Abstract
Multiplex molecular techniques can detect a diversity of respiratory viruses and bacteria that cause childhood acute respiratory infection rapidly and conveniently. However, currently available techniques show high variation in performance. We sought to compare the diagnostic accuracy of the novel multiplex NxTAG respiratory pathogen panel (RPP) RUO test versus a routine multiplex Anyplex II RV16 assay in respiratory specimens collected from children <18 years of age hospitalized with nonspecific symptoms of acute lower respiratory infection. Parallel testing was performed on nasopharyngeal aspirates prospectively collected at referral Children's Hospital Sant Joan de Déu (Barcelona, Spain) between June and November 2015. Agreement values between the two tests and kappa coefficients were assessed. Bidirectional sequencing was performed for the resolution of discordant results. A total of 319 samples were analyzed by both techniques. A total of 268 (84.0%) of them yielded concordant results. Positive percent agreement values ranged from 83.3 to 100%, while the negative percent agreement was more than 99% for all targets except for enterovirus/rhinovirus (EV/RV; 94.4%). Kappa coefficients ranged from 0.83 to 1.00. Discrepancy analysis confirmed 66.0% of NxTAG RPP RUO results. A total of 260 viruses were detected, with EV/RV (n = 105, 40.4%) being the most prevalent target. Viral coinfections were found in 44 (14.2%) samples. In addition, NxTAG RPP RUO detected single bacterial and mixed viral-bacterial infections in seven samples. NxTAG RPP RUO showed high positive and negative agreement with Anyplex II RV16 for main viruses that cause acute respiratory infections in children, coupled with an additional capability to detect some respiratory bacteria.
INTRODUCTION
Childhood acute lower respiratory infections (ALRI) remain as the most important single cause of global burden of disease among pediatric populations and a major cause of child mortality (1–3). It has been estimated that approximately 165 million episodes of ALRI occur globally each year in children aged 0 to 4 years, resulting in 2.1 million deaths (4). Management of these infections requires extensive use of health care and economic resources: viral ALRI alone has been described to account for an expenditure of approximately 2.4 billion dollars annually (3). Rapid etiological diagnosis of ALRI, followed by timely and specific treatment, has the potential to improve clinical outcomes and reduce the duration of hospital stay, as well as reduce unnecessary laboratory testing and antibiotic use (5, 6). However, discerning the causal agent of the infection is challenging in younger children, who typically present with nonspecific clinical symptoms (7, 8). Frequent occurrence of mixed viral and bacterial-viral infections at early ages (9, 10) makes etiological diagnosis even more difficult.
Nucleic acid amplification techniques are widely acknowledged to contribute improved sensitivity and rapidness over traditional methods such as culture and direct fluorescent antibody detection for diagnosing ALRI (11, 12). In recent years, the introduction of novel molecular assays that allow the performance of multiple analyses in one reaction has widened the spectrum of detectable respiratory pathogens while increasing throughput and convenience (13–15). The NxTAG respiratory pathogen panel (RPP) RUO (Luminex Molecular Diagnostics, Toronto, Ontario, Canada) is a qualitative reverse transcriptase PCR assay capable of detecting and differentiating the following 22 targets in a single patient sample: adenovirus (ADV), bocavirus (BoV), coronavirus (CoV) types 229E/NL63/OC43/HKU1, influenza A virus (IFV-A; including differentiating subtypes H1/H1N1/H3), influenza B virus (IFV-B), metapneumovirus (MPV), parainfluenza virus (PIV) types 1/2/3/4, respiratory syncytial virus (RSV) types A/B, enterovirus/rhinovirus (EV/RV), Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila (16). Similarly, Anyplex II RV16 (Seegene, Inc., Seoul, South Korea) is a qualitative multiplex PCR able to detect 16 targets, including AdV, BoV CoV types 229E/NL63/OC43, EV, IFV-A, IFV-B, MPV, PIV types 1 to 4, RSV types A/B, and RV (17). Both NxTAG RPP and Anyplex II RV16 RUO are Conformité Européene (CE)-marked assays. However, while the former has obtained U.S. Food and Drug Administration clearance (except for the Legionella target), the latter is not available in the United States.
The primary objective of this study was to evaluate the diagnostic accuracy of NxTAG RPP RUO in comparison to Anyplex II RV16 using respiratory specimens prospectively collected from pediatric patients hospitalized at a referral medical center out of the influenza season. A secondary objective was to describe the distribution of respiratory pathogens in the study sample.
