Rifampin-Isoniazid Oligonucleotide Typing: an alternative format for the rapid detection of multidrug resistant Mycobacterium tuberculosis

A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampicin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in genes rpoB, katG and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formation at a single annealing temperature irrespective of different GC content of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampicin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus, sensitivity and specificity reached 96.4% and 95.5% for rifampicin and 92.7 and 100% for isoniazid. Altogether, 40/45 (89%) multidrug resistant isolates were correctly identified. Advantages of this in house development include versatility, capacity to run up to 41 samples by triplicate in a single run and reuse of the membrane at least 10 times. These features reduce substantially cost per reaction and make the assay an attractive tool for use in reference laboratories of countries with high burden of multidrug-resistant tuberculosis and low resources, which cannot afford expensive commercial tests.