Underutilization of nontuberculous mycobacterial drug susceptibility testing in Ontario, Canada, 2010–2015

Abstract

Background

Drug susceptibility testing (DST) in nontuberculous mycobacterial pulmonary disease (NTM-PD) is useful for some Mycobacterium species. International guidelines recommend routine use of DST for clinically relevant mycobacteria. DST use and results are poorly studied at the population level. We sought to identify the frequency of DST utilization for nontuberculous mycobacteria (NTMs) and describe the potential relevance of these results in Ontario.

Methods

Using public health laboratory data, we performed a population-based retrospective analysis of NTM DST utilization in Ontario from May 2010 to June 2015. We determined the proportion of incident NTM-PD infections for which DST was performed and analyzed minimum inhibitory concentration (MIC) distributions from NTM testing overall, using thresholds recommended by the Clinical and Laboratory Standards Institute.

Results

The proportion of incident cases of NTM-PD tested for DST was 6.3% (240/3,806) for Mycobacterium avium complex (MAC), 36.2% (67/185) for M. abscessus, and 1.8% (19/1,057) for M. xenopi. Among specimens from all body sites, MAC resistance to clarithromycin occurred in 8.0% of specimens (21/262) and MAC resistance to amikacin (intravenous, MIC > 64 µg/mL) occurred in 22.6% (19/84). M. abscessus resistance occurred as follows: to amikacin, 3.8% (3/79); cefoxitin, 14.0% (11/79); imipenem, 30.4% (14/46); linezolid, 39.2% (31/79); clarithromycin, 54.2% (13/24); ciprofloxacin, 92.4% (73/79); and moxifloxacin, 91.1% (51/56). M. xenopi analysis was limited by few DST requests and a lack of DST clinical correlation.

Conclusions

We found that NTM DST is underutilized in Ontario and observed a very high frequency of amikacin resistance among MAC isolates.

Nontuberculous mycobacteria (NTM) are environmental organisms that can cause significant pulmonary disease (PD). Increasing prevalence of NTM-PD has been documented globally (1). In Ontario, a mean annual increase in NTM-PD of 6.5% was seen between 1998 and 2010, with an increase in prevalence from 29.3 per 100,000 during 1998–2002 to 41.3 per 100,000 during 2006–2010 (2). NTM-PD is difficult to treat, requiring prolonged courses of multiple antibiotics (3,4), and it has eradication rates as low as 30% (3) and disease recurrence rates approaching 50% within 4 years (5).

Selection of antibiotics for NTM-PD is challenging. The role of in vitro drug susceptibility testing (DST) is not fully understood (3,4). Minimum inhibitory concentration (MIC) breakpoints are recommended for only some of the drugs used for NTM-PD (4,6), and even for the drugs with recommended MICs clinical correlation is lacking for many.

It has been established that for Mycobacterium avium complex (MAC) pulmonary disease (MAC-PD), macrolides are critical antibiotics and in vitro resistance portends a poor prognosis (7). Macrolide resistance is uncommon in wild-type MAC (4,8). Aminoglycosides are important agents in MAC-PD (9), especially in organisms that show macrolide resistance (10–12) or in advanced disease, and knowledge regarding the utility of DST in this context is accumulating (10,13). For MAC, distinct MIC thresholds defining amikacin resistance have recently been recommended for intravenous (≥64 μg/mL) and inhaled liposomal (≥128 μg/mL) preparations, but recommendations for clarithromycin remain unchanged (14). For some other Mycobacterium species, for example M. abscessus, DST is recommended to assist with antibiotic drug selection. A functional erm(41) gene in M. abscessus ssp abscessus and ssp bolletii confers inducible resistance to macrolides that has been shown to be of critical clinical relevance (15–17). This understanding has led to the use of prolonged incubation with clarithromycin to provide meaningful DST to predict whether macrolides will have clinically significant antimicrobial effect. With this understanding, guidelines now recommend erm(41) gene sequencing to accurately and rapidly assess clarithromycin susceptibility (18). In contrast, published studies have highlighted the difficulties associated with DST and interpretation for M. xenopi (3,19,20).

