Recommendations for first-line and second-line drug testing and organism group, specific methodologies, and reporting recommendations have been addressed by the Clinical and Laboratory Standards Institute (CLSI) and are important in the selection of appropriate antimicrobial treatment regimens for nontuberculous mycobacteria (NTM) disease.
KEYWORDS: mycobacteria, nontuberculous mycobacteria, susceptibility
ABSTRACT
Recommendations for first-line and second-line drug testing and organism group, specific methodologies, and reporting recommendations have been addressed by the Clinical and Laboratory Standards Institute (CLSI) and are important in the selection of appropriate antimicrobial treatment regimens for nontuberculous mycobacteria (NTM) disease. This review also includes recent information on new antimicrobials proposed for the treatment of NTM but not yet addressed by the CLSI and molecular (gene sequencing) methods associated with the detection of antimicrobial resistance of two major therapeutic antimicrobials, clarithromycin and amikacin.
INTRODUCTION
The original guidelines for antimycobacterial susceptibility testing (AST) set by the Clinical and Laboratory Standards Institute (CLSI) were updated and expanded in 2011 (1) and more recently in December 2018. The current standard (CLSI M24, 3rd edition) includes recommendations for AST of slowly growing nontuberculous mycobacteria (NTM), including Mycobacterium avium complex (MAC), M. kansasii, and M. marinum, and the rapidly growing mycobacteria (RGM) (2). As in the previous edition (1), breakpoints for some NTM are based on clinical data, comparative breakpoints used for other organisms, population distribution data, and the experience of experts in the field of mycobacteriology.
Briefly, molecular methods (i.e., gene sequencing to identify antimicrobial mechanisms of resistance) associated with AST of some antimicrobials, especially amikacin and macrolides, have been included in the third edition. There are also updated breakpoints and quality control recommendations in table format which have been moved to a newly created supplement, CLSI document M62 (3).
The recommended standard AST method is broth microdilution using antimicrobial concentrations derived from serial 2-fold dilutions indexed to the base 2 (e.g., 1, 2, 4, 8, 16 μg/ml). With a few exceptions (i.e., for drugs for which no standardized NTM breakpoints have been described), an MIC value and its interpretation (i.e., susceptible [S], intermediate [I], or resistant [R]) should be reported.
It is the general consensus of experts that NTM AST should be performed only for isolates believed to be clinically significant. Isolates from blood, other sterile body fluids, and skin and soft tissue infections are usually considered clinically significant. Because NTM may colonize the respiratory tract and their isolation does not always correlate with clinical disease, specific criteria for establishing the clinical significance of NTM in respiratory samples have been developed (4, 5).
These criteria typically include the following:
-
•
At least two NTM culture-positive sputa (acid-fast bacilli [AFB] smear positive or negative) or one bronchial wash or lavage sample.
-
•
A transbronchial or lung biopsy specimen with supporting mycobacterial histopathology and a positive NTM culture.
A major caveat is that single positive sputum cultures are not sufficient for the establishment of a diagnosis of NTM. Moreover, single positive sputum cultures that are AFB smear-negative or contain low numbers of organisms are not likely to be clinically significant, and follow-up cultures should be requested if the diagnosis remains uncertain.
Testing skills for NTM AST are acquired through rigorous training and experience with all types of NTM. Test performance should be evaluated on initial validation and at regular intervals in order to demonstrate continued proficiency. Currently, we are unaware of any commercial source for proficiency testing for NTM AST only; therefore, laboratories that perform NTM AST should consider comparing in-house test results with an experienced referral lab. Thus, laboratories that encounter NTM infrequently should submit cultures for AST to a reference laboratory with expertise with NTM.
SLOWLY GROWING NONTUBERCULOUS MYCOBACTERIA
Mycobacterium avium complex (MAC).
MAC is an environmental opportunistic NTM pathogen that causes disease in susceptible hosts. Two major types of disease, chronic pulmonary disease in patients with bronchiectasis or other underlying lung disease and disseminated disease in HIV-infected hosts, are recognized (5, 6). Therapeutic antimicrobial regimens typically used for treatment of MAC include a macrolide, a rifamycin (e.g., rifampin or rifabutin), and ethambutol with or without a fourth agent, such as amikacin (5). However, there is no clinical correlation with in vitro MICs except for the macrolide (e.g., clarithromycin) and, most recently, amikacin (7).
