There are several convenient and accurate molecular assays to detect respiratory bacterial infection. The NeoPlex RB-8 detection kit (NeoPlex RB-8) is a new multiplex real-time PCR assay that simultaneously detects Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Haemophilus influenzae, Bordetella pertussis, Bordetella parapertussis, and Moraxella catarrhalis in a single test.
KEYWORDS: respiratory bacterial pathogen, community-acquired pneumonia, multiplex real-time PCR, clinical evaluation
ABSTRACT
There are several convenient and accurate molecular assays to detect respiratory bacterial infection. The NeoPlex RB-8 detection kit (NeoPlex RB-8) is a new multiplex real-time PCR assay that simultaneously detects Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Haemophilus influenzae, Bordetella pertussis, Bordetella parapertussis, and Moraxella catarrhalis in a single test. This study compared the clinical concordance of NeoPlex RB-8 with another method, Seeplex PneumoBacter ACE detection assay (Seeplex PB ACE), which simultaneously detects S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis. We tested 2,137 nasopharyngeal swab and sputum specimens using both assays. For discordant Bordetella parapertussis and M. catarrhalis specimens, we also performed bidirectional sequencing. For S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis, which are detected by both NeoPlex RB-8 and Seeplex PB ACE, the positive and negative agreement between the two assays ranged from 91.7 to 100% (κ = 0.918 to 1). S. pneumoniae and H. influenzae were the most discordant targets and measured with higher sensitivity and specificity by NeoPlex RB-8 than Seeplex PB ACE. For Bordetella parapertussis and M. catarrhalis, which are not detected by Seeplex PB ACE, NeoPlex RB-8 sensitivity and specificity were >99%. Overall, NeoPlex RB-8 was highly comparable to Seeplex PB ACE, but NeoPlex RB-8 was more clinically accurate, with higher throughput and more convenience.
INTRODUCTION
Acute respiratory infections (ARI), especially community-acquired pneumonia (CAP), are major causes of morbidity and mortality worldwide (1). Acute respiratory infections are responsible for 18 to 33% of all deaths in children less than 5 years old (2). Although the mortality rate of CAP is less than 1 to 5% for outpatients, the mortality rate increases to 12% in high-risk patients, such as elderly or immunocompromised patients, who require hospitalization due to CAP (3).
Many causative pathogens can lead to ARI, including legionellosis and pertussis (4–6). Because respiratory disease symptoms are very similar, whether caused by pathogenic virus or bacteria, accurate diagnosis of respiratory bacterial pathogens is required to select an appropriate antibiotic treatment (7–10). To meet this need, many nucleic acid-based methods to detect respiratory bacterial pathogens using conventional or real-time PCR are under development, because these methods are more convenient, with faster turnaround time and better clinical performance than traditional culture methods. Seeplex PneumoBacter ACE detection (Seeplex PB ACE; Seegene Inc., Seoul, South Korea) is a commonly used in vitro diagnostic reagent based on multiplex PCR with dual priming oligonucleotide (DPO) technology. It can simultaneously detect six respiratory bacterial pathogens: Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis.
The NeoPlex RB-8 detection kit (NeoPlex RB-8; Genematrix Inc., Seongnam, South Korea) is a similar real-time PCR-based molecular diagnostic assay certified by CE for in vitro diagnosis (CE-IVD). It can simultaneously identify eight respiratory bacterial pathogens: S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumoniae, L. pneumophila, B. pertussis, Bordetella parapertussis, and Moraxella catarrhalis. NeoPlex RB-8 utilizes C-Tag platform technology, which is a novel proprietary technology from Genematrix (Fig. 1). The goal of this study was to compare the clinical performance of the NeoPlex RB-8 detection kit and Seeplex PB ACE detection assay using nasopharyngeal swab (NPS) and sputum specimens.
FIG 1.
