We evaluated the GenoType NTM-DR (NTM-DR) line probe assay for identifying Mycobacterium avium complex (MAC) species and Mycobacterium abscessus subspecies and for determining clarithromycin and amikacin resistance. Thirty-eight reference strains and 145 clinical isolates (58 MAC and 87 M. abscessus isolates), including 54 clarithromycin- and/or amikacin-resistant strains, were involved.
KEYWORDS: GenoType NTM-DR, nontuberculous mycobacteria, amikacin, antibacterial susceptibility, clarithromycin, identification, mutation
ABSTRACT
We evaluated the GenoType NTM-DR (NTM-DR) line probe assay for identifying Mycobacterium avium complex (MAC) species and Mycobacterium abscessus subspecies and for determining clarithromycin and amikacin resistance. Thirty-eight reference strains and 145 clinical isolates (58 MAC and 87 M. abscessus isolates), including 54 clarithromycin- and/or amikacin-resistant strains, were involved. The performance of the NTM-DR assay in rapid identification was evaluated by comparison with results of multigene sequence-based typing, whereas performance in rapid detection of clarithromycin and amikacin resistance was evaluated by comparison with sequencing of the erm(41), rrl, and rrs genes and drug susceptibility testing (DST). The accuracies of MAC and M. abscessus (sub)species identification were 92.1% (35/38) and 100% (145/145) for the 38 reference strains and 145 clinical isolates, respectively. Three MAC strains other than M. intracellulare were found to cross-react with the M. intracellulare probe in the assay. Regarding clarithromycin resistance, NTM-DR detected rrl mutations in 52 isolates and yielded 99.3% (144/145) and 98.6% (143/145) concordant results with sequencing and DST, respectively. NTM-DR sensitivity and specificity in the detection of clarithromycin resistance were 96.3% (52/54) and 100% (91/91), respectively. The NTM-DR yielded accurate erm(41) genotype results for all 87 M. abscessus isolates. Regarding amikacin resistance, NTM-DR detected rrs mutations in five isolates and yielded 99.3% (144/145) and 97.9% (142/145) concordant results with sequencing and DST, respectively. Our results indicate that the NTM-DR assay is a straightforward and accurate approach for discriminating MAC and M. abscessus (sub)species and for detecting clarithromycin and amikacin resistance mutations and that it is a useful tool in the clinical setting.
INTRODUCTION
The incidence of infection with nontuberculous mycobacteria (NTM) in humans is steadily increasing although the clinical significance and treatment outcomes of NTM infections differ according to etiologic organism and resistance profile (1–3). Thus, differential and accurate identification of NTM species, together with drug susceptibility testing (DST), is of great interest in the clinical setting. Specifically, precise Mycobacterium avium complex (MAC) species identification and Mycobacterium abscessus subspecies identification in clinical isolates are becoming increasingly important due to different treatment outcomes and epidemiological implications (4–7). However, identification of these organisms is currently limited to specialized laboratories (8). Furthermore, the gold standard method for NTM DST is broth microdilution (BMD), which is a time-consuming method, especially with slow-growing mycobacteria (9).
GenoType NTM-DR (NTM-DR; Hain Lifescience, Nehren, Germany) is a line probe assay (LPA) that enables species- or subspecies-level identification of the major clinically encountered NTM, including MAC species (M. avium, M. intracellulare, and M. chimera), M. chelonae, and subspecies belonging to M. abscessus, i.e., M. abscessus subspecies abscessus, M. abscessus subspecies massiliense, and M. abscessus subspecies bolletii (10–12). The NTM-DR assay also allows for detection of antibiotic resistance to macrolides and aminoglycosides. Specifically, macrolide resistance is identified by polymorphisms (T28 or C28) at position 28 in the erm(41) gene and mutations at positions 2058/2059 in the rrl gene (11). Likewise, aminoglycoside resistance is detected by examining positions 1406 to 1408 of the rrs gene (10, 11). The mutation probes for the NTM-DR assay were designed to hybridize to alleles containing specific mutations: four mutations in the rrl gene, including A2058C (MUT1 probe), A2058G (MUT2), A2059C (MUT3), and A2059G (MUT4), and one mutation in the rrs gene (A1408G).
