Background: A multiplex reverse transcription (RT) polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) was previously developed to detect nine different microorganisms: enterovirus (EV), influenza virus type A (IVA) and type B (IVB), respiratory syncytial virus (RSV), parainfluenzavirus type 1 (PIV1) and type 3 (PIV3), adenovirus (AV), Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn) in a single test. These organisms do not usually colonize the respiratory tract of humans, but, if present, it may be assumed they are involved in respiratory disease. Objectives and study design: The m-RT-PCR-ELISA was tested on (i) culture supernatants of unknown contents, (ii) by determining the analytical sensitivity of 10-fold serial dilutions of culture supernatants and (iii) by determining clinical sensitivity in a retrospective study on 411 clinical specimens. The specimens were re-tested in parallel by m-RT-PCR-ELISA versus the gold standard culture and immunfluorescence, and versus individual RT-PCR. Results: (i) The 9-valent m-RT-PCR-ELISA shows 83% to 100% concordant results on 103 culture supernatants containing different organisms. (ii) The analytical sensitivity was as follows: higher sensitivity of the 9-valent m-RT-PCR-ELISA in comparison to culture in the cases of PIV3, IVA and IVB (factor 10) and AV and EV (factor 100), and lower sensitivity in case of RSV and PIV1 (factor 10). (iii) The agreement with the gold standard in the kappa statistic was excellent for RSV (κ=0.937), IVA (κ=0.940), very good for PIV1 (κ=0.914), IVB (κ=0.907) and satisfactory for PIV3 (κ=0.410). For AV, EV and Mpn the m-RT-PCR-ELISA preliminary could be qualified as very good, based on the data derived on culture supernatants. Information about the validity for Cpn is limited. Conclusion: The m-RT-PCR-ELISA is a feasible, sensitive and specific method for detection of a broad spectrum of organisms. It is suitable for individual as well as epidemiological diagnosis.

Kappa-statistics, m-RT-PCR-ELISA, Respiratory tract infections, Sensitivity, Validation,
Journal of Clinical Virology
Department of Virology

Puppe, W, Weigl, J, Aron, G.I, Gröndahl, B, Schmitt, H.J, Niesters, H.G.M, & Groen, J.M. (2004). Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory tract pathogens. Journal of Clinical Virology, 30(2), 165–174. doi:10.1016/j.jcv.2003.10.003