Rotavirus group A genotype circulation patterns across Kenya before and after nationwide vaccine introduction, 2010-2018.

BACKGROUND: Kenya introduced the monovalent G1P [8] Rotarix® vaccine into the infant immunization schedule in July 2014. We examined trends in rotavirus group A (RVA) genotype distribution pre- (January 2010-June 2014) and post- (July 2014-December 2018) RVA vaccine introduction. METHODS: Stool samples were collected from children aged < 13 years from four surveillance sites across Kenya: Kilifi County Hospital, Tabitha Clinic Nairobi, Lwak Mission Hospital, and Siaya County Referral Hospital (children aged < 5 years only). Samples were screened for RVA using enzyme linked immunosorbent assay (ELISA) and VP7 and VP4 genes sequenced to infer genotypes. RESULTS: We genotyped 614 samples in pre-vaccine and 261 in post-vaccine introduction periods. During the pre-vaccine introduction period, the most frequent RVA genotypes were G1P [8] (45.8%), G8P [4] (15.8%), G9P [8] (13.2%), G2P [4] (7.0%) and G3P [6] (3.1%). In the post-vaccine introduction period, the most frequent genotypes were G1P [8] (52.1%), G2P [4] (20.7%) and G3P [8] (16.1%). Predominant genotypes varied by year and site in both pre and post-vaccine periods. Temporal genotype patterns showed an increase in prevalence of vaccine heterotypic genotypes, such as the commonly DS-1-like G2P [4] (7.0 to 20.7%, P < .001) and G3P [8] (1.3 to 16.1%, P < .001) genotypes in the post-vaccine introduction period. Additionally, we observed a decline in prevalence of genotypes G8P [4] (15.8 to 0.4%, P < .001) and G9P [8] (13.2 to 5.4%, P < .001) in the post-vaccine introduction period. Phylogenetic analysis of genotype G1P [8], revealed circulation of strains of lineages G1-I, G1-II and P [8]-1, P [8]-III and P [8]-IV. Considerable genetic diversity was observed between the pre and post-vaccine strains, evidenced by distinct clusters. CONCLUSION: Genotype prevalence varied from before to after vaccine introduction. Such observations emphasize the need for long-term surveillance to monitor vaccine impact. These changes may represent natural secular variation or possible immuno-epidemiological changes arising from the introduction of the vaccine. Full genome sequencing could provide insights into post-vaccine evolutionary pressures and antigenic diversity.

Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples.

Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.

Multiple Introductions and Predominance of Rotavirus Group A Genotype G3P[8] in Kilifi, Coastal Kenya, 4 Years after Nationwide Vaccine Introduction.

Globally, rotavirus group A (RVA) remains a major cause of severe childhood diarrhea, despite the use of vaccines in more than 100 countries. RVA sequencing for local outbreaks facilitates investigation into strain composition, origins, spread, and vaccine failure. In 2018, we collected 248 stool samples from children aged less than 13 years admitted with diarrheal illness to Kilifi County Hospital, coastal Kenya. Antigen screening detected RVA in 55 samples (22.2%). Of these, VP7 (G) and VP4 (P) segments were successfully sequenced in 48 (87.3%) and phylogenetic analysis based on the VP7 sequences identified seven genetic clusters with six different GP combinations: G3P[8], G1P[8], G2P[4], G2P[8], G9P[8] and G12P[8]. The G3P[8] strains predominated the season (n = 37, 67.2%) and comprised three distinct G3 genetic clusters that fell within Lineage I and IX (the latter also known as equine-like G3 Lineage). Both the two G3 lineages have been recently detected in several countries. Our study is the first to document African children infected with G3 Lineage IX. These data highlight the global nature of RVA transmission and the importance of increasing global rotavirus vaccine coverage.

Molecular Epidemiology of Human Rhinovirus From 1-Year Surveillance Within a School Setting in Rural Coastal Kenya.

