Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae
ABSTRACT
We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae. The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.
INTRODUCTION
Mycoplasma pneumoniae is an important etiologic agent in pediatric and adult tracheobronchitis and community-acquired pneumonia (CAP). Centers for Disease Control and Prevention (CDC) investigations found that M. pneumoniae was the most common bacterial pathogen detected in children with CAP (8%) and in 2% of adult CAP cases during periods when M. pneumoniae was endemic (1, 2). Up to 20 to 40% of CAP in the general population and up to 70% in closed populations can be caused by M. pneumoniae during cyclic epidemic periods, with an estimated two million annual cases of CAP resulting in 100,000 hospitalizations of adults in the United States (3–6).
Macrolides have been the recommended treatment of choice for M. pneumoniae infections, especially in children, due to relative contraindications for tetracyclines and fluoroquinolones. In Asia, macrolide resistance rates in M. pneumoniae have surged in the past 2 decades to >90%, resulting in increased patient morbidity, greater cost of care, and prolonged hospital stays (3). Data from a recent U.S. national surveillance study demonstrated an overall M. pneumoniae macrolide resistance rate of 7.5%, but in some areas of the northeast and southeast, the rate exceeded 20% (7).
M. pneumoniae infection can be diagnosed by culture, serology, or molecular methods (3). Culture is relatively insensitive, requires specialized media, significant expertise, and takes several days to weeks to complete due to the fastidious nature and slow growth of the organism. Serology lacks both sensitivity and specificity due to various reasons, which include the high prevalence of background antibodies in healthy persons and the lack of an IgM response in many older individuals (3). Optimum use of serology requires testing acute and convalescent specimens, which is not practical for diagnosis in an ambulatory setting.
Molecular methods for M. pneumoniae detection were first developed in the 1980s and were refined further over the past decade with the advent of real-time PCR and the ability to detect macrolide resistance directly in clinical specimens without the need for culture (8, 9). Despite U.S. Food and Drug Administration (FDA) approval of the first molecular test for M. pneumoniae in 2011, there have been no specific recommendations from regulatory or professional organizations to perform such testing, and most infections due to M. pneumoniae remain undiagnosed. Inclusion of M. pneumoniae as a target on FDA-approved respiratory pathogen panels enables detection of this organism, but the clinical sensitivity of these assays has not been thoroughly evaluated.
Since 2011, there have been five FDA-cleared commercial molecular tests targeting M. pneumoniae in clinical specimens. Four of these are multianalyte PCR platforms that simultaneously detect several respiratory viruses, M. pneumoniae, and other bacterial pathogens such as Chlamydia pneumoniae and Bordetella spp. The other FDA-cleared commercial assay is a single-analyte loop-mediated isothermal amplification (LAMP) system that detects only M. pneumoniae. Since molecular testing has become more widely available in recent years and there are now several FDA-cleared assays, this diagnostic approach can now be considered the method of choice (3). Some molecularly based assays require separate DNA extraction, while others perform extraction, amplification, and detection on specimens inoculated directly into the instruments with results available in about 1 to 4 h. Although there are several previous publications describing the performance of various commercial molecularly based assays that include statistics on their ability to detect M. pneumoniae, most studies involving multiplex platforms have focused primarily on detection of respiratory viruses and included a relatively small number of specimens testing positive for M. pneumoniae (10–26). No previous studies have evaluated large numbers of M. pneumoniae-positive specimens to enable thorough head-to-head comparisons of commercial molecular diagnostic platforms available in the United States.
The University of Alabama at Birmingham (UAB) Diagnostic Mycoplasma Laboratory and the CDC independently developed, validated, and currently utilize PCR assays for rapid detection of macrolide resistance directly in respiratory specimens. However, there are no commercial assays sold in the United States that enable rapid detection of macrolide-resistant M. pneumoniae. SpeeDx, Inc. (Austin, TX), has developed the Resistance Plus MP, an investigational PCR assay for M. pneumoniae detection and designation of macrolide resistance. This assay is similar in format to their Resistance Plus MG assay that is now sold in some countries for detection and designation of macrolide resistance in Mycoplasma genitalium (27). Determination of macrolide resistance via molecular identification of rRNA mutations known to confer macrolide resistance is more rapid and practical than determination via culture and broth microdilution MICs (3, 8, 28). There are no published studies evaluating the Resistance Plus MP assay for detection of macrolide resistance directly in clinical specimens. To our knowledge, there are no other alternatives under commercial development in the United States for rapid detection of macrolide-resistant M. pneumoniae at this time.
