Temporal changes in the positivity rate of common enteric viruses among paediatric admissions in coastal Kenya, during the COVID-19 pandemic, 2019-2022.

BACKGROUND: The non-pharmaceutical interventions (NPIs) implemented to curb the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) early in the coronavirus disease 2019 (COVID-19) pandemic, substantially disrupted the activity of other respiratory viruses. However, there is limited data from low-and-middle income countries (LMICs) to determine whether these NPIs also impacted the transmission of common enteric viruses. Here, we investigated the changes in the positivity rate of five enteric viruses among hospitalised children who presented with diarrhoea to a referral hospital in coastal Kenya, during COVID-19 pandemic period. METHODS: A total of 870 stool samples from children under 13 years of age admitted to Kilifi County Hospital between January 2019, and December 2022 were screened for rotavirus group A (RVA), norovirus genogroup II (GII), astrovirus, sapovirus, and adenovirus type F40/41 using real-time reverse-transcription polymerase chain reaction. The proportions positive across the four years were compared using the chi-squared test statistic. RESULTS: One or more of the five virus targets were detected in 282 (32.4%) cases. A reduction in the positivity rate of RVA cases was observed from 2019 (12.1%, 95% confidence interval (CI) 8.7-16.2%) to 2020 (1.7%, 95% CI 0.2-6.0%; p < 0.001). However, in the 2022, RVA positivity rate rebounded to 23.5% (95% CI 18.2%-29.4%). For norovirus GII, the positivity rate fluctuated over the four years with its highest positivity rate observed in 2020 (16.2%; 95% C.I, 10.0-24.1%). No astrovirus cases were detected in 2020 and 2021, but the positivity rate in 2022 was similar to that in 2019 (3.1% (95% CI 1.5%-5.7%) vs. 3.3% (95% CI 1.4-6.5%)). A higher case fatality rate was observed in 2021 (9.0%) compared to the 2019 (3.2%), 2020 (6.8%) and 2022 (2.1%) (p < 0.001). CONCLUSION: Our study finds that in 2020 the transmission of common enteric viruses, especially RVA and astrovirus, in Kilifi Kenya may have been disrupted due to the COVID-19 NPIs. After 2020, local enteric virus transmission patterns appeared to return to pre-pandemic levels coinciding with the removal of most of the government COVID-19 NPIs.

New SARS-CoV-2 Omicron Variant with Spike Protein Mutation Y451H, Kilifi, Kenya, March-May 2023.

We report a newly emerged SARS-CoV-2 Omicron subvariant FY.4 that has mutations Y451H in spike and P42L in open reading frame 3a proteins. FY.4 emergence coincided with increased SARS-CoV-2 cases in coastal Kenya during April-May 2023. Continued SARS-CoV-2 genomic surveillance is needed to identify new lineages to inform COVID-19 outbreak prevention.

Genomic epidemiology of the rotavirus G2P[4] strains in coastal Kenya pre- and post-rotavirus vaccine introduction, 2012-8.

The introduction of rotavirus vaccines into the national immunization programme in many countries has led to a decline in childhood diarrhoea disease burden. Coincidentally, the incidence of some rotavirus group A (RVA) genotypes has increased, which may result from non-vaccine-type replacement. Here, we investigate the evolutionary genomics of rotavirus G2P[4] which has shown an increase in countries that introduced the monovalent Rotarix® vaccine. We examined sixty-three RVA G2P[4] strains sampled from children (aged below 13 years) admitted to Kilifi County Hospital, coastal Kenya, pre- (2012 to June 2014) and post-(July 2014 to 2018) rotavirus vaccine introduction. All the sixty-three genome sequences showed a typical DS-1-like genome constellation (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Pre-vaccine G2 sequences predominantly classified as sub-lineage IVa-3 and co-circulated with low numbers of sub-lineage IVa-1 strains, whereas post-vaccine G2 sequences mainly classified into sub-lineage IVa-3. In addition, in the pre-vaccine period, P[4] sub-lineage IVa strains co-circulated with low numbers of P[4] lineage II strains, but P[4] sub-lineage IVa strains predominated in the post-vaccine period. On the global phylogeny, the Kenyan pre- and post-vaccine G2P[4] strains clustered separately, suggesting that different virus populations circulated in the two periods. However, the strains from both periods exhibited conserved amino acid changes in the known antigenic epitopes, suggesting that replacement of the predominant G2P[4] cluster was unlikely a result of immune escape. Our findings demonstrate that the pre- and post-vaccine G2P[4] strains circulating in Kilifi, coastal Kenya, differed genetically but likely were antigenically similar. This information informs the discussion on the consequences of rotavirus vaccination on rotavirus diversity.