BACKGROUND: Human rhinovirus (HRV) is the most common cause of the common cold but may also lead to more severe respiratory illness in vulnerable populations. The epidemiology and genetic diversity of HRV within a school setting have not been previously described. The objective of this study was to characterize HRV molecular epidemiology in a primary school in a rural location of Kenya. METHODS: Between May 2017 and April 2018, over 3 school terms, we collected 1859 nasopharyngeal swabs (NPS) from pupils and teachers with symptoms of acute respiratory infection in a public primary school in Kilifi County, coastal Kenya. The samples were tested for HRV using real-time reverse transcription polymerase chain reaction. HRV-positive samples were sequenced in the VP4/VP2 coding region for species and genotype classification. RESULTS: A total of 307 NPS (16.4%) from 164 individuals were HRV positive, and 253 (82.4%) were successfully sequenced. The proportion of HRV in the lower primary classes was higher (19.8%) than upper primary classes (12.2%; P < .001). HRV-A was the most common species (134/253; 53.0%), followed by HRV-C (73/253; 28.9%) and HRV-B (46/253; 18.2%). Phylogenetic analysis identified 47 HRV genotypes. The most common genotypes were A2 and B70. Numerous (up to 22 in 1 school term) genotypes circulated simultaneously, there was no individual re-infection with the same genotype, and no genotype was detected in all 3 school terms. CONCLUSIONS: HRV was frequently detected among school-going children with mild acute respiratory illness symptoms, particularly in the younger age groups (<5-year-olds). Multiple HRV introductions were observed that were characterized by considerable genotype diversity.

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.

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.

Airway response to respiratory syncytial virus has incidental antibacterial effects.

RSV infection is typically associated with secondary bacterial infection. We hypothesise that the local airway immune response to RSV has incidental antibacterial effects. Using coordinated proteomics and metagenomics analysis we simultaneously analysed the microbiota and proteomes of the upper airway and determined direct antibacterial activity in airway secretions of RSV-infected children. Here, we report that the airway abundance of Streptococcus was higher in samples collected at the time of RSV infection compared with samples collected one month later. RSV infection is associated with neutrophil influx into the airway and degranulation and is marked by overexpression of proteins with known antibacterial activity including BPI, EPX, MPO and AZU1. Airway secretions of children infected with RSV, have significantly greater antibacterial activity compared to RSV-negative controls. This RSV-associated, neutrophil-mediated antibacterial response in the airway appears to act as a regulatory mechanism that modulates bacterial growth in the airways of RSV-infected children.

Author Correction: Airway response to respiratory syncytial virus has incidental antibacterial effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human rhinovirus spatial-temporal epidemiology in rural coastal Kenya, 2015-2016, observed through outpatient surveillance.

Background: Human rhinovirus (HRV) is the predominant cause of upper respiratory tract infections, resulting in a significant public health burden. The virus circulates as many different types (168), each generating strong homologous, but weak heterotypic, immunity. The influence of these features on transmission patterns of HRV in the community is understudied. Methods: Nasopharyngeal swabs were collected from patients with symptoms of acute respiratory infection (ARI) at nine out-patient facilities across a Health and Demographic Surveillance System between December 2015 and November 2016. HRV was diagnosed by real-time RT-PCR, and the VP4/VP2 genomic region of the positive samples sequenced. Phylogenetic analysis was used to determine the HRV types. Classification models and G-test statistic were used to investigate HRV type spatial distribution. Demographic characteristics and clinical features of ARI were also compared. Results: Of 5,744 NPS samples collected, HRV was detected in 1057 (18.4%), of which 817 (77.3%) were successfully sequenced. HRV species A, B and C were identified in 360 (44.1%), 67 (8.2%) and 390 (47.7%) samples, respectively. In total, 87 types were determined: 39, 10 and 38 occurred within species A, B and C, respectively. HRV types presented heterogeneous temporal patterns of persistence. Spatially, identical types occurred over a wide distance at similar times, but there was statistically significant evidence for clustering of types between health facilities in close proximity or linked by major road networks. Conclusion: This study records a high prevalence of HRV in out-patient presentations exhibiting high type diversity. Patterns of occurrence suggest frequent and independent community invasion of different types. Temporal differences of persistence between types may reflect variation in type-specific population immunity. Spatial patterns suggest either rapid spread or multiple invasions of the same type, but evidence of similar types amongst close health facilities, or along road systems, indicate type partitioning structured by local spread.