The Clinical and Laboratory Standards Institute (CLSI) recommends only broth micro-dilution DST for NTM (18). The American Thoracic Society–Infectious Diseases Society of America (ATS–IDSA) guidelines recommend broth micro-dilution DST of clinically significant isolates when susceptibility is variable or when there is risk of acquired mutational resistance (4). Macrolide testing for MAC-PD is recommended for clinically significant isolates from patients with previously untreated disease, patients previously treated with macrolides, and patients who relapsed while on macrolide therapy (4). For M. abscessus lung disease, testing of all clinically significant isolates is recommended (4).

Recent guidelines from the British Thoracic Society (BTS) add amikacin DST to recommendations for MAC-PD and offer more explicit guidance on drugs to be tested against M. abscessus (21). Moreover, the BTS guidelines acknowledge the limitations of DST in NTM in general, advising that aside from examples with established clinically significant drug MIC–species combinations (e.g., clarithromycin for MAC and a few others), results of DST should be used to guide or recommend treatment regimens, not dictate them (21). The very limited data regarding interpretation of DST for NTM not only make optimal drug selection challenging, but also relegate the concept of antibiotic stewardship to a seemingly distant future prospect.

In Ontario, NTM DST is performed at the request of the clinician, whose discretion determines when this information is required. We investigated the frequency of requested NTM DST and profiles of antibiotic resistance in NTM isolates that are commonly clinically significant in Ontario. Some of this work has been published in abstract form (22).

Methods

We used Public Health Ontario Laboratory (PHOL) data for a retrospective analysis of NTM DST in Ontario from May 2010 to June 2015. PHOL identifies approximately 95% of all NTM isolated from culture in Ontario. Treating clinicians must request NTM DST, which is performed at the National Reference Centre for Mycobacteriology (NRCM), National Microbiology Laboratory (Public Health Agency of Canada, Winnipeg, Manitoba). We identified all DST for NTM during the study period. For each patient with DST performed on an isolate, we included only the first test performed for each species. In other words, patients with more than one NTM species isolated and tested, we included results for each species. If a patient had DST performed repeatedly for the same NTM species, we included only results of the first test for that species. We analyzed MIC distributions and the resistance frequencies for M. avium, M. intracellulare, M. abscessus, and M. xenopi and resistance frequencies for M. fortuitum, M. chelonae, and M. kansasii. Although clinical, radiological, and treatment data were not available, we compared the frequency of DST performance against the contemporary frequency of incident PD for MAC, M. abscessus, and M. xenopi (23), reasoning that for new MAC and M. abscessus cases, a substantial proportion should have had DST performed and for M. xenopi, in which the role of DST is less clear, likely a smaller proportion had it performed.

Mycobacteria were detected using the BACTEC MGIT 960 system (Becton, Dickinson and Company, Franklin Lakes, NJ), Lowenstein-Jensen solid media, or both. NTM DST was performed according to contemporary guidelines (6) by broth micro-dilution using Sensititre Trek SLOMYCO and RAPMYCO panels (Thermo Fisher Scientific, Oakwood Village, OH). M. abscessus clarithromycin DST data analysis was restricted to data from November 2013 onward, when subspeciation and functional erm(41) gene detection from PHOL and 14-day extended incubation with clarithromycin from NRCM were available.

Resistance was defined using the revised guidelines’ MIC breakpoints when available (14). For amikacin, MAC isolates were analyzed separately using the CLSI thresholds for both intravenous amikacin (≤16 µg/mL, 32 µg/mL, and ≥64 µg/mL for susceptible, intermediate, and resistant, respectively) and inhaled liposomal amikacin (≤64 µg/mL and ≥128 µg/mL for susceptible and resistant, respectively) (14).

Data were analyzed using Microsoft Excel (Microsoft Office 365, Redmond, WA). This study was approved by the Ethics Review Board at Public Health Ontario (Toronto, Ontario).

Results

Overall, 30,759 NTM isolates were identified from 14,155 unique patients between May 2010 and June 2015: 16,774 M. avium from 7,803 patients; 4,444 M. xenopi from 2,698 patients; 2,513 M. intracellulare from 1,087 patients; 1,256 M. abscessus from 428 patients; 918 M. fortuitum from 635 patients; 423 M. chelonae from 326 patients; 382 M. kansasii from 182 patients; and 4,049 other NTM species from 3,206 patients.