Genomic epidemiology of human adenovirus F40 and F41 in coastal Kenya: A retrospective hospital-based surveillance study (2013-2022).

Human enteric adenovirus species F (HAdV-F) is a leading cause of childhood diarrhoeal deaths. The genomic analysis would be key to understanding transmission dynamics, potential drivers of disease severity, and vaccine development. However, currently, there are limited HAdV-F genomic data globally. Here, we sequenced and analysed HAdV-F from stool samples collected in coastal Kenya between 2013 and 2022. The samples were collected at Kilifi County Hospital in coastal Kenya from children <13 years of age who reported a history of three or more loose stools in the previous 24 hours. The genomes were analysed together with the data from the rest of the world by phylogenetic analysis and mutational profiling. Types and lineages were assigned based on phylogenetic clustering consistent with the previously described criteria and nomenclature. Participant clinical and demographic data were linked to genotypic data. Of ninety-one cases identified using real-time Polymerase Chain Reaction, eighty-eight near-complete genomes were assembled, and these were classified into HAdV-F40 (n = 41) and HAdV-F41 (n = 47). These types co-circulated throughout the study period. Three and four distinct lineages were observed for HAdV-F40 (Lineages 1-3) and HAdV-F41 (Lineages 1, 2A, 3A, 3C, and 3D). Types F40 and F41 coinfections were observed in five samples and F41 and B7 in one sample. Two children with F40 and 41 coinfections were also infected with rotavirus and had moderate and severe diseases as defined using the Vesikari Scoring System, respectively. Intratypic recombination was found in four HAdV-F40 sequences occurring between Lineages 1 and 3. None of the HAdV-F41 cases had jaundice. This study provides evidence of extensive genetic diversity, coinfections, and recombination within HAdV-F40 in a rural coastal Kenya that will inform public health policy, vaccine development that includes the locally circulating lineages, and molecular diagnostic assay development. We recommend future comprehensive studies elucidating on HAdV-F genetic diversity and immunity for rational vaccine development.

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century.

Differences in epidemiology of enteropathogens in children pre- and post-rotavirus vaccine introduction in Kilifi, coastal Kenya.