In the present study, we have sought to examine the comparative diagnostic performance of the following three FDA-cleared, multianalyte molecularly based platforms for detection of M. pneumoniae in clinical specimens: the FilmArray respiratory panel (RP) (bioMérieux/BioFire Diagnostics, Inc., Salt Lake City, UT), ePlex respiratory pathogen panel (RPP) (GenMark Diagnostics, Inc., Carlsbad, CA), and the NxTAG RPP (Luminex Corporation, Austin, TX). We also evaluated the single-analyte FDA-cleared Alethia Mycoplasma Direct (formerly illumigene Mycoplasma Direct; Meridian Bioscience, Inc., Cincinnati, OH) LAMP assay and the InGenius MGB Mycoplasma pneumoniae research use only (RUO) PCR (ELITechGroup, Inc., Bothell, WA). The SpeeDx Resistance Plus MP assay was evaluated for its ability to codetect M. pneumoniae and mutations associated with macrolide resistance. Data obtained from each commercial method were compared against results obtained by reference methods consisting of validated laboratory-developed PCR assays at the UAB Diagnostic Mycoplasma Laboratory and the CDC and by Sanger sequencing.
MATERIALS AND METHODS
Study design and specimen collection.
Investigators at 8 hospitals around the United States (Children’s Healthcare of Atlanta, Emory University, Atlanta, GA; Children’s Hospital Los Angeles, Los Angeles, CA; Children’s Mercy Medical Center, Kansas City, MO; Hackensack University Medical Center, Hackensack, NJ; Ann and Robert H. Lurie Children’s Hospital Chicago, Chicago, IL; Memorial Sloan Kettering Cancer Center, New York, NY; Seattle Children’s Hospital, Seattle, WA; and University of Texas Health Science Center, San Antonio, TX) froze remnant nasal swab respiratory specimens that were collected in universal transport medium between 2015 and 2019 for testing on the FilmArray RP. Specimens were maintained frozen at −80°C and shipped on dry ice to the UAB Diagnostic Mycoplasma Laboratory. Samples of <1.5 ml had insufficient volume to perform all tests and were excluded. A subset of specimens with sufficient volume from our recent national macrolide resistance surveillance study was also evaluated (7, 29). Extracted DNA from a single specimen obtained at Children’s of Alabama (Birmingham, AL) was also included for determination of macrolide resistance. Since all but one of these hospitals is a pediatric facility, most specimens were obtained from children with respiratory disease of sufficient severity to warrant an attempt to obtain a microbiological diagnosis. Patient demographic information was not a component of this study.
Specimen processing.
Following arrival at UAB, specimens were maintained frozen at −80°C until the day of processing. Nucleic acids were extracted from 400 μl of each sample utilizing the MagNA Pure LC large-volume DNA isolation kit on the MagNA Pure 2.0 instrument (Roche Diagnostics, Indianapolis, IN). DNA was eluted into 100 μl Tris-EDTA (TE) buffer and stored at −20°C until evaluation by the ELITe InGenius MGB Mycoplasma pneumoniae RUO commercial assay and two well-established reference laboratory-developed test methods, the UAB repMp1 quantitative PCR (qPCR) (28, 30) and the CDC multiplex Mp181 qPCR (31). Likewise, the ability of the SpeeDx Resistance Plus MP commercial assay to detect point mutations conferring macrolide resistance was evaluated against that of two well-established reference laboratory developed test methods, the UAB MRMp multiplex qPCR (8) and the CDC light upon extension (LUX) method (9), with adjudication by Sanger sequencing.
For the Luminex NxTAG RPP, 200 μl of original patient sample was spiked with internal control phage per the manufacturer’s specifications. Nucleic acids were extracted from spiked samples utilizing the Roche MagNA Pure 2.0 (Roche Diagnostics, Indianapolis, IN) and Luminex NxTAG kit specifications. The final elution volume of 100 μl was stored at −20°C until analysis on the Luminex MagPix instrument.
During the initial thaw, samples were aliquoted into multiple cryovials and maintained frozen until analysis by the Alethia Mycoplasma Direct and ePlex platforms. An additional aliquot was inoculated into SP4 culture medium to isolate and further categorize viable M. pneumoniae cells from each sample.
Culture and MIC determination.
Approximately 400 μl of each original specimen was subjected to culture for M. pneumoniae as previously described (32). Species-level identification of isolates was confirmed by repMp1 qPCR (30). The Clinical and Laboratory Standards Institute M43A broth microdilution method was used to determine susceptibility to the macrolide erythromycin. Erythromycin MICs of ≥1 μg/ml were designated resistant (33).
Detection of M. pneumoniae and macrolide resistance by laboratory-developed PCR assays at UAB and CDC.
The UAB repMp1 qPCR originally described by Dumke et al. (30), later adapted for use at UAB (28), and the UAB MRMp qPCR (8) were performed using isolated DNA extracted from original patient specimens as described above. The UAB repMp1 qPCR amplifies an 89-bp product within the 14 noncoding repetitive elements present in multiple copies in the genome and can detect 4.5 × 10−4 CFU/ml in PCR mixture, or 9.0 × 10−3 CFU/test (34). In its initial validation on the LightCycler 480 instrument, this PCR assay amplified 36 M. pneumoniae reference strains but did not amplify 103 species of other mycoplasmas, viruses, and bacteria common to the respiratory tract. The repMp1 PCR assay also demonstrated 100% sensitivity and 97.7% specificity compared to culture for detection of M. pneumoniae in throat swabs from 180 children with respiratory disease in a previous intralaboratory evaluation. The UAB MRMp PCR is a multiplex assay utilizing fluorescence resonance energy transfer (FRET) hybridization probes and high-resolution melt curve analysis on the Roche LightCycler platform to detect point mutations in domain V of the 23S rRNA gene conferring macrolide resistance, with a limit of detection (LoD) of 7 mutant molecules/μl in PCR mixture or 140 genome copies/test (8).
