Rapid assessment of drug susceptibility and mutation to resistance in mycobacterium tuberculosis Beijing type

The increasing rates of multidrug resistant tuberculosis (MDR-TB) worldwide, are seriously threatening the TB control efforts. The present thesis was aimed at increasing the knowledge about the role of different factors, such as the development of drug resistance and the efficacy of drug susceptibility assessments, in the problem with MDR-TB. We showed that the two novel commercial techniques, BacT/ALERT (bioMerieux) and INNO-LiPA Rif TB (Innogenetics), are sensitive and reliable methods for the determination of drug susceptibility in Mycobacterium tuberculosis (TB). Two different test protocols of the culture-based BacT/ALERT system were evaluated at separate times in our laboratory. After the first test episode (2002-2003), we concluded this system to be applicable for isolation of M. tuberculosis but that further optimization was needed for the assessment of drug susceptibility, since the system had too low sensitivity in detecting resistance to rifampicin and streptomycin. The results from the second test episode (2005-2006), using a novel protocol, showed improvement of detecting drug resistance to the three first line drugs; rifampicin, isoniazid and ethambutol. Apart from the high accuracy in determining drug resistance, advantages like the automated standardization of inoculum, recording and saving of data make this system a valid alternative for drug susceptibility testing of M. tuberculosis. The LiPA detects resistance to rifampicin by the identification of mutations in the rifampicin resistance-determining region of the rpoB gene. We evaluated this assay since a rapid detection of rifampicin resistance, and indirectly multidrug resistance (MDR), is of great importance to ensure an effective treatment that will break the chain of transmission and prevent the development of drug resistance. When using the BACTEC 460 methodology as the reference, the LiPA correctly determined all 27 rifampicin resistant strains and 24 of the 26 susceptible strains. The two aberrant samples were both determined to harbour a Leu511Pro mutation, thus confirming LiPAs correctness in finding mutations. MIC determination did, however, reveal susceptibility to rifampicin of the two strains. Due to LiPAs rapidity, easiness and high accuracy, it is a valuable tool that offers an early warning system for MDR-TB. The detection and identification of M. tuberculosis strains with increased potential to develop and spread drug resistance is important to notice, since such clones challenge the control and treatment regimens of TB. During the last decade attention has been paid to the M. tuberculosis Beijing genotype in particular. Strains belonging to the Beijing genotype have been responsible for massive spread and outbreaks of MDR-TB, especially in the regions of the Former Soviet Union. Suggestions that these strains easier gain drug resistance and/ or are more virulent have been made. We determined the rate at which 13 M. tuberculosis strains (including Beijing) mutated to rifampicin resistance. All strains, irrespective the genotype, showed a mutation rate of ∼ 1 × 10 mutations/cell/generation. Furthermore, we characterized the type and frequency of rpoB mutations in 189 rifampicin resistant mutants selected in vitro. Eighty-nine mutants were of the Beijing genotype. In summary, the Ser531Leu and His526Tyr mutations were predominant in both groups of genotypes, which also mirror the distribution in clinically rifampicin resistant strains. Interestingly, the only multiple mutations we selected, were exclusively present in the Beijing mutants. From this work new knowledge about rifampicin resistance in Beijing strains has been achieved on the genomic level. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I. Angeby K, Werngren J, Toro JC, Hedstrom G, Petrini B and Hoffner SE. Evaluation of the BacT/ALERT 3D System for Recovery and Drug Susceptibility Testing of Mycobacterium tuberculosis. Clinical Microbiology and Infection, 2003; 9:1148-1152. II. Werngren J, Klintz L and Hoffner SE. Evaluation of a Novel Kit for Use with the BacT/ALERT 3D System for Drug Susceptibility Testing of Mycobacterium tuberculosis. Journal of Clinical Microbiology, 2006; 44:2130-2132. III. Jureen P, Werngren J and Hoffner SE. Evaluation of the Line Probe Assay (LiPA) for Rapid Detection of Rifampicin Resistance in Mycobacterium tuberculosis. Tuberculosis, 2004; 84:311-316. IV. Werngren J and Hoffner SE. Drug-Susceptible Mycobacterium tuberculosis Beijing Genotype Does Not Develop Mutation-Conferred Resistance to Rifampin at an Elevated Rate. Journal of Clinical Microbiology, 2003; 41:1520-1524. V. Huitric E, Werngren J, Jureen P and Hoffner SE. Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants. Antimicrobial Agents and Chemotherapy, 2006; 50:2860-2862.