Of the 30,759 isolates, 546 (1.8%) were submitted for DST. Repeat patient–species combination isolates were removed, leaving 478 unique patient–species isolate combinations from 466 unique patients with a median (quartiles) age of 51.5 (29.3–71.6) years, of whom 59% were female. Of the 478 isolates, 44.4% were M. avium; 16.5%, M. abscessus; 10.5%, M. intracellulare; 8.4%, M. fortuitum; 6.7%, M. chelonae; 4.2%, M. xenopi; 2.9%, M. kansasii; and 6.5%, other. Pulmonary specimens made up 81.4%, nonpulmonary made up 12.3%, and the source was unknown for 6.3%.

Among unique patient–isolate pairs, DST was performed for pulmonary isolates in 240 patients with MAC, 67 patients with M. abscessus, and 19 patients with M. xenopi. From prior work, we identified contemporary incident cases of NTM-PD by species as follows: MAC, 3,806; M. abscessus, 185; and M. xenopi, 1,057 (23). Therefore, the proportion of incident cases of PD tested for DST was 6.3% (240/3,806) for MAC, 36.2% (67/185) for M. abscessus, and 1.8% (19/1,057) for M. xenopi.

Resistance data are presented for isolates from all anatomic sources. Table 1 outlines the frequency of resistance for M. avium and M. intracellulare to clarithromycin, moxifloxacin, linezolid, and amikacin. Among MAC isolates combined, macrolide resistance occurred in 21 of 262 (8.0%), resistance to intravenous amikacin (≥64 µg/mL) occurred in 19 of 84 (22.6%), and resistance to liposomal inhaled amikacin (≥128 µg/mL) occurred in 2 of 84 (2.4%). Corresponding MIC distributions are illustrated in Figures 1 and 2. Restricting to pulmonary MAC isolates yielded similar results; macrolide resistance occurred in 19 of 240 (7.9%), intravenous amikacin resistance (≥64 µg/mL) occurred in 19 of 77 (24.7%), and liposomal inhaled amikacin (≥128 µg/mL) resistance occurred in 2 of 77 (2.6%). Table 2 and Table 3 outline frequency of resistance for M. xenopi isolates and M. abscessus isolates, respectively. Among M. abscessus isolates, resistance occurred in the minority to amikacin (3/79; 3.8%), cefoxitin (11/79; 13.9%), imipenem (14/46; 30.4%), and linezolid (31/79; 39.2%), with resistance in the majority for other agents. Resistance to macrolides occurred in most subspecies abscessus and bolletii isolates (11/16; 68.8%), and few subspecies massiliense isolates (2/8; 25%). Restriction to pulmonary M. abscessus isolates yielded similar results, with resistance in the minority of cases to amikacin (3/67; 4.5%), cefoxitin (11/67; 16.4%), imipenem (13/37; 35.1%), and linezolid (27/67; 40.3%) and resistance in the majority for other agents.

Table 1:

Frequency of resistance observed for Mycobacterium avium and Mycobacterium intracellulare

	Breakpoint,* µg/mL			Isolates, no.	%
Species and Drug	S	I	R	S	I	R
M. avium
Clarithromycin	≤8	16	≥32	212	90.1	0.9	9.0
Moxifloxacin	≤1	2	≥4	210	40.0	27.1	32.9
Linezolid	≤8	16	≥32	212	10.4	25.0	64.6
Amikacin, IV	≤16	32	≥64	69	43.5	30.4	26.1
Amikacin, inhaled	≤64	N/A	≥128	69	97.1	0	2.9
M. intracellulare
Clarithromycin	≤8	16	≥32	50	96.0	0	4.0
Moxifloxacin	≤1	2	≥4	50	0	18.0	82.0
Linezolid	≤8	16	≥32	50	2.0	30.0	68.0
Amikacin, IV	≤16	32	≥64	15	73.3	20.0	6.7
Amikacin, inhaled	≤64	N/A	≥128	15	100.0	0	0

^* Clinical and Laboratory Standards Institute breakpoints (14) in vivo effectiveness of moxifloxacin and linezolid is described as unproven for MAC disease

S = Susceptible; I = Intermediate; R = Resistant; N/A = Not applicable; IV = Intravenous

Figure 1:

Distribution of clarithromycin MIC values for Mycobacterium avium and M. intracellulare isolates (14)

Arrow indicates CLSI resistant breakpoint ≥32 µg/mL.

