Abstract
BackgroundConventional reverse transcription-polymerase chain reaction (RT-PCR) amplification of the RNA-dependent RNA polymerase (RdRp) gene remains a used method for the rapid detection of norovirus (NV) in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated.Methods/Principal FindingsAfter an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6%) samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91–97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17–37.92, P = .003) and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21–24.73, P = .027) as significant and independent factors associated with false positives.ConclusionConventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.
Highlights
Norovirus (NV) is recognised as the leading cause of acute non-bacterial gastroenteritis and is frequently responsible for foodborne gastroenteritis outbreaks worldwide [1,2]
Conventional reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source
The region in ORF2 has been used as the target site for RT-PCR detection of norovirus by CaliciNet, a national laboratory surveillance network for norovirus outbreaks coordinated by CDC [1]
Summary
Norovirus (NV) is recognised as the leading cause of acute non-bacterial gastroenteritis and is frequently responsible for foodborne gastroenteritis outbreaks worldwide [1,2]. NV-associated disease in previous healthy individuals is self-limiting and usually characterised by vomiting, watery diarrhoea, abdominal cramps and low-grade fever. Severe outcomes, such as hospitalisation and mortality, are uncommon and estimated to occur in 0.54% and 0.06%, respectively, of infected cases during NV outbreaks [3]. A number of patients with acute gastroenteritis in this outbreak might have been falsely reported as having NV infection. The results of this study allowed a comprehensive evaluation of the incidence of and factors associated with false positivity in the RT-PCR assay in the laboratory diagnosis of human NV infections
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