ABSTRACT
The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis.
Subject(s)
Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Mycobacterium tuberculosis/genetics , Polymerase Chain Reaction/methods , Rifampin/pharmacology , Tuberculosis, Multidrug-Resistant/microbiology , Bacterial Proteins/genetics , Catalase/genetics , DNA-Directed RNA Polymerases , Humans , Microbial Sensitivity Tests/methods , Mutation, Missense , Mycobacterium tuberculosis/drug effects , Oxidoreductases/genetics , Sensitivity and SpecificityABSTRACT
A total of 196 rifampicin (RMP) resistant Mycobacterium tuberculosis isolates were searched for mutations in the cluster I region of the rpoB gene. Seventeen different types of mutations were observed in 184 (93.9%) isolates. The remaining isolates searched also had no mutations in the N-terminal region of this gene. Mutations were most frequently detected at single codons Ser531 (47.4%), His526 (20.9%), and Asp516 (20.4%). Our data indicate that genotypic drug susceptibility assays based on detection of mutations at the specific codons of the rpoB gene should be effective for predicting resistance to RMP and rifabutin and multidrug resistance in Lithuania.
Subject(s)
Antibiotics, Antitubercular/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Rifampin/pharmacology , Bacterial Proteins/physiology , Codon , DNA Mutational Analysis , DNA, Bacterial/analysis , DNA-Directed RNA Polymerases , Humans , Lithuania , Mycobacterium tuberculosis/pathogenicity , Polymerase Chain ReactionABSTRACT
BACKGROUND: Nations of the former Soviet Union have the world's highest reported levels of resistance to anti-tuberculosis drugs. We conducted the first national survey of anti-tuberculosis drug resistance in the Republic of Lithuania. METHODS: We tested Mycobacterium tuberculosis isolates from all incident culture-positive pulmonary TB patients registered in 2002. New patients were those treated for <1 month with any first-line anti-tuberculosis drug (isoniazid [INH], rifampin [RMP], ethambutol, or streptomycin); previously treated patients were those treated for > or =1 month. RESULTS: Of 1163 isolates, 475 (41%) were resistant to at least one first-line drug, and 263 (23%) were resistant to at least INH and RMP (MDR); this included 76/818 (9.3%) from new patients and 187/345 (54%) from previously treated patients. Of 52 MDR isolates randomly selected for extended testing at an international reference laboratory, 27 (51%, 95%CI 38-66) had resistance to pyrazinamide, 21 (40%, 95%CI 27-55) to kanamycin, and 9 (17%, 95%CI 8-30) to ofloxacin. CONCLUSIONS: The prevalence of MDR-TB in Lithuania is among the world's highest. Among MDR-TB isolates, aminoglycoside and fluoroquinolone resistance were common. To combat drug-resistant TB, Lithuania has implemented the WHO global TB control strategy (DOTS), and is developing an MDR-TB treatment program (DOTS-Plus).
Subject(s)
Tuberculosis, Multidrug-Resistant/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Ethambutol/pharmacology , Female , Humans , Isoniazid/pharmacology , Lithuania/epidemiology , Male , Middle Aged , Mycobacterium tuberculosis/drug effects , Prevalence , Random Allocation , Rifampin/pharmacology , Streptomycin/pharmacologyABSTRACT
In the first attempt to establish a quality assurance programme for susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones, 20 strains with different fluoroquinolone susceptibility patterns were distributed by the Supranational Reference Laboratory in Stockholm to the other mycobacterial reference laboratories of the Nordic and Baltic countries. Susceptibility testing to fluoroquinolones was performed according to routine procedures in each laboratory. Results were compared to sequence analysis of the gyrA gene and minimal inhibitory concentration determination. Most laboratories found identical susceptibility patterns. The two resistant strains were correctly identified by all laboratories, but three laboratories each falsely reported one susceptible strain as resistant. These results indicate that the participating laboratories yield reliable results in detection of fluoroquinolone-resistant strains, although the need for a standardised quality assurance programme for drug susceptibility testing for fluoroquinolones is stressed by the strains falsely reported as resistant.
