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Partial comparison of the NxTAG Respiratory Pathogen Panel Assay with the Luminex xTAG Respiratory Panel Fast Assay V2 and singleplex real-time polymerase chain reaction for detection of respiratory pathogens

In this study, 185 nasopharyngeal swabs were tested to compare the sensitivity and specificity of the Luminex NxTAG (NxTAG) Respiratory Pathogen Panel (RPP) Assay with those of the Luminex Respiratory Virus Panel (RVP) Fast Assay v2 and singleplex real-time polymerase chain reaction (PCR). The NxTAG Assay identified at least one infectious agent in 164 (88.7%) of the swabs. In 91 (6.2%) tests with negative results with the RVP Fast Assay v2, a virus was identified by the NxTAG (P < 0.001). With the NxTAG Assay, the detection rates were significantly higher for respiratory syncytial virus (P = 0.003), human metapneumovirus (P < 0.001), human rhinovirus/human enterovirus (P = 0.009) and human adenovirus (P < 0.001). Finally, the NxTAG Assay identified M. pneumoniae in 32 of 44 (72.7%) PCR-positive samples. However, the concordance with real-time PCR results was low for both assays. In conclusion, the results indicate that the NxTAG Assay overcomes some of the limitations of previous Luminex assays, although further studies are needed for a more complete evaluation of the new assay.


Introduction
Epidemics of different respiratory infectious agents occur simultaneously each winter season or at intervals of several years. Viruses are most frequently involved , but atypical bacterial pathogens, mainly Mycoplasma pneumoniae, can also play a role (Principi and Esposito, 2013;Brown et al., 2016). When available, the etiologic diagnosis of a respiratory infection is essential for planning adequate therapy to prevent the diffusion of infection, to minimize radiological or laboratory investigations and to predict the clinical course of the disease more precisely. Unfortunately, the clinical symptoms of diseases caused by several infectious agents are very similar regardless of the infectious agent, and only laboratory assays can identify the causative virus involved in each epidemic Esposito and Principi, 2012;.
In the past, viral and bacterial cultures have been used for identification, in addition to the direct fluorescence assay (DFA) for viruses. However, conventional methods have several limitations: the cultures are time-and labor-intensive, and the DFA (that detects viral antigens) although rapid, has poor sensitivity for the detection of most viruses (Ginocchio, 2007). The advent of nucleic acid amplification tests has significantly improved the etiologic diagnosis of respiratory infections. However, testing for all respiratory viral or atypical bacterial targets by using singleplex polymerase chain reaction (PCR) is expensive and laborious. Moreover, multiplex real-time PCR can be technologically challenging and can result in a loss of sensitivity. Multiplex assays involving the amplification and detection of a panel of respiratory viruses using suspension microarrays might provide a practical solution (Pabbaraju et al., 2008;Esposito et al., 2016a).
The Luminex Respiratory Virus Panel (RVP) Fast Assay was the first version of an assay with the ability to simultaneously identify targets of 19 different viral types and subtypes in respiratory secretions. Although some studies have reported that the RVP Fast Assay may provide results comparable or superior to those of culture and DFA for the diagnosis of respiratory viral infections (Mahony et al., 2007;Pabbaraju et al., 2011), the RVP Fast Assay has been criticized because direct comparisons with real-time PCR have shown that discordant results may occur for some viruses, and reduced sensitivity occurs mainly in samples with low viral loads (Choudhary et al., 2016;Esposito et al., 2016b;Gadsby et al., 2010). To overcome these limitations, a new assay, the Luminex RVP Fast Assay v2, has been developed. This assay includes two controls to ensure increased assay performance; however, it exhibits some of the limits of the first version. Esposito et al. have Diagnostic Microbiology and Infectious Disease 86 (2016) (Esposito et al., 2016b). To further improve viral detection and to simultaneously identify atypical bacteria, a third assay has been developed with optimized primer design and PCR protocols. Using respiratory samples, the present study compared the sensitivity and specificity of this new assay, the Luminex NxTAG Respiratory Pathogen Panel (RPP) Assay, to those of the Luminex RVP Fast Assay v2. The results obtained with both assays were compared to those from singleplex real-time PCR assays specifically designed for the identification of single infectious agents.