Extracted DNA from each specimen was shipped to the CDC and tested at the Pneumonia Response and Surveillance Laboratory using a multiplex real-time PCR assay that detects M. pneumoniae, Chlamydia pneumoniae, Legionella species, and human nucleic acids. The M. pneumoniae gene target is a 73-bp sequence of the gene encoding community-acquired respiratory distress syndrome (CARDS) toxin, and the assay’s analytical sensitivity has been reported as 1 to 5 CFU per test (35). This PCR assay was performed as previously described (31) but with replacement of oligonucleotides for improved detection of Legionella species (36). High-resolution melt (HRM) analysis for identification of the macrolide resistance genetic profile was performed with a slight modification of the original procedure for the LUX technique (9); the modification consisted of using a nested PCR approach to allow standardization of input material into the HRM reactions. The HRM profiles for clinical specimens were compared to those of the macrolide-resistant and macrolide-susceptible reference strains that were included in each run; the reference strains had been previously verified by sequencing and determination of MICs. To confirm macrolide resistance as detected by PCR and phenotypic MICs, the specific 23S rRNA point mutations known to be associated with macrolide resistance (3) were identified by Sanger sequencing of real-time PCR amplicons obtained in the UAB MRMp PCR assay.
Detection of M. pneumoniae using commercial molecularly based assays.
(i) BioFire FilmArray respiratory panel. Table 1 provides a description of the main distinguishing features of these six commercial assays. The FilmArray RP assay is an automated system that performs nucleic acid extraction, reverse transcription, DNA amplification, nested PCR, and melt curve analyses on 300 μl of original specimen to simultaneously detect 20 pathogens, including M. pneumoniae, within 60 min (37). The assay targets the M. pneumoniae CARDS toxin gene (MPN_372) with an LoD of 30 CFU/ml, which is equivalent to 3 CFU/test (37). Participating hospitals performed the initial FilmArray RP testing in accordance with manufacturer’s instructions using approximately 300 μl of original specimen. FilmArray positive and negative samples were shipped to UAB for evaluation on other commercial test platforms and by reference PCR methods.
TABLE 1
| Assay | Manufacturer | No. targets | FDA cleared | Mp gene target | Inline nucleic acid extraction | Sample throughputa | Vol required | Limit of detectione | Run time (h) | |
|---|---|---|---|---|---|---|---|---|---|---|
| Concn | Amt (CFU/test) | |||||||||
| FilmArray RP | Biofire | 20 | Yes | CARDS Txn | Yes | 12 per tower | ∼200 μl sample | 30 CFU/ml | 3b | 1 |
| ePlex RPP | GenMark | 17 | Yes | P1 adhesin | Yes | 24 per 4-tower configuration | ∼300 μl sample | 300 CCU/ml (300–3,000 CFU/mlc) | NA | 1.5 |
| NxTAG RPP | Luminex | 20 | Yes | P1 adhesin | No | 96-well plate | 210 μl sample; 35 μl DNA | 142 CCU/ml (142–1,420 CFU/mlc) | NA | 4 |
| InGenius MP RUO | ELITech | 1 | No | repMp4 | Available but not used in this study | 12 per run | 200 μl sample; >15 μl DNA | 8 CFU/ml | 0.16 | 2 |
| Mycoplasma Direct | Meridian | 1 | Yes | Protease-like protein | Yes | 10 per run | 25–50 μl sample | 200–2,350 CFU/ml | 7.5–88 | 1 |
| Resistance Plus MP | SpeeDx | 2 (Mp detection; macrolide resistance) | No | CARDS toxin, 23S rRNA | No | 96-well plate | >5 μl DNA | NA | NA | 2 |
a
Sample throughput is dependent on the unique instrument configurations incorporated into a lab workflow (for example, 1 ePlex tower enables simultaneous processing of 6 samples, whereas the 4-tower configuration enables processing of 24).
b
Calculated equivalent number.
c
d
Mp, Mycoplasma pneumoniae.
e
NA, not available.
(ii) Luminex NxTAG respiratory pathogen panel. The NxTAG RPP assay simultaneously detects 20 viral and bacterial pathogens, including M. pneumoniae, by utilizing multiplex PCR amplification, bead hybridization, and fluorescence imaging with the Luminex MagPix instrument. Unlike the FilmArray, this assay utilizes a 96-well plate format, enabling evaluation of 1 to 96 samples within 4 h. Extracted nucleic acids (35 μl) are added to rehydrate lyophilized reagents enabling multiplexed PCR and bead hybridization utilizing the Veriti thermocycler (Applied Biosystems/Thermo Fisher Scientific). After thermocycling, assay vessels were transferred to the MagPix instrument for fluorescence imaging, data acquisition, and analysis in accordance with the manufacturer’s specifications. The NxTAG RPP targets M. pneumoniae P1 adhesin with a LoD of 142 color change units (CCU)/ml (https://www.accessdata.fda.gov/cdrh_docs/reviews/K152386.pdf), corresponding to ∼142 to 1,420 CFU/ml (38).