AST MAC breakpoints for amikacin have been revised to include separate values for intravenous and the newer inhaled liposomal formulation of amikacin. A recent study of isolates from a clinical trial evaluating inhaled liposomal amikacin revealed that the typical acquired mutational amikacin resistance mechanism in the 16S rRNA gene was not present in isolates treated with inhaled amikacin with MICs of ≤64 μg/ml but was present in isolates with MICs of >64 μg/ml (8, 9). Additionally, patients with isolates with amikacin MICs of >64 μg/ml did not respond to treatment, suggesting that the resistance breakpoints for inhaled amikacin should be >64 μg/ml (9). Likewise, patients treated with intravenous amikacin that had amikacin MICs of 64 μg/ml harbored the amikacin mutation in the 16S rRNA gene, and the patients did not respond to treatment with amikacin (10).
Previous studies have shown that the major amikacin (i.e., 16S rRNA gene) mutations are in base pairs 1408, 1409, or 1411 of the 16S rRNA gene (8). If any of these mutations are noted, the isolate may be reported as resistant without performing phenotypic AST. However, because there may be as yet undescribed mechanisms of amikacin resistance, the absence of one of the 16S rRNA gene mutations does not preclude the presence of amikacin resistance (8).
The macrolides have been considered the cornerstone of treatment for MAC (5, 11). Macrolide AST breakpoints for MAC are unchanged in the CLSI third edition (MICs of ≤8 [S], 16 [I], and ≥32 [R] μg/ml) (see Table 1). As previously stated, only clarithromycin should be tested in the macrolide class. A MAC isolate with an intermediate MIC may suggest emerging resistance, and the patient should be monitored carefully, with subsequent isolates of MAC tested for possible macrolide resistance. Clarithromycin resistance mutations are detected in the 23S rRNA gene at adenine position 2058 or 2059 (11, 12). Isolates that have these mutations are clarithromycin resistant at the initial reading (i.e., usually 7 days). If MICs are questionable, testing should be repeated and/or the isolate sent to a referral laboratory which can sequence for the mutation (2, 12).
TABLE 1.
Antimycobacterial agents and susceptibility breakpoints (MICs) for testing slowly growing nontuberculous mycobacteria
| Species/complex | MIC (μg/ml) |
Comment | ||
|---|---|---|---|---|
| Susceptible | Intermediate | Resistant | ||
| M. avium complex | ||||
| First line | ||||
| Clarithromycin | ≤8 | 16 | ≥32 | Clarithromycin is class drug for macrolides |
| Amikacin (IV) | ≤16 | 32 | ≥64 | |
| Amikacin (liposomal inhaled) | ≤64 | ≥128 | ||
| Second line | ||||
| Moxifloxacin | ≤1 | 2 | ≥4 | The clinical efficacy of these agents for MAC disease remains uncertain |
| Linezolid | ≤8 | 16 | ≥32 | |
| M. kansasiia | ||||
| First line | ||||
| Clarithromycin | ≤8 | 16 | ≥32 | |
| Rifampin | ≤1 | ≥2 | ||
| Second line | ||||
| Amikacin | ≤16 | 32 | ≥64 | |
| Ciprofloxacin | ≤1 | 2 | ≥4 | Ciprofloxacin and levofloxacin are interchangeable, but both are less active than moxifloxacin |
| Doxycycline | ≤1 | 2–4 | ≥8 | |
| Linezolid | ≤8 | 16 | ≥32 | |
| Minocycline | ≤1 | 2–4 | ≥8 | |
| Moxifloxacin | ≤1 | 2 | ≥4 | |
| Rifabutin | ≤2 | ≥4 | ||
| TMP-SMX | ≤2/38 | ≥4/76 | ||
| M. marinum | ||||
| Amikacin | ≤16 | 32 | ≥64 | |
| Ciprofloxacin | ≤1 | 2 | ≥4 | |
| Clarithromycin | ≤8 | 16 | ≥32 | |
| Doxycycline | ≤1 | 2–4 | ≥8 | |
| Linezolid | ≤8 | 16 | ≥32 | |
| Minocycline | ≤1 | 2–4 | ≥8 | |
| Moxifloxacin | ≤1 | 2 | ≥4 | |
| Rifabutin | ≤2 | ≥4 | ||
| Rifampin | ≤1 | ≥2 | ||
| TMP-SMX | ≤2/38 | ≥4/76 | ||
For M. kansasii, second-line agents should be tested if isolates are rifampin resistant.
Moxifloxacin and linezolid may show limited activity against some isolates of MAC (13). AST may be performed, but the clinical efficacy of these agents for MAC has not been established (2).
Mycobacterium kansasii.
M. kansasii, like MAC, is an environmental opportunistic organism that causes disease in a susceptible host. The most frequent type of infection is pulmonary. The current standard of care is a multidrug antimicrobial regimen consisting of rifampin, ethambutol (EMB), and isoniazid (INH). A short-course therapy using a macrolide in place of INH has also been suggested, but only small studies have assessed its efficacy (14). In HIV-infected patients being treated with protease inhibitors, rifabutin is usually substituted for rifampin (5). Treatment failure is generally associated with rifampin resistance; therefore, all isolates should be tested for susceptibility to rifampin (2, 5). All rifampin-susceptible isolates are susceptible to rifabutin, and thus, special testing of rifabutin is not necessary (2).