Materials and methods
Nasopharyngeal swabs collected from otherwise healthy children admitted to the Pediatric Highly Intensive Care Unit of the Fondazione IRCCS Ca′ Granda Ospedale Maggiore Policlinico, Milan, Italy, for respiratory tract infections in February 2015 were evaluated. Respiratory secretions were collected immediately after admission to the hospital, by using a nasopharyngeal flocked swab (one swab per child), which was stored in a tube containing 1 mL of universal transport medium (Copan, Brescia, Italy). The analysis also included a number of nasopharyngeal samples, collected from children with communityacquired pneumonia, that had previously been found positive for M. pneumoniae with a validated real-time PCR method  and had been stored at −80°C for no longer than 6 months. The study was approved by the Ethics Committee of the Fondazione IRCCS Ca′ Granda Ospedale Maggiore Policlinico, Milan, Italy. Written informed consent from a parent or legal guardian was obtained, and children ≥8 years of age were asked to provide their written consent.
The general characteristics and clinical findings of the enrolled children were collected and archived on a previously prepared electronic chart.
Nucleic acids were extracted from the swabs by using the NucliSens easyMAG automated extraction system (Biomeriéux, Craponne, France). A portion of the extract was tested for infectious agents using the Luminex NxTAG Assay and the Luminex RVP Fast Assay v2 (both produced by Luminex Molecular Diagnostics, Inc., Toronto, ON, Canada). Both of these assays simultaneously detect, influenza A viruses (non-specific influenza A, A/H1N1, A/H3N2, and influenza A/H1N1 2009), influenza B virus, respiratory syncytial virus (RSV), parainfluenza viruses (types 1-4), hADV, human metapneumovirus (hMPV), human coronaviruses (229E, NL63, OC43 and HKU1), human enterovirus/rhinovirus (hEV/hRV) and human bocavirus, according to the manufacturer's instructions (Luminex Molecular Diagno stics Inc.). However, the NxTAG Assay is slightly less time consuming in comparison to RVP Fast Assay v2 (about 3.5 hours vs more than 4 hours). Moreover, the NxTAG Assay can differentiate RSV type A from RSV type B and can detect Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. The remaining extracts were tested for RSV A and B, hRV, hMPV, hADV and M. pneumoniae by using validated real-time PCR specific for each of the agents (Bosis et al., 2008;Esposito et al., 2015Esposito et al., , 2016bPrincipi et al., , 2015. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen's kappa coefficients with 95% confidence intervals (95% CI) of the NxTAG Assay were calculated; in agreement with previous definition (Cyr and Francis, 1992), a kappa coefficient higher than 0.80 was considered to indicate agreement. McNemar's test was applied to compare paired proportions. The analyses were performed using SAS version 9.2 (Cary, NC, USA).

Results
A total of 185 nasopharyngeal swabs were tested; 142 were from children admitted to the hospital, and 43 were positive for M. pneumoniae. Table 1 summarizes the results obtained using the NxTAG Assay. At least one infectious agent was identified in 164 (88.7%) of the swabs (Table 1).
Comparison with the RVP Fast Assay v2 was possible only for 184 samples, because this assay produced un "undetermined" result for one sample. The NxTAG Assay detected a significantly higher number of viruses (Table 2). In 91 (6.2%) tests with a negative result with the RVP Fast Assay v2, a virus was identified by the NxTAG Assay (P b 0.001). In contrast, in only 4 tests in which a virus was not detected by the NxTAG Assay, a viral agent was identified by the RVP Fast Assay v2. The detection rates were higher for all of the most common viruses, with differences that were statistically significant for RSV (P = 0.003), hMPV (P b 0.001), hRV/hEV (P = 0.009) and hADV (P b 0.001). No differences were found for influenza and parainfluenza 3 virus. Finally, the NxTAG Assay identified M. pneumoniae in 32 of 44 (72.7%) PCR-positive samples.
Tables 3 and 4 present the sensitivity, specificity, PPV, and NPV of the NxTAG assay and the RVP Fast Assay v2 compared to real-time PCR specific for RSV A and B, hMPV, hRV, hADV and M. pneumoniae. For RSV, the two multiplex assays had similar sensitivity, specificity, PPV and NPV. Moreover, both exhibited good concordance with real-time PCR, as evidenced by Cohen's kappa coefficients higher than 0.80 in

