Detection of Isoniazid-Resistant Clinical isolates of Mycobacterium tuberculosis from India using Ser۳۱۵Thr marker by Comparison of molecular methods

In this study, Substitution at codon Ser۳۱۵ of katG gene, a reliable marker for isoniazid (INH) resistance was analyzed and compared by three molecular methods such as DNA  sequencing, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and PCR-single strand conformation polymorphism (PCR-SSCP) in ۱۰۵ phenotypically resistant isolates obtained from various parts of India. Out of the ۱۰۵ resistant isolates, ۶۴ (۶۱%) were found to be resistant by DNA sequencing, ۵۴ (۵۱%) by PCR-RFLP and ۵۷ (۵۴%) by PCR-SSCP methods. The results obtained using PCR-SSCP and PCR-RFLP methods were compared with those from DNA sequencing (gold standard). The sensitivity and specificity of PCR-RFLP were ۸۴% and ۱۰۰% respectively and corresponding values for PCR-SSCP method were ۸۹% and ۹۵% respectively.  The study has shown the comparison of the simple, rapid and cost effective methods with DNA sequencing targeting codon Ser۳۱۵ of katG gene and suggests that PCR-RFLP and PCR-SSCP may be performed as alternative inexpensive methods in settings with a high prevalence of INH-resistant M. tuberculosis strains where sequencing cannot be afforded.

Keywords:

Mycobacterium tuberculosis , isoniazid resistance , DNA sequencing , PCR-SSCP , PCR-RFLP