Although INH and EMB are part of the treatment regimen for M. kansasii, MIC values for these agents do not correlate well with clinical response data; therefore, CLSI does not recommend reporting results for them. INH MICs for untreated (wild-type) strains are generally 0.5 to 5 μg/ml; thus, the standard Mycobacterium tuberculosis complex (MTBC) critical concentration of 0.2 μg/ml almost always indicates resistance, and the 1.0 μg/ml concentration gives variable results even upon repeat cultures of the same strain of M. kansasii (1, 2). EMB is a technically difficult agent to test in the laboratory, as results are not reproducible by broth microdilution. EMB MIC results may be misleading, and thus, the new CLSI recommendation is not to report EMB MICs (2).
If isolates of M. kansasii are rifampin resistant (MIC, >1 μg/ml), the following second-line drugs should be tested: amikacin, ciprofloxacin, linezolid, moxifloxacin, doxycycline/minocycline, rifabutin, and trimethoprim-sulfamethoxazole (TMP-SMX) (3) (see Table 1).
Mycobacterium marinum.
M. marinum is a photochromogenic NTM often found in freshwater and salt water. Originally, it was thought to infect only marine fish, and thus, the name M. marinum was given. However, it is now known to be ubiquitous in the environment (5, 15). The names “fish tank granuloma” and “swimming pool granuloma” have been used to describe M. marinum infection associated with home aquariums and water-related activities, including swimming and fishing (5).
Treatment regimens involve a variety of antimicrobials, most often including doxycycline, minocycline, TMP-SMX, rifampin plus ethambutol, and clarithromycin (5, 14). Established guidelines recommend treatment with at least two active agents (5) typically for about 1 to 2 months following resolution of symptoms.
Antimicrobials that are recommended for in vitro AST of M. marinum include the same as for rifampin-resistant M. kansasii as listed in CLSI M62 (3). As with M. kansasii, although used for treatment, EMB should not be tested, because results can be misleading.
If a patient is not culture negative after 3 months of appropriate treatment, AST should be repeated and the treatment regimen adjusted based on the in vitro MICs. A follow-up culture should be performed after 6 months of total treatment. If the patient remains culture positive at that time, an expert in the treatment of NTM infections should be consulted (2, 5).
Miscellaneous slowly growing NTM.
Several slowly growing mycobacteria other than MAC, M. kansasii, and M. marinum are known to cause disease. The most commonly encountered (worldwide) nonfastidious species include the species within the M. terrae complex (most often M. arupense, M. heraklionense, and M. virginiense), M. simiae, M. malmoense, and M. xenopi (5, 15). M. gordonae is often recovered but rarely considered clinically significant as noted by the absence of substantial clinical reports (5). Thus, AST is rarely necessary. AST of the same antimicrobials as recommended for rifampin-resistant M. kansasii should be tested. AST should be repeated after 3 to 6 months of therapy if clinically indicated or if the patient remains culture positive.
Slowly growing species, including M. haemophilum, M. genavense, and M. ulcerans are considered fastidious, requiring either supplementation of media, prolonged incubation, or both for recovery in the laboratory (1, 2, 16). AST may also be challenging and in some cases not possible for these species.
M. haemophilum is most often associated with skin and soft tissue infections, especially in the extremities due to its predilection for cooler temperatures. Growth of the species on solid medium supplemented with ferric ammonium citrate or hemin usually requires several weeks at temperatures of ≤32°C (16). Isolates are resistant to ethambutol. However, AST should include amikacin, ciprofloxacin, clarithromycin, doxycycline, linezolid, minocycline, rifampin, and TMP-SMX. Usually, at least two of these agents are used for treatment (16). Testing is difficult in broth microdilution, as supplementation must be used (16). However, the agar disk elution method, using commercial antimicrobial disks initially eluted into oleic acid dextrose catalase (OADC) in a six-cell tissue culture plate has been used successfully. Subsequently, molten Middlebrook agar is added to the plate, which is incubated for at least 2 weeks.
AST for M. genavense is often not possible, due to the organism’s extreme fastidiousness. The organism requires an acid pH and mycobactin J supplementation and at least 6 weeks of incubation (2, 15). Due to its prolonged incubation, antimicrobial degradation usually precludes AST, although some molecular studies have shown resistance to INH and susceptibility to rifampin, clarithromycin, amikacin, ciprofloxacin, and pyrazinamide, with variable results for EMB. The incidence of M. genavense in HIV-infected individuals is declining but appears to be increasing in other immunocompromised individuals, including transplant recipients (17). Optimal treatment has not been established, but most experts recommend at least three active agents, similar to for other NTM infections (5).