MATERIALS AND METHODS
Study design and setting.
A cross-sectional study was conducted to analyze nasopharyngeal aspirates collected consecutively from children and adolescents <18 years of age with nonspecific symptoms of ALRI admitted to Children's Hospital Sant Joan de Déu during the period from June to November 2015. ALRI was defined as the presence of at least one specific lower-respiratory-tract sign (fast or difficulty breathing, chest wall indrawing) and/or abnormal auscultatory findings (crackles/crepitations or bronchial breath sounds). Hospital Sant Joan de Déu is a 345-bed teaching reference medical center that covers the Barcelona South metropolitan area (Catalonia region, Spain) and provides health care services to a pediatric population of ≈200,000 subjects representing 18% of the total population of Catalonia within this age group.
Sample collection and microbiological methods.
Nasopharyngeal aspirates were prospectively collected in phosphate-buffered saline according to standard operational procedures of the hospital's clinical laboratory. When we collected repeated samples from the same patient, only the first sample was considered for evaluation. Specimens were aliquoted into two parts, frozen at −80°C, and thawed prior to testing. Sample batches were run two to three times a week to optimize the use of reagents. The first aliquot of each sample was routinely processed by Anyplex II RV16. The second aliquot was also tested by NxTAG RPP RUO for the purposes of this study. Tests were performed according to the respective manufacturers' instructions as previously described (16, 17).
DNA/RNA was isolated by Magna Pure extraction method (Roche Diagnostics, Indianapolis, IN) on a total of 200 μl of the sample with the addition of a volume of 10 μl of internal control (MS2) directly to the sample before performing the extraction. Isolated DNA/RNA was eluted in 100 μl, and each extraction run included a negative control. Anyplex II RV16 requires a separate cDNA synthesis step before performing the multiplex PCR step whereas NxTAG RPP RUO is a one-step technique that undergoes reverse transcription to generate cDNA that is amplified using PCR. Each run included at least one negative control from the extraction method and one water negative control. All samples, DNA/RNA extracts, controls, and reagents were kept cold during the assay performance. Weekly and daily calibrations of the equipment were performed before assay testing.
Discordant results were resolved by direct sequencing of specific pathogen targets with M13-tagged analyte specific primers. PCR and Sanger sequencing primers were designed not to overlap with the primers used in the NxTAG RPP RUO panel. Dye-labeled terminator cycle sequencing was performed using a BigDye Terminator v3.1 cycle sequencing kit (Thermo-Fisher, Waltham, MA). Sample electrophoresis and sequence analysis were performed on the 3730xl Analyzer (Thermo-Fisher) using 3730xl data collection software (v3.1.1) and sequencing analysis software (v5.4). Sequences that were at least 200 bases in length, with a QV value ≥20 for at least 90% of the bases, and that contained fewer than 5% ambiguous base calls were further analyzed using NCBI BLAST.
Data collection and analysis.
Diagnostic results were retrieved from the hospital's clinical laboratory database and registered in a specific study database that also included the corresponding sample identification codes and dates of analyses. Demographic and epidemiological data of patients were kept anonymous and not included in the study database. In the absence of a reference standard, comparison of diagnostic performance between NxTAG RPP RUO and Anyplex II RV16 was assessed measuring the positive and negative percent agreement values and the kappa coefficients for each target. The positive percent agreement (PPA) was calculated with the formula PPA = 2a/(2a + b + c), and the negative percent agreement (NPA) was calculated according to the formula NSA = 2d/(2d + b + c), where a and d are the numbers of positive and negative samples determined by both NxTAG RPP RUO and Anyplex II RV16, respectively, b is the number of samples determined to be positive by NxTAG RPP RUO and negative by Anyplex II RV16, and c is the number of samples determined to be positive by Anyplex II RV16 and negative by NxTAG RPP RUO. Kappa coefficients were determined as previously reported (18).
Targets only included in the NxTAG RPP RUO panel (CoV-HKU1, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila) were not evaluated. Since NxTAG RPP RUO has combined EV and RV targets and Anyplex II RV16 detects them separately, EV and RV results for Anyplex II RV16 were pooled to allow a comparative analysis. In the same way, given that NxTAG RPP RUO distinguishes between IFV-A subtypes H1/H1N1/H3 and Anyplex II RV16 targets IFV-A without differentiation, IFV-A subtype results for NxTAG RPP were also grouped in a single category. Confidence intervals (CI) were set at 95%, and significance was set at a two-sided P value of <0.05 for all statistical analyses, which were conducted using Stata v13.1 (Stata Corp., College Station, TX).