So far, studies on the performance of NTM-DR (10–12), especially ones examining a large number of isolates and antibiotic-resistant clinical strains, have been limited. The aim of this study was to evaluate the analytical and clinical performance of the NTM-DR assay for identifying (sub)species of MAC and M. abscessus and determining their resistance to macrolides and aminoglycosides. We evaluated the performance of the assay by comparison of the results to those with multigene sequence-based identification, BMD for clarithromycin (CLR) and amikacin (AMK), and sequencing of the erm(41), rrl, and rrs genes.
MATERIALS AND METHODS
NTM isolates.
This study was carried out at a tertiary care hospital in Seoul, South Korea, and was approved by the Institutional Review Board of Samsung Medical Center. The study involved 145 clinical NTM isolates and a total of 38 reference strains, including 33 mycobacterial strains and 5 strains from the Gordonia, Nocardia, Rhodococcus, and Tsukamurella genera. The clinical NTM isolates consisted of 58 MAC and 87 M. abscessus isolates that were used in our previous studies and subcultured from frozen pure cultures stored at −70°C (13–16). All clinical isolates were obtained from patients who fulfilled the diagnostic criteria of NTM pulmonary disease (1).
Multigene sequencing for identification and molecular detection of antibiotic resistance.
Subcultures grown in 1 ml of liquid mycobacteria growth indicator tube culture medium (Becton, Dickinson, Sparks, MD, USA) were centrifuged at 20,000 × g for 15 min. The precipitates were resuspended in 100 to 300 μl of distilled water and heated to 95°C for 20 min to extract the DNA. After centrifugation, the supernatants were used for PCR and sequencing. NTM species were identified by multigene sequencing analysis of rrs, rpoB, and hsp65, as described previously (17–19). The amplified sequences were analyzed using the GenBank database with the Basic Local Alignment Search Tool (BLAST), available from the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The regions of genes involved in CLR and AMK resistance were evaluated by PCR and sequencing as described previously (20–22). Each M. abscessus isolate was genotyped for the presence of a T-to-C mutation at position 28 of the erm(41) gene, a mutation associated with loss of inducible macrolide resistance (20). The presence of point mutations at position 2058 or 2059 (Escherichia coli numbering) in the rrl gene was also investigated (21). Last, the rrs gene was sequenced from position 1200 to 1501 (22).
GenoType NTM-DR assay.
After DNA extraction as described above, amplification and hybridization were performed using the NTM-DR assay according to the manufacturer’s recommended protocol. Briefly, a master mix containing amplification mixes A and B was prepared, and 5 μl of extracted DNA was added for PCR. PCR amplification was carried out under the following conditions: 1 cycle at 95°C for 15 min, followed by a first round of amplification consisting of 10 cycles of 30 s at 95°C and 120 s at 65°C. The second round of amplification consisted of 20 cycles of 25 s at 95°C, 40 s at 50°C, and 40 s at 70°C, with a final extension at 70°C for 8 min. Reverse hybridization and detection were carried out in a shaking water bath (TwinCubator; Hain) according to the manufacturer’s instructions. The developed strips were attached to an evaluation sheet and interpreted with the help of the chart provided by the manufacturer. Three separate observers independently interpreted the results in a blinded fashion.
Drug susceptibility testing.
DST for CLR and AMK was performed at the Korean Institute of Tuberculosis, a World Health Organization-designated supranational reference laboratory, using BMD as described by the Clinical and Laboratory Standards Institute (CLSI) (23). The tested concentration ranges were 0.5 to 64 μg/ml and 1 to 256 μg/ml for CLR and AMK, respectively. The MICs were interpreted according to the CLSI M64 protocol (24). The breakpoints for CLR were as follows: (i) for MAC, ≤8 μg/ml, 16 μg/ml, and ≥32 μg/ml indicated susceptible, intermediate, and resistant isolates, respectively; (ii) for M. abscessus, ≤2 μg/ml, 4 μg/ml, and ≥8 μg/ml indicated susceptible, intermediate, and resistant isolates, respectively. For both MAC and M. abscessus, isolates with AMK MICs of ≤16 μg/ml, 32 μg/ml, and ≥64 μg/ml were considered susceptible, intermediate, and resistant, respectively.