M. ulcerans causes a serious progressive destructive infection of skin and soft tissue mostly in Africa and Australia, where it is known as Buruli and Bairnsdale ulcer, respectively (18). M. ulcerans is an extremely slowly growing (6 to 12 weeks) NTM species which, like M. genavense, is typically diagnosed in the laboratory by molecular methods. Also like M. genavense, AST is difficult (1, 2).
Culture of M. ulcerans on laboratory medium usually has a low yield of the organisms, and PCR or other molecular methods are preferred for laboratory detection due to lower sensitivity and extended incubation time for its recovery (1, 2, 18).
RAPIDLY GROWING MYCOBACTERIA
The major nonpigmented rapidly growing mycobacteria (RGM) groups/complexes responsible for clinical disease in humans are the M. abscessus complex (M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii), the M. fortuitum group (M. fortuitum, M. houstonense, M. porcinum, M. neworleansense, M. senegalense/M. conceptionense, M. brisbanense, M. boenickei, and M. septicum), the M. mucogenicum group (M. mucogenicum, M. phocaicum, and M. aubagnense), and the M. chelonae complex (M. chelonae, M. immunogenum, M. salmoniphilum, M. saopaulense, and M. franklinii) (19).
A few species of pigmented RGM, including M. bacteremicum and M. neoaurum, have also been associated with clinical disease, mainly mycobacterial sepsis related to long-term catheterization. The nonpigmented M. mucogenicum group is rarely a cause of pulmonary infection but, like M. bacteremicum and M. neoaurum, has been associated with catheter-related sepsis (5, 19).
The M. abscessus complex is the primary cause of pulmonary disease among RGM. The M. abscessus complex has also been the causative agent of skin, soft tissue, and disseminated disease. The M. fortuitum group is primarily associated with skin and soft tissue infections, especially postsurgical or posttraumatic injury. M. chelonae is also most often a cause of skin and soft tissue infections, usually in immune-suppressed patients, especially those on high-dose steroids (5). Like the M. fortuitum group, M. chelonae is not a common cause of pulmonary infection. M. fortuitum may be associated with pulmonary infection in patients with achalasia or lipid pneumonia (5, 19). M. immunogenum is phenotypically similar to M. chelonae but is more drug resistant and is often associated with hypersensitivity pneumonitis (5, 19).
The antimicrobials recommended by CLSI for testing of the RGM include amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline (or minocycline), imipenem, linezolid, moxifloxacin, trimethoprim-sulfamethoxazole, and tobramycin (for the M. chelonae/M. immunogenum complex only) (see Table 2). Tigecycline and clofazimine may be tested, but there are insufficient data to establish MIC breakpoints. Therefore, for these three agents, if requested, an MIC without interpretation should be given (2).
TABLE 2.
Antimicrobial agents and susceptibility breakpoints (MICs) for testing rapidly growing mycobacteriaa
| Antimicrobial agent | MIC (μg/ml) |
Comment | ||
|---|---|---|---|---|
| Susceptible | Intermediate | Resistant | ||
| Amikacin | 16 | 32 | ≥64 | M. abscessus complex isolates with MIC of ≥64 μg/ml should be retested and/or the 16S rRNA gene sequenced to check for mutation |
| Cefoxitin | 16 | 32–64 | ≥128 | |
| Ciprofloxacin | ≤1 | 2 | ≥4 | Ciprofloxacin and levofloxacin are interchangeable, but both are less active than the newer B-methoxy-fluoroquinolones |
| Clarithromycin | ≤2 | 4 | ≥8 | See text for information on the erm gene; clarithromycin and azithromycin are interchangeable clinically |
| Doxycycline | ≤1 | 2–4 | ≥8 | |
| Imipenem | ≤4 | 8–16 | ≥32 | All isolates of M. fortuitum, M. smegmatis, and the M. mucogenicum group are presumed imipenem susceptible; imipenem MICs do not predict meropenem or ertapenem susceptibility |
| Linezolid | ≤8 | 16 | ≥32 | |
| Meropenem | ≤4 | 8–16 | ≥32 | |
| Moxifloxacin | ≤1 | 2 | ≥4 | |
| Tigecycline | Insufficient data to establish breakpoints; only MIC should be reported | |||
| Tobramycin | ≤2 | 4 | ≥8 | Predominantly for M. chelonae; if MIC > 4 μg/ml, the test should be repeated and/or the identification confirmed by rpoβ gene sequencing |
| TMP-SMX | ≤2/38 | ≥4/76 | MIC is 80% inhibition | |
CLSI M24, 3rd ed., 2018 (2).