M. avium MIC50 = 2 µg/mL (69%); M. intracellulare MIC50 = 2 µg/mL (84%). MIC = Minimum inhibitory concentration; CLSI = Clinical and Laboratory Standards Institute

Figure 2:

Distribution of amikacin MIC values for Mycobacterium avium and M. intracellulare isolates (14)

CLSI breakpoints are R ≥64 µg/mL and I = 32µg/mL (intravenous amikacin) and R ≥128 µg/mL (liposomal inhaled).

M. avium MIC50 = 32 µg/mL (74%); M. intracellulare MIC50 = 16 µg/mL (73%) MIC = Minimum inhibitory concentration; CLSI = Clinical and Laboratory Standards Institute; R = Resistant; I = Intermediate

Table 2:

Frequency of resistance observed for Mycobacterium xenopi isolates

			No. (%)
Drug	Breakpoint,* µg/mL	Isolates, no.	S	R
Clarithromycin	R >16	19	19 (100)	0
Rifampin	R >1	19	14 (74)	5 (26)
Amikacin	R >32	19	19 (100)	0
Ciprofloxacin	R >2	19	19 (100)	0
Moxifloxacin	R >2	7	6 (86)	1 (14)
Rifabutin	R >2	19	19 (100)	0

* Clinical and Laboratory Standards Institute breakpoints (14)

R = Resistant; S = Susceptible

Table 3:

Frequency of resistance observed for Mycobacterium abscessus isolates

	Breakpoint,* µg/mL				No. (%)
Drug	S	I	R	Isolates, no. (%)	S	I	R
Amikacin	≤16	32	≥64	79	68 (86)	8 (10)	3 (4)
Cefoxitin	≤16	32–64	≥128	79	4 (5)	64 (81)	11 (14)
Ciprofloxacin	≤1	2	≥4	79	2 (3)	4 (5)	73 (92)
Doxycycline	≤1	2–4	≥8	79	0	1 (1)	78 (99)
Imipenem	≤4	8–16	≥32	46	0	32 (70)	14 (30)
Linezolid	≤8	16	≥32	79	26 (33)	22 (28)	31 (39)
Moxifloxacin	≤1	2	≥4	56	2 (4)	3 (5)	51 (91)
Clarithromycin by ssp†	≤2	4	≤8
All ssp				24	11 (46)	0	13 (54)
ssp abscessus				13 (54)	4 (31)	0	9 (69)
ssp massiliense				8 (33)	6 (75)	0	2 (25)
ssp bolletii				3 (13)	1 (33)	0	2 (67)

^* Breakpoints as per the Clinical and Laboratory Standards Institute for rapidly growing mycobacteria (14)

^† Analysis of clarithromycin drug susceptibility testing data for M. abscessus were restricted to November 2013–June 2015 (extended incubation for clarithromycin instituted November 2013 at the National Reference Centre for Mycobacteriology)

S = Susceptible; I = Intermediate; R = Resistant

Discussion

In our comprehensive analysis of unique patient–isolate NTM DST in Ontario between May 2010 and June 2015, we found that DST was performed for only 6.3% and 1.8% of incident MAC-PD and M. xenopi-PD cases, respectively, and for 36.2% of M. abscessus-PD cases. This divergence from guidelines leads to inadequate patient care and, moreover, could contribute to increases in macrolide-resistant isolates. Although DST is guideline recommended and available on request (turnaround time approximately 4 wk), it is possible that clinicians are unaware of its availability or the recommendations. Alternatively, clinicians may elect to treat empirically and request DST only in the event of inadequate response.

Regardless, the 8% proportion of macrolide resistance among MAC isolates (7.9% for pulmonary isolates) implies that a significant minority of patients would receive grossly inadequate therapy in the absence of DST results, that DST is underused for MAC, and that clinicians should test all patients who may be treated. It was recently observed that 24.3% of MAC-PD patients in Ontario during 2001–2013 were treated with anti-MAC regimens (24). Applying this proportion of treated cases to the 3,806 incident cases during the current study period yields an expectation that 925 patients would have been treated. Our observation in the current study, that 240 patients with MAC-PD had DST, in a period when we expected 925 patients to be treated for MAC-PD, suggests that only about one-fourth of patients treated for MAC-PD had DST to guide therapy.

