C-211. Rapid Detection of Mycobacterium spp. Resistance to Antimicrobial Agents by Real-Time Reverse Transcription PCR

J-M. Balada-Llasat1, C. K. Wallis2, B. A. Brown-Elliott3, R. J. Wallace, Jr3, F. C. Fang1,2;
1Univ. of Washington, Seattle, WA, 2Harborview Med. Ctr., Seattle, WA, 3Univ. of Texas Hlth. Sci. Ctr., Tyler, TX.

Antibiotic-resistant Mycobacterium spp. pose a considerable challenge for clinicians, laboratories, and public health officials. Conventional methodologies for the detection of resistance rely on bacterial growth, resulting in a considerable delay in diagnosis. Molecular methods have the potential to expedite susceptibility testing. In the present study, we monitored 16s rRNA by real-time reverse transcription PCR (RT-PCR) to distinguish clarithromycin-resistant and -susceptible Mycobacterium avium complex (MAC) strains. Twenty-six resistant (MIC > 32 mg/L) and twenty-four susceptible clinical MAC isolates were grown in the absence or presence of 32 mg/L clarithromycin, with rRNA measured at 48h post-inoculation. The difference in Ct values (ΔCt = Ct [32 clarithromycin] - Ct [0 clarithromycin]) was measured in the absence or presence of clarithromycin. The rRNA levels in resistant strains were relatively indifferent to the presence of 32 mg/L clarithromycin, with mean ΔCt 2 ± 2, whereas susceptible strains exhibited reduced rRNA levels following clarithromycin treatment with mean ΔCt 9.4 ± 2. Strains were defined as resistant when ΔCt < 4 and susceptible when ΔCt > 7. Using these criteria, 44 of 50 strains were correctly identified as susceptible or resistant as corroborated by classical macrodilution and E-test methods. RT-PCR reduced the time required for the detection of resistance from 7-14 to 2 days. The remaining 6 strains were indeterminate by the RT-PCR method and consisted primarily of resistant strains in which significant clarithromycin-dependent changes in rRNA levels were observed. In conclusion, RT-PCR has the potential to substantially reduce the time required to determine the susceptibility of Mycobacterium spp. to antimicrobial agents. Further methodological modifications may be required to reduce the number of indeterminate strains.

165/C. Diagnostic Mycology and Parasitology

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