Five-antituberculosis Drug-resistance Genes Detection Using Array System.

Detection of resistance to drugs for Mycobacterium tuberculosis takes about two months from the sample collection using culture-based methods. To test a rapid method for detection of resistance for five antituberculosis drugs using DNA microarray and to examine its potential for clinical use, we employed a DNA microarray for detection of seven mutations genes related to resistance of five kinds of antituberculous drugs using Mycobacterium tuberculosis DNA isolated from sputum. The results of microarray analysis were compared with the results of a standard culture method of Lowenstein-jensen drug sensitivity testing system. DNA microarray analysis showed a high sensitivity (>90%) for all five drugs. Specificity of rifampicin and ethambutol were nearly 90%, however specificity of isoniazid (60%) and kanamycin (67%) were not enough. The amount of Mycobacterium tuberculosis DNA required for microarray analysis corresponded to at least 1-9 Acid-Fast Bacilli per 10 fields by carbolfuchsin staining. DNA microarray analysis appears to be useful for estimation of drug resistances, nevertheless its limitations. To minimize misunderstanding, it is necessary to confirm the number of bacilli in the sputum, and culture method is needed for comparison when use the PCR-based array system.

DNA microarray genotyping of N-acetyltransferase 2 polymorphism using carbodiimide as the linker for assessment of isoniazid hepatotoxicity.

The antituberculous drug isoniazid (INH) is acetylated by N-acetyltransferase 2 (NAT2), and the frequency of INH-induced hepatotoxicity is determined by the NAT2 genotype. NAT2 genotyping is not done routinely in hospital laboratories because of its difficulty. Use of microarrays for research is becoming common and its expectations of clinical application are increasing. In this study, we attempted to develop an easier method of NAT2 genotyping for clinical use. We devised a novel oligonucleotide-based DNA microarray for NAT2 genotyping using a recently developed technique for attaching oligonucleotide probes to poly carbodiimide-coated glass slides, which achieves a stronger hybridization signal and better specificity than the more widely used aminosilane-coated slides. To assess the validity of this microarray, four clones with NAT2 mutations and DNA from 42 tuberculosis patients were investigated by the microarray method and by restriction fragment length polymorphism analysis. The results of genotyping by these two methods were in agreement. Analysis of the relationship between the NAT2 phenotype determined by the DNA microarray and the risk of INH-induced hepatotoxicity revealed that slow acetylators had a significantly higher risk. These findings suggest that our microarray may be clinically useful for predicting drug reactions to INH.