Evaluation of the Dako IDEIA norovirus EIA assay for detection of norovirus using faecal specimens from Australian gastroenteritis outbreaks.
OBJECTIVE
New techniques for detection of norovirus, a major cause of gastroenteritis, require ongoing evaluation. The aim of this study was to use material from gastroenteritis outbreaks in Victoria, Australia, to evaluate the sensitivity and specificity of the Dako IDEIA norovirus EIA assay, using both photometric and visual analysis.


METHODS
A total of 130 faecal specimens from 41 gastroenteritis outbreaks were tested for norovirus by electron microscopy (EM), a two-round multiplex reverse transcription-polymerase chain reaction (RT-PCR) method and the IDEIA norovirus assay. All specimens with sufficient amplified product were sequenced to determine their norovirus genotype. In addition, six well-established RT-PCR protocols were used to test four EIA-positive, multiplex RT-PCR/EM-negative specimens. Also, a range of RT-PCR protocols was used to test a specimen positive for GII only by the multiplex RT-PCR but positive for GI and GII by the EIA. The effect of multiple freezing-thawing cycles on EIA positivity was tested on seven additional specimens. A further seven specimens, known to contain the gastroenteritis viruses sapovirus, adenovirus, astrovirus and rotavirus were also tested by the IDEIA norovirus assay.


RESULTS
The IDEIA norovirus assay gave a single-specimen sensitivity and specificity of 66% and 85%, respectively (visual analysis compared with the multiplex RT-PCR), 63% and 88% (photometric analysis compared with the multiplex RT-PCR), 65% and 87% (visual analysis compared with the multiplex RT-PCR and/or EM) and 62% and 90% (photometric analysis compared with the multiplex RT-PCR and/or EM). None of the four EIA-positive specimens negative by the multiplex RT-PCR and/or EM was positive by any of the six alternative RT-PCR protocols. The specimen positive for GI and GII by EIA but for GII only by the multiplex RT-PCR was not positive for GI by any of the alternative RT-PCR protocols. A minimum of three specimens per outbreak had to be tested by the EIA to ensure that norovirus-positive outbreaks (multiplex RT-PCR and/or EM) were classified as positive for norovirus by the IDEIA norovirus assay (visual or photometric analysis). However, one specimen from a norovirus-negative outbreak (multiplex RT-PCR and/or EM) for which four specimens were provided was positive for norovirus by the IDEIA norovirus assay. Seven norovirus genotypes were identified by open reading frame 1 sequencing analysis and specimens from all seven norovirus genotypes (as well as an EM-positive/multiplex RT-PCR-negative specimen) were detected by the IDEIA norovirus assay by both visual and photometric analysis. Repeated freezing-thawing cycles (up to six) for faecal specimens did not reduce the sensitivity of the EIA assay but could render an EIA-negative specimen EIA-positive. The specimens positive for sapovirus, adenovirus, astrovirus and rotavirus were EIA-negative.


CONCLUSIONS
The IDEIA norovirus assay lacks the sensitivity and specificity to ascribe a particular result to a particular specimen, but could be useful for detecting norovirus in a gastroenteritis outbreak where specimens are plentiful, although it is difficult to avoid a risk of false positives. Since visual analysis can be used for result assessment almost as reliably as photometric analysis, the test kit would be useful for laboratories lacking specialist equipment such as a photometric microplate reader.
Dimitriadis
Anna
A
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia.
Bruggink
Leesa D
LD
Marshall
John A
JA
D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D004196:Disease Outbreaks; D005243:Feces / Q000821:virology*; D005759:Gastroenteritis / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D029322:Norovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014739:Victoria / Q000453:epidemiology