A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea

A novel multiplex polymerase chain reaction assay was developed to identify 10 viruses in a single tube. The assay was targeted to detect group A and C rotaviruses, adenovirus, norovirus GI, norovirus GII, sapovirus, astrovirus, Aichi virus, parechovirus, and enterovirus. A total of 235 stool samples were collected from infants and children with acute gastroenteritis in Kyoto, Japan, from 2008 to 2009, then tested by this novel multiplex PCR and compared with a multiplex PCR described previously, which used 3 primer sets. The novel multiplex PCR could detect the targeted viruses in 111 of the 235 (47.2%) stool samples. Of these, 9 out of 10 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, sapovirus, adenovirus, parechovirus, group C rotavirus, astrovirus, and norovirus GI. In contrast, the multiplex PCR that used 3 sets of primers could detect the targeted viruses in 109 of the 235 (46.4%) stool samples. Among these, 8 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, adenovirus, parechovirus, group C rotavirus, sapovirus, and astrovirus. The results suggested that the new multiplex PCR is useful as a rapid and cost effective diagnostic tool for the detection of major pathogenic viruses causing diarrhea.     

Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples

Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6%, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.     

Norovirus genotypes involved in the outbreaks of gastroenteritis in Croatia during the winter season 2004--2005

Seven outbreaks and four sporadic cases of the non-bacterial gastroenteritis caused by a norovirus (NoV) were detected in Croatia between November 2004 and February 2005. An enzyme immunoassay (EIA) and three different RT-PCRs for the viral polymerase (ORF1 RT-PCR) and genogroup I (GI) or II (GII) of capsid gene regions (GI-ORF2 RT-PCR; GII-ORF2 RT-PCR) were performed to detect NoV in 21 stool samples. To characterize NoVs, sequencing of the ORF1 region was performed on 12 RT-PCR positive samples, whereas the ORF2 region was sequenced for 5 cases. Four outbreaks were caused by the genotype GII.4 (Lordsdale) and one outbreak was caused by the genotype GI.1 (Norwalk). One of the outbreaks was characterized as potentially mixed GII.4 and GI.1 infection. In the monitored period, genotype GII.4 dominated as the cause of noroviral infections in adults.     

Assessment of a rapid immunochromatographic test for the diagnosis of norovirus gastroenteritis

Noroviruses are the leading cause of acute gastroenteritis in people of all ages. Since the viruses are highly infectious, rapid and early diagnosis is important to prevent and control the disease. The present study aimed to evaluate the commercial immunochromatographic test RIDA? QUICK Norovirus for the detection of norovirus in stool samples from patients with acute gastroenteritis in Thailand. As compared with reference RT-PCR results, the RIDA? QUICK Norovirus assay provided a sensitivity of 48.2 and 83.3% with a specificity of 87.5%. False positive results were observed in 12.5% of norovirus-negative stool samples. Based on commercial quantitative real-time RT-PCR, the RIDA? QUICK Norovirus assay revealed a highly significant association, p-value <0.001, and good agreement (kappa?=?0.6). The assay could detect norovirus in stool samples ranging from 3.22?×?10(6) to 3.26?×?10(8) copies/ml. False negative results occurred in the stool samples containing 5.9?×?10(6) copies/ml of norovirus GI or 1.85?×?10(4)?-?4.28?×?10(5) copies/ml of GII. The immunochromatographic RIDA? QUICK Norovirus assay may be useful for rapid screening of norovirus infections in patients with acute gastroenteritis in both developed and developing countries where the RT-PCR method has not been established for routine diagnosis.     

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.     

Comparison of the RIDASCREEN norovirus enzyme immunoassay to IDEIA NLV GI/GII by testing stools also assayed by RT-PCR and electron microscopy

RIDASCREEN norovirus enzyme immunoassay (EIA) detected 80.3% of norovirus-infected feces samples compared to 60.6% by IDEIA NLV GI/GII from 228 patients with no false positives by either assay. RT-PCR and electron microscopy percent sensitivity and specificity were 98.5, 100 and 36.4 and 96.9, respectively.     

Human caliciviruses detected in Mexican children admitted to hospital during 1998–2000, with severe acute gastroenteritis not due to other enteropathogens

Few studies exist regarding the frequency of human caliciviruses as single etiologic agents in sporadic cases, or in outbreaks occurring in children hospitalized for acute gastroenteritis. In this study, a total of 1,129 children of <5 years of age and hospitalized due to acute diarrhea were enrolled from three main hospitals in Mexico City during a period of 3 years (March 1998 to December 2000). After analyzing all fecal samples for several enteropathogens, 396 stools that remained negative were further screened for human caliciviruses by RT‐PCR using a primer set specific to norovirus and sapovirus. Human caliciviruses were detected in 5.6% (22/396) of the children. The minimum incidence rate for 1999 were 5.3% (7/132) for 1999 and 7.8% (13/167) for 2000, since only fecal specimens that tested negative to other enteric pathogens were examined. Positive samples were further characterized using specific GI and GII primers and sequencing. Norovirus GII was detected in 19/22 samples, most of them were GII/4, while sapovirus GI/2 was detected in one sample. Associations between the presence of human calicivirus and clinical and epidemiological data revealed that diarrhea occurred with a seasonal pattern, and that children hospitalized due to human calicivirus disease scored an average of 13 ± 3.2 (SD) points on the Vesikari scale, which corresponded to severe episodes. These results highlight that human caliciviruses, by themselves, are enteropathogens of acute severe diarrhea among young Mexican children requiring hospitalization and that their detection is important in order to reduce the diagnosis gap. J. Med. Virol. 82:632–637, 2010. © 2010 Wiley‐Liss, Inc.