RESULTS
NTM identification.
The analytical specificity test results for the NTM-DR assay using reference strains are summarized in Table 1. The accuracy for the 38 reference strains was 92.1% (35/38; 95% confidence interval [CI], 77.5 to 97.9%); three mycobacterial strains, namely, M. marseillense, M. paraintracellulare, and M. yongonense, were all misidentified as M. intracellulare.
TABLE 1.
Analytical specificity of the GenoType NTM-DR assay
| Microbial species | Reference straina | GenoType NTM-DR assay |
|
|---|---|---|---|
| Band patterns | Interpretationc | ||
| Mycobacterium spp. | |||
| M. abscessus subsp. abscessus | ATCC 19977 | CC, UC, SP 4, 5, 6, 9, 10b | M. abscessus subsp. abscessus |
| M. alvei | ATCC 51304 | CC, UC | Other species |
| M. avium | KMRC 00136-41011 | CC, UC, SP 1 | M. avium |
| M. abscessus subsp. bolletii | CCUG 50184 | CC, UC, SP 4, 5, 6, 7, 9, 10 | M. abscessus subsp. bolletii |
| M. celatum | ATCC 51131 | CC, UC, SP 4 | Other species |
| M. chelonae | ATCC 35752 | CC, UC, SP 4, 5 | M. chelonae |
| M. conceptionense | CCUG 50187 | CC, UC, SP 8, 9 | Other species |
| M. cosmeticum | ATCC BAA-878 | CC, UC, SP 4 | Other species |
| M. flavescens | ATCC 14474 | CC, UC | Other species |
| M. fortuitum | KMRC 00136-60004 | CC, UC | Other species |
| M. gordonae | ATCC 14470 | CC, UC, SP 4 | Other species |
| M. interjectum | ATCC 51457 | CC, UC, SP 2, 4 | Other species |
| M. intracellulare | ATCC 13950 | CC, UC, SP 2 | M. intracellulare |
| M. kansasii | ATCC 12478 | CC, UC, SP 4 | Other species |
| M. marinum | KMRC 00136-21108 | CC, UC, SP 4 | Other species |
| M. marseillense | KMRC 00136-83001 | CC, UC, SP 2 | M. intracellulare |
| M. abscessus subsp. massiliense | CCUG 48898 | CC, UC, SP 4, 5, 8, 9 | M. abscessus subsp. massiliense |
| M. mucogenicum | ATCC 49650 | CC, UC, SP 4 | Other species |
| M. neoaurum | ATCC 25795 | CC, UC, SP 4 | Other species |
| M. nonchromogenicum | KMRC 00136-46002 | CC, UC, SP 4 | Other species |
| M. obuense | ATCC 27023 | CC, UC | Other species |
| M. paraintracellulare | KCTC 29084 | CC, UC, SP 2 | M. intracellulare |
| M. parascrofulaceum | ATCC BAA-614 | CC, UC, SP 2, 3, 4 | Other species |
| M. paraterrae | KCTC 19556 | CC, UC, SP 4 | Other species |
| M. peregrinum | KMRC 00136-75003 | CC, UC | Other species |
| M. phlei | ATCC 11758 | CC, UC | Other species |
| M. senuense | KCTC 19147 | CC, UC, SP 4 | Other species |
| M. smegmatis | ATCC 19420 | CC, UC | Other species |
| M. szulgai | ATCC 35799 | CC, UC, SP 4 | Other species |
| M. terrae | ATCC 15755 | CC, UC | Other species |
| M. tuberculosis | ATCC 27294 | CC, UC, SP 4 | Other species |
| M. vaccae | ATCC 15483 | CC, UC, SP 4 | Other species |
| M. yongonense | KCTC 19555 | CC, UC, SP 2 | M. intracellulare |
| Nonmycobacterial Corynebacterineae spp. | |||
| Gordonia polyisoprenivorans | ATCC BAA-14 | CC, UC | Other species |
| Gordonia terrae | ATCC 25594 | CC, UC | Other species |
| Nocardia brasiliensis | ATCC 19296 | CC, UC | Other species |
| Rhodococcus ruber | ATCC 27863 | CC, UC | Other species |
| Tsukamurella pulmonis | ATCC 700081 | CC, UC | Other species |
ATCC, American Type Culture Collection; CCUG, Culture Collection University of Göteborg; KCTC, Korean Collection for Type Cultures; KMRC, Korea Mycobacterium Resource Center.