Because susceptibility testing results for some agents may apply to specific species, the CLSI and most mycobacterial experts strongly advise identification to the species level or, in the case of the M. abscessus complex, to the subspecies level (2, 15, 19, 20). This is also necessary for proper selection of treatment options (5, 15, 19). In the future, prediction of clarithromycin resistance by whole-genome sequencing may be a reasonable approach as the method becomes more accessible to clinical laboratories (21).
Incubation times for isolates of the RGM range from 2 to 5 days. Results for some isolates, predominantly those in the M. fortuitum group, may be reported at 48 hours, but the majority of the RGM require 3 to 5 days for adequate growth in broth. Generally, the final MIC reading should be at no more than 5 days due to instability of some of the antimicrobials being tested (e.g., carbapenems and tetracyclines) (2).
There are two major exceptions to the recommendation for no more than 5 days of incubation. Isolates of the M. abscessus complex, especially those from patients treated with multiple antimicrobial agents over time, such as those with cystic fibrosis, often grow very slowly in broth, even after multiple passages of the organism onto solid medium. In some cases, varying the incubation temperature or growing the organisms in broth first, especially in a shaker incubator, may be helpful. If incubation beyond 5 days is required for adequate growth, only clarithromycin and amikacin can be reliably reported due to the instability of most of the antimicrobials (2). When extension of incubation is required, the CLSI recommends a comment such as “This isolate required extended incubation and results for only clarithromycin and amikacin are reliable after incubation for >5 days” (2).
Another exception to the ≤5-day incubation period is clarithromycin. It is important to detect inducible macrolide resistance in RGM (22, 23). The erm gene, which has been noted in several RGM species, confers inducible resistance to macrolides (22). Phenotypical detection of inducible macrolide resistance is accomplished by extending the incubation of clarithromycin to 14 days unless the MIC is ≥16 μg/ml at an earlier time point, at which time the final report should be issued. If the clarithromycin MIC is 4 or 8 μg/ml at 14 days, the test should be repeated, and if it remains 4 or 8 μg/ml, the isolate should be sent to a referral laboratory that has experience in testing RGM and the ability to perform sequencing of the erm gene and confirm identification by rpoβ gene sequencing of the isolate if needed (2, 20).
Gene sequencing is required for accurate identification to the species/subspecies level of most RGM to better understand the basis of macrolide resistance. The rpoβ gene sequence is necessary in order to identify the subspecies of the M. abscessus complex, including in order of prevalence, subsp. abscessus, subsp. massiliense, and subsp. bolletii. In many cases, sequencing of the erm(41) gene, harbored by approximately 80% of subsp. abscessus isolates, will predict inducible macrolide resistance (20). Approximately 20% of the isolates of subsp. abscessus are known to have a nonfunctional erm(41) gene, and these isolates will remain macrolide susceptible even after 14 days of incubation (20). Although the matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) method is gaining popularity in clinical laboratories, it cannot identify the M. abscessus complex to the subspecies level, which is critically important for selection of treatment (24, 25).
Macrolide resistance in the M. abscessus complex may also be related to mutations in the 23S rRNA gene just as is true for MAC. Mutational resistance may occur during or after treatment due to a single base-pair mutation at adenine 2058 or 2059 (using the Escherichia coli numbering system) in the 23S rRNA gene. This type of acquired resistance is usually readily seen at the initial (3 to 5 days) MIC reading, at which time the macrolide result can be finalized as resistant (2). Like mutations in MAC, if the clarithromycin MIC is questionable, testing should be repeated and/or the isolate sent to a referral laboratory that can use sequencing to detect the base-pair mutation in the 23S rRNA gene (2, 11).
Several species of RGM are known to have a nonfunctional or absent erm gene and are therefore expected to be susceptible to clarithromycin. These include M. abscessus subsp. massiliense, which has a truncated erm gene (22), M. chelonae (22, 26), M. immunogenum (27), the M. mucogenicum group (27), M. peregrinum, and M. senegalense (23). For these groups, if identification by sequencing of the rpoβ gene has been performed and no clarithromycin mutational resistance is present, a clarithromycin result of “susceptible” may be reported at the initial MIC reading, and extended incubation is not required.
