Direct Detection of Rifampin-ResistantMycobacterium tuberculosisin Respiratory Specimens by PCR-DNA Sequencing

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Abstract
This study evaluated the feasibility of a molecular strategy based on identification ofMycobacterium tuberculosisby IS6110PCR or Cobas Amplicor PCR, andrpoBPCR-DNA sequencing of the 81-bp rifampin resistance determining region (RRDR) for direct detection of rifampin resistance in respiratory specimens. A collection of 2,138 respiratory specimens and 352 nonduplicateM. tuberculosisisolates (including 233 isolates from the evaluated respiratory specimens and an additional collection of 119 stored isolates) from Southern China was investigated. Using culture as the reference test, the overall diagnostic sensitivities of an acid-fast bacillus (AFB) smear, Cobas Amplicor PCR, IS6110PCR were 54.5% (156 of 286), 86.7% (248 of 286), and 89.2% (255 of 286), respectively. The sensitivities of therpoBPCR for the specimens with positive AFB smears and with positive PCR results in the IS6110PCR and/or Cobas Amplicor PCR were 100% (156 of 156) and 92.3% (239 of 259), respectively. Of the 352 nonduplicateM. tuberculosisisolates, the agar proportion method for rifampin reported 39 resistant strains. Full agreement (352 of 352) was found with the agar proportion method and the genotype inferred from therpoBDNA sequencing data for rifampin. Thirty-nine mutations of nine distinct kinds, eight point mutations, and one deletion within the RRDR were found in the 39 resistant strains. For the direct DNA sequencing performed onrpoBPCR-positive respiratory specimens, the concordance with the agar proportion method and the subsequent PCR-sequencing for the culture isolate was 100%. This strategy has potential application for direct and rapid diagnosis of rifampin-resistantM. tuberculosisin IS6110PCR or Cobas Amplicor PCR-positive respiratory specimens.

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