RESULTS
A total of 320 nasopharyngeal aspirates were collected, but one specimen yielded invalid results by NxTAG RPP RUO due to a low bead count. Of the remaining 319 samples, 268 (84.0%) showed concordant results, and 51 had 56 discordant results: 46 for one target and 5 for two targets. RSV-A showed an optimal PPA of 100% (95% CI = 90.8 to 100.0). PPA values could not be assessed for CoV-229E, CoV-NL63, MPV, PIV-1, and PIV-2 due to the low number of positives found. The rest of viruses targeted by both techniques gave values of PPA in a range from 83.3 to 100.0. On the other hand, NPA was over 99% with a lower bound 95% CI of at least 97.8% being met for all targets except for EV/RV (94.4%, 95% CI = 91.8 to 96.2). Kappa coefficients were high, ranging from values of 0.83 to 1.00. Measurements of agreement are recorded in Table 1. After excluding eight discordant samples with insufficient volume for retesting, discrepancy analysis confirmed 31 of 47 (66.0%) NxTAG RPP RUO results in the remaining 43 samples, as described in Table 2.
TABLE 1.
Agreement between Luminex NxTAG RPP RUO and Seegene Anyplex II RV16 testsa
| Target | No. of tests |
% agreement (95% CI) |
Kappa coefficient (95% CI) | ||||
|---|---|---|---|---|---|---|---|
| RPP+ RVP+ | RPP+ RVP– | RPP– RVP+ | RPP– RVP– | PPA | NPA | ||
| Adenovirus | 12 | 1 | 3 | 303 | 85.7 (68.5–94.3) | 99.3 (98.3–99.4) | 0.85 (0.71–0.99) |
| Bocavirus | 22 | 4 | 2 | 292 | 88.0 (76.2–94.4) | 99.0 (97.8–99.5) | 0.87 (0.77–0.97) |
| Coronavirus | |||||||
| 229E | 1 | 0 | 0 | 318 | NA | 100.0 (99.4–100.0) | 1.00 (1.00–1.00) |
| NL63 | 1 | 0 | 0 | 318 | NA | 100.0 (99.4–100.0) | 1.00 (1.00–1.00) |
| OC43 | 12 | 0 | 3 | 304 | 88.9 (71.9–96.2) | 99.5 (98.6–99.8) | 0.88 (0.75–1.00) |
| Enterovirus/rhinovirus | 84 | 14 | 11 | 210 | 87.1 (81.6–91.1) | 94.4 (91.8–96.2) | 0.81 (0.74–0.88) |
| Influenza virus | |||||||
| Ab | 9 | 1 | 1 | 308 | 90.0 (69.9–97.2) | 99.7 (98.8–99.9) | 0.90 (0.75–1.00) |
| B | 1 | 0 | 0 | 318 | NA | 100.0 (99.4–100.0) | 1.00 (1.00–1.00) |
| Metapneumovirus | 20 | 5 | 0 | 294 | 88.9 (76.5–95.2) | 99.2 (98.0–99.6) | 0.88 (0.78–0.98) |
| Parainfluenza virus | |||||||
| 1 | 1 | 0 | 0 | 318 | NA | 100.0 (99.4–100.0) | 1.00 (1.00–1.00) |
| 2 | 1 | 0 | 0 | 318 | NA | 100.0 (99.4–100.0) | 1.00 (1.00–1.00) |
| 3 | 10 | 4 | 0 | 305 | 83.3 (64.2–93.3) | 99.4 (98.3–99.8) | 0.83 (0.66–0.99) |
| 4 | 6 | 0 | 2 | 311 | 85.7 (60.1–96.0) | 99.7 (98.8–99.9) | 0.85 (0.65–1.00) |
| Respiratory syncytial virus | |||||||
| A | 19 | 0 | 0 | 300 | 100.0 (90.8–100.0) | 100.0 (99.4–100.0) | 1.00 (1.00–1.00) |
| B | 24 | 4 | 1 | 290 | 90.6 (79.8–95.9) | 99.2 (98.0–99.6) | 0.90 (0.81–0.99) |
RPP, Luminex NxTAG RPP RUO; RVP, Seegene Anyplex II RV16; PPA, positive percent agreement; NPA, negative percent agreement; CI, confidence interval; NA, not assessed.
That is, the sum of all Luminex NxTAG RPP RUO results for influenza virus A subtypes (H1, H1N1, and H3).
TABLE 2.