(iii) GenMark ePlex respiratory pathogen panel. The ePlex RPP is a cartridge-based assay that utilizes electrowetting microfluidic technology to perform multiplexed nucleic acid amplification and probe hybridization, enabling simultaneous detection of 17 viral and bacterial pathogens, including M. pneumoniae via electrical impedance within 1.5 h. It targets the M. pneumoniae P1 adhesin with an LoD of 300 CCU/ml (https://www.accessdata.fda.gov/cdrh_docs/reviews/K163636.pdf), corresponding to ∼300 to 3,000 CFU/ml (38). Specimen aliquots (200 μl) were vortexed, added to the sample delivery tube, vortexed a second time, and inverted prior to application of 350 μl into the RPP cartridge. The cartridge was then sealed, inserted into the ePlex instrument, and processed.
(iv) Alethia (illumigene) Mycoplasma Direct. The Mycoplasma Direct assay is an automated sample-to-result single-analyte loop-mediated isothermal amplification (LAMP) system with a Clinical Laboratory Improvement Amendment moderate complexity classification that targets a 208-bp DNA sequence found in the intracellular protease-like protein gene with a reported LoD of 200 to 2,350 CFU/ml or 7.5 to 88 CFU/test (26). Although the manufacturer’s specifications suggest utilization of a primary patient throat swab in nonnutritive transport medium with a pledget (liquid Amies without charcoal or liquid Stuart; maximum volume, 1.2 ml), this study evaluated nasal swab specimens collected in universal transport medium (>3 ml). To optimize compatibility, we aliquoted 25 μl of primary specimen into the SMP Prep tubes provided with the kit. As this input volume is within the range of volume released from swabs used in clinical diagnostic work (∼10 to 60 μl), this alteration to the procedure was considered acceptable (39). SMP Prep tubes were then vortexed for 10 s, and ∼150 μl of sample-buffer mixture was added to the heat inactivation tubes. Tubes were sealed and incubated on a dry heat block at 90 to 100°C for 10 min, then vortexed for another 10 s. Subsequently, 50 μl heat-inactivated sample was added to the test, and control chambers and the test device was sealed and inserted into the Alethia device/reader in accordance with manufacturer’s specifications. Results were displayed as positive or negative at the conclusion of the run (∼40 min.)
(v) ELITe InGenius Mycoplasma pneumoniae MGB RUO. The ELITech InGenius is a fully automated sample-to-result PCR system that integrates nucleic acid extraction, real-time PCR amplification, and reporting directly from primary patient samples in either single-analyte or multiplex formats. Although it is not FDA-cleared, the InGenius MP RUO assay targeting the noncoding DNA sequence repMp4 has been previously validated in the UAB Diagnostic Mycoplasma Laboratory and was shown to be sensitive and specific for M. pneumoniae in a variety of respiratory specimens with an analytic sensitivity of 8 CFU/ml or 0.16 CFU/test (34). Extracted nucleic acid (15 μl) was analyzed utilizing the instrument’s qPCR-only option in accordance with manufacturer’s specifications and as previously described; results were available within 3 h (setup to analysis) (34).
(vi) SpeeDx Resistance Plus MP. The Resistance Plus MP is an investigational non-FDA cleared reverse transcription-PCR (RT-PCR) commercial kit for the detection of M. pneumoniae and macrolide resistance mutations. This multiplex kit utilizes three independent fluorescent channels for detection of (i) the CARDS toxin, (ii) probes specific for the most commonly reported point mutations in the 23S rRNA gene conferring macrolide resistance, and (3) an internal control. In accordance with the manufacturer’s specifications, color compensation and additional optimizations were performed on a LightCycler 480 instrument. Once assay conditions were optimized, 5 μl of extracted nucleic acids was added to 15 μl of a proprietary master mix. Assay plates were then sealed and transferred to the LightCycler to perform real-time PCR. Sample analyses were performed per the manufacturer’s specifications.
Statistical analysis.
All specimens previously designated M. pneumoniae positive or negative by the FilmArray assay were tested by the UAB and CDC PCRs to establish a confirmed true-positive (TP) baseline, defined as any specimen testing positive by both PCRs. Any specimen that yielded discrepant results with these two PCRs was retested by both assays. If results were still discrepant, the specimen was removed from the analysis. A confirmed true negative (TN) was defined as any specimen that tested negative by both reference PCRs. A false positive (FP) was defined as any specimen that tested positive by a commercial assay that was negative by both reference PCRs. A false negative (FN) was defined as any specimen that tested negative by a commercial test that was positive by both reference tests. When any specimen gave an indeterminate result for M. pneumoniae detection by a commercial assay, the specimen was not included in performance calculations, given insufficient volume for repeat testing.
