Rapid screening of mutations for second-line-drug-resistant genes in Mycobacterium tuberculosis culture isolates by in-house developed DNA bio-chip.

BACKGROUND: The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing and is a major setback to TB control in India. The availability of quick and reliable methods for detecting second-line drug resistance (SLDR) is vital to managing patients satisfactorily. A rapid molecular technique to detect SLDR in Mycobacterium tuberculosis (M. tuberculosis) has been developed using DNA biochip.

METHODS: Specific probes containing wild-type region or specific mutations were designed for immobilization on DNA bio-chip. DNA bio-chip was developed in-house on polycarbonate track-etched membranes (PC-TEM). DNA bio-chip allows the identification of mutations in gyrA gene for fluoroquinolone (FQ) resistance, in rrs gene and the eis promoter region for resistance to second-line injectable drugs (SLID). An asymmetric multiplex PCR was standardized for gyrA, rrs and eis genes. A chemiluminescence based biochip assay was optimized. Bio-chip was tested on 112 M. tuberculosis clinical isolates with different resistance spectra.

RESULTS: Isolates analyzed using bio-chip shows that 61 (61%) samples were wild-type. Twelve samples show mutations in gyrA gene, 11 samples in rrs gene, 12 samples in eis gene and 4 samples show double mutation in rrs and eis genes. The sensitivity and specificity of bio-chip for detection of FQ resistance ranged from 75 to 100% and 96.7%-100%, respectively. The sensitivity and specificity of SLID detection ranged from 90.9 to 100% and 96.7-100% respectively. The analytical sensitivity of the bio-chip was ~ 250 genome copies per assay.

CONCLUSION: The biochip has high sensitivity and specificity and could be useful for clinical microbiology studies and epidemiological surveillance of drug resistant (DR) M. tuberculosis. It is a highly accurate tool for screening for SLDR, significantly reducing the time for phenotypic drug susceptibility test (DST) results from weeks to a single day.