Principles of C-Tag platform technology. (A) PCR amplification and probe hybridization using C-Tag primer and probe. Multiplex PCR uses multiple C-Tag primers to incorporate C-Tag sequence in amplicons. After a second PCR cycle, the complementary strand of the C-Tag sequence is amplified and immediately cleaved by a thermostable restriction endonuclease. The complementary fragment of each C-Tag sequence then is hybridized to its respective fluorescent C-Tag probe (R, reporter; Q, quencher). (B) Multiple melt peaks from C-Tag probes with the same fluorescent dye (reporter) in melting curve analysis. The different melting temperatures (Tm) of each C-Tag probe causes the unique melt peak. When three probes have the same reporter but different Tm (65°C, 75°C, and 85°C for targets A, B, and C, respectively), three melt peaks are shown in one fluorescent channel for the reporter in the presence of targets A, B, and C.
MATERIALS AND METHODS
Clinical specimens.
The Institutional Review Board of Samkwang Medical Laboratories approved this study (SML-IRB-16-10-17). We collected 2,137 clinical specimens, including 945 NPS and 1,192 sputum specimens, between June 2017 and April 2018. NPS specimens were placed into UTM transport medium and sputum into a sputum container. DNA then was extracted using the MagNA Pure 96 system (Roche Molecular Systems, Inc., NJ, USA), according to the manufacturer’s instructions. Extracted DNA was stored at –20°C if not used immediately.
NeoPlex RB-8 detection testing.
The extracted DNA from NPS and sputum specimens was analyzed with the NeoPlex RB-8 for detecting respiratory bacterial pathogens, according to the manufacturer’s instructions. Briefly, 5 μl of DNA was mixed with 5 μl of DW (distilled water), 5 μl of RB-8 PPM, and 5 μl of 4× master mix. DNA was then amplified using a CFX96 real-time PCR system (Bio-Rad Inc., Hercules, CA, USA) under the following conditions: 50°C for 4 min, 95°C for 15 min, and 40 cycles for two steps at 95°C for 20 s, 65°C for 1 min 30 s, 73°C for 10 min, and 55°C for 30 s. Melting curve analysis began with a stepwise increase of 0.5°C/step from 55°C to 90°C with a 5-s pause between steps. Melting peaks were derived from the initial fluorescence (F) versus temperature (T) curves by plotting the negative derivative of fluorescence over temperature versus temperature (dF/dT versus T) by CFX96 Manager software, ver.1.6 (Bio-Rad Inc., Hercules, CA, USA). Each amplification reaction mixture contained human beta-globin DNA as an internal control, and each run was accompanied by positive and negative controls.
Seeplex PB ACE detection testing.
We conducted Seeplex PB ACE detection testing according to the manufacturer’s instructions (11). Briefly, we performed PCRs in a 20-μl volume composed of 3 μl DNA and 17 μl PCR premix under the following conditions: 94°C for 15 min, 40 cycles of three steps (94°C for 30 s, 60°C for 1 min 30 s, and 72°C for 1 min 30), and a final extension at 72°C for 10 min. PCR products were visualized after agarose gel electrophoresis on ethidium bromide-stained 2% agarose gels. The estimated sizes of amplicons were the following: M. pneumoniae, 583 bp; L. pneumophila, 472 bp; S. pneumoniae, 350 bp; H. influenzae, 257 bp; B. pertussis, 200 bp; and C. pneumoniae, 146 bp. Each amplification reaction mixture contained internal control DNA, and each run was accompanied by positive and negative controls.
Concordance analysis.
The NeoPlex RB-8 testing result was considered concordant when it agreed with results from the Seeplex PB ACE. Concordant results were designated true positive (TP) or true negative (TN). Discordant results between two assays (i.e., false positive [FP] or false negative [FN]) and all results for B. parapertussis and M. catarrhalis, which cannot be detected by Seeplex PB ACE, were further tested and resolved by target-specific bidirectional sequencing, a gold standard validation method (see Table S1 in the supplemental material). We sequenced PCR products that amplified with the target-specific sequencing primers using the BigDye Terminator v3.1 cycle sequencing kit with the target-specific primers and analyzed the products with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were identified using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) databases (http://blast.ncbi.nlm.n-ih.gov/BLAST).
Statistical analysis.
