Abstract
BackgroundA broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel.MethodsThe analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP), were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens.ResultsTo compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml) and RSV (103 copies/ml). The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found.The incidence of respiratory viruses was compared in tracheal secretion (TS) samples (n = 100) of mechanically ventilated patients in winter (n = 50) and summer (n = 50). In winter, respiratory viruses were detected in 32 TS samples (64%) by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32%) and PIV-2 (20%). Multiple infections were detected in 16 TS samples (32%) by RespiFinder-19. Fewer infections were found in summer (RespiFinder-19: 20%; RVP: 6%). All positive results were verified using monoplex PCR.ConclusionsMultiplex PCR tests have a broad spectrum of pathogens to test at a time. Analysis of multiple inoculated samples revealed a different focus of the detected virus types by the three assays. Analysis of clinical samples showed a high concordance of detected viruses by the RespiFinder-19 compared to monoplex tests.
Highlights
A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar
This study presents the first comparison of the analytical sensitivity of three novel multiplex PCR methods, the RespiFinder-19 assay, RespiFinder-SMART(Single tube Multiplex Amplification in Real-Time)-22 assay and the xTAG Respiratory Virus Panel Fast Assay (Abbott Molecular, Wiesbaden, Germany), with quantified virus control material
The Respiratory Virus Panel Fast Assay (RVP) showed the detection of influenzavirus A (INF-A, 1.78E + 02 copies/ml), respiratory syntical virus A and B (RSV-A/B, 3.94E + 02 copies/ml), coronavirus OC43 (CoV OC43) and human metapneumovirus even in a high dilution ratio (Table 2)
Summary
A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. The identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. A high rate of respiratory tract infections is caused by viruses (approximately 80%) [1,2]. Due to the similarity in clinical presentation of respiratory tract infections, causative pathogens could not be identified on the basis of symptoms alone. To overcome limitations concerning the use of several monoplex tests in parallel and the resulting shortage of sample volume, the development of multiplex tests for a fast and exact identification is necessary. Several multiplex tests are commercially available [9,10,11,12,13,14,15,16]. A number of studies have already compared the detection frequencies of multiplex assays with conventional monoplexPCR assays in clinical samples [12,17,18,19], but a comparison of the analytical sensitivity of these multiplex assays with quantified standardized control material does not exist
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