ABSTRACT
Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.
Subject(s)
Humans , Mycobacterium tuberculosis , Nucleic Acid Hybridization/methods , Polymerase Chain Reaction/methods , Sputum , Tuberculosis, Pulmonary , Colorimetry , DNA, Bacterial , Mycobacterium tuberculosis , Oligonucleotide Probes , Reagent Kits, Diagnostic , Sensitivity and SpecificityABSTRACT
Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mycobacterium tuberculosis , Mutation/genetics , Colorimetry/methods , DNA, Bacterial/analysis , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Nucleic Acid Hybridization , Polymerase Chain ReactionABSTRACT
BACKGROUND: Mycobacterium abscessus is the most pathogenic and drug-resistant rapid-growing mycobacterium. Clarithromycin or azithromycin are the only regular oral antimycobacterial agents that have an effect on M. abscessus. We tried to detect the clarithromycin-resistant strains from the clinical isolates of M. abscessus. METHODS: We tried to isolate the clarithromycin-resistant strains from 220 clinical isolates of M. abscessus by performing using reverse hybridization assay (RHA) and the broth microdilution test (BMT). RESULTS: Seven resistant strains (3.2%) from all the tested clinical isolates were detected by BMT. Three of these resistant strains were also detected by RHA and it was confirmed that they had point mutants. CONCLUSION: These results showed that clarithromycin resistance in M. abscessus clinical isolates is related to a point mutation and other unknown mechanisms.