Michael Catton

Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens

ABSTRACT A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.

Heminested Multiplex Reverse Transcription-PCR for Detection and Differentiation of Norwalk-Like Virus Genogroups 1 and 2 in Fecal Samples

ABSTRACT The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 μl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

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 characterization of measles viruses isolated in Victoria, Australia, between 1973 and 1998

Molecular epidemiology studies have made significant contributions to the control of measles virus infection through the identification of source and transmission pathways of the virus. These studies allow observation of changes in measles virus genotypes over time in a particular geographical location, clarification of epidemiological links during measles outbreaks, separation of indigenous strains from newly imported strains and distinction between vaccine- and wild-type virus-associated illness. A total of 35 wild-type measles viruses identified in Victoria, Australia, between 1973 and 1998 were characterized by nucleic acid sequence analysis of the nucleoprotein gene and, in some cases, the haemagglutinin gene. Relatedness between the viruses was studied and genotypes were assigned using a classification scheme recently proposed by the World Health Organization. Five recognized genotypes (C2, D1, D4, D5 and H) and one previously undescribed genotype, which we propose to be D7, were identified. Successive replacement of measles virus genetic lineages occurred in Victoria, with no evidence of temporal overlap, during this 25 year period. This pattern of circulation is likely to represent serial importation of wild-type measles virus strains from overseas foci of measles virus infections.

Diagnosis of enteric pathogens in children with gastroenteritis

Summary The aim of this study was to determine the isolation trends of common and emerging pathogens in children over a 12‐month period. The study group included 412 children under 6 years with diarrhoea who were either hospitalised, or seen in the outpatients department of The Sydney Childrens Hospital. Pathogens were detected in 137 (33%) samples, with rotavirus most common (40%), followed by adenovirus (26%), astrovirus (12%), Campylobacter jejuni (12%), Salmonella spp. (10%) and Giardia lamblia (< 1%). Giardia‐specific antigen (GSA) was detected in 11 of 382 (3%) using an enzyme immunoassay (EIA), and this included four samples in which cysts of G. lamblia were detected by microscopy. Using electron microscopy (EM), viruses were detected in 29 of 120 (24%) samples from hospitalised children and 53 of 171 (31%) outpatients ( P = 0.23). Amongst this subset, Norwalklike viruses (NLVs) were detected by RT‐PCR in 10 samples including six of 14 with small round viruses, one of seven with small viral‐like particles (SVLPs), and three of 126 EMnegative samples. Lactoferrin, detected by EIA, was 59% more likely to be positive in samples infected with salmonella/ campylobacter than in samples in which bacterial pathogens were not isolated. As an indicator for infection with these bacterial agents, the assay showed a sensitivity and specificity of 95 and 40.3%, respectively. A routine microbiological analysis of stools from children of this age group should include a screen for foodborne bacterial agents and rotavirus. Tests for adenovirus, astrovirus and NLVs should be secondary.The cost‐effectiveness of including the EIAs for lactoferrin and G. lamblia in the routine testing protocol needs to be evaluated.

Studies of measles viruses circulating in Australia between 1999 and 2001 reveals a new genotype

Nineteen distinct measles virus (MV) strains associated with nine different genotypes were identified in five Australian states (Victoria, New South Wales, Queensland, Northern Territory and Western Australia) between 1999 and 2001. One of the strains identified is likely to represent a new genotype within the clade D viruses (proposed to be d9). No evidence for an indigenous MV strain was found. When epidemiologic information associated with the index case was available for the outbreaks, it usually supported introduction of the virus from overseas, with the main source being South East Asia. Changes in the circulation of MV in Australia since the early 1970s were also observed. Prior to the introduction of measles vaccine, the majority of the population acquired immunity through infection with wild-type virus in early childhood. Nowadays in Australia, young adults are at most risk of infection. The age range of cases in the study period was from 1 month to 48 years, with the majority (59%) of cases from individuals aged 18-30 years.

Evaluation of the RIDA®QUICK immunochromatographic norovirus detection assay using specimens from Australian gastroenteritis incidents

A range of laboratory methods is now available for the detection of norovirus, a major cause of gastroenteritis. Recently, a commercial immunochromatographic assay for norovirus detection, the RIDA(®)QUICK assay, has become available, but there is still only limited information on its efficacy. This study examined the sensitivity and specificity of the RIDA(®)QUICK assay, using faecal material received for testing in a major diagnostic/reference laboratory in Australia. The sensitivity of the assay was found to be 83% and the specificity was 100%. No false positive norovirus results were found and the assay did not cross-react with common faecal viruses such as rotavirus, astrovirus, sapovirus and adenovirus. The assay was less reliable for genogroup I (GI) noroviruses than for genogroup II (GII) noroviruses. Genotypes detected by the assay included GII.1, GII.2, GII.3, GII.4, GII.6 and GII.7. The assay failed to detect any GI specimens in the test group. Genotypes not detected included GI.4 and GI.6. The assay was simple and quick to perform. It is valuable in a point-of-care situation or as a backup in a laboratory where a rapid initial norovirus result is required.

A comparative evaluation of the sensitivity of two automated and two manual nucleic acid extraction methods for the detection of norovirus by RT-PCR

The aim of the study was to compare the sensitivity of a norovirus RT-PCR method using two manual RNA extraction methods (Qiagen and Roche) and two automated RNA extraction methods (Qiagen and Corbett). All four RNA extraction methods gave similar sensitivities although the automated methods, especially the Corbett, required significantly less labour than the manual methods. The automated methods also enabled RNA extraction of approximately two to three times the number of specimens in a given time period compared to manual methods.

High level excretion of norwalk-like virus following resolution of clinical illness

Summary We report the case of an elderly woman excreting high levels (about 5 2 10 5 virions per gram of faeces) of Norwalk‐like virus (NLV) in the absence of any clinical symptoms of gastroenteritis. Analysis by reverse transcription, polymerase chain reaction and DNA sequencing was carried out on a 342‐nucleotide region of open reading frame 1. This indicated that the NLV belonged to genogroup 2 and was more closely related to the Camberwell subgroup, the most common circulating in southeast Australia at present, than to the Norwalk and Mexico viruses.

Investigation of Optimal Specimen Type and Sampling Time for Detection of Measles Virus RNA during a Measles Epidemic

ABSTRACT At various times postonset of rash, 74 patients positive for measles virus-specific immunoglobulin M provided samples for detection of measles virus RNA by a reverse transcriptase PCR. Of lymphocytes, urine, throat swab, and serum specimens, throat swab specimens were optimal for detection of measles virus RNA during the first 2 weeks after the rash.