We analyzed positive/negative percent agreement (PPA/NPA), Cohen’s kappa coefficients, sensitivity, specificity, and positive/negative predictive value (PPV/NPV) with a 95% confidence interval (CI) (12). All statistical analyses used Med-Calc, ver. 18.10.2 (MedCalc Software, Ostend, Belgium).
RESULTS
We analyzed 945 NPS and 1,192 sputum specimens using NeoPlex RB-8 and Seeplex PB ACE. We found that NeoPlex RB-8 detected respiratory bacterial pathogens in 559 NPS (59%) and 733 sputum (61%) specimens, whereas Seeplex PB ACE detected respiratory bacterial pathogens in 500 NPS (53%) and 726 sputum (61%) specimens. Of these, 559 (26%) NeoPlex RB-8- and 471 (22%) Seeplex PB ACE-tested specimens were found to be multiple target infections (see Table S2 in the supplemental material).
We analyzed the PPA/NPA and kappa coefficient of the six targets commonly shared between NeoPlex RB-8 and Seeplex PB ACE detection (Table 1). S. pneumoniae and H. influenzae had the most positives. However, the PPA/NPA in NPS samples was 91.7%/97.7% (κ = 0.918; 95% CI, 0.899 to 0.934) for S. pneumoniae and 94.9%/99.1% (κ = 0.967; 95% CI, 0.953 to 0.974) for H. influenzae, and in sputum samples it was 95.2%/98.6% (κ = 0.947; 95% CI, 0.933 to 0.958) for S. pneumoniae and 95.7%/97.1% (κ = 0.940; 95% CI, 0.925 to 0.952) for H. influenzae. C. pneumoniae and L. pneumophila were 100% concordant between NeoPlex RB-8 and Seeplex PB ACE methods (κ = 1.000). Notably, neither assay detected a positive L. pneumophila result in NPS samples, but 29 specimens were L. pneumophila positive in sputum samples. For M. pneumoniae and B. pertussis, the NPA of both assays was 100% for all specimen types. However, the PPA of B. pertussis was 96.4% in NPS samples, and that of M. pneumoniae was 99.0% in sputum specimens.
TABLE 1.
Comparative respiratory bacterial pathogen detection by NeoPlex RB-8 and Seeplex PB ACE
| Target | N+/S+a | N+/S− | N−/S+ | N−/S− | PPA (%) (95% CI) | NPA (%) (95% CI) | κ (95% CI) |
|---|---|---|---|---|---|---|---|
| NPS (n = 945) | |||||||
| Streptococcus pneumoniae | 311 | 14 | 28 | 592 | 91.7 (88.3–94.2) | 97.7 (96.2–98.6) | 0.918 (0.899–0.934) |
| Mycoplasma pneumoniae | 74 | 0 | 0 | 871 | 100 (95.1–100) | 100 (99.6–100) | 1.000 (0.996–1.000) |
| Chlamydophila pneumoniae | 29 | 0 | 0 | 916 | 100 (88.3–100) | 100 (99.6–100) | 1.000 (0.996–1.000) |
| Legionella pneumophila | 0 | 0 | 0 | 945 | NAb | 100 (99.6–100) | 1.000 (0.996–1.000) |
| Haemophilus influenzae | 242 | 6 | 13 | 684 | 94.9 (91.5–97.0) | 99.1 (98.1–99.6) | 0.967 (0.953–0.976) |
| Bordetella pertussis | 27 | 0 | 1 | 917 | 96.4 (82.3–99.4) | 100 (99.6–100) | 0.999 (0.994–0.999) |
| Sputum (n = 1,192) | |||||||
| Streptococcus pneumoniae | 457 | 10 | 23 | 702 | 95.2 (92.9–96.8) | 98.6 (97.4–99.2) | 0.947 (0.933–0.958) |
| Mycoplasma pneumoniae | 96 | 0 | 1 | 1,095 | 99.0 (94.4–99.8) | 100 (99.7–100) | 0.999 (0.995–0.999) |
| Chlamydophila pneumoniae | 28 | 0 | 0 | 1,164 | 100 (87.9–100) | 100 (99.7–100) | 1.000 (0.997–1.000) |
| Legionella pneumophila | 29 | 0 | 0 | 1,163 | 100 (88.3–100) | 100 (99.7–100) | 1.000 (0.997–1.000) |
| Haemophilus influenzae | 376 | 23 | 17 | 776 | 95.7 (93.2–97.3) | 97.1 (95.7–98.1) | 0.940 (0.925–0.952) |
| Bordetella pertussis | 26 | 0 | 0 | 1,166 | 100 (87.1–100) | 100 (99.7–100) | 1.000 (0.997–1.000) |
N, NeoPlex RB-8; S, Seeplex PB ACE; N+/S+, positive with both assays; N+/S−, NeoPlex RB-8 positive and Seeplex PB ACE negative; −/+, NeoPlex RB-8 negative and Seeplex PB ACE positive; N−/S−, negative with both assays.