Specimens deemed to harbor macrolide-resistant M. pneumoniae either grew an isolate that tested resistant to erythromycin (MIC, ≥1 μg/ml) or tested positive for macrolide resistance by both the UAB and CDC PCRs. Specimens testing positive by one but not both PCRs were adjudicated by Sanger sequencing of the 23S rRNA gene. Specimens deemed to harbor macrolide-susceptible M. pneumoniae tested negative by both reference PCRs, with isolates (if available) testing susceptible to erythromycin (MIC, <1 μg/ml.) The clinical sensitivity and clinical specificity of each assay were calculated using standard methods for proportions and exact binomial 95% confidence intervals. McNemar’s test was used to determine statistically significant differences between the commercial assays and the reference PCRs, with a P value of ≤ 0.05 being designated significant. All statistical analysis used the SAS software program, version 9.4 (SAS Institute, Inc., Cary, NC). Our sample size of 428, which included 212 M. pneumoniae-positive and 216 M. pneumoniae-negative specimens, allowed for precise estimates of sensitivity and specificity for the various commercial assays.
Ethics.
This study was performed with the approval of the UAB Institutional Review Board for Human Use.
RESULTS
Specimen summary.
A total of 434 nasopharyngeal (NP) swab specimens were received from 8 institutions throughout the United States. Two of these specimens grew M. pneumoniae in culture but tested negative by all molecular tests, indicating either culture contamination or low organism burden with only a subset of sample aliquots harboring bacteria. In addition, consensus between the UAB and CDC reference PCRs could not be obtained for 4 specimens. Exclusion of these six samples from this study yielded a total of 428 specimens, which included 212 TPs and 216 TNs. Among the 212 TPs, there were 177 (83.5%) that were also culture positive for M. pneumoniae.
Detection of M. pneumoniae by commercial molecularly based assays.
Table 2 summarizes the test performance characteristics of the six commercial assays in comparison to the UAB and CDC reference methods. There were no statistically significant differences in clinical specificity between commercial and reference test methods ranging from 99.5% to 100%. There were only two FNs in the entire study, one each with the InGenius and Resistance Plus MP. Likewise, there was no statistically significant difference in clinical sensitivity between the FilmArray RP (98.1%) and InGenius MP RUO (99.5%) and reference test methods. In contrast, statistically significantly differences from reference methods were noted for the Resistance Plus MP (93.3%), ePlex RPP (83.6%), NxTAG RPP (64.6%), and the Mycoplasma Direct (55.7%) assays. The following invalid results were obtained for each platform: ePlex RPP (34; 7.9%), Resistance Plus MP (9; 2%), NxTAG RPP (7; 1.6%), InGenius (2; 0.5%), and Mycoplasma Direct (1; 0.2%). Invalid result rates for the FilmArray RP are not available, as these tests were performed on fresh rather than frozen samples and attainment of valid FilmArray RP test results was required for specimen inclusion in this study.
TABLE 2
| Assay | Total no. of specimens | No. of positives/no. of true positives | Sensitivity (%) | 95% confidence range (%) | No. of negatives/no. of true negatives | Specificity (%) | 95% confidence range | P valueb |
|---|---|---|---|---|---|---|---|---|
| FilmArray RP | 428 | 208/212 | 98.1 | 96.3–99.9 | 216/216 | 100 | 98.3–100 | 0.13 |
| ePlex RPP | 394c | 163/195 | 83.6 | 77.6–88.5 | 199/199 | 100 | 98.2–100 | <0.0001 |
| NxTAG RPP | 421d | 135/209 | 64.6 | 57.7–71.1 | 212/212 | 100 | 98.3–100 | <0.0001 |
| InGenius MP RUO | 426e | 209/210 | 99.5 | 97.4–100 | 215/216 | 99.5 | 97.5–100 | 1.0 |
| Mycoplasma Direct | 427f | 118/212 | 55.7 | 48.7–62.5 | 215/215f | 100 | 98.3–100 | <0.0001 |
| Resistance Plus MP | 419g | 195/209 | 93.3 | 89.0–96.3 | 209/210 | 99.5 | 97.0–100 | 0.001 |
a
Laboratory-validated PCR assays at UAB and CDC were used as the reference standards. A true positive was defined as one for which both reference PCRs were positive. A true negative was defined as one for which both tests were negative.
b
Determined by McNemar’s test.
c
There were 34 invalid tests on the GenMark ePlex.
d
There were 7 invalid tests on the Luminex NxTAG.
e
There were 2 invalid tests on the ELITech ELITe InGenius.
f
There was 1 invalid test on the Alethia Mycoplasma Direct.
g
There were 9 invalid tests on the SpeeDx Resistance Plus MP. These data refer only to the detection of M. pneumoniae and not to the designation of macrolide resistance.
