Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Abstract

cTreating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization. Regional increases in the prevalence of tuberculosis (TB) with drug resistance and a broad distribution of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB (1, 2) may reverse recent gains in global TB control (1). Molecular detection of mutations associated with drug resistance has facilitated rapid detection of drug resistance in the Mycobacterium tuberculosis complex (MTBC) (3-7), and the use of such molecular tools has become increasingly important in TB control and TB patient management (5, 7-10). Recognizing the advantages and disadvantages of various molecular methods will help in the selection of optimal methods for improved prediction of drug resistance in MTBC, which is critical in advancing molecular diagnostic approaches for this defined purpose.In 2003, the Microbial Diseases Laboratory (MDL) at the California Department of Public Health developed a real-time, probe-based assay using molecular beacons for screening for MDR TB (11). Remarkable improvements in turnaround times for predicting resistance to isoniazid (INH) and rifampin (RIF) and significant impacts on the management of MDR TB (9) were realized. However, several incidents of detection of rpoB mutations not conferring RIF resistance resulted in erroneous reporting of false RIF resi...

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.…”

“…To validate the Sanger sequencing results, we repeated the sequencing of a subset of isolates using pyrosequencing (PSQ), according to the standard manufacturer procedures adapted for the sequencing of XDR-TB-associated mutations (19,20). Briefly, we used the PyroMark Q96 ID system (Qiagen, Valencia, CA) to perform PSQ of five of the eight Sanger sequencing gene targets identified in Table 1 (katG, inhA promoter, rpoB, gyrA, and rrs).…”

MTBDR plus and MTBDR sl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

“…Pyrosequencing (PSQ), the reference sequencing standard used for this study, has previously been described in detail (20,26). In brief, PSQ methods include DNA extraction, amplification by PCR, and real-time sequencing.…”

“…Primers were used to synthesize nucleotides into growing DNA chains of no larger than 30 bp (5). The gene regions sequenced included codons 507 to 521 and 522 to 533 of the rpoB gene, codons 312 to 316 of katG, the inhA promoter region Ϫ4 to Ϫ20, gyrA codons 88 to 95, and the rrs gene region from nucleotides 1397 to 1406 (20,26). Each gene target was initially sequenced once.…”

Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

“…Pyrosequencing (PSQ), a real-time method for rapid sequencing of small segments of genomic DNA (10), is capable of reliably detecting mutations that confer first (11-13)-and second-line drug resistance (14,15) in M. tuberculosis. PSQ not only deter-mines the presence or absence of these mutations, but also displays detailed sequence data, which enables users to distinguish mutations conferring resistance from silent mutations as well as from those conferring different levels of resistance (16,17).…”