Numbers refer to species-specific probes (SP). CC, conjugate control; UC, universal control.
Bold type indicates species misidentification.
A total of 145 clinical isolates were studied, of which 100% (145/145; 95% CI, 96.8 to 100%) of the species or subspecies identification results obtained from the NTM-DR assay were concordant with the results obtained by multigene sequence-based identification. Among the 58 MAC isolates tested, 23 and 35 isolates were identified as M. avium and M. intracellulare, respectively. A total of 87 M. abscessus isolates were identified at the subspecies levels, consisting of 47 M. abscessus subsp. abscessus, 38 M. abscessus subsp. massiliense, and two M. abscessus subsp. bolletii.
Detection of CLR resistance.
The results of the NTM-DR assay, sequencing, and DST for detection of CLR resistance are presented in Table 2. Among 58 MAC isolates, 25 (9 M. avium and 16 M. intracellulare) showed mutations in the rrl gene by sequencing. The NTM-DR assay identified a corresponding genotypic pattern for all 58 MAC isolates (Table 2 and Table 3). Two mutant (MUT) bands (MUT1 and MUT3) were observed for one M. avium isolate harboring three mutations: A2058C, A2058T, and A2059C (Table 3). One M. intracellulare isolate harboring the A2059T mutation revealed neither wild-type (WT) nor MUT bands by the NTM-DR assay, which was attributed to the lack of a specific MUT probe for this mutation. The rrl genotype results obtained from the NTM-DR assay were 100% concordant (58/58; 95% CI, 92.2 to 100%) with the CLR DST results. NTM-DR sensitivity and specificity in the detection of CLR resistance of MAC were 100% (25/25; 95% CI, 83.4 to 100%) and 100% (33/33; 95% CI, 87.0 to 100%), respectively.
TABLE 2.
Comparison of GenoType NTM-DR assay results with rrl gene sequencing and DST for detecting clarithromycin resistance
| GenoType NTM-DR identification |
rrl gene sequencing result (no. of isolates)a
|
Clarithromycin drug susceptibility testing (no. of isolates)a
|
|||
|---|---|---|---|---|---|
| Wild type | Mutant | Susceptible | Inducibly resistant | Resistant | |
| MAC | |||||
| M. avium | |||||
| Wild type | 14 | 0 | 14 | 0 | |
| Mutant | 0 | 9 | 0 | 9 | |
| M. intracellulare | |||||
| Wild type | 19 | 0 | 19 | 0 | |
| Mutant | 0 | 16 | 0 | 16 | |
| M. abscessus | |||||
| M. abscessus subsp. abscessus | |||||
| Wild type | 37 (13, 24) | 1 (0, 1) | 12 (12, 0) | 25 (1, 24) | 1 (0, 1) |
| Mutant | 0 | 9 (1, 8) | 0 | 0 | 9 (1, 8) |
| M. abscessus subsp. massiliense | |||||
| Wild type | 20 (0, 20) | 0 | 20 (0, 20) | 0 | 0 |
| Mutant | 0 | 18 (0, 18) | 0 | 0 | 18 (0, 18) |
| M. abscessus subsp. bolletii | |||||
| Wild type | 2 (0, 2) | 0 | 0 | 1 (0, 1) | 1 (0, 1) |
| Mutant | 0 | 0 | 0 | 0 | 0 |
| Total | |||||
| Wild type | 92 | 1 | 65 | 26 | 2 |
| Mutant | 0 | 52 | 0 | 0 | 52 |
Values in parentheses represent the number of erm(41) C28 sequevars and the number of erm(41) T28 sequevars, respectively.