The rate of DST performance for M. abscessus was much higher, with more than one-third of patients having an isolate tested, compared with 6.3% and 1.8% for MAC and M. xenopi, respectively. On one hand, it is perhaps reassuring that M. abscessus was tested more frequently, given that macrolide resistance is common and its presence is of critical importance (16). On the other hand, the 36.2% request rate appears to be low, unless providers are reserving DST request until treatment is deemed necessary. However, the wisdom of such a practice could be questioned given the DST turnaround time (for M. abscessus, typically 2.5–5 wk depending on whether the isolate is recent or already archived [frozen]) and the potentially rapid deterioration with M. abscessus-PD, highlighting the utility of the rapidly available erm(41) and rrl gene sequencing, which is now routinely reported by PHOL (rrl since 2016) for M. abscessus isolates. It is suggested that patients with M. abscessus-PD should have DST performed, even if antimicrobial treatment is not immediately indicated (4). Better communication with health care providers regarding recommendations for DST and availability of testing may improve the use of NTM DST in our setting. Promotion of guidance documents such as the Best Practices for Pulmonary Nontuberculous Mycobacteria (25) may aid this communication.

The substantial macrolide resistance among MAC isolates likely reflects macrolide use in the absence of adequate companion drugs to prevent resistance. A previous study has shown that macrolide monotherapy for 4 months was followed by resistance in 20% of patients, whereas standard three-drug therapy—a macrolide, ethambutol, and a rifamycin—was associated with a 0.7% risk of macrolide resistance within the first 6 months of therapy and a 4% risk of macrolide resistance at any time in the future (11). Although ethambutol is thought to be a particularly important companion drug in protecting against macrolide resistance (11,26), fluoroquinolones are felt to provide no protection against the development of macrolide resistance. Two-drug therapy with a macrolide and a fluoroquinolone is believed to impart a risk of macrolide resistance similar to that of macrolide monotherapy (11,26). Macrolide resistance is an important issue in MAC-PD in that it is an extremely challenging, and not infrequently lethal, infection that is largely iatrogenic and thus preventable (4,11,12). Aminoglycosides are important agents in MAC-PD (9) in cases of macrolide resistance (10–12) and in advanced disease (4).

The interpretation of MAC resistance to amikacin in our study varies substantially depending on which resistance breakpoint is used. Focusing on pulmonary isolates, based on the CLSI threshold for intravenous amikacin (R MIC ≥ 64 μg/mL), resistance in 24.7% of M. avium–M. intracellulare isolates is truly alarming for this important agent. However, applying the recommended threshold for liposomal inhaled amikacin (R MIC ≥ 128 μg/mL) to pulmonary M. avium–M. intracellulare isolates identifies resistance in 2.6%, a reassuringly small proportion. It is not clear which threshold is most relevant in routine clinical care. In the work of Brown-Elliott et al (10), a threshold of more than 64 μg/mL was reported to be associated in all cases with a 16S rRNA gene A1408G mutation and, when clinical records were available, extensive prior treatment that usually included amikacin. However, the gene mutation was absent in all isolates with MIC ≥ 64 μg/mL. On the basis of Brown-Elliott et al’s data (10), perhaps a higher MIC threshold (>64 μg/mL) might be preferred for intravenous amikacin in MAC lung disease, but this requires additional clinical data. The higher MIC threshold recommended for inhaled liposomal amikacin (≥128 μg/mL) seems consistent with the data from Brown-Elliott et al (10) as well as with those from Olivier et al (13), the latter of which was a study of inhaled liposomal amikacin.

The lack of clinical information prevents speculation as to whether high MICs may have been generated through prior extensive use of amikacin or other aminoglycosides for gram-negative infections. Validating breakpoints with the best clinical correlation is important to guide therapy and may depend on the route of administration, as per the current CLSI guidelines, because inhaled amikacin and intravenous amikacin would each be expected to deliver significantly different drug concentrations depending on regional pulmonary perfusion and ventilation (10). In our setting, with the lack of ready availability of liposomal inhaled amikacin and easily available intravenous amikacin, we have concerns that the CLSI MIC for amikacin in MAC identifies a very large proportion of patients with amikacin resistance.