BACKGROUND: Kenya introduced Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) vaccination into its national immunization programme beginning July 2014. The impact of this vaccination program on the local epidemiology of various known enteropathogens is not fully understood. METHODS: We used a custom TaqMan Array Card (TAC) to screen for 28 different enteropathogens in 718 stools from children aged less than 13 years admitted to Kilifi County Hospital, coastal Kenya, following presentation with diarrhea in 2013 (before vaccine introduction) and in 2016-2018 (after vaccine introduction). Pathogen positivity rate differences between pre- and post-Rotarix® vaccination introduction were examined using both univariate and multivariable logistic regression models. RESULTS: In 665 specimens (92.6%), one or more enteropathogen was detected, while in 323 specimens (48.6%) three or more enteropathogens were detected. The top six detected enteropathogens were: enteroaggregative Escherichia coli (EAggEC; 42.1%), enteropathogenic Escherichia coli (EPEC; 30.2%), enterovirus (26.9%), rotavirus group A (RVA; 24.8%), parechovirus (16.6%) and norovirus GI/GII (14.4%). Post-rotavirus vaccine introduction, there was a significant increase in the proportion of samples testing positive for EAggEC (35.7% vs. 45.3%, p = 0.014), cytomegalovirus (4.2% vs. 9.9%, p = 0.008), Vibrio cholerae (0.0% vs. 2.3%, p = 0.019), Strongyloides species (0.8% vs. 3.6%, p = 0.048) and Dientamoeba fragilis (2.1% vs. 7.8%, p = 0.004). Although not reaching statistical significance, the positivity rate of adenovirus 40/41 (5.8% vs. 7.3%, p = 0.444), norovirus GI/GII (11.2% vs. 15.9%, p = 0.089), Shigella species (8.7% vs. 13.0%, p = 0.092) and Cryptosporidium spp. (11.6% vs. 14.7%, p = 0.261) appeared to increase post-vaccine introduction. Conversely, the positivity rate of sapovirus decreased significantly post-vaccine introduction (7.8% vs. 4.0%, p = 0.030) while that of RVA appeared not to change (27.4% vs. 23.5%, p = 0.253). More enteropathogen coinfections were detected per child post-vaccine introduction compared to before (mean: 2.7 vs. 2.3; p = 0.0025). CONCLUSIONS: In this rural Coastal Kenya setting, childhood enteropathogen infection burden was high both pre- and post-rotavirus vaccination introduction. Children who had diarrheal admissions post-vaccination showed an increase in coinfections and changes in specific enteropathogen positivity rates. This study highlights the utility of multipathogen detection platforms such as TAC in understanding etiology of childhood acute gastroenteritis in resource-limited regions.

Transmission networks of SARS-CoV-2 in Coastal Kenya during the first two waves: A retrospective genomic study.

Background: Detailed understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) regional transmission networks within sub-Saharan Africa is key for guiding local public health interventions against the pandemic. Methods: Here, we analysed 1139 SARS-CoV-2 genomes from positive samples collected between March 2020 and February 2021 across six counties of Coastal Kenya (Mombasa, Kilifi, Taita Taveta, Kwale, Tana River, and Lamu) to infer virus introductions and local transmission patterns during the first two waves of infections. Virus importations were inferred using ancestral state reconstruction, and virus dispersal between counties was estimated using discrete phylogeographic analysis. Results: During Wave 1, 23 distinct Pango lineages were detected across the six counties, while during Wave 2, 29 lineages were detected; 9 of which occurred in both waves and 4 seemed to be Kenya specific (B.1.530, B.1.549, B.1.596.1, and N.8). Most of the sequenced infections belonged to lineage B.1 (n = 723, 63%), which predominated in both Wave 1 (73%, followed by lineages N.8 [6%] and B.1.1 [6%]) and Wave 2 (56%, followed by lineages B.1.549 [21%] and B.1.530 [5%]). Over the study period, we estimated 280 SARS-CoV-2 virus importations into Coastal Kenya. Mombasa City, a vital tourist and commercial centre for the region, was a major route for virus imports, most of which occurred during Wave 1, when many Coronavirus Disease 2019 (COVID-19) government restrictions were still in force. In Wave 2, inter-county transmission predominated, resulting in the emergence of local transmission chains and diversity. Conclusions: Our analysis supports moving COVID-19 control strategies in the region from a focus on international travel to strategies that will reduce local transmission. Funding: This work was funded by The Wellcome (grant numbers: 220985, 203077/Z/16/Z, 220977/Z/20/Z, and 222574/Z/21/Z) and the National Institute for Health and Care Research (NIHR), project references: 17/63/and 16/136/33 using UK Aid from the UK government to support global health research, The UK Foreign, Commonwealth and Development Office. The views expressed in this publication are those of the author(s) and not necessarily those of the funding agencies.

Genomic Epidemiology of SARS-CoV-2 in Seychelles, 2020-2021.

