Broad reactivity of the Luminex xTAG Respiratory Virus Panel (RVP) assay for the detection of human rhinoviruses.

Human rhinoviruses (HRV) are a major cause of the common cold and a variety of both upper and lower respiratory tract infections including sinusitis, otitis media, bronchitis, primary pneumonia and have been associated with the worsening of asthma and chronic obstructive pulmonary disease. With the advent of new antivirals directed at the treatment of serious HRV infections it is becoming increasingly important to detect a broad range of HRV strains so that targeted antiviral therapy can be appropriated instituted.2, 3

The xTAG Respiratory Virus Panel (RVP, Luminex Molecular Diagnostics, Toronto, Canada) is a US Food and Drug Administration (US FDA) cleared multiplex nucleic acid amplification test for the detection of respiratory viruses including adenovirus (ADV), human metapneumovirus (hMPV), influenza A (influA with subtyping of seasonal H1 and seasonal H3), influenza B (influB), parainfluenza types 1 (PIV-1), 2 (PIV-2) and 3 (PIV-3), respiratory syncytial virus (RSV) A and B, and HRV. Some enteroviral (EV) strains are also detected due to cross reactivity with HRV and therefore the assay does not distinguish between HRV and EV.4, 5 Additional viruses detected by the full RVP (Research Use Only in US) include PIV-4, coronaviruses (corona) NL63, OC43, HKU-1 and 229E.

A multicenter study demonstrated that RVP can detect culture isolates of HRV serotypes 39 and 54 and HRV strains of phylogenetic groups A, B, C, E and F from clinical samples. The aim of this study was to further assess the analytical reactivity of RVP for the detection of a wide variety of HRV serotypes and in clinical samples previously identified as HRV/EV positive by RVP. Differentiation of HRV from EV in RVP HRV/EV positive samples was established using a laboratory developed HRV assay (LDTHRV) based on a modified version of the method described by Lu et al., and the NucliSENS easyQ Enterovirus assay (EVA, bioMérieux, Durham, NC). A 50 μl aliquot from cultures of HRV species A and B (n  = 98, serotypes 1–100, with the exception of serotypes 53 and 87), influA, influB, RSV A and B, and ADV, and 200 μl aliquots from discard clinical samples (nasopharyngeal swabs in universal transport media [UTM, Copan, Brescia, Italy]) were added to 2.0 ml NucliSENS lysis buffer tubes (bioMérieux). Nucleic acids (NA) were extracted using the NucliSENS easyMAG (bioMérieux) and tested with LDTHRV, EVA and RVP according to the manufacturer's instructions.

All 98 HRV isolates were detected by RVP and LDTHRV. EVA was negative for 86/98 HRV isolates tested. The remaining 13 HRV isolates (serotypes 5, 12, 15, 16, 22, 26, 33, 38, 42, 65, 69, 88, 99) gave positive EVA results of varying intensity. The 13 HRV isolates had culture titers ranging from a TCID50 of 1.1 × 10−3 to 4.7 × 10−5 and demonstrated varying levels of RVP reactivity with MFI values ranging from 716 to 4734. EVA results were negative for isolates after testing ten-fold dilutions of the original culture. Nucleic acids derived from 3 clinical samples that contained HRV species C tested positive with RVP, LDTHRV and negative with EVA. Discard clinical samples, previously determined by RVP to be only HRV/EV positive (+) (n  = 98), HRV/EV(+) with an additional virus (n  = 6; 3 hMPV, 1 PIV-2, 1 PIV-3, 1 RSV-B), HRV/EV negative (−) but other respiratory virus (+) (n  = 25; 1 ADV, 4 corona NL63, 2 influA, 2 influB, 3 hMPV, 2 PIV-1, 3 PIV-2, 2 PIV-3, 2 PIV-4, 2 RSV-A, 2 RSV-B), gave the expected results. All samples positive for HRV/EV by RVP were detected by LDTHRV and were negative by EVA. All samples negative for HRV/EV by RVP were negative by LDTHRV and EVA.

In conclusion, the xTAG RVP can detect a broad range of HRV serotypes, including HRV A, B and C strains. Although the assay cannot differentiate between HRV and EV, supplemental assays can be used to differentiate HRV and EV when clinically indicated. The detection of HRV will facilitate the use of appropriate targeted antiviral therapy.

Funding

This study was funded in part by Biota Holdings, Australia.

Competing interests

CCG has received research funding from Luminex and is a member of the Luminex Scientific Advisory Board.

Ethical approval

Not required.