TABLE 3.
Isolates with an rrl mutation detected by the GenoType NTM-DR assay and/or sequencing
| Species | GenoType NTM-DR assay resultb | erm(41) sequevar | No. of isolates | Sequencing result | Clarithromycin MIC (μg/ml)a |
|---|---|---|---|---|---|
| M. avium | MUT1 (A2058C) | 3 | A2058C | ≥64 | |
| MUT2 (A2058G) | 2 | A2058G | >64 | ||
| 1 | A2058G, A2058T | >64 | |||
| MUT3 (A2059C) | 2 | A2059C | ≥64 | ||
| MUT1 (A2058C), MUT3 (A2059C) | 1 | A2058C, A2058T, A2059C | >64 | ||
| M. intracellulare | MUT1 (A2058C) | 2 | A2058C | >64 | |
| MUT2 (A2058G) | 4 | A2058G | ≥64 | ||
| MUT3 (A2059C) | 1 | A2059C | >64 | ||
| MUT4 (A2059G) | 8 | A2059G | ≥64 | ||
| Other | 1 | A2059T | >64 | ||
| M. abscessus subsp. abscessus | MUT1 (A2058C) | T28 | 4 | A2058C | >64 |
| MUT2 (A2058G) | T28 | 3 | A2058G | ≥64 | |
| C28 | 1 | A2058G | ≥64 | ||
| MUT4 (A2059G) | T28 | 1 | A2059G | 64 | |
| WT | T28 | 1 | A2058T, WT | >64 | |
| M. abscessus subsp. massiliense | MUT1 (A2058C) | T28 | 2 | A2058C | >64 |
| MUT2 (A2058G) | T28 | 5 | A2058G | ≥64 | |
| MUT4 (A2059G) | T28 | 8 | A2059G | ≥64 | |
| T28 | 1 | A2059G | 8 | ||
| Other | T28 | 1 | A2058T | 16 | |
| T28 | 1 | A2057G, A2058G | 32 |
All isolates were resistant.
Other, wild-type band missing.
Among the 87 M. abscessus isolates, 28 (10 M. abscessus subsp. abscessus and 18 M. abscessus subsp. massiliense) showed mutations in the rrl gene by sequencing. Of 47 M. abscessus subsp. abscessus isolates, 14 had the erm(41) C28 sequevar, including 13 isolates with the WT rrl gene and a single isolate with an rrl mutation, and the remaining 33 were of the erm(41) T28 sequevar, including 24 isolates with the WT rrl gene, a single isolate with an rrl mutation identified by gene sequencing, and an additional eight isolates with rrl mutations detected by both the NTM-DR assay and sequencing (Table 2). The NTM-DR assay matched 100% (87/87; 95% CI, 94.7 to 100%) of the erm(41) sequencing results and 98.9% (86/87; 95% CI, 92.9 to 99.9%) of the rrl sequencing results (Table 2). The M. abscessus subsp. abscessus isolate with a discordant rrl result harbored an A2058T mutation in a heterogeneous pattern with the WT allele, from which only the rrl WT band was detected by the NTM-DR assay (Table 3). One M. abscessus subsp. massiliense isolate harboring the A2057G and A2058G mutations revealed an absence of the WT band by the NTM-DR assay.
The rrl genotype of M. abscessus isolates obtained with the NTM-DR assay was 97.7% (85/87; 95% CI, 91.2 to 99.6%) concordant with the CLR DST results (Table 2). NTM-DR sensitivity and specificity in the detection of CLR resistance of M. abscessus were 93.1% (27/29; 95% CI, 75.8 to 98.8%) and 100% (58/58; 95% CI, 92.3 to 100%), respectively. Two isolates, one each of M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, were WT by the NTM-DR assay but resistant to CLR (MIC, >64 μg/ml). Although the M. abscessus subsp. abscessus isolate harbored an A2058T mutation, no rrl mutations were detected by sequencing in the M. abscessus subsp. bolletii isolate. Phenotypic inducible CLR resistance was detected in 26 M. abscessus isolates (25 M. abscessus subsp. abscessus and 1 M. abscessus subsp. bolletii), which all had the WT rrl gene accompanied by the erm(41) T28 genotype, except for one isolate with the C28 genotype.