Seychelles, an archipelago of 155 islands in the Indian Ocean, had confirmed 24,788 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the 31st of December 2021. The first SARS-CoV-2 cases in Seychelles were reported on the 14th of March 2020, but cases remained low until January 2021, when a surge was observed. Here, we investigated the potential drivers of the surge by genomic analysis of 1056 SARS-CoV-2 positive samples collected in Seychelles between 14 March 2020 and 31 December 2021. The Seychelles genomes were classified into 32 Pango lineages, 1042 of which fell within four variants of concern, i.e., Alpha, Beta, Delta and Omicron. Sporadic cases of SARS-CoV-2 detected in Seychelles in 2020 were mainly of lineage B.1 (lineage predominantly observed in Europe) but this lineage was rapidly replaced by Beta variant starting January 2021, and which was also subsequently replaced by the Delta variant in May 2021 that dominated till November 2021 when Omicron cases were identified. Using the ancestral state reconstruction approach, we estimated that at least 78 independent SARS-CoV-2 introduction events occurred in Seychelles during the study period. The majority of viral introductions into Seychelles occurred in 2021, despite substantial COVID-19 restrictions in place during this period. We conclude that the surge of SARS-CoV-2 cases in Seychelles in January 2021 was primarily due to the introduction of more transmissible SARS-CoV-2 variants into the islands.

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol.

INTRODUCTION: The ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs. METHODS: We utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'. RESULTS: We observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60-70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery. CONCLUSION: Utilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa.

The progression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Africa has so far been heterogeneous, and the full impact is not yet well understood. In this study, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations predominantly from Europe, which diminished after the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1, and C.1.1. Although distorted by low sampling numbers and blind spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a source for new variants.

Near-Complete Genome Sequences of Eight Human Astroviruses Recovered from Diarrheal Stool Samples of Hospitalized Children in Coastal Kenya in 2019.

Here, using a sequence-independent sequencing approach (M. V. Phan, P. Hong Anh, N. Van Cuong, B. Oude Munnink, et al., Virus Evol 2:vew027, 2016, https://doi.org/10.1093/ve/vew027), we determined human astrovirus (HAstV) genome sequences from eight diarrheal stool samples collected in coastal Kenya in 2019. Phylogenetic analysis identified the following 4 genotypes: HAstV-1 (n = 4), HAstV-2 (n = 1), HAstV-3 (n = 1), and HAstV-5 (n = 2).

Detection of SARS-CoV-2 variant 501Y.V2 in Comoros Islands in January 2021.

Background. Genomic data is key in understanding the spread and evolution of SARS-CoV-2 pandemic and informing the design and evaluation of interventions. However, SARS-CoV-2 genomic data remains scarce across Africa, with no reports yet from the Indian Ocean islands. Methods. We genome sequenced six SARS-CoV-2 positive samples from the first major infection wave in the Union of Comoros in January 2021 and undertook detailed phylogenetic analysis. Results. All the recovered six genomes classified within the 501Y.V2 variant of concern (also known as lineage B.1.351) and appeared to be from 2 sub-clusters with the most recent common ancestor dated 30 th Oct-2020 (95% Credibility Interval: 06 th Sep-2020 to 10 th Dec-2020). Comparison of the Comoros genomes with those of 501Y.V2 variant of concern from other countries deposited into the GISAID database revealed their close association with viruses identified in France and Mayotte (part of the Comoros archipelago and a France, Overseas Department). Conclusions. The recovered genomes, albeit few, confirmed local transmission following probably multiple introductions of the SARS-CoV-2 501Y.V2 variant of concern during the Comoros's first major COVID-19 wave. These findings demonstrate the importance of genomic surveillance and have implications for ongoing control strategies on the islands.

Epidemiological Trends of Five Common Diarrhea-Associated Enteric Viruses Pre- and Post-Rotavirus Vaccine Introduction in Coastal Kenya.

