024 and Gopalkrishna et al., 2021, each listed under separate norovirus/rotavirus rows). We would also flag that nearly half of included studies (20/45) originate from India, which limits generalizability to other LMIC regions; that the English-only search restriction may have compounded the detected publication bias by excluding French/Spanish/Portuguese-language literature; and that pooling cross-sectional and surveillance designs together, despite differing methodologies, risks obscuring real differences in burden. Finally, if the Data Availability Statement restricts data to “available upon request,” this would not meet PLOS ONE’s minimal-dataset policy we could not verify this wording against the material provided and would ask the editor to confirm with the authors.

None of these issues undermine the manuscript’s core validity or contribution; they are correctable through text and data reconciliation, consistent with Accept with Text Revisions. However, several of the items above are substantive data-accuracy checks, not merely stylistic ones, and should be resolved before production.

2. Line-by-Line Revisions & Language Corrections

Representative examples are given for each manuscript section; this is not an exhaustive list. Citation-bracket spacing (e.g., “word[12]” instead of “word [12]”) recurs dozens of times throughout and should be corrected globally.

Response: All language-related concerns have been corrected in accordance with your comments. Please see the attached “Response to Reviewers” document for detailed responses to each comment.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0359886.s006.docx^{(31.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0359886.r003

Decision Letter 1

Ricardo Q Gurgel

15 Sep 2026

Dear Dr. Ayele,

Thank you for submitting your manuscript to PLOS One. After careful consideration, we feel that it has merit but does not fully meet PLOS One’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Ricardo Q. Gurgel, PhD

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comment:

Please do these changes and the manuscript will be accetpted. The changes are: My recommendation: Accept with following minor revision : Most changes are satisfactory; however, please (1) submit a clean file free of tracked-changes artifacts, (2) reconcile the I² value throughout, (3) either add the promised diagnostic-method meta-regression or remove that from the response letter, and (4) proofread the specific lines noted, several of which were marked 'Corrected' but remain unchanged.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

Reviewer #1: I thank the authors for their careful consideration of my comments and for the revisions made to the manuscript. After reviewing the authors’ responses and the revised manuscript, I consider that my concerns have been adequately addressed.

The revisions have improved the clarity, methodological rigor, and overall quality of the manuscript. I have no further major comments or concerns.

I therefore recommend acceptance of the manuscript in its revised form.

Reviewer #2: My recommendation: Accept with following minor revision : Most changes are satisfactory; however, please (1) submit a clean file free of tracked-changes artifacts, (2) reconcile the I² value throughout, (3) either add the promised diagnostic-method meta-regression or remove that from the response letter, and (4) proofread the specific lines noted, several of which were marked 'Corrected' but remain unchanged.

**********

what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes: CATHARINA CORREA COSTA

Reviewer #2: No

**********

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

PLoS One. 2026 Oct 13;21(10):e0359886. doi: 10.1371/journal.pone.0359886.r004

Author response to Decision Letter 2

15 Sep 2026

Response to Reviewers

We sincerely thank the reviewers and the Academic Editor for their careful evaluation of our revised manuscript and for their constructive and valuable comments. We greatly appreciate Reviewer 1’s recommendation for acceptance and Reviewer 2’s positive assessment of the revised manuscript. We have carefully considered the remaining minor issues raised by Reviewer 2 and have addressed each point as detailed below.

Response to Reviewer 2

Comment 1:

“Please submit a clean file free of tracked-changes artifacts.”

Response: Thank you for pointing this out. We have carefully reviewed the revised manuscript and prepared a clean version free of tracked changes, comments, and other revision artifacts. The clean manuscript has been uploaded as requested.

Comment 2:

“Reconcile the I² value throughout.”

Response: Thank you for raising this point. We carefully checked all reported I² values throughout the manuscript. We confirmed that the differences in I² values do not represent inconsistencies or errors; rather, they reflect the different levels of heterogeneity observed across the overall meta-analysis and the various subgroup analyses. Because each subgroup analysis is based on a different set of studies, the corresponding I² values naturally differ. We have nevertheless carefully reviewed the presentation of all I² values throughout the manuscript and ensured that each value corresponds correctly to its respective overall or subgroup analysis and is reported consistently with the statistical results.

Comment 3:

“Either add the promised diagnostic-method meta-regression or remove that from the response letter.”

Response: We sincerely thank the reviewer for this important clarification and apologize for any confusion caused by our previous response. Following the reviewer’s suggestion, we have removed the statement regarding the promised diagnostic-method meta-regression from the response letter. We have also carefully reviewed the manuscript and confirmed that it contains no unsupported claims regarding this analysis. We greatly appreciate the reviewer’s careful attention to this matter, which has helped us improve the clarity and accuracy of our response.

Comment 4:

“Proofread the specific lines noted, several of which were marked ‘Corrected’ but remain unchanged.”