Overall, the concordance between the NTM-DR assay results and those of sequencing was 99.3% (144/145; 95% CI, 95.6 to 99.9%), and the concordance with DST results was 98.6% (143/145; 95% CI, 94.6 to 99.8%) for detection of CLR resistance. NTM-DR sensitivity and specificity in the detection of CLR resistance were 96.3% (52/54; 95% CI, 86.2 to 99.4%) and 100% (91/91; 95% CI, 95.0 to 100%), respectively.
Detection of AMK resistance.
The results of the NTM-DR assay and corresponding sequencing and DST results for detection of AMK resistance are presented in Table 4. Among 58 MAC isolates, 3 (2 M. avium and 1 M. intracellulare) showed mutations in the rrs gene by sequencing. The NTM-DR assay showed a concordant genotypic pattern for two MAC isolates with an A1408G mutation. One M. avium isolate produced a discordant rrs result and comprised a heterogeneous population carrying the T1406A mutation and WT alleles, from which only the WT band was observed (Table 5). The rrs genotype obtained with the NTM-DR assay was 96.6% (56/58; 95% CI, 87.0 to 99.4%) concordant with the results of AMK DST (Table 4). NTM-DR sensitivity and specificity in the detection of AMK resistance of MAC were 50% (2/4; 95% CI, 9.2 to 90.8%) and 100% (54/54; 95% CI, 91.7 to 100%), respectively. The MICs for the two discordant M. avium isolates were both 64 μg/ml, while those for isolates with the A1408G mutation (detected as a positive MUT1 probe) were >256 μg/ml.
TABLE 4.
Comparison of GenoType NTM-DR assay results with rrs gene sequencing and DST for detecting amikacin resistance
| GenoType NTM-DR rrs gene result |
rrs gene sequencing result (no. of isolates) |
Amikacin drug susceptibility testing (no. of isolates) |
|||
|---|---|---|---|---|---|
| Wild type | Mutant | Susceptible | Intermediate | Resistant | |
| MAC | |||||
| M. avium | |||||
| Wild type | 21 | 1 | 12 | 8 | 2 |
| Mutant | 0 | 1 | 0 | 1 | |
| M. intracellulare | |||||
| Wild type | 34 | 0 | 19 | 15 | 0 |
| Mutant | 0 | 1 | 0 | 1 | |
| M. abscessus | |||||
| M. abscessus subsp. abscessus | |||||
| Wild type | 46 | 0 | 34 | 12 | 0 |
| Mutant | 0 | 1 | 0 | 0 | 1 |
| M. abscessus subsp. massiliense | |||||
| Wild type | 36 | 0 | 25 | 10 | 1 |
| Mutant | 0 | 2 | 0 | 0 | 2 |
| M. abscessus subsp. bolletii | |||||
| Wild type | 2 | 0 | 2 | 0 | 0 |
| Mutant | 0 | 0 | 0 | 0 | 0 |
| Total | |||||
| Wild type | 139 | 1 | 92 | 45 | 3 |
| Mutant | 0 | 5 | 0 | 0 | 5 |
TABLE 5.
Isolates with an rrs mutation detected by the GenoType NTM-DR assay and/or sequencing
| Species | GenoType NTM-DR assay result | No. of isolates | Sequencing result | Amikacin MIC (μg/ml) |
|---|---|---|---|---|
| M. avium | MUT1 (A1408G) | 1 | A1408G | >256 |
| WT | 1 | T1406A, WT | 32 | |
| M. intracellulare | MUT1 (A1408G) | 1 | A1408G | >256 |
| M. abscessus subsp. abscessus | MUT1 (A1408G) | 1 | A1408G | >256 |
| M. abscessus subsp. massiliense | MUT1 (A1408G) | 2 | A1408G | >256 |
Among the 87 M. abscessus isolates, 3 (1 M. abscessus subsp. abscessus and 2 M. abscessus subsp. massiliense) showed an A1408G mutation in the rrs gene by sequencing. The NTM-DR assay revealed a corresponding genotypic pattern for all M. abscessus isolates (Table 4 and Table 5). The rrs genotype determined by the NTM-DR assay was concordant with AMK DST results with the exception of one M. abscessus subsp. massiliense isolate with a WT rrs gene and an MIC of 64 μg/ml. NTM-DR sensitivity and specificity in the detection of AMK resistance of M. abscessus were 75% (3/4; 95% CI, 21.9 to 98.7%) and 100% (83/83; 95% CI, 94.5 to 100%), respectively.