Using real-time RT-PCR, we screened stool samples from children aged <5 years presenting with diarrhea and admitted to Kilifi County Hospital, coastal Kenya, pre- (2003 and 2013) and post-rotavirus vaccine introduction (2016 and 2019) for five viruses, namely rotavirus group A (RVA), norovirus GII, adenovirus, astrovirus and sapovirus. Of the 984 samples analyzed, at least one virus was detected in 401 (40.8%) patients. Post rotavirus vaccine introduction, the prevalence of RVA decreased (23.3% vs. 13.8%, p < 0.001) while that of norovirus GII increased (6.6% vs. 10.9%, p = 0.023). The prevalence of adenovirus, astrovirus and sapovirus remained statistically unchanged between the two periods: 9.9% vs. 14.2%, 2.4% vs. 3.2 %, 4.6% vs. 2.6%, (p = 0.053, 0.585 and 0.133), respectively. The median age of diarrhea cases was higher post vaccine introduction (12.5 months, interquartile range (IQR): 7.9-21 vs. 11.2 months pre-introduction, IQR: 6.8-16.5, p < 0.001). In this setting, RVA and adenovirus cases peaked in the dry months while norovirus GII and sapovirus peaked in the rainy season. Astrovirus did not display clear seasonality. In conclusion, following rotavirus vaccine introduction, we found a significant reduction in the prevalence of RVA in coastal Kenya but an increase in norovirus GII prevalence in hospitalized children.

Pooled testing conserves SARS-CoV-2 laboratory resources and improves test turn-around time: experience on the Kenyan Coast.

Background. International recommendations for the control of the coronavirus disease 2019 (COVID-19) pandemic emphasize the central role of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent, at scale. The availability of testing reagents, laboratory equipment and qualified staff are important bottlenecks to achieving this. Elsewhere, pooled testing (i.e. combining multiple samples in the same reaction) has been suggested to increase testing capacities in the pandemic period. Methods. We discuss our experience with SARS-CoV-2 pooled testing using real-time reverse transcription polymerase chain reaction (RT-PCR) on the Kenyan Coast. Results. In mid-May, 2020, our RT-PCR testing capacity for SARS-CoV-2 was improved by ~100% as a result of adoption of a six-sample pooled testing strategy. This was accompanied with a concomitant saving of ~50% of SARS-CoV-2 laboratory test kits at both the RNA extraction and RT-PCR stages. However, pooled testing came with a slight decline of test sensitivity. The RT-PCR cycle threshold value (ΔCt) was ~1.59 higher for samples tested in pools compared to samples tested singly. Conclusions. Pooled testing is a useful strategy to increase SARS-CoV-2 laboratory testing capacity especially in low-income settings.

An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams.

Background: The global COVID-19 outbreak relies on a quantitative real-time polymerase chain reaction (qRT-PCR) for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2), to facilitate the roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in our setting, where the supply of testing reagents is limited. Methods: Standards generated from a serially-diluted positive control and previously identified positive/negative samples were used to determine the optimal volumes of the qRT-PCR reagents and to evaluate the validity and performance of four assays: Charité Berlin and European Virus Archive - GLOBAL (EVAg) primer-probe sets, and DAAN and Beijing Genomics Institute (BGI) premixed commercial kits. A multiplex and singleplex RT-PCR kit was used with the two primer-probe sets and the recommended assay volumes of the two premixed kits were altered. Results: In comparison to the multiplex RT-PCR kit, the singleplex RT-PCR kit combined with the primer-probe sets yielded consistent cycle threshold (Ct) values across the different titrations tested. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit showed incomparable Ct values and inconsistent results between batches using the manufacturer's recommended volumes. Conclusion: We achieved a 2.5-fold and 4-fold increase in the number of tests/kit for the premixed kits and the primer-probe sets, respectively. The primer-probe set assays were reliable and consistent, and we preferred a combination of an EVAg and a Berlin target. Any inconclusive result was repeated by different individuals following the same protocol. DAAN was a consistent and reliable assay even at lower concentrations from the stated recommendations. BGI in contrast, required dilution to improve its performance and was hence an assay that was used in combination with EVAg or Berlin targets.