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

Abstract

The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of drug resistance, including multidrug and extensive drug resistance to TB (M/XDRTB). Rapid diagnosis of resistance to fluoroquinolones (FQs) using molecular assays is essential for the implementation of appropriate drug regimens and prevention of the transmission of XDR strains. A total of 51 individual MDRTB strains were tested by pyrosequencing of the quinolone resistance determining region of the gyrA gene and the GenoType MTBDRsl assay (Hain Lifescience, GmbH, Nehren, Germany), and the results were evaluated against those obtained by phenotypic drug susceptibility testing (DST). Mutations were detected in 25 (78.1%) FQ-resistant strains, with the majority of mutations (n ‫؍‬ 19 [73.0%]) found in codon 94 of the gyrA gene; the novel mutation 1457 C3⌻ was found in the gyrB gene. Three mixed allelic variants were detected, which is a well-known phenomenon in areas with high TB and drug-resistant TB rates. The sensitivity and specificity of pyrosequencing (86.2 and 100%, respectively) and MTBDRsl (86.2 and 100%, respectively) were high; however, the results for 5.9% of the analyzed strains were unreadable when MTBDRsl was used. The MTBDRsl and pyrosequencing assays offer a rapid and accurate means for diagnosing resistance to FQs in high-TB-burden areas.Tuberculosis (TB) remains a major threat to public health worldwide and in the developing world in particular, causing almost 2 million deaths annually. The real magnitude of drug resistance is not yet known, although the Indian subcontinent, China, and Russia are believed to account for the majority of cases globally (48).Multidrug-resistant TB (MDRTB), caused by TB bacilli resistant to both isoniazid (INH) and rifampin (RIF), poses difficulties in diagnosis and treatment, with lower survival rates (especially in HIV-infected persons) and the associated high costs of TB control programs. The World Health Organization (WHO) estimates current MDRTB rates in new and previously treated cases globally to be 2.9 and 15.3%, respectively (50).The Russian Federation is a high-TB-burden country with high rates of TB drug resistance, dominated by TB strains of the Beijing family reported to be associated with MDRTB (7, 42). The Samara Oblast (Central Russia) is a hot spot for both TB and HIV epidemics; ca. 20% of new TB cases are MDR, with rates even higher in previously treated cases and in the prison sector (8). Converging TB, drug-resistant TB, and HIV epidemics pose a serious problem for low-and middle-income countries such as Russia where access to second-and third-line drug therapy is limited (2, 8).The rate of extensively drug-resistant TB (XDRTB) (which is caused by MDRTB bacilli with additional resistance to a fluoroquinolone [FQ] and one or more injectable drugs) has been increasing in Russia (36) due to multiple incomplete treatment regimens and poor infection control practice. Cases of XDRTB show a high rate of treatment failure and mortality especially in XDRTB patients with c...

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

“…The presence of a mixed wild-type and resistant population with regard to this drug has also been reported in previous studies (7,13). Specificity values were around 80% and similar to values in the literature (7,8,13,14,18,20). A natural polymorphism at gyrA codon 95 (ACC) unrelated to FLQ resistance has been described (31), and by pyrosequencing, we found it in 9 clinical samples (1 per patient) and 28 clinical strains.…”

“…Surprisingly, these values are lower than the ones reported in previous studies, which ranged from 70 to 90% (7,8,13,14,18,20). One possible explanation is that the total number of FLQ-resistant specimens was 15, which is low in comparison to the number of specimens tested in other published studies.…”

Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

“…As GyrA houses the most common substitutions, GenoType MTBDRsl is designed to detect those substitutions (A90V, S91P, D94A, D94N/Y, D94G, and D94H). The sensitivity of this test for FQ resistance detection ranges from to 75.6% to 90.6% (4,10,12,14,17), the gap being mainly due to the absence of detection of GyrB substitutions (4). Substitutions in GyrB in M. tuberculosis clinical strains are being increasingly reported (2, 4, 6, 8, 9, 16, 19, 21, 25-27, 29, 32).…”

High Proportion of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates with Novel Gyrase Polymorphisms and a gyrA Region Associated with Fluoroquinolone Susceptibility

“…It should be noted that most of the patient populations studied to date with this diagnostic test have had multidrug-resistant tuberculosis (MDR-TB; defined as resistance to at least isoniazid and rifampin) (6,16). In MDR-TB patients, the MTBDRsl assay has been 76 to 91% sensitive in detecting genotypic fluoroquinolone resistance (6,16,17,19,20). Many such patients likely received extensive prior treatment, including fluoroquinolones.…”