NA, not applicable.
We then performed bidirectional sequencing as a validation method for the 136 discordant specimens, including 62 discordant results in NPS and 74 in sputum (Table 2). We also performed this validation for B. parapertussis- and M. catarrhalis-positive and -negative specimens obtained using NeoPlex RB-8 (Table 3).
TABLE 2.
Bidirectional sequencing results for samples discordant between NeoPlex RB-8 and Seeplex PB ACE
| Target | N−/S+a | Bidirectional sequencing |
N+/S− | Bidirectional sequencing |
||
|---|---|---|---|---|---|---|
| Positive | Negative | Positive | Negative | |||
| NPS (n = 62) | ||||||
| Streptococcus pneumoniae | 28 | 5 | 23 | 14 | 14 | 0 |
| Mycoplasma pneumoniae | 0 | 0 | 0 | 0 | 0 | 0 |
| Chlamydophila pneumoniae | 0 | 0 | 0 | 0 | 0 | 0 |
| Legionella pneumophila | 0 | 0 | 0 | 0 | 0 | 0 |
| Haemophilus influenzae | 13 | 2 | 11 | 6 | 6 | 0 |
| Bordetella pertussis | 1 | 1 | 0 | 0 | 0 | 0 |
| Sputum (n = 74) | ||||||
| Streptococcus pneumoniae | 23 | 5 | 18 | 10 | 8 | 2 |
| Mycoplasma pneumoniae | 1 | 1 | 0 | 0 | 0 | 0 |
| Chlamydophila pneumoniae | 0 | 0 | 0 | 0 | 0 | 0 |
| Legionella pneumophila | 0 | 0 | 0 | 0 | 0 | 0 |
| Haemophilus influenzae | 17 | 5 | 12 | 23 | 21 | 2 |
| Bordetella pertussis | 0 | 0 | 0 | 0 | 0 | 0 |
N, NeoPlex RB8; S, Seeplex PB ACE; N−/S+, NeoPlex RB8 negative and Seeplex PB ACE positive; +/−, NeoPlex RB-8 positive and Seeplex PB ACE negative.
TABLE 3.
Comparison of bidirectional sequencing results to detect B. parapertussis and M. catarrhalis using NeoPlex RB-8
| Target | N+a | Bidirectional sequencing |
N− | Bidirectional sequencing |
||
|---|---|---|---|---|---|---|
| Positive | Negative | Positive | Negative | |||
| NPS | ||||||
| Bordetella parapertussis | 16 | 16 | 0 | 929 | 0 | 929 |
| Moraxella catarrhalis | 226 | 225 | 1 | 719 | 0 | 719 |
| Sputum | ||||||
| Bordetella parapertussis | 17 | 17 | 0 | 1,175 | 0 | 1,175 |
| Moraxella catarrhalis | 33 | 32 | 1 | 1,159 | 0 | 1,159 |
N+, NeoPlex RB-8 positive; N−, NeoPlex RB-8 negative.
