Pyrosequencing for Rapid Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Specimens

Abstract

The aim of this study was to evaluate a pyrosequencing method for the detection of Mycobacterium tuberculosis isolates resistant to rifampin and isoniazid using both clinical strains and clinical samples, comparing the results with those of the Bactec 460TB and GenoType MTBDRplus assays. In comparison to Bactec 460TB as the gold standard, the sensitivity of pyrosequencing for detecting isoniazid and rifampin resistance was 76.9% and 97.2%, respectively, for clinical strains, and the specificity was 97.2 and 97.9%, respectively. For clinical specimens, the sensitivity and specificity for both drugs were 85.7% and 100%, respectively. The overall concordance between pyrosequencing and the GenoType MTBDRplus assay for clinical strains was 99.1%, and for clinical samples, it was 98.2%. Pyrosequencing is a valuable tool for rifampin and isoniazid resistance detection.Tuberculosis control and prevention programs are based on an early diagnosis followed by rapid identification of drug resistance (26). The detection of drug-resistant Mycobacterium tuberculosis strains is usually performed by time-consuming phenotypic methods. In this sense, new molecular methods for the detection of mutations conferring drug resistance to the two main first-line antituberculous drugs, rifampin (RIF) (rpoB) (18,20,23) and isoniazid (INH) (katG and the inhA promoter region), have been developed (8,18,19,21,22).Pyrosequencing is a semiautomated sequencing assay based on real-time monitoring of DNA synthesis, optimized to analyze single-nucleotide polymorphisms (SNPs) and short DNA sequences (9). The detection of single polymorphisms by pyrosequencing has allowed the identification, resistance detection, and typing of a wide range of microorganisms, including M. tuberculosis (2,3,5,11,12,14,17,27).The objective of this study was to evaluate the ability of pyrosequencing to detect the most frequent mutations in rpoB, katG, and inhA in clinical strains of M. tuberculosis and directly from clinical specimens, comparing the results with phenotypic and genotypic standard methods (genotyping and GenoType MTBDRplus; Hain Lifescience GmbH, Nehren, Germany). To our knowledge, this is the first study comparing pyrosequencing results with those obtained using GenoType MTBDRplus.Clinical strains. Ninety well-characterized M. tuberculosis strains were phenotypically studied using the radiometric method of the Bactec 460TB system (Becton Dickinson, Towson, MD). Among the 90 characterized strains, 11 were fully susceptible and 79 were resistant (43 RIF s /INH r and 36 RIF r / INH r ). All clinical strains were genotypically characterized with the GenoType assay. In addition, for 67 of 79 INH-and/or RIF-resistant strains, genotypic characterization by sequencing was also available. Commercial methods were performed following the manufacturer's instructions.Clinical specimens. Forty-eight clinical specimens were retrospectively selected from 26 patients during clinical routine (Fig. 1). The Ziehl-Neelsen smears were graded on a scale from 0 to 4ϩ (15). The s...

GenoType MTBDR sl for Molecular Detection of Second-Line-Drug and Ethambutol Resistance in Mycobacterium tuberculosis Strains and Clinical Samples

“…At the moment, there are different molecular tests that identify MDR and XDR M. tuberculosis, and these are mainly DNA sequencing and line probe assays (11,21,29,37). Interestingly, in recent years pyrosequencing has also appeared as a molecular test for the detection of M. tuberculosis strains resistant to INH and RIF (4,12,23).…”

Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

“…Based on our findings, it is clear that molecular diagnostics based on the detection of Ͻ30 SNPs in 6 genes should be able to predict clinically relevant phenotypic resistance to INH, RIF, FQ, AMK, KAN, and CAP with 90 to 98% sensitivity and almost 100% specificity in most regions. The existing Hain Lifescience LPAs (MTBDRplus and MTBDRsl) detect most of these mutations, with some limitations noted above, and new sequencing-based platforms, such as PSQ, have the ability to detect and discriminate all of these SNPs (19,20,43). Given that Ͻ25% of incident MDR-TB cases are currently being diagnosed based on phenotypic DST alone, it seems that there is a clear clinical role for molecular diagnostics, at least in early treatment decisions while waiting for phenotypic DST results.…”

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

“…The identification of mycobacteria to the species level cannot be performed with traditional automated identification systems and instead requires more complex and labor-intensive molecular assays, including nucleic acid amplification strategies, molecular probes, high-performance liquid chromatography (HPLC) analysis of mycolic acids from the bacterial cell wall, or DNA sequencing methods (142,(145)(146)(147)(148). Moreover, the general biology of the organism slows the time to diagnosis due to its requirements for specialized growth media, fastidious nature, and exceedingly slow generation time.…”