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

Everlyn Kamau,Charles N Agoti,Clement S Lewa,John Oketch,Betty E Owor,Grieven P Otieno,Anne Bett,Patricia A Cane,D James Nokes

doi:10.1016/j.jcv.2016.12.011

Everlyn Kamau, Charles N Agoti + Show 7 more

Open Access

https://doi.org/10.1016/j.jcv.2016.12.011

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Abstract

BackgroundDirect 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. ObjectivesEstablish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Study designNucleoprotein (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. ResultsN 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. ConclusionsAn 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.

Highlights

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to Respiratory syncytial virus (RSV) diagnosis in Kilifi, Kenya
False negatives can arise due to mismatches in primers or probes due to new polymorphisms in viral genomes [9] leading to subsequent underestimation of disease burden, unclear epidemiology and misguided clinical management
Phylogenetic analyses of the matching N and G gene sequences showed that the reverse transcription-PCR (rRT-PCR) negative RSV-B viruses are a separate phylogenetic clade distinct from the positive viruses (Fig. 2)

Summary

Introduction

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. These two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Study design: Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Results: N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. RSV diagnosis in pediatric admissions at KCH has throughout included a direct immuno-fluorescence test (IFAT) (RSV DFA kit, Light DiagnosticsTM), and in 2008, a custom multiplex realtime reverse transcription-PCR (rRT-PCR) [6,7] was implemented to screen for a range of respiratory viruses and for improved diagnostic sensitivity. We observed a discordance of RSV-B detection between IFAT and rRT-PCR methods in the recent seasonal epidemics (2014/15 and 2015/16) and postulated that this was due to probe-template nucleotide mismatches

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