Among the 136 discordant specimens, 64 of 83 FN specimens were determined to be TN, and 49 of 53 FP specimens were TP. For S. pneumoniae validation in NPS, 23 of 28 FN specimens were confirmed as TN, and all 14 FP specimens were confirmed as TP. Similarly, for H. influenzae validation in NPS, 11 of 13 FN specimens were confirmed as TN, and all 6 FP specimens were confirmed as TP. S. pneumoniae validation in sputum found that 18 of 23 FN specimens were TN and 8 of 10 FP specimens were TP. H. influenzae validation in sputum found that 12 of 17 FN specimens were TN, and 21 of 23 FP specimens were TP. Each false-negative result in M. pneumoniae and B. pertussis tests was confirmed as a TP by the sequencing assay. No discordant results were observed for C. pneumoniae and L. pneumophila in either sample type (Table 2).
Table 3 compares results of B. parapertussis and M. catarrhalis detection with NeoPlex RB-8 and the bidirectional sequencing method. Thirty-three B. parapertussis-positive results using RB-8 detection for both sample types were confirmed positive by sequencing. For M. catarrhalis validation, 225 of 226 NPS specimens and 32 of 33 sputum specimens that were positive in the RB-8 assay were confirmed positive by sequencing. All B. parapertussis- and M. catarrhalis-negative results obtained using NeoPlex RB-8 were confirmed as negative by sequencing.
Combining all test results, we calculated the clinical sensitivity, specificity, and PPV/NPV of eight targets for NeoPlex RB-8 and six targets for Seeplex PB ACE (Tables 4 and 5). The sensitivity and specificity for detecting S. pneumoniae and H. influenzae by NeoPlex RB-8 were 98.5% or higher; however, for Seeplex PB ACE, sensitivity and specificity were 98.1% or lower in both NPS and sputum. The sensitivity and specificity for detecting C. pneumoniae and L. pneumophila was 100% for both assays in NPS and sputum (excepting any sensitivity result for L. pneumophila detection in NPS due to a lack of positive results). The sensitivities for detecting M. pneumoniae and B. pertussis were 99% and 96.4%, respectively, using NeoPlex RB-8 and 100% for both bacteria using Seeplex PB ACE. B. parapertussis and M. catarrhalis had sensitivities and specificities over 99% when detected by NeoPlex RB-8.
TABLE 4.
Clinical performance of NeoPlex RB-8 for nasopharyngeal swab and sputum specimens
| Target | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | PPV (%) (95% CI) | NPV (%) (95% CI) |
|---|---|---|---|---|
| NPS | ||||
| Streptococcus pneumoniae | 98.5 (96.5–99.4) | 100 (99.4–100) | 100 (98.8–100) | 99.2 (98.1–99.7) |
| Mycoplasma pneumoniae | 100 (95.1–100) | 100 (99.6–100) | 100 (95.1–100) | 100 (99.6–100) |
| Chlamydophila pneumoniae | 100 (88.3–100) | 100 (99.6–100) | 100 (88.3–100) | 100 (99.6–100) |
| Legionella pneumophila | NAa | 100 (99.6–100) | NA | 100 (99.6–100) |
| Haemophilus influenzae | 99.2 (97.1–99.8) | 100 (99.5–100) | 100 (98.5–100) | 99.7 (99.0–99.9) |
| Bordetella pertussis | 96.4 (82.3–99.4) | 100 (99.6–100) | 100 (87.5–100) | 99.9 (99.4–99.9) |
| Bordetella parapertussis | 100 (80.6–100) | 100 (99.6–100) | 100 (80.6–100) | 100 (99.6–100) |
| Moraxella catarrhalis | 100 (98.3–100) | 99.9 (99.2–99.9) | 99.6 (97.5–99.9) | 100 (99.5–100) |
| Sputum | ||||
| Streptococcus pneumoniae | 98.9 (97.5–99.5) | 99.7 (99.0–99.9) | 99.6 (98.5–99.9) | 99.3 (98.4–99.7) |
| Mycoplasma pneumoniae | 99.0 (94.4–99.8) | 100 (99.7–100) | 100 (96.2–100) | 99.9 (99.5–99.9) |
| Chlamydophila pneumoniae | 100 (87.9–100) | 100 (99.7–100) | 100 (87.9–100) | 100 (99.7–100) |
| Legionella pneumophila | 100 (88.3–100) | 100 (99.7–100) | 100 (88.3–100) | 100 (99.7–100) |
| Haemophilus influenzae | 98.8 (97.1–99.5) | 99.8 (99.1–99.9) | 99.5 (98.2–99.9) | 99.4 (98.5–99.7) |
| Bordetella pertussis | 100 (87.1–100) | 100 (99.7–100) | 100 (87.1–100) | 100 (99.7–100) |
| Bordetella parapertussis | 100 (81.6–100) | 100 (99.7–100) | 100 (81.6–100) | 100 (99.7–100) |
| Moraxella catarrhalis | 100 (89.3–100) | 99.9 (99.5–99.9) | 97.0 (84.7–99.5) | 100 (99.7–100) |
NA, not applicable.
