Pyrosequencing identification of Mycobacterium tuberculosis W-Beijing

The worldwide expanding Mycobacterium tuberculosis W-Beijing family is associated with treatment failure and relapse. Its identification currently relies on spoligotyping and conventional sequencing. We developed pyrosequencing as an alternative method for its identification.

Pyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 M. tuberculosis isolates as W-Beijing isolates. In addition, pyrosequencing found a previously unreported TGC deletion in the Rv0927c gene of W-Beijing isolates. Total concordance was found between the pyrosequencing data and conventional sequencing, as well as reference molecular identification. Multispacer Sequence Typing assigned the W-Beijing isolates to the Asian lineage and the 96 non-W-Beijing isolates to the Euro-American lineage (P < 10-5). The W-Beijing isolates were all susceptible to streptomycin, rifampin, isoniazid, ethambutol, and pyrazinamide; no resistance-associated mutations were detected in these eight W-Beijing isolates. There were no statistically significant differences in the antibiotic susceptibility of W-Beijing and non-W-Beijing isolates (p = 0.2, X2 test). Pyrosequencing correctly identified M. tuberculosis organisms in 26/26 sputum specimens exhibiting acid-fast bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 euro/test.

Pyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.