In contrast, M. abscessus subsp. bolletii is known to have a functional erm gene (20). For this subspecies, clarithromycin should be reported as resistant (with no MIC value) on the initial reading. However, an isolate identified as M. abscessus subsp. abscessus by rpoβ gene sequence analysis requires erm(41) gene sequencing to accurately assess macrolide susceptibility. Currently, all sequevars (approximately 20 sequence variants) of M. abscessus subsp. abscessus except sequevar II (also known as Mab30) have functional erm(41) genes (20, 22). Mab30 has a nonfunctional erm (28T to C substitution) gene. An isolate molecularly identified as sequevar II may also be reported as “susceptible” upon the initial reading (no MIC value given). Recently, other isolates of M. abscessus subsp. abscessus with a nonfunctional erm(41) gene (currently designated “hybrid types”) have been recognized. These isolates are identified by rpoβ as subspecies abscessus but have a truncated erm gene as for M. abscessus subsp. massiliense. These isolates should also be (inducible) macrolide susceptible.
M. fortuitum, M. houstonense, M. porcinum, M. neworleansense, M. goodii, M. smegmatis, M. mageritense, and M. wolinskyi are known to harbor erm genes and should be considered macrolide resistant (22, 23). However, currently, there are insufficient published data to justify this assessment without phenotypic susceptibility testing. Thus, the full 14-day incubation (unless the MIC value is ≥16 μg/ml earlier) is required for accurate macrolide assessment.
In addition to macrolide resistance, detection of amikacin resistance is also important in the selection of antimicrobial treatment. Aminoglycosides, including amikacin, are major components of therapy for many NTM, including M. abscessus. Previous studies show that a single substitution (A→G) at position 1408 in the 16S rRNA gene is responsible for amikacin resistance in isolates of the M. abscessus complex and the M. chelonae complex (8).
NEW ANTIMICROBIALS FOR TREATMENT OF NTM
Other antimicrobials which may be useful for treatment of certain NTM infections include clofazimine, bedaquiline, and tedizolid. These are currently being investigated as possible agents for treatment of isolates of the M. abscessus complex and MAC. Clofazimine is a member of the riminophenazine group of antibiotics, primarily used for the treatment of leprosy (28). Tedizolid is a new oxazolidinone antimicrobial which, like its predecessor, linezolid, has shown in vitro activity against some isolates of NTM, including some strains of the M. abscessus complex, M. chelonae, M. mucogenicum, the M. fortuitum group, the M. avium complex, M. arupense, M. kansasii, M. simiae, and M. marinum (29).
Bedaquiline is a novel diarylquinolone antibiotic which is active against a variety of mycobacteria, including multiple drug-resistant M. tuberculosis (MDRTB) (30, 31). In fact, the drug was approved in 2012 by the U.S. Food and Drug Administration (FDA) for the treatment of MDRTB (30). Bedaquiline offers a unique mechanism of action (i.e., inhibition of ATP synthase) with rare cross-resistance to other agents except clofazimine (30).
At this time, there are no standardized procedures, breakpoints, or interpretive criteria for AST of clofazimine, tedizolid, or bedaquiline. Therefore, if testing of any of these agents is requested, the recommendation is to use broth microdilution and report only an MIC without interpretation.
ACKNOWLEDGMENTS
We thank the members of the Antimycobacterial Susceptibility Testing Committee of the CLSI and the laboratory staff of the Mycobacteria/Nocardia Research Laboratory at the University of Texas Health Science Center at Tyler for their laboratory expertise. We appreciate the support of our Laboratory Director, Richard J. Wallace, Jr., and our administrative specialist, Joanne Woodring, for typing the manuscript.
REFERENCES
- 1.Clinical and Laboratory Standards Institute. 2011. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes; approved standard, 2nd ed CLSI document M24-A2 Clinical and Laboratory Standards Institute, Wayne, PA. [PubMed] [Google Scholar]
- 2.Clinical and Laboratory Standards Institute. 2018. Susceptibility testing of mycobacteria, Nocardia spp., and other aerobic actinomycetes, 3rd ed, CLSI standard document M24 Clinical and Laboratory Standards Institute, Wayne, PA. [PubMed] [Google Scholar]