Overall, the concordance of results of the NTM-DR assay with those of sequencing was 99.3% (144/145), and the concordance with DST results was 97.9% (142/145; 95% CI, 93.6 to 99.5%) for detection of AMK resistance. NTM-DR sensitivity and specificity in the detection of AMK resistance were 62.5% (5/8; 95% CI, 25.9 to 89.8%) and 100% (137/137; 95% CI, 96.6 to 100%), respectively. The MIC for isolates harboring the A1408G mutation was >256 μg/ml.
DISCUSSION
In this study, the NTM-DR assay exhibited excellent performance for identifying M. abscessus (sub)species in clinical isolates and good performance with most MAC species. However, we demonstrated that the assay has limitations in identifying MAC isolates at the species level. The MAC includes a total of 12 validly published species: M. avium, M. intracellulare, M. chimera, M. colombiense, M. arosiense, M. vulneris, M. marseillense, M. timonense, M. bouchedurhonense, M. yongonense, M. paraintracellulare, and M. lepraemurium (25). Among these, three MAC reference strains (M. marseillense, M. paraintracellulare, and M. yongonense) other than M. avium and M. intracellulare were included in our study, and all three strains were misidentified as M. intracellulare. Probes specific to these species that cross-reacted with the M. intracellulare probe are not available on the test strip. Similar misidentification of MAC species as M. intracellulare by LPAs has been reported previously (12, 25–27). Specifically, Mok et al. showed that M. arosiense, M. timonense, M. bouchedurhonense, and M. marseillense all cross-reacted with the M. intracellulare probe using the NTM-DR assay (12). These MAC species are infrequently encountered NTM species and are rarely associated with human infection, and thus the clinical spectra for these MAC species are not fully understood (28–30). However, the possibility of such misidentifications should be taken into account.
Acquired CLR resistance is almost always associated with mutations of the rrl gene at positions 2058/2059 (31). Indeed, these point mutations are present at high frequency in CLR-resistant MAC and M. abscessus isolates (32–35). In addition, inducible resistance to CLR has been reported in M. abscessus subsp. abscessus and M. abscessus subsp. bolletii due to induced synthesis of an RNA methylase encoded by the erm(41) gene (36). M. abscessus subsp. abscessus and M. abscessus subsp. bolletii exhibit a T/C polymorphism at position 28 of the erm(41) gene, with T28 strains showing inducible resistance and C28 strains lacking inducible resistance (36, 37). Furthermore, inducible resistance does not occur in M. abscessus subsp. massiliense because it has a partially deleted, nonfunctional erm(41) gene (35–37). Therefore, detection of acquired and intrinsic resistance is necessary prior to starting treatment or at recurrence of NTM disease. However, the time needed to perform NTM DST for detecting CLR resistance can be as long as 6 weeks for MAC and 14 days for M. abscessus (24). The results of our study indicate that the NTM-DR assay is a rapid and accurate tool for detecting resistance to both CLR and AMK. So far, two other studies have reported the results of the NTM-DR assay for molecular detection of antibiotic resistance in MAC and M. abscessus (10, 11). However, based on these previous studies, it was difficult to reliably assess the sensitivity of the assay for detecting antibiotic resistance among NTM isolates overall and with regard to MAC and M. abscessus isolates specifically due to their small number of CLR-resistant isolates. The study of Mougari et al. included nine and seven CLR-resistant MAC and M. abscessus isolates, respectively (11), and the study of Kehrmann et al. included only M. abscessus isolates, of which 11 were CLR resistant (10). In the present study, we included a sufficient number of CLR-resistant MAC and M. abscessus isolates (total of 54; 25 MAC and 29 M. abscessus isolates) to demonstrate that the sensitivity and specificity of the NTM-DR assay were excellent for detection of CLR resistance in both MAC and M. abscessus. However, we did observe two false WT results for two of the isolates. They were found to comprise a heterogeneous population with a WT allele and a resistance allele, as determined by sequencing. In order to check for reproducibility, the NTM-DR assay and sequencing were repeated, and the results were concordant with the original results. Consistent with this observation, Mougari et al. observed that a heterogeneous population containing a resistance mutation that is not screened for by a specific probe can be missed if a WT population is also present (11). Therefore, it is recommended that the NTM-DR assay should be used in tandem with phenotypic DST since this assay has the inherent limitations of LPAs: (i) the inability to exclude the possibility that a strain is resistant when the strain has a WT pattern and (ii) the inability to detect other mechanisms of resistance to CLR and AMK.
