Evaluation of the Dako IDEIA norovirus EIA assay for detection of norovirus using faecal specimens from Australian gastroenteritis outbreaks.

AIMS: 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.

Rotavirus detection and characterisation in outbreaks of gastroenteritis in aged-care facilities.

BACKGROUND: Although rotavirus is a major cause of gastroenteritis in children, its role in adult gastroenteritis and the sensitivity of different methods for its detection in specimens collected from adults are less well understood. OBJECTIVES: (1) To examine the frequency and seasonality of rotavirus-associated gastroenteritis outbreaks in aged-care facilities in Victoria, Australia. (2) To determine rotavirus type in these outbreaks. (3) To determine whether other enteropathogenic agents are present in specimens from these outbreaks. (4) To examine the sensitivity of different methods (electron microscopy (EM), reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA) and latex agglutination (LA)) for the detection of rotavirus in specimens from adults. STUDY DESIGN: Specimens from gastroenteritis outbreaks in aged-care facilities forwarded to this laboratory for the years 1997-2000 were tested for enteropathogenic agents by various methods. Epidemiological, clinical and seasonal data from the rotavirus-positive outbreaks were analysed. RESULTS: Rotavirus was detected by EM in 18 out of 29 individuals associated with seven out of 53 (13%) gastroenteritis outbreaks in aged-care facilities; norovirus was detected in 22 outbreaks (42%) and astrovirus in one outbreak (2%). No mixed viral infection was found in any outbreak. All rotaviruses were typed as Group A by RT-PCR. The rotaviruses in the seven outbreaks were G-typed as follows: G2 (three outbreaks), G4 (two outbreaks), G1 (one outbreak) and G9 (one outbreak). The rotavirus-associated outbreaks were concentrated in mid-winter to mid-spring. The relative sensitivities of the Group A rotavirus detection methods (for the 29 specimens tested) were EM (18), first-round RT-PCR (11), second-round PCR (19), EIA-visual (19), EIA-photometric (19) and LA (13). CONCLUSIONS: In Victoria, Australia, outbreaks of gastroenteritis associated with rotavirus are quite common in aged-care facilities. They involve Group A rotavirus and have a winter/spring seasonality. G-types G1, G2, G4 and G9 were all detected. EIA, second-round PCR and EM proved sensitive methods for rotavirus detection whereas first-round RT-PCR and LA did not.

Molecular and epidemiological features of norovirus-associated gastroenteritis outbreaks in Victoria, Australia in 2001.

Norovirus was identified in 30 of 59 gastroenteritis outbreaks occurring in the state of Victoria, Australia in 2001 by RT-PCR and/or electron microscopy (EM). Norovirus outbreaks occurred in hostels/nursing homes (27%), hospitals (13%), youth refuges (3%), social gatherings associated with food consumption (27%), school outings/camps (13%), and pre-school/child-minding centers (17%). Norovirus outbreaks tended to occur in the warmer months. Phylogenetic analysis identified six clusters, one within genogroup 1 (G1) and five within genogroup 2 (G2). Cluster 1, which incorporates the G2 Camberwell/Lordsdale strains, was the most common (39% of outbreaks). In 2 of 27 outbreaks, strains from two G2 clusters, 1 and 5, occurred. Norovirus G2 was more common in the young and very old than in those in intermediate years. Norovirus G2 detection rate was higher in females than in males for adults (>15 years) and the susceptibility of adult females to norovirus G2 infection relative to males increased with age. In one outbreak analyzed, some sequences had a single base substitution, but this did not result in an amino acid (aa) change. The two most common norovirus clusters (G2 clusters 1 and 4) occurred in the capital of Victoria, Melbourne, and regional Victoria, but the least common clusters (G2 clusters 2 and 3 and G1 cluster 8) only occurred in inner Melbourne. Norovirus was occasionally detected by EM but not by RT-PCR. The occurrence of norovirus outbreaks is modulated by a large group of factors, which will have to be considered in any epidemiological model.