Incidence of and Factors Associated with False Positives in Laboratory Diagnosis of Norovirus Infection by Amplification of the RNA-Dependent RNA Polymerase Gene

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.

Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

“…the lack of reliance on larger, more expensive equipment for amplification and detection; (ii.) the use of recombinase enzymes that reduce the likelihood of false positive results due to their inherent proofreading capabilities6736; (iii.) quicker time-to-result; and (iv) the potential for reduced impact of matrix-associated inhibitors.…”

Surveillance of Food- and Smear-Transmitted Pathogens in European Soldiers with Diarrhea on Deployment in the Tropics: Experience from the European Union Training Mission (EUTM) Mali

“…The inability of genotyping in initially positive tested samples is a consequence of the high mutation rate in Norovirus. In contrast, depending on the targeted sequence, false positive results of Norovirus PCR tests may occur [ 29 ]. However, this was not confirmed in this study.…”

Genotype distribution of norovirus around the emergence of Sydney_2012 and the antigenic drift of contemporary GII.4 epidemic strains

“…The VP1 of GII.4 strains is evolving via epochal evolution, a pattern of change characterized by circulating strain replacement with a newly emergent strain, followed [26]. This new variant has also disseminated widely across China and has replaced Den Haag_2006b as the predominant variant since September 2012 [27][28][29].…”