Only 19 M. xenopi patients had DST performed despite the fact that this species is the second most common cause of NTM-PD in Ontario, with 1,057 incident cases previously identified during the study period (23). Assuming that 15% of M. xenopi-PD patients in Ontario are treated (24), we estimate that 158 incident cases were treated during the study period. The lack of data supporting the utility of DST for M. xenopi may explain the very low level of request for DST. The ATS–IDSA currently do not provide strong recommendations for first-line therapy of M. xenopi-PD while emphasizing that an effective treatment regimen has yet to be established (4). It has been stated that isoniazid, a rifamycin, ethambutol, and clarithromycin ± adjunctive streptomycin in the first months may comprise a reasonable regimen, and moxifloxacin could be substituted for one of the antituberculous agents (4). The recent BTS recommendations consist of rifampin, ethambutol, and a macrolide, with either a quinolone or isoniazid (21).

In our study, M. xenopi showed no resistance to clarithromycin, amikacin, ciprofloxacin, and rifabutin; low resistance (14.3%) to moxifloxacin; and higher levels of resistance to rifampin (26.3%). We observed resistance to rifampin in 26.3% (5/19) of isolates and resistance to moxifloxacin in 14.3% (1/7) of isolates. We did not observe any resistance to the other drugs tested. Prior studies reporting DST in M. xenopi isolates include a 15-year laboratory survey from one institution in England that included 219 isolates (27), a clinical series from the Netherlands that included 42 isolates (19), and a clinical series from France that included 136 patients but that did not indicate the proportion for whom testing was performed (20). Resistance rates to clarithromycin were reported in the study from England (1/219; <1%) and France (2.5%), similar to our finding of no resistance. Resistance to rifampin was very uncommon in the study from England (3/217[1.2%], using a higher MIC threshold of 2), but comparable to ours (5/19; 26.3%,) and the studies from France (40%) and the Netherlands (13/42; 31.0%). Resistance to amikacin, ciprofloxacin, and rifabutin was very uncommon, in both our study and the study from England, with rates of 0%–2% for all cases. The interpretation of these findings is difficult, however, in that clinical studies to support relevant MIC thresholds are absent (4,19,20).

Regarding M. abscessus, the ATS–IDSA guidelines suggest that therapy with a macrolide and amikacin with 2–4 months of cefoxitin or imipenem has been shown to provide some clinical and microbiological improvements and that a combination of parenteral drugs selected on the basis of DST may be useful in cases of macrolide resistance (4). The BTS recommends that DST for M. abscessus should include clarithromycin, cefoxitin, and amikacin (and preferably also tigecycline, imipenem, minocycline, doxycycline, moxifloxacin, linezolid, co-trimoxazole, and clofazimine if a validated method is accessible) to guide, but not dictate, treatment regimens (21). We should note that this recommendation is not consistent with ATS–IDSA guidelines, which do not recommend minocycline, doxycycline, moxifloxacin, or co-trimoxazole for M. abscessus and that, as of yet, there are no accepted interpretive guidelines for clofazimine MICs (4,14).

The first-line treatment recommendation by the BTS includes three intravenous agents plus a macrolide (in the absence of constitutive macrolide resistance), with treatment guided by DST results, relying particularly on the results regarding clarithromycin and amikacin and to a lesser extent on those for the other agents (21). In our study, M. abscessus isolates were usually resistant to moxifloxacin (91%), ciprofloxacin (92%), and doxycycline (99%) but less often resistant to linezolid (39%), rates that are similar to those presented in a 2012 review by Brown-Elliott et al (8). Resistance to amikacin was infrequent (4%), similar to the results of Brown-Elliott et al (8) and Griffith et al (11), although higher rates of resistance to amikacin were identified in a large DST study in the Netherlands (7). For M. abscessus, imipenem DST demonstrated intermediate resistance in 70% of our isolates and resistance in 30%. Many laboratories do not report MICs for this drug–species combination as a result of issues with interpretation and reproducibility of testing (8). Although this rationale is understandable, this approach is questionable from the perspective of the now long-established recommendations made in 2011 regarding interpretation of imipenem MICs for M. abscessus (6). We think it is appropriate to present the MICs despite limitations but, given the lack of studies demonstrating clinical correlation with MICs, are uncertain of their utility in patient management. In our study, M. abscessus was frequently found to have intermediate resistance to cefoxitin (81%), consistent with a previous review demonstrating typically low to intermediate cefoxitin MICs (8).