The LoDs for each commercial assay varied widely (Table 1) and may explain statistically significant decreases in clinical sensitivity observed for the Resistance Plus MP (93.3%), ePlex RPP (83.6%), NxTAG RPP (64.6%), and Mycoplasma Direct (55.7%) assays. Consistent with this hypothesis, Fig. 1 shows statistically significant differences in repMp1 threshold cycle (CT) values for concordant positives (lower CT values; higher organism load) and false negatives (higher CT values; lower organism load) for each commercial assay listed above.
FIG 1
Detection of macrolide-resistant M. pneumoniae by Resistance Plus MP.
The UAB MRMp PCR designated 21/212 M. pneumoniae-positive specimens in the original data set as macrolide resistant (9.9%). The addition of 12 more specimens from our previous surveillance study (7) brought the total number to 33. Twenty-one out of 33 specimens designated macrolide-resistant by the UAB MRMp PCR were culture positive and had erythromycin MICs determined, all of which were >32 μg/ml. There were 4 specimens reported by the CDC macrolide resistance PCR as indeterminant, 2 of which were culture-positive and had erythromycin MICs of >32 μg/ml. Macrolide resistance mutations were detected in all 4 of these specimens by Sanger sequencing. One other specimen was designated wild type (WT) by the CDC PCR, but it also had an erythromycin MIC of >32 μg/ml and had the A2063G mutation. Sanger sequencing determined that the A2063G mutation was most common, occurring in 24 specimens; A2064G was found in 6 specimens, with the remaining 3 specimens having mixtures of A2063T/G, A2063G/WT, and A2064G/WT. The Resistance Plus MP detected 27/33 macrolide-resistant M. pneumoniae isolates, resulting in a sensitivity of 81.8% (95% confidence interval [CI] = 64.5 to 93.0%). Among these 33 proven macrolide-resistant specimens, 1 tested negative for M. pneumoniae and 5 designated the specimen as WT. Among the 5 misclassified mutations, 2 were A2063G, 2 were A2064G, and 1 was A2063G/WT. The specificity was 185/186 (99.45%; 95% CI = 97.0 to 100%) with 1 FP. As noted above, the Resistance Plus MP performed better for M. pneumoniae detection overall, identifying 93.3% of the true-positive specimens.
DISCUSSION
Although other studies have evaluated the test performance characteristics of molecular diagnostic assays targeting M. pneumoniae, these assays are not commercially available in the United States (40–42). Despite FDA approval of several diagnostic tests targeting M. pneumoniae that are included in this evaluation, there have been minimal head-to-head comparisons with limited sample sizes to draw definitive conclusions on the relative utility of each assay. This study is the first to recruit and evaluate a large number of samples from 8 sites throughout the United States to compare the performance of six commercial assays, including FDA-approved diagnostics targeting M. pneumoniae. Detailed descriptions of the multianalyte FilmArray RP, ePlex RPP, and NxTAG RPP, including basic information regarding function, throughput, turnaround times, and overall performance data, with particular emphasis on detection of respiratory viruses, have been discussed in depth by Ramanan et al. (43). Therefore, our discussion of these assays, as well as of the Alethia Mycoplasma Direct, ELITe InGenius MP RUO, and Resistance Plus MP assays, is limited primarily to their performance for the detection of M. pneumoniae and macrolide resistance.
The FilmArray RP was the first multiplex PCR FDA approved for the detection of bacterial and viral respiratory pathogens (17, 18, 44). We found the FilmArray RP compared extremely well with the reference PCRs, with 98.1% sensitivity and 100% specificity. This is consistent with other studies showing high concordance for many pathogens on the panel relative to laboratory-developed PCRs (23, 25). We also found that the FilmArray RP performed significantly better than the ePlex RPP and NxTAG RPP multiplex panels. Published head-to-head comparisons of multiplex panels show significant variability in detection of M. pneumoniae, with some publications indicating that the ePlex is more sensitive than the FilmArray RP (15), that the NxTAG RPP exhibits similar sensitivity to the FilmArray RP (16, 18), and that the NxTAG RPP is not as sensitive as the FilmArray RP (17, 21) or a laboratory-developed PCR assay (24). A significant limitation of all of these publications is the small number of M. pneumoniae-positive specimens that were available (typically <10). Our study, using a much larger sample size of 212 M. pneumoniae-positive specimens distributed throughout the United States, shows a clear difference with respect to the performance of the three multiplex panels. Similar test performance evaluations of the most recent FDA-approved molecular diagnostic test, the QIAstat-Dx respiratory panel (Qiagen, Germantown, MD) are needed.
As Mycoplasma pneumoniae is only one of several targets in multiplex assays, additional consideration of reported sensitivities to other pathogens, organism coverage, workflow, costs, and additional tests available on the platform should be taken into consideration when exploring diagnostic test options. As M. pneumoniae disproportionately affects children and adolescents, laboratory directors of pediatric hospitals should focus particular attention on platform differences in test performance characteristics for organisms of interest to their patient population. In addition, test manufacturers should note opportunities for optimization of assay conditions and actively promote large multicenter evaluations of individual targets included on syndromic panels.
