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Journal of Clinical Microbiology, April 2005, p. 2030-2031, Vol. 43, No. 4
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.4.2030-2031.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Comparison of Two Real-Time PCR Methods for Diagnosis of Norovirus Infection in Outbreak and Community Settings


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LETTER
 
In a recent edition of the Journal of Clinical Microbiology, a real-time PCR test was described that used TaqMan technology to detect and differentiate between group 1 and group 2 noroviruses (NV) (2). We have previously described a SYBR green-based real-time PCR method based on primers Ni and E3, which has been our frontline test for outbreaks of NV since 2000 (1). We compared both assays on 217 stool samples sent to the West of Scotland Specialist Virology Centre from 6 nosocomial outbreaks plus 153 sporadic cases of acute gastroenteritis. All samples from outbreaks had previously tested negative for bacteria and parasites. However, microbiological data for the sporadic cases of gastroenteritis was absent.

Overall, the method described by Kageyama et al. detected more NV-positive samples than the SYBR green-based method (41 versus 13) (Table 1). Of the sporadic cases, the TaqMan assay detected more NV-positive samples (15 versus 6). The TaqMan assay detected NV in five of the six outbreaks investigated, while the SYBR green-based assay detected NV in two of the six outbreaks. Using the criteria described by Richards et al. (4), which state that the definition of an outbreak requires the investigation of a minimum of six samples of which at least two should be positive by a laboratory method for NV or other pathogen, this method identified NV as the cause of four outbreaks (outbreaks 1, 3, 4, and 5). In comparison, the SYBR green-based method identified NV as the cause of one outbreak only (outbreak 3). Although both methods detected a single positive sample in an outbreak (one sample was positive by the TaqMan assay in outbreak 2 and one sample was positive by the SYBR green-based assay in outbreak 4), neither result means that NV could be considered as the definite cause until more positive samples are obtained. One outbreak was negative for NV by both real-time PCR methods (outbreak 6).


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  		TABLE 1. Comparison of TaqMan PCR and a SYBR green-based assay for 217 samples taken from outbreaks and sporadic cases of IID

Studies of hospital outbreaks of NV have shown that rapid implementation of infection control procedures can prevent spread to further wards (3). Once an outbreak has been detected it is our experience that clinicians and microbiologists will not close a ward or form an outbreak control team without a positive virological confirmation. Consequently, for infection control to be successful, hospital outbreaks of NV need to be confirmed using rapid and sensitive diagnostic tests. Real-time PCR is ideally suited for NV diagnosis in this setting as it is as sensitive as nested gel methods and results are available within the working day. There are now a number of real-time PCR methods available for the detection of NV. At present comparisons between the various assays are lacking.

Overall, our results show that, of the two methods, the method described by Kageyama et al. offers more sensitive real-time diagnosis of NV in sporadic cases and the outbreak setting. The use of the SYBR green-based method as the frontline test would have resulted in three false-negative outbreaks. The reduced sensitivity of the SYBR green-based method is most likely a consequence of primers Ni and E3. In a previous edition of this journal, Vinje et al. showed a heminested gel-based PCR assay (utilizing primers Ni, E3, and Ando) to have a low end-point detection limit when compared to other gel-based assays (5). This assay also failed to detect ~30% of the NV strains examined.

Although both methods are rapid, we recommend the use of the TaqMan assay for NV diagnosis in the sporadic and outbreak setting as it offers increased sensitivity and easy interpretation (melting peak variation can be observed when using the SYBR green method). The ability to use the TaqMan assay to type NV-positive samples without the need for further molecular analysis will allow laboratories to monitor general epidemiological patterns and may highlight areas that may warrant further investigation (e.g., sudden increases in outbreaks attributable to G1). Although the TaqMan assay may be slightly more expensive in terms of reagents (it utilizes more primers and three dual-labeled probes), the SYBR green-based method will be more expensive overall as there will be a greater need for alternative NV testing on negative outbreaks and sporadic samples. It should also be noted that the improved sensitivity of the TaqMan assay would also lead to significant improvements in the sensitivity and thus utility of national outbreak surveillance data. This will allow a more accurate assessment of the burden, in terms of both morbidity and healthcare costs of NV outbreaks. This will also aid the planning and assessment of public health prevention policies, which will in time contribute to healthcare cost savings.


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REFERENCES
 
    1
  1. Gunson, R. N., J. Miller, and W. F. Carman. 2003. Comparison of real-time PCR and EIA for the detection of outbreaks of acute gastroenteritis caused by norovirus. Commun. Dis. Public Health 6:297-299.[Medline]
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  2. Kageyama, T., S. Kojima, M. Shinohara, K. Uchida, S. Fukushi, F. B. Hoshino, N. Takeda, and K. Katayama. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J. Clin. Microbiol. 41:1548-1557.[Abstract/Free Full Text]
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  3. McCall, J., and R. Smithson. 2002. Rapid response and strict control measures can contain a hospital outbreak of Norwalk-like virus. Commun. Dis. Public Health 5:243-246.[Medline]
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  4. Richards, A. F., B. Lopman, A. Gunn, A. Curry, D. Ellis, H. Cotterill, S. Ratcliffe, M. Jenkins, H. Appleton, C. I. Gallimore, J. J. Gray, and D. W. Brown. 2003. Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces. J. Clin. Virol. 26:109-115.[CrossRef][Medline]
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  5. Vinjé, J., H. Vennema, L. Maunula, C.-H. von Bonsdorff, M. Hoehne, E. Schreier, A. Richards, J. Green, D. Brown, S. S. Beard, S. S. Monroe, E. de Bruin, L. Svensson, and M. P. G. Koopmans. 2003. International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses. J. Clin. Microbiol. 41:1423-1433.[Abstract/Free Full Text]
R. N. Gunson*
W. F. Carman
West of Scotland Specialist Virology Centre
Gartnavel General Hospital
Great Western Road
Glasgow G12 OYN, United Kingdom

* Phone: 44 141 211 0080, Fax: 44 141 211 0082, E-mail: Rory.Gunson{at}NorthGlasgow.Scot.NHS.UK

Journal of Clinical Microbiology, April 2005, p. 2030-2031, Vol. 43, No. 4
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.4.2030-2031.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Adamson, W. E., Gunson, R. N., Maclean, A., Carman, W. F. (2007). Emergence of a New Norovirus Variant in Scotland in 2006. J. Clin. Microbiol. 45: 4058-4060 [Abstract] [Full Text]  

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