In this report, we describe a new multiplex real-time PCR assay for the most commonly detected respiratory viral pathogens, namely, influenza viruses (A and B) and HRSV. The key features of this novel assay include its rapidity (turnaround time, approximately 2 h 30 min, including 30 min for sample preparation, 60 min for reverse transcription, and 60 min for real-time PCR including viral identification), and its sensitivity (50 copies per assay for each viral target). Our results clearly demonstrate that our multiplex real-time PCR assay is more sensitive than commercially available antigenic detection tests for both influenza A virus and HRSV, with the greatest difference in sensitivity noted for influenza viruses.
Multiplex RT-PCR assays targeting as many as nine different respiratory pathogens have been reported previously (6
). More recently, a TaqMan-based real-time PCR assay for simultaneous detection of influenza A and B viruses has also been described (31
). Compared to conventional multiplex PCR assays, our real-time PCR test is much more rapid because it avoids additional nested amplification and/or hybridization steps required for identification of viral products. Furthermore, the real-time amplification procedure minimizes the chances of contamination, because there is no post-PCR processing of the samples. Although our multiplex real-time PCR assay based on melting-curve analysis of amplicons does not permit absolute quantification of the viral targets as in the TaqMan PCR procedure (31
), it allows for a larger number of viral targets to be detected simultaneously, since there is no limitation related to the capability of the system to detect multiple dyes linked to detection probes with distinct emission wavelengths. Eventually, more than three viral targets could be detected simultaneously in the LightCycler assay, assuming the absence of interaction between PCR primers and a reproducible and discriminatory Tm
for each viral amplicon.
Evaluation of our multiplex real-time PCR assay in a pediatric population with severe ARTI clearly illustrated its clinical potential. Indeed, our assay identified a viral pathogen (influenza virus or HRSV) in two-thirds of the hospitalized children. These numbers are similar to those previously reported by our group for the same population, which were determined by use of individual real-time assays for these viral pathogens (1
). Notably, we were able to show that 6% of hospitalized children had dual viral infections, a finding that has been reported, albeit at a lower frequency, by other investigators (8
). Rapid identification of these viral pathogens (influenza virus and HRSV) is of paramount importance for purposes of isolation in the hospital setting and early institution of specific antiviral therapy (32
). We are currently designing additional multiplex assays based on the same technology to cover the whole spectrum of respiratory pathogens including parainfluenza viruses, adenoviruses, enteroviruses, and coronaviruses (27
). To this list, we should also add the recently described human metapneumovirus, which has been reported by our group and others to be present in as many as 10% of hospitalized children with ARTI (1
A specific evaluation of the real-time multiplex PCR assay for influenza virus A revealed that it was twice as sensitive as the rapid antigen detection test in use at our institution during the winter of 2001 to 2002. None of the two PCR-negative, antigen-positive samples were confirmed as positive by a second PCR targeting another conserved gene (M2) of influenza virus A, whereas most (22 of 25 [88.0%]) multiplex PCR-positive, antigen-negative samples were. In general, the false-negative antigenic test results could be explained by small amounts of viral RNA in those NPA samples, as demonstrated by high CT
values in the corresponding real-time PCR assays. Three multiplex PCR test results were not confirmed by the influenza A virus M2 PCR test and were thus considered to be false-positive results. However, since we have not formally evaluated the sensitivity of the M2 PCR test and since viral cultures were not routinely done, we cannot rule out the possibility that such discrepant results were indeed true positives. We and other investigators have shown the superiority of RT-PCR tests over conventional methods (antigenic tests and viral culture) for detecting influenza viruses in clinical samples, including those sent to the laboratory by mail (2
). It is now common procedure at our institution to confirm any negative antigenic test results for influenza A and B viruses by either viral culture or RT-PCR, considering the high specificity but relatively poor sensitivity of the former assays.
For HRSV, the multiplex real-time PCR assay was also found to be more sensitive (by approximately 15%) than the rapid antigenic test when a second RT-PCR assay for the HRSV gG gene was used to resolve discrepancies. Most of the false-negative antigenic test results could be explained by small amounts of viral RNA in the NPA samples, as shown by mean CT
values of 32.7 for discordant results compared to 28.6 for concordant positive results. The specificity of the multiplex assay was excellent, with only one positive result not confirmed by the second PCR test. In contrast, the multiplex PCR assay missed six cases of HRSV which tested positive in both the antigenic test and the second PCR assay for HRSV gG. Notably, four of those six samples tested positive for influenza A virus in the multiplex PCR assay. Thus, although our multiplex PCR test has the capability of detecting dual infections (as seen in 6% of our cases), it is possible that large amounts of one virus could inhibit amplification of other pathogens. It is noteworthy that the multiplex PCR assay had the ability to amplify both HRSV genotypes, as shown by the detection of 15 B and 11 A genotypes by use of specific PCR assays aimed at detecting variable regions of the HRSV gG gene (17
). Although the rapid antigenic test performed relatively well (sensitivity, 82%; specificity, 95%) for detection of HRSV in young children, the real advantage of PCR probably lies in testing of the adult population, whose viral titers are lower and for whom antigenic tests with upper respiratory tract samples are not recommended (5
Some limitations of our study are worth mentioning. First, the absence of circulation of influenza B viruses during the study period prevented adequate validation of our multiplex assay for this pathogen and in particular for its ability to detect mixed (influenza B virus and HRSV) infections. Second, the performance of our multiplex PCR assay cannot be generalized to other types of samples (e.g., throat swabs and sputum) or to other populations (e.g., outpatients and adults) at the present time. Also, the absence of serological testing in our study may have underestimated the rate of viral infections, especially for those children who presented late after the onset of symptoms. Finally, although it was relatively easy to distinguish between the different viral pathogens based on their specific Tm only (Table ), we sometimes had to confirm viral amplicons by gel electrophoresis, which increased the assay's turnaround time.
In conclusion, we have described a rapid and sensitive multiplex real-time PCR assay for detection of influenza viruses and HRSV in children's NPA samples. This new assay is as specific as, and much more sensitive than, currently available antigen detection tests; it could complement the latter in the hospital setting when there is high clinical suspicion despite negative results. Future work is needed to expand the panel of viral pathogens detected by such rapid molecular methods in order to eventually circumvent the need for viral cultures. Also, future evaluation of the multiplex PCR assay is warranted in adult populations with severe ARTI.