The performance of the new ePlex Respiratory Pathogen (RP) panel (GenMark Diagnostics) for the simultaneous detection of 19 viruses (influenza A virus; influenza A H1 virus; influenza A 2009 H1 virus; influenza A H3 virus; influenza B virus; adenovirus; coronaviruses [HKU1, OC43, NL63, and 229E]; human rhinovirus/enterovirus; human metapneumovirus; parainfluenza viruses 1, 2, 3, and 4; and respiratory syncytial virus [RSV] [RSV subtype A and RSV subtype B]) and 2 bacteria (Mycoplasma pneumoniae and Chlamydia pneumoniae) was evaluated. Prospectively and retrospectively collected nasopharyngeal swab (NPS) specimens (n = 2,908) were evaluated by using the ePlex RP panel, with the bioMérieux/BioFire FilmArray Respiratory Panel (BioFire RP) as the comparator method. Discordance analysis was performed by using target-specific PCRs and bidirectional sequencing. The reproducibility of the assay was evaluated by using reproducibility panels comprised of 6 pathogens. The overall agreement between the ePlex RP and BioFire RP results was >95% for all targets. Positive percent agreement with the BioFire RP result for viruses ranged from 85.1% (95% confidence interval [CI], 80.2% to 88.9%) to 95.1% (95% CI, 89.0% to 97.9%), while negative percent agreement values ranged from 99.5% (95% CI, 99.1% to 99.7%) to 99.8% (95% CI, 99.5% to 99.9%). Additional testing of discordant targets (12%; 349/2,908) confirmed the results of ePlex RP for 38% (131/349) of samples tested. Reproducibility was 100% for all targets tested, with the exception of adenovirus, for which reproducibilities were 91.6% at low virus concentrations and 100% at moderate virus concentrations. The ePlex RP panel offers a new, rapid, and sensitive “sample-to-answer” multiplex panel for the detection of the most common viral and bacterial respiratory pathogens.

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

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The sequence of the HAl region of the he-

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.