Rapid speciation of 15 clinically relevant mycobacteria with simultaneous detection of resistance to rifampin, isoniazid, and streptomycin in Mycobacterium tuberculosis complex.
OBJECTIVE
To design and standardize an in-house reverse line blot hybridization (RLBH) assay for the accurate identification of 15 clinically relevant species of mycobacteria and for the detection of drug resistance to rifampin (RIF), isoniazid (INH), and streptomycin (STR) in Mycobacterium tuberculosis complex (MTB).


METHODS
Oligonucleotides specific for 15 different species of mycobacteria and wild type and mutant alleles of selected codons in the rpobeta, inhA, katG, rpsL, and rrs genes were designed and immobilized on a membrane. A multiplex PCR was standardized to amplify all target genes. The assay was optimized using ATCC and known mutant strains. Three hundred MTB isolates, 85 non-tuberculous mycobacteria (NTM) isolates, and 48 smear-positive specimens were analyzed. Results were confirmed by PCR restriction enzyme assay and sequencing.


RESULTS
Upon RLBH analysis, among the NTM, 14% were identified as Mycobacterium fortuitum, 16% were identified as Mycobacterium abscessus, 20% showed 99% homology with Mycobacterium intracellulare, and 31% showed 98% homology with Mycobacterium simiae. Of the 300 MTB isolates analyzed, 75% RIF-resistant isolates had Ser531Leu mutation in the rpobeta gene. Of the INH-resistant isolates, 89% showed Ser315Thr mutation in the katG gene, whereas 16% showed -15 C-->T mutation in the promoter region of the inhA gene. Among STR-resistant isolates, 75% had A-->G mutation in the rpsL gene at codon 43. RLBH results showed 96-99% concordance with phenotypic culture results.


CONCLUSIONS
This is a first attempt at combining speciation with detection of drug resistance to RIF, INH, and STR in MTB for accurate and rapid management of mycobacterial infections as well as for compiling genotypic epidemiological data.
P.D. Hinduja National Hospital and Medical Research Centre, Maharashtra, India.
Shubhada
S
Shenai
Camilla
C
Rodrigues
Ajita
A
Mehta
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D015342:DNA Probes; D024881:Drug Resistance, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000187:drug effects* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012293:Rifampin / Q000494:pharmacology; D013307:Streptomycin / Q000494:pharmacology; D013997:Time Factors; D014376:Tuberculosis / Q000453:epidemiology* / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000453:epidemiology* / Q000382:microbiology
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D015342:DNA Probes; D007538:Isoniazid; D012293:Rifampin; D013307:Streptomycin