TABLE 5.
Clinical performance of Seeplex PB ACE for nasopharyngeal swab and sputum specimens
| Target | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | PPV (%) (95% CI) | NPV (%) (95% CI) |
|---|---|---|---|---|
| NPS | ||||
| Streptococcus pneumoniae | 95.8 (93.0–97.5) | 96.3 (94.5–97.5) | 93.2 (90.0–95.4) | 97.7 (96.2–98.6) |
| Mycoplasma pneumoniae | 100 (95.1–100) | 100 (99.6–100) | 100 (95.1–100) | 100 (99.6–100) |
| Chlamydophila pneumoniae | 100 (88.3–100) | 100 (99.6–100) | 100 (88.3–100) | 100 (99.6–100) |
| Legionella pneumophila | NAa | 100 (99.6–100) | NA | 100 (99.6–100) |
| Haemophilus influenzae | 97.6 (94.8–98.9) | 98.1 (96.8–98.9) | 94.9 (91.5–97.0) | 99.1 (98.1–99.6) |
| Bordetella pertussis | 100 (87.9–100) | 100 (99.6–100) | 100 (87.9–100) | 100 (99.6–100) |
| Sputum | ||||
| Streptococcus pneumoniae | 97.9 (96.1–98.8) | 96.8 (95.3–97.9) | 95.2 (92.9–96.8) | 98.6 (97.4–99.2) |
| Mycoplasma pneumoniae | 100 (96.2–100) | 100 (99.7–100) | 100 (96.2–100) | 100 (99.7–100) |
| Chlamydophila pneumoniae | 100 (87.9–100) | 100 (99.7–100) | 100 (87.9–100) | 100 (99.7–100) |
| Legionella pneumophila | 100 (88.3–100) | 100 (99.7–100) | 100 (88.3–100) | 100 (99.7–100) |
| Haemophilus influenzae | 94.2 (91.5–96.1) | 97.9 (96.6–98.7) | 95.7 (93.2–97.3) | 97.1 (95.7–98.1) |
| Bordetella pertussis | 100 (87.1–100) | 100 (99.7–100) | 100 (87.1–100) | 100 (99.7–100) |
NA, not applicable.
DISCUSSION
In this study, we compared the clinical performance of the NeoPlex RB-8 detection kit with that of the Seeplex PB ACE detection assay to detect respiratory bacterial pathogens in NPS and sputum specimens. Both assays are based on PCR technology. NeoPlex RB-8 can detect eight CAP-causative bacteria, whereas Seeplex PB ACE detects six.
For the six targets detected by both assays, the kappa coefficient was over 0.91. Specifically, the concordance values of M. pneumoniae, C. pneumoniae, L. pneumophila, and B. pertussis were over 0.99, indicating a good correlation between the two assays. Likewise, the clinical sensitivity and specificity for those four targets were over 99% in both assays. In summary, the performance of NeoPlex RB-8 and Seeplex PB ACE was equivalent for M. pneumoniae, C. pneumoniae, L. pneumophila, and B. pertussis.
S. pneumoniae and H. influenzae were the most discordant targets between assays. Among the 2,137 samples, 75 S. pneumoniae results and 59 H. influenzae results were discordant (Table 1). This result may be caused by multiple serotypes of those pathogens and high genetic similarity within each genus. S. pneumoniae has at least 92 known serotypes, and these have specific genome sequences; therefore, the well-known cps gene could amplify up to 84 serotypes (13, 14). In addition, because the viridans group in genus Streptococcus has a genome sequence highly similar to that of S. pneumoniae, some species, such as S. mitis and S. oralis, are often misdiagnosed as S. pneumoniae (15, 16).
