This study compares the performance of four commercial multiplex PCR assays (Resplex II Panel v2.0, Seeplex RV15, xTAG RVP and xTAG RVP Fast) and direct fluorescent antibody (DFA) staining and viral isolation. Seven hundred and fifty nasopharyngeal swabs were tested for 17 viral agents. In each assay, the sensitivity and specificity for each target were determined against a composite reference standard. Two hundred and eighty-eight out of 750 (38.4%) specimens were positive by DFA or viral isolation, while an additional 214 (28.5%) were positive by multiplex PCR, for a total positivity rate of 66.9%. Of 502 positive specimens, one virus was detected in 420 specimens (83.7%), two in 77 (15.3%), three in four (0.8%) and four in one case (0.2%). Compared with a composite reference standard, the inter-assay accuracy of the multiplex PCR assays varied, but all were superior to conventional diagnostic methods in detecting a broad range of respiratory viral agents in children. In addition, the sensitivity of two commercial assays, Resplex II Plus PRE and Seeplex Influenza A/B Subtyping, was determined relative to the Astra influenza Screen & Type assay for detection of influenza A viruses, including seasonal influenzas and pandemic H1N1 2009 influenza A virus. Using 75 positive and 55 negative nasopharyngeal swabs for influenza A by the Astra assay, the sensitivity of Seeplex and Resplex was 95.9% and 91.8%, respectively, with a specificity of 100% for both.

Compared to non-viral exacerbations, viral-related exacerbations were associated with worse severity and quality of life scores but similar pulmonary inflammation.

The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) and its spread throughout the world catalyzed the development of rapid diagnostic tests. SARS CoV has been shown to replicate in the gastrointestinal tract (4), and consistent with this, stool samples were shown to be appropriate for diagnostic testing for SARS CoV. Peiris et al. (7) found a positivity rate of 97% (65/67 samples) for detection of SARS CoV nucleic acid in stool samples at 14 days after the onset of symptoms. By contrast, Chan et al. (1) found a lower overall stool positivity rate of 26.2% (70/267), with a 42.9% (9/21) positivity rate within 1 week of the onset of symptoms, a 68.0% (17/25) positivity rate between 1 and 2 weeks of onset, and a 70.8% (34/48) positivity rate between 2 and 4 weeks of onset. Preliminary studies performed in our laboratories indicated that variations in RNA extraction methods could explain the differences seen in these studies. We anticipated that the optimization of extraction methods for stool samples could potentially increase the sensitivity of amplification detection of SARS CoV, especially early in infection. Stool samples have been recognized to be heterogeneous and difficult samples for use for molecular analysis. Bile salts, he...