Simultaneous Identification of Mycobacterial Isolates to the Species Level and Determination of Tuberculosis Drug Resistance by PCR Followed by Electrospray Ionization Mass Spectrometry

Abstract

Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.

Broad-range PCR coupled with mass-spectrometry for the detection of Mycobacterium tuberculosis drug resistance

“…This low proportion of PGG1 and consequently of Beijing type observed in the studied group is similar to that reported in Central and Western Europe 3 and in contrast with the higher prevalence rates from neighbouring Ukraine and North-Western Russia. 5 This observation needs to be confirmed by further studies, since the present study was not designed for prevalence estimation.…”

“…5 A large study performed on 1340 samples originating from different populations showed very good results for drug resistance detection with PCR/ESI-MS, with sensitivities of 96.4% for RIF and 89.2% for INH and specificities of 98.6% and 95.8% for RIF and INH, respectively. 5 In another study performed on 48 samples the sensitivity and specificity values were 100% and 93.8% for INH and 100% and 92.3% for RIF when PCR/ESI-MS was compared to phenotypic DST. 7 Similarly, in the present study PCR/ESI-MS had sensitivities in detecting INH and RIF resistance of 100% and 92% respectively and specificities exceeding 95% to both RIF and INH.…”

“…Discordant results between phenotypic and genotypic results were described for several genotypic methods, including PCR-ESI/MS. 5,7 Several causes might explain these discordances, like the limited number of targeted genes and mutations, the detection of mutations associated with an elevated MIC but lower than the critical concentration, the challenges of phenotypic DST for some drugs, like ETB, and the incomplete understanding of the molecular mechanisms of drug resistance.…”

“…5 Briefly, the test detects a number of mutations in genes associated with resistance to INH (katG, inhA and its promoter, ahpC promoter), RIF (rpoB), EMB (embB) or FQ (gyrA). Detection of polymorphisms at katG codon 463 and gyrA codon 95 was used for identification of the PGG.…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…The use of mass spectrometry for the identification of Mtb complex from culture isolates may replace current standards such as biochemical analysis, nucleic acid hybridization probes, and DNA sequencing due to the ability of mass spectrometry to rapidly and accurately identify Mtb in a cost-effective manner while minimizing technologist hands-on time and effort (Saleeb et al, 2011). Although still in it's infancy in the diagnostic microbiology laboratory, this technology may also have utility in predicting drug resistance patterns and evaluating epidemiologic groups (Bouakaze et al, 2011;Massire et al, 2011;Schurch et al, 2011).…”