Similarly, H. influenzae includes six capsulated serotypes, from A to F. Type B (Hib) is well known to be the main cause of not only pneumonia but also meningitis. The incidence of diseases from Hib infection has decreased since the development of Hib vaccine. However, infection with unencapsulated H. influenzae serotypes (nontypeable Haemophilus influenzae, or NTHi) has increased, and the NTHi collection includes more than 90 similar isolates (17–19). Furthermore, the genomic sequence of NTHi is very similar to that of Haemophilus parainfluenzae, Haemophilus haemolyticus, and Aggregatibacter aphrophilus, which can cause misdiagnosis as H. influenzae (20, 21).
It is possible that these characteristics of S. pneumoniae and H. influenzae caused high discordance between NeoPlex RB-8 and Seeplex PB ACE methods. Thus, we conducted bidirectional sequencing as the gold standard for validation to determine whether discordant samples of H. influenzae were truly positive or negative. Overall, NeoPlex RB-8 showed more than 98% sensitivity and specificity, whereas Seeplex PB ACE showed approximately 94% sensitivity and specificity, indicating that NeoPlex RB-8 is superior to Seeplex PB ACE in diagnosing S. pneumoniae and H. influenzae in NPS and sputum.
B. parapertussis and M. catarrhalis bacteria cannot be detected by Seeplex PB ACE, but these infections require clinical attention. Although B. parapertussis is less prevalent and severe than B. pertussis, it can cause pertussis-associated pneumonia in infants or elderly patients with lower immunity and who have not received the DTaP or Tdap vaccine (22). M. catarrhalis is associated with exacerbation of chronic obstructive pulmonary disease (COPD) (23) and is a main causative pathogen of COPD-associated pneumonia (24). Despite the clinical importance of accurately diagnosing B. parapertussis and M. catarrhalis, there are only a few commercially available B. parapertussis and M. catarrhalis molecular diagnostic assays.
In this study, we compared the ability of the NeoPlex RB-8 method with that of a bidirectional sequencing method to assess the performance of NeoPlex RB-8 in detecting B. parapertussis and M. catarrhalis. The NeoPlex RB-8 assay was over 99% sensitive and specific in detecting B. parapertussis and M. catarrhalis. Thus, NeoPlex RB-8 may be useful for diagnosing B. parapertussis- or M. catarrhalis-infected patients.
In this study, we used only NPS and sputum specimens to compare NeoPlex RB-8 and Seeplex PB ACE assays. In general, NPS and sputum are not the only specimens used to analyze or diagnose respiratory tract infections. Nasopharyngeal aspirate and bronchoalveolar lavage (BAL) specimens are also used. Thus, to better analyze the clinical performance of these assays, future studies with aspirate and BAL specimens are needed.
In conclusion, our study shows that the NeoPlex RB-8 assay is highly comparable to the Seeplex PB ACE detection assay and has high clinical sensitivity and specificity to detect respiratory bacterial pathogens in NPS and sputum specimens. In particular, the clinical performance of NeoPlex RB-8 in detecting S. pneumoniae and H. influenzae, the most important CAP-causative pathogens, is superior to that of the Seeplex PB ACE assay. Furthermore, the NeoPlex RB-8 assay can detect B. parapertussis and M. catarrhalis, which most commercially available assays cannot detect. Thus, the NeoPlex RB-8 assay may be a convenient and effective tool for clinical laboratories to simultaneously detect eight major respiratory bacterial pathogens using a single convenient real-time PCR test.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by the Technology Innovation Program (10067274, Modular Type Precision Molecular Diagnostics to Detect Wide Spectrum of Respiratory Pathogen Panels), funded by the Ministry of Trade, Industry & Energy (MOTIE, South Korea).
J.W.K., S.S.H., S.-O.K., and S.P.H. are employees of GeneMatrix, Inc. The other authors have no conflict of interest to declare.
Footnotes
Supplemental material is available online only.
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