Resolution of discrepant results
| Discrepant result seta | Test results and resolutionb |
No. of discrepancies | ||
|---|---|---|---|---|
| Luminex NxTAG RPP RUO | Seegene Anyplex II RV16 | Resolution | ||
| 1 | Adenovirus | NEG | NA | 1 |
| 2 | NEG | Adenovirus | NA | 1 |
| 3 | NEG | Adenovirus | Adenovirus | 2 |
| 4 | Bocavirus | NEG | NA | 2 |
| 5 | Bocavirus | NEG | Bocavirus | 2 |
| 6 | NEG | Bocavirus | NEG | 2 |
| 7 | NEG | Coronavirus OC43 | NEG | 3 |
| 8 | EV/RV | NEG | EV/RV | 13 |
| 9 | EV/RV | NEG | NEG | 1 |
| 10 | NEG | EV/RV | NA | 1 |
| 11 | NEG | EV/RV | EV/RV | 8 |
| 12 | NEG | EV/RV | NEG | 2 |
| 13 | Influenza virus A | NEG | Influenza virus A | 1 |
| 14 | NEG | Influenza virus A | NA | 1 |
| 15 | Metapneumovirus | NEG | NA | 3 |
| 16 | Metapneumovirus | NEG | Metapneumovirus | 2 |
| 17 | PIV-3 | NEG | PIV-3 | 4 |
| 18 | NEG | PIV-4 | PIV-4 | 2 |
| 19 | RSV-B | NEG | RSV-B | 2 |
| 20 | RSV-B | NEG | NEG | 2 |
| 21 | NEG | RSV-B | RSV-B | 1 |
| Total no. | ||||
| Discrepant results resolved by sequencing | 47 | |||
| Discrepant results | 56 | |||
The various observed combinations of discrepant test results and resolutions are listed from 1 to 21.
NEG, negative; NA, not applicable (insufficient sample volume for sequencing).
A total of 260 viruses were detected: EV/RV (n = 105, 40.4%) was the most prevalent target, followed by RSV-B (n = 27, 10.4%), and BoV (n = 24, 9.2%). Viral coinfections were found in 44 (14.2%) samples. Of these, 37 samples were coinfected by 2 viruses, and 7 contained more than 2 viruses, as indicated in Table 3. Specifically, AdV and BoV were more involved in coinfections (78.6 and 70.8%, respectively) than in single infections, whereas PIV-3, RSV-B, and EV/RV were more commonly associated with single infections (100.0, 77.8, and 69.2%, respectively). It is noteworthy that NxTAG RPP RUO also detected targets not included in the Anyplex II RV16 panel, including Mycoplasma pneumoniae (n = 6), Chlamydophila pneumoniae (n = 1), and CoV-HKU1 (n = 1).
TABLE 3.
Distribution of infections according to detection techniques
| No. and type of target | No. of positive results (%) |
||
|---|---|---|---|
| NxTAG RPP RUO | Anyplex II RV16 | Discrepancy analysis | |
| No infection | 115 (36.1) | 125 (39.2) | 113 (36.3) |
| Single target | |||
| Viral | 151 (47.3) | 149 (46.7) | 154 (49.5) |
| Bacterial | 3 (1.0) | NAa | |
| Two targets | |||
| Viral coinfection | 40 (12.5) | 39 (12.2) | 37 (11.9) |
| Viral-bacterial coinfection | 2 (0.6) | NA | |
| Three targets | |||
| Viral coinfection | 4 (1.3) | 5 (1.6) | 6 (2.0) |
| Viral-bacterial coinfection | 2 (0.6) | NA | |
| Four targets | |||
| Viral coinfection | 2 (0.6) | 1 (0.3) | 1 (0.3) |
| Viral-bacterial coinfection | |||
| Total no. | |||
| Coinfections | 50 (15.7) | 45 (14.1) | 44 (14.2) |
| Infections and coinfections | 204 (63.9) | 194 (60.8) | 198 (63.7) |
| Samples | 319 (100.0) | 319 (100.0) | 311 (100.0) |
NA, not applicable.
DISCUSSION
To our knowledge, this study is the first to compare the RUO version of NxTAG RPP and Anyplex II RV16 in nasopharyngeal aspirates sampled on pediatric patients hospitalized with ALRI. The overall results obtained by both techniques demonstrated high agreement when compared in our study sample.