Of the 24 isolates of M. abscessus that underwent extended incubation with clarithromycin, resistance was seen in 69% (9/13) of ssp abscessus, 25% (2/8) of ssp massiliense, and 67% (2/3) of ssp bolletii. As expected, a minority of M. abscessus ssp abscessus and ssp bolletii were found to be susceptible to macrolides, making up 31% of M. abscessus ssp abscessus and 33% of M. abscessus ssp bolletii isolates (28,29). Presumably this is due to high rates of inactivating mutations in the erm(41) gene in the presence of a C28 sequevar (15,17). Although routine erm(41) gene sequencing was implemented at PHOL in July 2013, not enough data were available for analysis because phenotypic DST was performed on request only. The finding that most (2/3) M. abscessus ssp massiliense isolates were macrolide resistant is surprising, given that isolates of this subspecies have a truncated inactive erm(41) gene and thus lack inducible macrolide resistance. The macrolide resistance identified in these isolates must therefore indicate mutational (rrl gene) resistance. Unfortunately, these data predate the PHOL introduction of rrl gene analyses for M. abscessus, and therefore rrl mutational status cannot be verified.

Our study had several limitations. First, because clinical and radiological data were unavailable, neither the clinical significance of isolates nor prior antibiotic exposure could be assessed. Although we believe it is likely that clinicians requested DST on clinically significant isolates, the lack of information regarding prior and current antibiotic therapy limits our ability to explore the potential effect of this exposure on our results. Second, our method of estimating the proportion of incident cases with DST was imperfect, given that DST may have been performed on a patient whose disease was incident before our study period, whereas we used measures of disease that were incident during the study period. The direction of bias from this limitation is not clear, but given the chronicity of NTM-PD, the magnitude is likely small. Third, when DST is only completed at the request of the submitting physician, there is potential for specimen submitter bias. Specifically, it is possible that DST is more likely to be requested by physicians with expertise in NTM-PD, who may be more likely to treat complex cases, and when patients are not responding well to standard therapy. Both of these situations seem likely to bias toward higher levels of drug resistance, so perhaps our findings represent overestimates in this regard.

In summary, NTM DST appears to be vastly underutilized in Ontario, with a significant proportion of macrolide resistance among tested MAC isolates and DST performed in only 36.2% of patients with incident M. abscessus-PD. Clinicians in Ontario should be strongly encouraged to increase appropriate utilization of DST for NTM-PD. In addition, new CLSI-recommended MIC thresholds for intravenous amikacin in MAC suggest that a large minority of MAC patients have amikacin-resistant strains in Ontario. Clinicians should carefully consider amikacin MICs for MAC isolates and perhaps request repeat testing when the MIC appears high. More data correlating amikacin MIC with response to this drug would be welcome. More data are needed regarding DST interpretation, especially for M. xenopi. Finally, data correlating DST for NTM species with clinical outcomes are sorely needed to facilitate optimal drug selection and antibiotic stewardship.

Funding Statement

This research was funded by Public Health Ontario.

Competing Interests:

Dr Brode reports grants from Insmed and personal fees from Boehringer Ingelheim and Astra-Zeneca outside the submitted work. Dr Marras reports grants and personal fees from Insmed and personal fees from Astra-Zeneca, Horizon, RedHill, and Novartis outside the submitted work;

Ethics Approval:

This study was approved by the Ethics Review Board at Public Health Ontario (Toronto, Ontario, Canada).

Informed Consent:

N/A

Registry and the Registration No. of the Study/Trial:

N/A

Animal Studies:

N/A

Funding:

This research was funded by Public Health Ontario.

Peer Review:

This article has been peer reviewed.