Feasibility of the GenoType MTBDR sl Assay for Fluoroquinolone, Amikacin-Capreomycin, and Ethambutol Resistance Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

Abstract

The new GenoTypeThe worldwide emergence of extensively drug-resistant tuberculosis (XDR TB, resistant at least to rifampin and isoniazid, a fluoroquinolone [FLQ], and one of the three injectable second-line drugs amikacin [AM], kanamycin [KM], and capreomycin [CM]) is a serious global health problem (20,25). In the World Health Organization fourth global report on drug resistance (25), it was documented that more than 45 countries have reported XDR cases. The actual incidence could be underestimated, because second-line drug susceptibility testing (DST) is not available in many countries. To avoid a progressive development similar to that observed in multidrugresistant TB (resistant at least to rifampin and isoniazid) worldwide, now having the highest rate ever at 5.3%, timely identification of resistant Mycobacterium tuberculosis complex (MTBC) strains is mandatory.Conventional DST for XDR strains is performed sequentially in a two-step procedure beginning with a culture and first-line drug testing, proceeding to further drug testing in the case of multidrug resistance. The time needed for testing, even with the most rapid liquid methods, is still around 1 week per test, constrained by the relatively slow growth of M. tuberculosis (15, 18). The required time can be shortened by fast molecular methods to 1 day per test (3,8,21). Since recently broad-based knowledge about mutations that cause resistance to ethambutol (EMB) and some second-line drugs is available. Resistance to FLQs, AM-CM, and EMB in M. tuberculosis is most frequently attributed to mutations in the gyrA, rrs, and embB genes, respectively. First investigations have shown that by targeting mutations in codons 90, 91, and 94 in the gyrA gene, approximately 70 to 90% of all FLQ-resistant strains can be correctly detected (2, 13, 24). Previous reports have linked mutations A1401G, C1402T, and G1484T in the rrs gene to AM, CM, and KAN resistance (1, 11, 12), each of them being responsible for a specific resistance pattern. Mutations G1484T and A1401G were found to cause high-level resistance to all drugs, whereas C1402T causes resistance to only CM and KAN.Furthermore, mutations at embB codon 306 are found in 30 to 68% of EMB-resistant clinical strains (16,17,26).PCR-based techniques provide new possibilities for the rapid diagnosis of first-and second-line drug resistance; however, not all mycobacterial laboratories have access to DNAsequencing facilities. As an alternative, DNA strip assays for the detection of rifampin (INNO-LiPA Rif. TB; Innogenetics, Ghent, Belgium) or rifampin and isoniazid resistance of M. tuberculosis in a single assay (GenoType MTBDR; Hain Lifescience, Nehren, Germany) are now commercially available. These assays have been evaluated for M. tuberculosis cultures and specimens (3,7,8,10,21). The DNA strip assays are based on PCR or multiplex PCR in combination with reverse hybridization. The existence of a resistant strain is signaled either by the omission of a wild-type band or the appearance of bands representing specific mut...

Evaluation of Two Molecular Assays for Rapid Detection of Mycobacterium tuberculosis Resistance to Fluoroquinolones in High-Tuberculosis and -Multidrug-Resistance Settings

“…This is similar to previously reported data (5,17), suggesting that this assay is feasible for use in areas with high rates of drug resistance. There was 95.5% concordance between results of conventional sequencing and the MTBDRsl assay, excluding unreadable results and mixed allelic variants, demonstrating a high specificity for the assay.…”

“…Bacteriological methods are highly sensitive and specific but very labor-intensive and time-consuming. Consequently, molecular diagnostic methods are being introduced into routine use for the rapid detection of FQ resistance by identification of mutations in specific gyrA and gyrB genes regions (QRDR) (17,46). This study is the first to assess the effectiveness of both a noncommercial DNA sequencing and a commercially available molecular diagnostics system (GenoType MTBDRsl) for the rapid detection of M. tuberculosis resistance to FQ, conducted in the Russian Federation (a middle-incidence region with high rates resistance to anti-TB drugs).…”

“…Assays for the detection of mutations in genes associated with resistance to ethambutol (EMB), aminoglycosides/cyclic peptides (AG/CP), and FQ have been developed by Hain Lifescience, GmbH (Nehren, Germany), evaluated in low-incidence settings in two recent studies. and demonstrated good sensitivity and specificity (5,17). Pyrosequencing assays have also proved effective for the detection of mutations in genes associated with the drug resistance (19).…”

Predictive Value of Molecular Drug Resistance Testing of Mycobacterium tuberculosis Isolates in Valle del Cauca, Colombia

“…Previous evaluations of the GenoType kits have documented their performance in detecting resistance to first-and second-line antituberculosis (anti-TB) drugs in different continents, like Africa, Asia, and Europe (2,3,4,5). From these studies, it became clear that there are geographic variations in the mutations that are associated with drug resistance in Mycobacterium tuberculosis (6).…”

A Systematic Review Of Gyrase Mutations Associated With Fluoroquinolone-Resistant Mycobacterium Tuberculosis And A Proposed Gyrase Numbering System

“…In addition to fluoroquinolone mono-resistant M. tuberculosis isolates, such tests could be used to rapidly identify extensively drug-resistant tuberculosis (XDR-TB; defined as resistance to at least isoniazid and rifampicin plus a fluoroquinolone and a second-line injectable agent such as capreomycin, amikacin and kanamycin). 28 -30 A line probe assay has been developed to detect fluoroquinolone-resistance in M. tuberculosis, but its sensitivity ranges from 75.6% to 89.5% when compared with conventional phenotypic susceptibility methods, 19,31,32 and 87% to 100% when compared with DNA sequencing. 31,33 The purpose of this review was to characterize all DNA gyrase gene mutations described in M. tuberculosis clinical strains and to distinguish those associated with fluoroquinolone resistance and those reported in fluoroquinolone-susceptible strains.…”