A recent evaluation of NxTAG RPP RUO against a composite standard of BioFire FilmArray respiratory panel or singleplex real-time PCR by Chen et al. reported ≥96% sensitivity and specificity rates for all respiratory targets except CoV-OC43 (19). Similarly, Beckmann et al. compared NxTAG RPP to a reference multiplex nucleic acid amplification testing (RespiFinder-22) and also determined high detection rates of >94.7% for all targets except for CoV-OC43 (88.9%), CoV-NL63 (83.3), and BoV (90%) (16). On the other hand, studies of performance of the previous xTAG RVP assay in relation to specific real-time PCR techniques (20, 21) reported moderate values of sensitivity for AdV. The results of our study are not comparable to the ones previously described since we analyzed agreement between tests in the absence of a gold standard. Nevertheless, we obtained high values of PPA, NPA, and kappa coefficient either for CoV-OC43, BoV, and AdV or the rest of the targets common to the two panels, which reflects a similar diagnostic performance of NxTAG RPP RUO and Anyplex II RV16. In this regard, there were minor differences in the number of positive results by NxTAG RPP RUO (n = 204) and Anyplex II RV16 (n = 194) compared to the number of positives confirmed after discrepancy analysis (n = 198). Of note, the important proportion of coinfected specimens that we detected (14.2%) could otherwise have been missed by traditional methods.
In addition to the targets common to the two panels, the new technique was able to yield positive results for Mycoplasma pneumoniae and Chlamydophila pneumoniae, which are bacterial agents frequently associated with pediatric respiratory infections (22, 23). Conversely, the fact that we did not detect any Legionella pneumophila infection in our study could be explained by the unusual incidence of this bacterium in children. On the other hand, NxTAG RPP RUO detected one case of CoV-HKU1 and distinguished the subtypes of influenza A virus, which has important implications for epidemiological surveillance and clinical purposes (24, 25).
The distribution of viruses, as well as the proportion of coinfected samples, was similar to values previously described for pediatric populations in our geographical area (9, 26). Even so, viral distribution showed some particularities compared to the epidemiological surveillance information reported by the Catalan Public Health Agency for the 2015-2016 influenza epidemic season (available on Gencat.cat website [http://canalsalut.gencat.cat/web/.content/home_canal_salut/professionals/temes_de_salut/vigilancia_epidemiologica/documents/arxius/spfi.pdf]). Of note, our study period covered weeks 26 to 45 of year 2015 and did not overlap with the 2015-2016 influenza epidemic season, which started at week 48. Surveillance data, including patients of all ages at the regional scale, indicated that the predominant viruses circulating out of the epidemic season were, in descending order of incidence rates, RV, PIV, EV, and ADV. In our pediatric study, EV/RV were the most prevalent targets (40.4%), but the detection rate was moderate for all PIV types (9.3%) and minor for AdV (5.4%). In a similar way, presence of BoV (9.2%) and MPV (8.5%) was noticeable in the present study, whereas the incidence of these viruses was not reported in the regional surveillance data. The lack of time overlapping between our study period and the epidemic season could explain the low proportions of IFV-A (3.8%) and IFV-B (0.4%) found. In turn, we observed a moderate prevalence of RSV-B (10.4%), despite the fact that the first onset of RSV infections in the general population was not registered until week 45. Differences between the viral distribution in our study and regional epidemiological data suggest that the circulation trends of certain respiratory viruses out of the seasonal period may have distinctive patterns in our pediatric population and our reference area compared to the general population of the region.
A limitation of the study was the low number of positive results observed for certain targets, such as IFV-B, CoV-229E, CoV-NL63, PIV-1, and PIV-2, which did not allow us to measure agreement values for these targets. Another potential limitation may be that batched testing was carried out after we performed a freezing-defrosting cycle on the samples. However, since all of the specimens were exposed to the same storage conditions before being processed by any technique, we assume that the potential confounding effect of the freezing-defrosting steps on values of diagnostic accuracy was controlled.
In conclusion, NxTAG RPP RUO showed diagnostic performance similar to Anyplex II RV16 for main targets that cause childhood ALRI, together with additional capability to detect some respiratory bacteria. Further studies should be conducted to assess the agreement between the two techniques for coronavirus, influenza virus, and parainfluenza virus.
ACKNOWLEDGMENTS
This study was supported in part by a research agreement between Luminex Molecular Diagnostics and Research Foundation Sant Joan de Déu and also by Fondo Europeo de Desarrollo Regional (FEDER) and the Ministry of Science and Innovation, Institute of Health Carlos III, Projects of Research on Health (grant PI13/01729).
Funding Statement
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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