Syndromic panels targeting many respiratory pathogens simplify testing algorithms and improve the speed of diagnosis with minimal hands-on time for laboratory technologists (43). However, by virtue of targeting multiple pathogens, the LoD and analytical sensitivity for individual targets are often reduced relative to those of singleplex assays. Additionally, multiplex panels exhibit high instrument costs and high costs per test, and they lack flexibility for customization. Although singleplex assays such as the Mycoplasma Direct LAMP only target one pathogen, instrument costs and costs per test are considerably lower, and assay optimization for the single target often yields enhanced analytical sensitivity for this target relative to that of multiplex tests.
Although, the InGenius MGB M. pneumoniae RUO PCR assay is not yet FDA approved, we chose to incorporate this assay into this study due to its commercial availability and a recent publication noting impressive test performance characteristics (34). Additionally, the ability of the InGenius platform to perform 12 independent and customizable tests per run offers clinical labs, particularly moderately sized complex clinical laboratories running many different molecular tests, an alternative solution for M. pneumoniae testing. This singleplex assay exhibited the highest clinical sensitivity (99.5%), outperforming all other tests. Although the ELITech InGenius platform can perform nucleic extraction directly from patient samples, our in-house studies showed equivalent nucleic acid extraction efficiency with the Roche MagNA Pure platform (data not shown). Therefore, we chose to run the InGenius MP RUO assay with extracted nucleic acids (not direct samples) as the input enabling thorough evaluation of the RT-PCR component of this test system. Future studies with large sample sizes are recommended to evaluate the relative performance of this assay when starting with direct specimens.
Unexpectedly, the Mycoplasma Direct singleplex assay detected only 55.7% of confirmed M. pneumoniae-positive specimens, yielding the poorest performance of all six commercial tests. In our previous evaluation of the illumigene Mycoplasma DNA amplification assay, the forerunner of Mycoplasma Direct (26), among 214 respiratory specimens from a variety of sources previously cultured for M. pneumoniae (indicative of a higher organism load), the sensitivity of the illumigene assay was 100% and the specificity was 99%. Chou and Zheng identified the sensitivity and specificity of the illumigene assay to be 100% compared with those of the FilmArray RP and also reported a greater analytic sensitivity for the illumigene (23.5 CFU/ml versus 235 CFU/ml for the FilmArray RP) (22). The Mycoplasma Direct assay does not require a separate DNA extraction step, making it similar in this respect to the multiplex systems. However, the gene target and assay LAMP chemistry are identical for both assays, and prior evaluations showed comparable test performances (10). To our knowledge, there have been no other studies performed with the new version of the Mycoplasma Direct comparing it to other methods since it was reformatted with an integrated DNA extraction procedure. The poor performance of the Mycoplasma Direct assay in this study may be attributable to modification of manufacturer-recommended specimen collection protocols. Although the Mycoplasma Direct protocol specifies collection of throat swabs in nonnutritive transport medium with a pledget (<1.2 ml), samples in this study were nasal swabs collected in universal transport medium (∼3 ml) as specified for BioFire testing. Given the lack of convincing evidence that throat swabs are superior to nasal swabs for detection of M. pneumoniae (45), we aliquoted 25 μl of specimen into SMP Prep tubes and processed the samples per the manufacturers’ instructions. Future studies strictly following manufacturer protocols are required to determine the true test performance characteristics of the Mycoplasma Direct assay.
The optimal approach to assess the clinical accuracy of various commercial molecularly based tests for detection of M. pneumoniae would be to prospectively collect specimens and run them on all of the platforms before any storage, freezing, or other manipulation took place. The challenge for performing such a study is that the low frequency for circulating M. pneumoniae during periods when it is not epidemic would mean that very large numbers of specimens would have to be collected for meaningful comparison, making such an endeavor very costly and time-consuming. Among 1,612 respiratory specimens analyzed by the FilmArray RP2, Leber et al. (11) found M. pneumoniae in only 23 specimens (1.4%). Moreover, no single hospital or laboratory has a need to have all of these instruments and assays in operation on-site for simultaneous testing. Future studies initiated during epidemic years may enable prospective evaluation of test performance characteristics lacking the caveats highlighted in the current study.
Given our study design, it is worthwhile to mention some potential factors that might have affected the results of this study. All specimens had been collected in universal transport medium for testing on the FilmArray RP and frozen prior to shipment to UAB. There was no attempt to determine whether the same type of transport system was used by all hospitals over the 4-year period of specimen accrual, and there was no way to know if there were variations in storage temperatures or length of storage prior to freezing. Additionally, there is a possibility that some organism loss may have occurred after freezing and thawing, which could have affected performance of the commercial assays other than the FilmArray RP performed on-site. However, the FilmArray RP concordantly detected M. pneumoniae in 208/212 (98.1%) confirmed-positive specimens, while all 216 remaining specimens were concordantly negative, resulting in an overall 99.1% agreement for the FilmArray RP and the reference PCRs. Since there were no instances in which the FilmArray RP was positive when the reference PCRs were negative, it is unlikely there was any significant loss of viable DNA as a result of sample handling, storage, freezing, and shipping.
