Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis by a Single Multiplex Allele-specific Polymerase Chain Reaction Assay

(Pages: 97-102)
Kimia Taghavi, M.Sc., 1Parissa Farnia, Ph.D., 1Mohammad Varahram, Ph.D., 1,*Fatemeh Maryam Sheikhoslami, Ph.D., 1Mojtaba Ahmadi, B.Sc., 1Mehdi Kazempoor, M.Sc., 2Mohammad Reza Masjedi, Ph.D., 3Ali Akbar Velayati, Ph.D., 4
* Corresponding Address:P.O. Box: 19575/154 Mycobacteriology Research Centre National Research Institute of Tuberculosis and Lung Disease (NRITLD) Shahid Beheshti Medical UniversityTehran Iran Email: mvarahram@nritld.ac.ir
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Taghavi Kimia, Farnia Parissa, Varahram Mohammad, Sheikhoslami Fatemeh Maryam, Ahmadi Mojtaba, Kazempoor Mehdi, Masjedi Mohammad Reza, Velayati Ali Akbar. Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis by a Single Multiplex Allele-specific Polymerase Chain Reaction Assay. Cell J. 2011; 13(2): 97-102.

Abstract

Objective:

Global surveillance has shown that drug resistant (DR) tuberculosis (TB) is widespread. Prompt detection of Mycobacterium tuberculosis drug resistance is essential for effective control of TB. The most frequent mutations associated with Isoniazid (INH) resistance in Mycobacterium are substitutions at codons 315 in the katG gene and the mabA-inhA promoter region (−15). This survey evaluated INH resistant-associated mutations in order to determine rapid detection of TB resistance.

Materials and Methods:

Through a descriptive cross- sectional study total of 96 sputum specimens were digested, examined microscopically for acid-fast bacilli and inoculated into Löwenstein-Jensen slants. Thereafter, the susceptibility and identification tests were performed on culture positive specimens. Subsequently, the strains were subjected to multiplex allele-specific polymerase chain reaction (MAS-PCR) targeting in the codons 315 in the katG gene and the mabA-inhA promoter region. Distinct PCR banding patterns were observed for different mutation profiles.

Results:

Drug susceptibility testing revealed that out of 96 available isolates, 30 (31.3%) were susceptible, 36 (37.5%) had multi-drug resistance (MDR-TB) and 30 (31.3%) showed mono- drug resistance. In comparison with the culture-based phenotypic drug susceptibility test, the sensitivity and specificity of MAS-PCR assay for drug resistance-related genetic mutations were 76.7% and 71.4%, respectively. The correlation between MAS-PCR and culture-based phenotypic drug susceptibility testing findings was 99.4%.

Conclusion:

The profile of the isolates suggests a significant number of different DR strains with a high frequency of mutations at codon 315 of the katG gene. MAS-PCR provides a rapid, simple and cost-effective method for detecting MDR-TB.