Mutations in the rrs gene responsible for AMK resistance occur mainly at position 1408 in both MAC and M. abscessus (35, 38). In our study, among eight isolates that were resistant to AMK, five harbored the A1408G mutation and had corresponding MICs of >256 μg/ml. On the other hand, the rrs A1408G mutation was not detected in isolates with an MIC of 64 μg/ml, in accordance with previous reports (10, 39, 40). Until recently, no AMK breakpoints for MAC had been established (23). Brown-Elliott et al. suggested a resistance breakpoint of ≥64 μg/ml, which was derived from a mutation study on the rrs gene (39). However, Griffith et al. reported that treatment success correlates with the following MIC breakpoints: susceptible, ≤64 μg/ml; resistant, >64 μg/ml (41, 42). In guidelines published in November 2018, new AMK breakpoints for MAC were proposed by the CLSI, which need to be clinically validated (24). The CLSI guidelines included two separate sets of breakpoints for AMK, as follows: (i) for intravenous AMK, breakpoints of ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant; (ii) for liposomal inhaled AMK, breakpoints of ≤64 μg/ml for susceptible and ≥128 μg/ml for resistant. Although two separate sets of breakpoints for determining resistance were suggested, MAC isolates with MICs of >64 μg/ml almost always had mutations in the rrs gene regardless of the formulation of AMK, as shown by previous studies (39, 42) and the present study. Thus, the efficacy of therapy for isolates with AMK MICs of 64 μg/ml would require further clinical validation.
Apart from the A1408G mutation in the rrs gene, Nessar et al. demonstrated that mutations at positions 1406, 1409, and 1491 of rrs (T1406A, C1409T, and G1491T) are associated with AMK resistance in M. abscessus (22). In the present study, we describe for the first time a T1406A mutation in an M. avium isolate, previously only described in M. abscessus. The isolate harboring the T1406A mutation presented an AMK-susceptible phenotype (MIC, 32 μg/ml). However, it is not clear whether this substitution contributes to AMK resistance since the substitution was found in a heterogeneous pattern with a WT allele (Table 5).
A limitation of the present study was that we did not evaluate M. chimera and tested only two M. abscessus subsp. bolletii isolates since they are rarely isolated in South Korea (43–46). Thus, we cannot be sure about the performance of the NTM-DR assay for these two (sub)species in this study.
In conclusion, NTM-DR is a straightforward and accurate assay for discriminating MAC and M. abscessus (sub)species and for detecting CLR and AMK resistance mutations. We expect that the NTM-DR assay will be a useful and routine tool in the clinical setting.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation of Korea (NRF) funded by the South Korean Government (MSIT) (NRF-2018R1A2A1A05018309).
Charles L. Daley has received grants from Insmed, Inc., and has served on Advisory Boards for Insmed Inc., Johnson and Johnson, Spero, and Horizon, which were not associated with the submitted work. Won-Jung Koh has received a consultation fee from Insmed, Inc., for the Insmed Advisory Board Meeting, which was not associated with the submitted work. We have no other conflicts of interest to declare.
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