Unlike in a clinical laboratory setting, limited sample volumes prevented repeat testing when a commercial diagnostic test gave an invalid result. In this study, high invalid result rates (34/428; 7.9%) were only problematic for the ePlex RPP. Although high relative to those of other test systems, ePlex RPP invalid rates for the study were within the range (2.2 to 8.4%; mean, 5.6%) typical for regular clinical diagnostic use at the Children’s of Alabama Pediatric Virology Laboratory. It is therefore highly unlikely that storage/handling affected the ePlex RPP invalid result rate. Although it is not possible to know how exclusion of 34 results affected our evaluation of test performance characteristics, an equal distribution of samples (17 positive and 17 negative) yielding invalid results suggests a minimal effect on test performance parameters had repeat testing been available.
The rate of macrolide-resistant isolates (21/212; 9.9%) and the predominance of A2063G point mutations in the current study is consistent with recently published prevalence rates in the United States (7). Implementation of a commercial molecular test capable of detecting macrolide resistance in clinical laboratories would enable the identification of infected patients whose treatment would benefit from an alternative antibiotic choice and help mitigate rising macrolide resistance rates (7). Although the Resistance Plus MP assay is not yet available in the United States, it is the only commercial assay targeting macrolide-resistant M. pneumoniae and was included in this study to evaluate its potential utility to meet this increasingly important diagnostic niche. The clinical sensitivity of the Resistance Plus MP to detect M. pneumoniae in clinical specimens was generally good, at 93.3%, compared to the reference PCRs. This sensitivity was not significantly different from that of the FilmArray (98.1%) but did differ from that of the ELITe InGenius (99.5%), since the 95% confidence intervals did not overlap, as shown in Table 2. The Resistance Plus MP accurately detected 27/33 (81.8%) of specimens confirmed as macrolide resistant by other methods. The Resistance Plus MP assay had different sensitivities for detection of different mutations. One-third of A2064G mutations (2/6) were missed. This PCR is still under development by SpeeDx, and attention needs to be given to the PCR design and the nature of the reagents in the PCR mixture, as well as the PCR program, in order to improve diagnostic accuracy. Future studies are needed to evaluate and identify optimized assays capable of accurately distinguishing patients infected with macrolide-resistant M. pneumoniae.
In summary, we have performed an evaluation of six commercial molecularly based methods for the detection of M. pneumoniae in clinical specimens using two laboratory developed PCRs as reference methods. Among the three multiplex PCRs, the FilmArray RP had the best overall performance and detected significantly more confirmed positive specimens than the ePlex RPP and NxTAG RPP. Although not yet FDA cleared, the single-analyte InGenius MP RUO assay had the highest sensitivity of all tests evaluated. The single-analyte Mycoplasma Direct is the closest to a point-of-care test for M. pneumoniae, but in this evaluation it detected only slightly more than half of the positive specimens. Despite an acceptable performance for the Resistance Plus MP in detection of M. pneumoniae, additional work is necessary to develop commercial assays capable of accurately identifying macrolide-resistant M. pneumoniae.
Our study is the first to address commercial systems and their differences in detection of M. pneumoniae, as well as documenting macrolide resistance in comparison with reference testing by two different laboratory-developed PCRs. These data may aid in selection of diagnostic testing for syndromic management of M. pneumoniae infections. Our laboratory at the University of Alabama at Birmingham currently performs PCR for detection of M. pneumoniae as a single analyte. Because of concerns for macrolide resistance and its frequent occurrence in this area (7), we perform our laboratory-developed PCR for detection of macrolide resistance on all specimens that test positive for the organism and report the results of both tests at the same time.
ACKNOWLEDGMENTS
Financial support was provided by U.S. Centers for Disease Control and Prevention contract 75D30119C02902 to K. B. Waites.
Melanie Fecanin provided technical assistance with M. pneumoniae culture and MIC testing. Jennifer Potter provided technical assistance in performance of GenMark PCRs. The support of the administrative staff at the CDC is gratefully acknowledged for their assistance in financial administration for this project, as well as for guidance in study design.
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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Information & Contributors
Information
Published In
Journal of Clinical Microbiology
Volume 58 • Number 6 • 26 May 2020
eLocator: 10.1128/jcm.00242-20
Editor: Geoffrey A. Land, Carter BloodCare & Baylor University Medical Center
PubMed: 32269102
Copyright
© 2020 American Society for Microbiology. All Rights Reserved.
History
Received: 7 February 2020
Returned for modification: 7 March 2020
Accepted: 1 April 2020
Published online: 26 May 2020
Keywords
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Editor
Geoffrey A. Land
Editor
Carter BloodCare & Baylor University Medical Center
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2020.
Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae. J Clin Microbiol
58:10.1128/jcm.00242-20.
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