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Diagnosing norovirus-associated infectious intestinal disease using viral load.

Phillips G, Lopman B, Tam CC, Iturriza-Gomara M, Brown D, Gray J - BMC Infect. Dis. (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastrointestinal, Emerging and Zoonotic Infections, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London, UK. gemma.phillips@hpa.org.uk

ABSTRACT

Background: Reverse 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.

Methods: Real-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.

Results: One 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.

Conclusion: We 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.

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Percentage distribution of real time RT-PCR Ct values in IID cases and controls. Low Ct values correspond to high viral loads; the viral load decreases with increasing Ct value. 'EM cases' are IID cases positive by electron microscopy, 'RT-PCR cases' are IID cases negative by electron microscopy and subsequently positive by RT-PCR. Sample sizes: EM cases = 92, RT-PCR cases = 497, controls = 159.
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Figure 1: Percentage distribution of real time RT-PCR Ct values in IID cases and controls. Low Ct values correspond to high viral loads; the viral load decreases with increasing Ct value. 'EM cases' are IID cases positive by electron microscopy, 'RT-PCR cases' are IID cases negative by electron microscopy and subsequently positive by RT-PCR. Sample sizes: EM cases = 92, RT-PCR cases = 497, controls = 159.

Mentions: The median Ct value was lower in IID cases (median 34) than in controls (median 38) (Table 2). The difference compared to controls was greatest for IID cases positive by electron microscopy (median 24); there was very little overlap in the distribution of Ct values in electron microscopy positive IID cases and controls (Figure 1). The distribution of Ct values for the IID cases who were negative by electron microscopy and subsequently RT-PCR positive overlaps substantially with the controls, although a small proportion have the higher viral loads seen in the electron microscopy positive IID cases (Figure 1, Table 2).


Diagnosing norovirus-associated infectious intestinal disease using viral load.

Phillips G, Lopman B, Tam CC, Iturriza-Gomara M, Brown D, Gray J - BMC Infect. Dis. (2009)

Percentage distribution of real time RT-PCR Ct values in IID cases and controls. Low Ct values correspond to high viral loads; the viral load decreases with increasing Ct value. 'EM cases' are IID cases positive by electron microscopy, 'RT-PCR cases' are IID cases negative by electron microscopy and subsequently positive by RT-PCR. Sample sizes: EM cases = 92, RT-PCR cases = 497, controls = 159.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698835&req=5

Figure 1: Percentage distribution of real time RT-PCR Ct values in IID cases and controls. Low Ct values correspond to high viral loads; the viral load decreases with increasing Ct value. 'EM cases' are IID cases positive by electron microscopy, 'RT-PCR cases' are IID cases negative by electron microscopy and subsequently positive by RT-PCR. Sample sizes: EM cases = 92, RT-PCR cases = 497, controls = 159.
Mentions: The median Ct value was lower in IID cases (median 34) than in controls (median 38) (Table 2). The difference compared to controls was greatest for IID cases positive by electron microscopy (median 24); there was very little overlap in the distribution of Ct values in electron microscopy positive IID cases and controls (Figure 1). The distribution of Ct values for the IID cases who were negative by electron microscopy and subsequently RT-PCR positive overlaps substantially with the controls, although a small proportion have the higher viral loads seen in the electron microscopy positive IID cases (Figure 1, Table 2).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastrointestinal, Emerging and Zoonotic Infections, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London, UK. gemma.phillips@hpa.org.uk

ABSTRACT

Background: Reverse 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.

Methods: Real-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.

Results: One 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.

Conclusion: We 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.

Show MeSH
Related in: MedlinePlus