- 3.Clinical and Laboratory Standards Institute. 2018. Performance standards for susceptibility testing of mycobacteria, Nocardia spp., and other aerobic actinomycetes, 1st ed CLSI document M62 Clinical and Laboratory Standards Institute, Wayne, PA. [PubMed] [Google Scholar]
- 4.Clinical and Laboratory Standards Institute. 2018. Laboratory detection and identification of mycobacteria. CLSI guideline M48 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 5.Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ Jr, Winthrop K. 2007. An official ATS/IDSA statement: diagnosis, treatment and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175:367–416. doi: 10.1164/rccm.200604-571ST. [DOI] [PubMed] [Google Scholar]
- 6.Chaisson RE, Benson CA, Dube MP, Heifets LB, Korvick JA, Elkin S, Smith T, Craft JC, Sattler FR. 1994. Clarithromycin therapy for bacteremic Mycobacterium avium complex disease. A randomized, double-blind, dose-ranging study in patients with AIDS. AIDS Clinical Trials Group Protocol 157 Study Team. Ann Intern Med 121:905–911. doi: 10.7326/0003-4819-121-12-199412150-00001. [DOI] [PubMed] [Google Scholar]
- 7.Brown-Elliott BA, Iakhiaeva E, Griffith DE, Woods GL, Stout JE, Wolfe CR, Turenne CY, Wallace RJ Jr. 2013. In vitro activity of amikacin against isolates of Mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rRNA gene mutation in treated isolates. J Clin Microbiol 51:3389–3394. (Erratum, 52:1311, 2014). doi: 10.1128/JCM.01612-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Prammananan T, Sander P, Brown BA, Frischkorn K, Onyi GO, Zhang Y, Böttger EC, Wallace RJ Jr. 1998. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae. J Infect Dis 177:1573–1581. doi: 10.1086/515328. [DOI] [PubMed] [Google Scholar]
- 9.Griffith DE, Eagle G, Thomson R, Aksamit TR, Hasegawa N, Morimoto K, Addrizzo-Harris DJ, O’Donnell AE, Marras TK, Flume PA, Loebinger MR, Morgan L, Codecasa LR, Hill AT, Ruoss SJ, Yim J-J, Ringshausen FC, Field SK, Philley JV, Wallace RJ, van Ingen J, Coulter C, Nezamis J, Winthrop KL. 2018. Amikacin liposome inhalation suspension for treatment-refractory lung disease caused by Mycobacterium avium complex (CONVERT). A prospective, open-label, randomized study. Am J Respir Crit Care Med 198:1559–1569. doi: 10.1164/rccm.201807-1318OC. [DOI] [PubMed] [Google Scholar]
- 10.Olivier KN, Griffith DE, Eagle G, McGinnis JP, Micioni L, Liu K, Daley CL, Winthrop KL, Ruoss S, Addrizzo-Harris DJ, Flume PA, Dorgan D, Salathe M, Brown-Elliott BA, Gupta R, Wallace RJ. 2017. Randomized trial of liposomal amikacin for inhalation in nontuberculous mycobacterial lung disease. Am J Respir Crit Care Med 195:814–823. doi: 10.1164/rccm.201604-0700OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Brown-Elliott BA, Nash KA, Wallace RJ Jr. 2012. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin Microbiol Rev 25:545–582. doi: 10.1128/CMR.05030-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Meier A, Kirschner P, Springer B, Steingrube VA, Brown BA, Wallace RJ Jr, Böttger EC. 1994. Identification of mutations in 23S rRNA gene of clarithromycin-resistant Mycobacterium intracellulare. Antimicrob Agents Chemother 38:381–384. doi: 10.1128/AAC.38.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Wallace RJ Jr, Brown-Elliott BA, Ward SC, Crist CJ, Mann LB, Wilson RW. 2001. Activities of linezolid against rapidly growing mycobacteria. Antimicrob Agents Chemother 45:764–767. doi: 10.1128/AAC.45.3.764-767.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Griffith DE, Brown-Elliott BA, Wallace RJ Jr. 2003. Thrice-weekly clarithromycin-containing regimen for treatment of Mycobacterium kansasii lung disease: results of a preliminary study. Clin Infect Dis 37:1178–1182. doi: 10.1086/378742. [DOI] [PubMed] [Google Scholar]
- 15.Brown-Elliott BA. 2018. Laboratory diagnosis and antimicrobial susceptibility testing of nontuberculous mycobacteria, p 15–59. In Griffith DE. (ed), Nontuberculous mycobacterial disease. Respiratory medicine Humana Press, Cham, Switzerland. [Google Scholar]
- 16.Lindeboom JA, Bruijnesteijn van Coppenraet LES, van Soolingen D, Prins JM, Kuijper EJ. 2011. Clinical manifestations, diagnosis, and treatment of Mycobacterium haemophilum infections. Clin Microbiol Rev 24:701–717. doi: 10.1128/CMR.00020-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Coelho R, Hanna R, Flagg A, Stempak LM, Ondrejka S, Procop GW, Harrington S, Zembillas A, Kusick K, Gonzalez BE. 2017. Mycobacterium genavense-induced spindle cell pseudotumor in a pediatric hematopoietic stem cell transplant recipient: case report and review of the literature. Transpl Infect Dis 19:e12656. doi: 10.1111/tid.12656. [DOI] [PubMed] [Google Scholar]
