Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR

Background Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Objectives Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Study design Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. Results N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. Conclusions An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy

Evolution of respiratory syncytial virus genotype BA in Kilifi, Kenya, 15 years on

Respiratory syncytial virus (RSV) is recognised as a leading cause of severe acute respiratory disease and deaths among infants and vulnerable adults. Clinical RSV isolates can be divided into several known genotypes. RSV genotype BA, characterised by a 60-nucleotide duplication in the G glycoprotein gene, emerged in 1999 and quickly disseminated globally replacing other RSV group B genotypes. Continual molecular epidemiology is critical to understand the evolutionary processes maintaining the success of the BA viruses. We analysed 735 G gene sequences from samples collected from paediatric patients in Kilifi, Kenya, between 2003 and 2017. The virus population comprised of several genetically distinct variants (n = 56) co-circulating within and between epidemics. In addition, there was consistent seasonal fluctuations in relative genetic diversity. Amino acid changes increasingly accumulated over the surveillance period including two residues (N178S and Q180R) that mapped to monoclonal antibody 2D10 epitopes, as well as addition of putative N-glycosylation sequons. Further, switching and toggling of amino acids within and between epidemics was observed. On a global phylogeny, the BA viruses from different countries form geographically isolated clusters suggesting substantial localized variants. This study offers insights into longitudinal population dynamics of a globally endemic RSV genotype within a discrete location

Genomic epidemiology of the rotavirus G2P[4] strains in coastal Kenya pre- and post-rotavirus vaccine introduction, 2012 – 2018

The introduction of rotavirus vaccines into the national immunization programme in many countries has led to a decline of 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 63 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 2014-2018) rotavirus vaccine introduction. All the 63 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

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

Respiratory Syncytial Virus genotype BA in Kilifi, Kenya : evolutionary dynamics, 15 years on

Respiratory syncytial virus (RSV) is recognised as a leading cause of severe acute respiratory disease and deaths among infants and vulnerable adults. Clinical RSV isolates can be divided into several known genotypes. RSV genotype BA, characterised by a 60-nucleotide duplication in the G glycoprotein gene, emerged in 1999 and quickly disseminated globally replacing other RSV group B genotypes. Continual molecular epidemiology is critical to understand the evolutionary processes maintaining the success of the BA viruses. We analysed 735 G gene sequences from samples collected from paediatric patients in Kilifi, Kenya, between 2003 and 2017. The virus population comprised of several genetically distinct variants (n = 56) co-circulating within and between epidemics. In addition, there was consistent seasonal fluctuations in relative genetic diversity. Amino acid changes increasingly accumulated over the surveillance period including two residues (N178S and Q180R) that mapped to monoclonal antibody 2D10 epitopes, as well as addition of putative N-glycosylation sequons. Further, switching and toggling of amino acids within and between epidemics was observed. On a global phylogeny, the BA viruses from different countries form geographically isolated clusters suggesting substantial localized variants. This study offers insights into longitudinal population dynamics of a globally endemic RSV genotype within a discrete location

Replication Data for: Genomic epidemiology of the rotavirus G2P[4] strains in coastal Kenya pre- and post-rotavirus introduction, 2012-2018

This is a replication dataset for the manuscript titled: "Genomic epidemiology of the rotavirus G2P[4] strains in coastal Kenya pre- and post-rotavirus introduction, 2012-2018." The dataset is part of the study investigating genomic epidemiology of rotavirus A strains circulating among children admitted to Kilifi County Hospital following routine vaccination. The dataset contains the participants' social demographic characteristics, vaccination status, narrows down to participants infected with the G2P[4] strain from 2012 to 2018. The dataset was used to generate genome sequences for G2P[4] strains from the G2P[4] genotyping of the VP7 and VP4 genes

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 COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate 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 a resource-limited setting. Methods: We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive – GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results: The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion: We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit’s performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results