Reduced sensitivity of a multiplex commercial respiratory panel for detection of Mycoplasma pneumoniae is due to specimen type

KEYWORDS Mycoplasma pneumoniae, molecular diagnosis M ycoplasma pneumoniae (MPN) is a major pathogen of community-acquired respiratory infections in children.This bacterium is fastidious, taking weeks to grow in culture.Nucleic acid amplification tests are now the method of choice for diagnosis of MPN-respiratory infection.Following the COVID-19 pandemic, a significant increase in MPN infection has been reported since Fall 2023 (1).The microbiology laboratory at Nationwide Children's Hospital offers two tests that detect MPN: (i) FilmArray Respiratory Panel v2.1 (RP2.1,BioFire Diagnostics, Salt Lake City, UT), a multiplex test that detects 22 respiratory pathogens including MPN, directly from nasopharyngeal swabs(NP) (2, 3); (ii) a standalone laboratory-developed PCR(MPN-PCR) that detects MPN from throat swabs (THT) (4).The limits of detection (LoD) are 460 copies/mL and 500 copies/mL for RP2.1 and MPN-PCR, respectively.While these LoDs are similar, we observed differences in the two assays' performance during quality assurance review.We aimed to compare the sensitivities of NP and THT specimen types for MPN detection in our patient population.From September 2023 to June 2024, 315 patients were identified who had both an NP tested on RP2.1 and a THT tested on MPN-PCR within two days of each other.Two hundred and fifty seven (81.6%) had negative results from both tests.Among the remaining 58 patients, 27(46.6%)had positive results from both tests, 31(53.4%)were positive by MPN-PCR only, and none were positive by RP2.1 only.The median Cycle threshold (Ct) value from MPN-PCR on THT was 29.4 [interquartile range (IQR): 26.1, 31.1] for patients positive by both tests and 30.1 (IQR: 26.5, 35.4) for patients positive by MPN-PCR only and were not significantly different (P = 0.36).Among the 31 patients whose NP were negative by RP2.1 and THT were positive by MPN-PCR, 28 remnant NP were available for further analysis and were tested with MPN-PCR; all but four were negative.The median Ct of these four positives was 38.6 (IQR: 37.8, 39.4).Among 27 patients whose NP and THT were both positive, 25 remnant NP were tested with MPN-PCR and all were positive.The median Ct for the NP was 34.0 (IQR: 30.6, 36.2),significantly higher than that of the corresponding THT (29.4,IQR: 36.5, 35.4,P = 0.0006, Fig. 1).In total, 53 NPs were tested with both RP2.1 and MPN-PCR; the positive percentage agreement, negative percent agreement, and overall concordance between the two assays on NP were 100%, 85.7%, and 92.5%, respectively.Here, we showed that NP tested with RP2.1 missed over half of the patients whose THT were positive for MPN.Testing the same NP with both tests showed that sensitivities between the two assays were similar and suggests the difference in detection was attributable to the specimen type and not differences in assay performance.For patients with MPN positive by both specimen types vs those positive by THT only, Ct values in THT were similar.However, Ct values in NP were significantly higher than that of the THT.No patient had a positive NP and a negative THT.Other studies also demonstrated that