- 18.O’Brien DP, Jenkin G, Buntine J, Steffen CM, McDonald A, Horne S, Friedman ND, Athan E, Hughes A, Callan PP, Johnson P. 2014. Treatment and prevention of Mycobacterium ulcerans infection (Buruli ulcer) in Australia: guideline update. Med J Aust 200:267–270. doi: 10.5694/mja13.11331. [DOI] [PubMed] [Google Scholar]
- 19.Brown-Elliott BA, Philley JV. 2017. Rapidly growing mycobacteria, p 703–723. In Schlossberg D. (ed), Tuberculosis and nontuberculous mycobacterial infections, 7th ed ASM Press, Washington, DC. [Google Scholar]
- 20.Brown-Elliott BA, Vasireddy S, Vasireddy R, Iakhiaeva E, Howard ST, Nash KA, Parodi N, Strong A, Gee M, Smith T, Wallace RJ Jr. 2015. Utility of sequencing the erm(41) gene in isolates of Mycobacterium abscessus subsp. abscessus with low and intermediate clarithromycin MICs. J Clin Microbiol 53:1211–1215. (Erratum, 54:1172, 2016). doi: 10.1128/JCM.02950-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Lipworth S, Hough N, Leach L, Morgan M, Jeffery K, Andersson M, Robinson E, Smith EG, Crook D, Peto T, Walker T. 2018. Whole-genome sequencing of predicting clarithromycin resistance in Mycobacterium abscessus. Antimicrob Agents Chemother 63:e01204-18. doi: 10.1128/AAC.01204-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Nash KA, Brown-Elliott BA, Wallace RJ Jr. 2009. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob Agents Chemother 53:1367–1376. doi: 10.1128/AAC.01275-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Nash KA, Andini N, Zhang Y, Brown-Elliott BA, Wallace RJ Jr. 2006. Intrinsic macrolide resistance in rapidly growing mycobacteria. Antimicrob Agents Chemother 50:3476–3478. doi: 10.1128/AAC.00402-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Body BA, Beard MA, Slechta ES, Hanson KE, Barker AP, Babady NE, McMillen T, Tang Y-W, Brown-Elliott BA, Iakhiaeva E, Vasireddy R, Vasireddy S, Smith T, Wallace RJ Jr, Turner S, Curtis L, Butler-Wu S, Rychert J. 2018. Evaluation of the Vitek MS v3.0 matrix-assisted laser desorption ionization–time of flight mass spectrometry system for identification of Mycobacterium and Nocardia species. J Clin Microbiol 56:e00237-18. doi: 10.1128/JCM.00237-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Buckwalter SP, Olson SL, Connelly BJ, Lucas BC, Rodning AA, Walchak RC, Deml SM, Wohlfiel SL, Wengenack NL. 2016. Evaluation of matrix-assisted laser desorption ionization–time of flight mass spectrometry for identification of Mycobacterium species, Nocardia species, and other aerobic actinomycetes. J Clin Microbiol 54:376–384. doi: 10.1128/JCM.02128-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Hanson KE, Slechta ES, Muir H, Barker A. 2014. Rapid molecular detection of inducible macrolide resistance in Mycobacterium chelonae and M. abscessus strains: a replacement for 14 day susceptibility testing? J Clin Microbiol 52:1705–1707. doi: 10.1128/JCM.03464-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Brown-Elliott BA, Hanson K, Vasireddy S, Iakhiaeva E, Nash KA, Vasireddy R, Parodi N, Smith T, Gee M, Strong A, Barker A, Cohen S, Muir H, Slechta ES, Wallace RJ Jr. 2015. Absence of a functional erm gene in isolates of Mycobacterium immunogenum and the Mycobacterium mucogenicum group, based on in vitro clarithromycin susceptibility. J Clin Microbiol 53:875–878. doi: 10.1128/JCM.02936-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Shen G-H, Wu B-D, Hu S-T, Lin C-F, Wu K-M, Chen J-H. 2010. High efficacy of clofazimine and its synergistic effect with amikacin against rapidly growing mycobacteria. Int J Antimicrob Agents 35:400–404. doi: 10.1016/j.ijantimicag.2009.12.008. [DOI] [PubMed] [Google Scholar]
- 29.Brown-Elliott BA, Wallace RJ Jr. 2017. In vitro susceptibility testing of tedizolid against nontuberculous mycobacteria. J Clin Microbiol 55:1747–1754. doi: 10.1128/JCM.00274-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Brown-Elliott BA, Philley JV, Griffith DE, Thakkar F, Wallace RJ Jr. 2017. In vitro susceptibility testing of bedaquiline against Mycobacterium avium complex. Antimicrob Agents Chemother 61:e01798-16. doi: 10.1128/AAC.01798-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Brown-Elliott BA, Wallace RJ Jr. 2018. In vitro susceptibility testing of bedaquiline against Mycobacterium abscessus complex. Antimicrob Agents Chemother 63:e01919-18. doi: 10.1128/AAC.01919-18. [DOI] [PMC free article] [PubMed] [Google Scholar]