Abstract
BackgroundReverse transcription-polymerase chain reaction (RT-PCR) is the main method for laboratory diagnosis of norovirus-associated infectious intestinal disease (IID). However, up to 16% of healthy individuals in the community, with no recent history of IID, may be RT-PCR positive; so it is unclear whether norovirus is actually the cause of illness in an IID case when they are RT-PCR positive. It is important to identify the pathogen causing illness in sporadic IID cases, for clinical management and for community based incidence studies. The aim of this study was to investigate how faecal viral load can be used to determine when norovirus is the most likely cause of illness in an IID case.MethodsReal-time RT-PCR was used to determine the viral load in faecal specimens collected from 589 IID cases and 159 healthy controls, who were infected with genogroup II noroviruses. Cycle threshold (Ct) values from the real-time RT-PCR were used as a proxy measure of viral load. Receiver-operating characteristic (ROC) analysis was used to identify a cut-off in viral load for attributing illness to norovirus in IID cases.ResultsOne hundred and sixty-nine IID cases and 159 controls met the inclusion criteria for the ROC analysis. The optimal Ct value cut-off for attributing IID to norovirus was 31. The same cut-off was selected when using healthy controls, or IID cases who were positive by culture for bacterial pathogens, as the reference negative group. This alternative reference negative group can be identified amongst specimens routinely received in clinical virology laboratories.ConclusionWe demonstrated that ROC analysis can be used to select a cut-off for a norovirus real time RT-PCR assay, to aid clinical interpretation and diagnose when norovirus is the cause of IID. Specimens routinely received for diagnosis in clinical virology laboratories can be used to select an appropriate cut-off. Individual laboratories can use this method to define in-house cut-offs for their assays, to provide the best possible diagnostic service to clinicians and public health workers. Other clinical and epidemiological information should also be considered for patients with Ct values close to the cut-off, for the most accurate diagnosis of IID aetiology.
Highlights
Reverse transcription-polymerase chain reaction (RT-PCR) is the main method for laboratory diagnosis of norovirus-associated infectious intestinal disease (IID)
Descriptive analysis Cycle threshold (Ct) values were generated for 589 IID cases and 159 healthy controls, who were infected with genogroup II noroviruses; 92 of the IID cases were positive by electron microscopy and 497 were negative by electron microscopy but subsequently positive by RT-PCR
By electron microscopy and subsequently RT-PCR positive overlaps substantially with the controls, a small proportion have the higher viral loads seen in the electron microscopy positive IID cases (Figure 1, Table 2)
Summary
Reverse transcription-polymerase chain reaction (RT-PCR) is the main method for laboratory diagnosis of norovirus-associated infectious intestinal disease (IID). It is important to identify the pathogen causing illness in sporadic IID cases, for clinical management and for community based incidence studies. Mixed gastrointestinal infections are frequently detected, especially in infants and young children and when polymerase chain reaction (PCR) assays are used for diagnosis [1,2]. It is important to determine which pathogen is the cause of illness, in order to direct clinical management for individual patients and to advance epidemiological understanding of IID. Reverse transcription- PCR (RT-PCR) is the method of choice for detecting norovirus in clinical specimens. RT-PCR detects a much wider range of norovirus genetic variants than enzyme-linked immunosorbent assays (ELISA) and may be more adaptable for detection of new strains [6]
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