Direct detection of isoniazid-resistant Mycobacterium tuberculosis in respiratory specimens by multiplex allele-specific polymerase chain reaction

• Published in: Diagnostic microbiology and infectious disease 2011, Vol.69 (1), p.51-58
• Main Authors: Siu, Gilman Kit Hang, Tam, Yuk Ho, Ho, Pak Leung, Lee, Ann Siew Gek, Que, Tak Lun, Tse, Cindy Wing Sze, Yip, Kam Tong, Lam, Jason Tsz Hin, Cheng, Vincent Chi Chung, Yuen, Kwok Yung, Yam, Wing Cheong
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Substitution - genetics; Bacterial Proteins - genetics; Bacteriology; Biological and medical sciences; Catalase - genetics; DNA, Bacterial - genetics; Drug Resistance, Bacterial; Fundamental and applied biological sciences. Psychology; Humans; Infectious Disease; Infectious diseases; INH resistance; Internal Medicine; Isoniazid - pharmacology; KatG; maba-inhA; Medical sciences; Microbial Sensitivity Tests - methods; Microbiology; Miscellaneous; Multiplex allele-specific PCR; Mutation, Missense; Mycobacterium tuberculosis; Mycobacterium tuberculosis - drug effects; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Oxidoreductases - genetics; Polymerase Chain Reaction - methods; Rapid diagnosis; Sputum - microbiology; Time Factors; Tuberculosis - microbiology
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2010.08.021

Abstract This study evaluated the feasibility of using 2 multiplex allele-specific polymerase chain reaction (MAS-PCR) assays targeting 2 mutations (codon 315 of the katG gene and the 15th nucleotide preceding the mabA-inhA operon) to directly detect isoniazid (INH)-resistant Mycobacterium tuberculosis in cultured isolates and respiratory specimens. A total of 203 M. tuberculosis isolates and 487 respiratory specimens were investigated. The MAS-PCR assays successfully amplified all M. tuberculosis isolates and acid-fast bacilli smear-positive specimens while only 49.2% of the smear-negative specimens exhibited positive MAS-PCR results. The MAS-PCR assays identified 83.4% and 79.2% of the resistant strains in the culture isolates and respiratory specimens, respectively. All the inferred genotypes were in complete accordance with subsequent DNA sequence analyses. This study suggested the application of our improved MAS-PCR protocols to provide the rapid identification of INH-resistant M. tuberculosis directly in respiratory specimens. The technical simplicity, short turnaround time, and low cost of this molecular strategy should facilitate routine diagnostic services in developing areas with a high prevalence of drug-resistant tuberculosis.