Response: We sincerely appreciate the reviewer for identifying this issue. We have rechecked all the specific lines and comments raised in the previous review round, particularly those for which we had indicated “Corrected.” We carefully compared the reviewer’s comments, our previous responses, and the revised manuscript, and have now made the necessary corrections wherever they were still missing. The manuscript has also been thoroughly proofread to ensure that all stated corrections are accurately reflected in the final version.

Attachment

Submitted filename: 2 Response to Reviewers.docx

pone.0359886.s007.docx^{(14.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0359886.r005

Decision Letter 2

Ricardo Q Gurgel

20 Sep 2026

Prevalence and Genotype Distribution of Norovirus and Rotavirus Among Children Under Five with Diarrheal Infection in Lower-Middle-Income Countries After Rotavirus Vaccine Introduction: A Systematic Review and Meta-analysis

PONE-D-26-34325R2

Dear Dr. Ayele,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ricardo Q. Gurgel, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0359886.r006

Acceptance letter

Ricardo Q Gurgel

PONE-D-26-34325R2

PLOS One

Dear Dr. Ayele,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

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You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

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Thank you for submitting your work to PLOS One and supporting open access.

Kind regards,

PLOS One Editorial Office Staff

on behalf of

Professor Ricardo Q. Gurgel

Academic Editor

PLOS One

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

(DOCX)

pone.0359886.s001.docx^{(22KB, docx)}

(DOCX)

pone.0359886.s002.docx^{(22.6KB, docx)}

S3 Table. PRISMA 2020 checklist.

(DOCX)

pone.0359886.s003.docx^{(267.7KB, docx)}

(DOCX)

pone.0359886.s004.docx^{(28.8KB, docx)}

Attachment

Submitted filename: Peer_Review_Norovirus_Rotavirus_Manuscript.pdf

pone.0359886.s005.pdf^{(278KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0359886.s006.docx^{(31.5KB, docx)}

Attachment

Submitted filename: 2 Response to Reviewers.docx

pone.0359886.s007.docx^{(14.9KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Prevalence and genotype distribution of norovirus and rotavirus among children under five with diarrheal infection in lower-middle-income countries after Rotavirus vaccine introduction: A systematic review and meta-analysis

Firayad Ayele

Tesfaye Assebe

Miheret Mandefro

Desalegn Admassu Ayana

Nega Assefa

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods and materials

Registration and reporting

Data source and study search strategy

Inclusion and exclusion criteria for studies

Study selection

Data extraction

Study risk of bias and quality assessment

Evidence synthesis and analysis

Results

Study selection process

Risk of bias and quality assessment

Characteristics of included studies

Table 1. Summary characteristics of the 45 study reports for the systematic review and meta-analysis on prevalence and genotype distribution of norovirus and rotavirus among children under five with acute diarrhea in lower-middle-income countries after rotavirus vaccine introduction, 2026.

Pooled prevalence of Norovirus and Rotavirus

Fig 2. Forest plot showing the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Subgroup analysis, sensitivity analysis, and publication bias

Fig 3. Forest plot showing leave-one-out meta-analysis for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Fig 4. Galbraith plot for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Fig 5. Forest plot showing the subgroup analysis by outcome variable for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Fig 6. Forest plot showing the subgroup analysis by region for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Fig 7. Forest plot showing the subgroup analysis by study design for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Fig 8. Forest plot showing the subgroup analysis by detection methods for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Fig 9. Funnel plot for the pooled prevalence of norovirus and rotavirus among under five children with diarrheal infection in LMICs.

Genotype distribution Norovirus and Rotavirus

Fig 10. Distribution of Norovirus Genotype among children under five with diarrheal infection in lower-middle-income country.

Fig 11. Distribution of Rotavirus Genotype among children under five with diarrheal infection in lower-middle-income countries.

Pooled prevalence of predominant genotype Norovirus and Rotavirus

Fig 12. Pooled prevalence of GII among children under five with diarrheal infection in lower-middle-income countries.

Fig 16. Pooled prevalence of G1P[8] among children under five with diarrheal infection in lower-middle-income countries.

Fig 13. Pooled prevalence of GII.4 among children under five with diarrheal infection in lower-middle-income countries.

Fig 14. Pooled prevalence of GI among children under five with diarrheal infection in lower-middle-income countries.

Fig 15. Pooled prevalence of G3P [8] among children under five with diarrheal infection in lower-middle-income countries.

Discussion

Limitations of the study

Conclusions and recommendations

Conclusions

Recommendations

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Ricardo Q Gurgel

Roles

Author response to Decision Letter 1

Decision Letter 1

Ricardo Q Gurgel

Roles

Author response to Decision Letter 2

Decision Letter 2

Ricardo Q Gurgel

Roles

Acceptance letter

Ricardo Q Gurgel

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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