Wild-type minimal inhibitory concentration distributions of secondline drugs in mycobacterium tubercolosis complex clinical isolated in relation to recommended critical concentrations in Limpopo Province, South Africa

Abstract

The reference phenotypic methods for Mycobacterium tuberculosis drug susceptibility testing are qualitative and based on drug critical concentrations. Limitations include lack of standardization and variations in laboratory preparation of drug stock solutions. The recommended critical concentrations are determined by consensus and experience rather than scientific data. Consequently incorrect and inadequate susceptibility breakpoints are used and patients receive ineffective antimicrobial therapy. The determination of wild-type minimal inhibitory concentration distribution is an important tool used by European Committee for antimicrobial susceptibility Testing (EUCAST) to establish clinical breakpoints in Europe. This could be applicable in South Africa. Aim To determine wild-type minimal inhibitory concentration distributions of first and secondline drugs against Mycobacterium tuberculosis complex clinical isolates and compare these with the recommended critical concentration in Limpopo province. Methods A sample of 101 Mycobacterium tuberculosis complex positive cultures were collected from National Health Laboratory Services in Polokwane (Limpopo province) and subcultured on BACTEC MGIT 960 system. The isolates were inoculated on MYCOTB MIC plates to determine the wild-type MIC distributions of first and second-line drugs. The data were compared with currently recommended critical concentrations. DNA was extracted and amplified by PCR. Genotypic drug susceptibility testing was performed using GenoType MTBDRplus version 2.0 and GenoType MTBDRsl version 2.0 for the first- and second-line drugs, respectively. Genotyping of clinical isolates was performed to determine M. tuberculosis strain families using spoligotyping. vi Results Wild-type MIC distributions range reported in this study are as follows rifampin (≤ 0.12 - 0.5 μg/μg/ml), isoniazid (≤ 0.3 - 2.00 μg/ml), rifabutin (≤ 0.12 - 0.25 μg/ml), ethionamide (≤ 0.12 - 5 μg/ml), ethambutol (≤ 0.5 - 2 μg/ml), streptomycin (≤ 0.25 - 0.5 μg/ml), paraaminosalicylic (≤ 0.5 - 4.0 μg/ml), cycloserine (≤ 2 -16 μg/ml), amikacin (≤ 0.12 - 0.5 μg/ml), kanamycin (≤ 0.6 -2.5 μg/ml), moxifloxacin (≤ 0.6 - 0.5 μg/ml), ofloxacin (≤ 0.25 - 1 μg/ml). GenoType MTBDRplus detected (n= 68, 67%) rifampin resistance (MUT 3=26, MUT 2=18, MUT 2B=8) on the rpoB gene. Isoniazid resistant (n=20, 19.8%) was detected katG MUT (n=20, 19.8%) on katG gene (S315T1). Genotypic resistance to second-line drugs determined by GenoType MTBRsl detected no mutations in (n= 98, 97%) isolates on gyrA, gyrB rrs and eis gene and (n=3, 2.9%) isolates non mycobacterium tuberculosis complex were detected. The frequency and percentage of Mycobacterium tuberculosis family strain were identified in (n= 81, 80%) of the clinical isolates which matched 18 pre-existing shared types. The results showed high genotype diversity with the Beijing strain (n= 30, 29.7%) and T family (n= 19, 18.8%) dominating. Twenty isolates (19.8%) had no shared types thus reported as orphan. Conclusion The findings obtained in this study suggest wild-type Minimal Inhibitory Concentration distributions may be considered when setting clinical breakpoints. Discordant results were observed between phenotypic and genotypic DST for rifampin, isoniazid, streptomycin, rifabutin and ethambutol, suggesting that breakpoint concentrations for some drugs are set too high while others are too low. The Mycobacterium tuberculosis clinical isolates displayed diverse family strain with Beijing and T strain predominate breakpoints for first-line and second-line drugs used in Mycobacterium tuberculosis treatments. Poster Presentations Poster presented at faculty of Health science first annual research day on Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using MYCOTB MIC plate. University of Limpopo Tiro hall 16th to 17th September 2014. Poster presented at National Health Laboratory Service Pathology Research and Development Congress (PathReD) on Determination of families strains of Mycobacterium tuberculosis circulating in Limpopo Province, South Africa. Emperors Palace 14th April-16th April 2015.