Evaluation of the MGIT 960/EpiCenter TB eXiST system for drug susceptibility testing for 1 Mycobacterium abscessus group

Background: Drug susceptibility test (DST) of the Mycobacterium abscessus group (MAG) and other 21 rapidly growing nontuberculous mycobacteria by conventional microplate techniques is complicated due 22 to inducible resistance to clarithromycin and other technical factors. This study evaluated the application 23 of the BACTEC MGIT 960/Epicenter TB eXiST for DST of MAG clinical isolates. 24 Methods: M. abscessus ATCC19977 was used as the reference strain for the standardizing the DST by 25 MGIT 960 and as the internal control for testing of 31 clinical isolates tests submitted to a reference 26 laboratory for DST and confirmed as MAG. Clarithromycin genotyping was performed for the loci in the rrl 27 and erm (41) genes known to impact resistance phenotype. 28 Results: The 31 MAG isolates included 14 M. abscessus , 8 M. massiliense , and 9 M. bolletii . Using 29 conventional microplate technique according to CLSI guideline, the isolates had a high percentage of 30 resistance for cefoxitin (93.5%) and imipenem (100%), and sensitivity for amikacin (96.7%). Comparing 31 microplate and MGIT 960 results across those 93 pairs of results (31 isolates x 3 antibiotics), 73 (80.6%) 32 were concordant and the remaining 18 (19.4%) represented minor errors; there were no major or very 33 major errors. Concordance was 100% for amikacin, 84% for imipenem and 58% for cefoxitin. susceptible susceptible 3 isolates as resistant after 3 days incubation, and 14 isolates inducible resistance from Day the latter isolates, MGIT 960 reported 8 as resistant and 6 as intermediate, without modifications to the protocol developed. all isolates, susceptibility the


Introduction
growing mycobacteria (RGM) causing clinically significant infection, accounting for 80% of lung disease 51 caused by RGM 1-4 . MAG isolates are widely distributed in the environment and have been associated with 52 both nosocomial and community-acquired opportunistic infections in compromised hosts and in persons 53 with underlying chronic lung disease, including patients with cystic fibrosis (CF) 1-4 . With advances in 54 diagnostic methods, there has been a worldwide increase in the reported incidence of MAG infections 5 .

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Treatment of these infections is substantially complicated by the high frequency of resistance to many       For all 31 isolates, the genotypes at erm(41) and rrl were compared with the results of CLR microplate susceptibility testing using the Day 14 reading and the subspecies identification (Table 3)

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Among the 17 resistant isolates, PCR indicated that all had an intact WT erm(41). Sequencing of rrl 149 identified only a single isolate with the A2058G mutation; that isolate also had intact erm(41) and was Initially, growth of ATCC 19977 T was assessed in CAMHB supplemented with OADC using dilutions of growth control to reach 400 growth units (GU) after 3.0 days. Growth of ATCC 19977 T in MGIT tube with 156 OADC was detected after one day of incubation for all dilutions up to 10 -4 , and after two and three days of 157 incubation for the 2x10 -5 and 10 -8 dilutions, respectively. Subsequently, the 10 -4 dilution was evaluated 158 with and without OADC enrichment and bacterial growth was detected after two and four days of 159 incubation, respectively. Similar results were obtained with a subset of the clinical isolates. Thus, for MAG 160 isolates, which have a faster intrinsic growth rate than MTB or slow-growing nontuberculous mycobacteria 161 (NTM), OADC enrichment resulted in accelerated growth rates inconsistent with the timeframes and 162 endpoints specified for the growth control (GC) in the MGIT 960 system. Consequently, in all subsequent 163 work OADC was omitted from both growth control and drug testing tubes.

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The appropriate dilution for DST of MAG by MGIT 960 was determined using three isolates with different 165 susceptibility profiles for CLR by REMA. The three isolates -ATCC 19977 T , which demonstrates 166 inducible resistance, plus two clinical isolates, one fully susceptible and one strictly resistant -were 167 evaluated using 10 -2 and 10 -3 dilutions prepared as described. Only the 10 -2 dilution consistently provided 168 the expected susceptibility profiles for all three CLR phenotypes (data not shown) and was subsequently 169 confirmed as satisfactory for the other antibiotics. dashed blue lines represent 10 -3 and 10 -2 dilutions, respectively, which reached 400 GU in <72 hours and, 176 as noted above, would not be valid controls. The other colored lines represent the growth curves in the the GC tube reaches 400 GU; in the example shown, GC reached 400 GU at 3.5 days, and so the assay endpoint is 10.5 days.

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The interpretation of the MGIT 960 system is based on the incubation time at which growth at the 182 breakpoint (also referred to as the critical concentration) for the antibiotic being tested (e.g., 2 mg/L for 183 CLA) reaches 100 GU relative to the time the GC reaches 400 GU and the time of the assay endpoint.

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The BACTEC MGIT 960 system is an automated system originally applied to drug susceptibility testing of has been enhanced by release of TB eXiST software that supports the use of multiple protocols, including

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The analysis of the CLR results is modestly complicated by the phenomenon of inducible resistance (IR).

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In conventional 3-day MIC testing, all isolates with IR would be falsely reported as susceptible. Detecting 254 IR requires a modified procedure in which the plates are read on multiple occasions over an extended 14-255 day incubation. Although this procedure is effort intensive, it is reliable, with genotyping indicating that all 256 isolates assessed as either R or IR had intact erm(41) and conversely, in all isolates assessed as S that clarithromycin promotes the activation of the erm(41) gene, leading to methylation of the drug binding site that were R or IR by the REMA method were reported as R or I by the MGIT 960 system, and all S 262 isolates gave concordant results with both systems. Thus, applying the standardized workflow described for the MGIT 960 system provided the clinically relevant result. Consequently, we suggest that, to provide