Discussion
To the best of our knowledge, this is the first study to measure the efficiency of the NxTAG Assay in the detection of respiratory viruses  and atypical bacteria. Unfortunately, this study has the following limitations: because of the relatively small number of nasopharyngeal samples tested and the poor circulation of some infectious agents during the study period, only the most common respiratory viruses and Mycoplasma pneumoniae could be adequately evaluated. As an example, although the same number of influenza and parainfluenza virus positive samples was detected, the total number of positive cases for both viruses is very small and it is not possible to draw firm conclusions regarding possible differences in the ability of the assays in detecting these viruses. Moreover, the remaining nucleic acid extract available for real-time PCR was not always sufficient to permit singleplex real-time PCR determinations in all the studied swabs. However, the available data seem to indicate that the NxTAG Assay has overcome some of the limitations of the previous Luminex assays, although it remains generally less sensitive and specific than singleplex real-time PCR assays designed to identify the different infectious agents. Moreover, differentiation of hRV from hEV remains a limitation that can be overcome only with specific real-time PCR, as demonstrated with this study.
The most important advances are related to the detection of hADV, differentiation between RSV A and B and the identification of M. pneumoniae. A previous study has reported that the sensitivity of the RVP Fast v2 assay for hADV detection is no greater than 50% (Esposito et al., 2015). In the present study, the sensitivity of this assay for hADV was even lower. In contrast, the NxTAG Assay identified approximately two-thirds of the hADV cases detected by real-time PCR. HADV can cause severe or even fatal respiratory disease, which can be associated with a high risk of long-term respiratory sequelae (Campbell et al., 2012). Thus, the early identification of severe respiratory infections caused by hADV during outbreaks may be useful for monitoring the circulation of the virus and for planning the development of adequate preventive and therapeutic measures. Similar conclusions can be drawn for M. pneumoniae, an atypical bacterium that is responsible for a high number of cases of community-acquired pneumonia in older children, adolescents and young adults  and can cause severe neurological complications in a substantial number of cases (Esposito et al., 2011). Epidemics usually occur every 4-7 years, and the early identification of patients infected by this pathogen is essential to ensure adequate antibiotic treatment and to limit the circulation of the pathogen. Less important is the possibility of differentiating RSV A from RSV B because in general, these RSV types do not have a very different global clinical impact. Differences in virulence have been reported between only different subtypes of the same viral type; RSV A/NA1 has been found to be more frequently associated with lower respiratory tract infections (P b 0.0001), and patients infected with RSV A/NA1 require hospitalization (P = 0.007) more often than those infected by genotype A/ON1 (Esposito et al., 2015).
However, further studies are needed for a more complete evaluation of the NxTAG Assay. More samples collected during the whole year should be evaluated, and all the viruses and atypical bacteria for which this assay was designed should be tested. Moreover, accurate cost analyses should be performed. -RSV 95% CI = 95% confidence interval; HADV = human adenovirus; hMPV = human metapneumovirus; hRV/hEV = human rhinovirus/human enterovirus; NPV = negative predictive value; PPV = positive predictive value; RSV = respiratory syncytial virus. a Real-time PCR analyses were not available for 42 subjects, and the Luminex Respiratory Virus Panel Fast assay v2 analyses produced "undetermined" results for one subject. b Specific real-time PCR identified all these viruses as hRV.