Rapid diagnosis of extensively drug-resistant tuberculosis by use of a reverse line blot hybridization assay

Abstract

Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.

Fig. 1

RLBH performed for rpoB, katG, inhA, gyrA, gyrB, and rrs. All the different probes are blotted vertically, and patient samples (PCR products) are applied horizontally. The probe in lane 1 is a common probe for all mycobacteria, and then there is a probe for the M. tuberculosis complex. Then there are 5 probes for the wild-type rpoB gene to check for rifampin-susceptible cases and 16 mutant probes for detection of rifampin resistance. Similarly, we targeted the inhA wild type and mutant with a probe for the promoter region, followed by probes for the wild type and mutant at codon 315 of the katG gene. This is further followed by probes for the wild type and mutant for gyrA mutations at codons 90, 91, 94, and 95. Similarly, we also targeted the gyrB codon 510 region with wild-type and mutant probes. For the second-line aminoglycosides, the rrs region at codons 1401, 1402, and 1484 were used. To illustrate an example, the patient sample marked by the arrow has mycobacterial infection due to M. tuberculosis complex and shows binding with probes 3 to 6, which are for the rpoB wild-type codon 509 to 529. It does not show binding with probe 7 but shows binding with probe 9, indicating RIF resistance due to a position 531 TTG mutation. Further, the same sample shows binding with probe 24, which is for the inhA promoter wild type, and shows binding with probe 27, which is the probe for a katG mutation at codon 315, thereby indicating INH resistance due to a katG mutation. The same sample shows binding with the 28th, 31st, 36th, 37th, 39th, and 42nd probes, which correspond to the gyrA codon 90 wild type, gyrA codon 94 wild type, gyrA codon 95 polymorphism, gyrB wild type, rrs codon 1401 wild type, and rrs codon 1484 wild type, respectively, thus giving a result for that particular sample indicating resistance to INH and RIF and susceptibility to FQs and KAN, AM, and CM. WT and wt, wild type; mt, mutant.