Evaluation of the MTBDRsl Test for Detection of Second-Line-Drug Resistance in Mycobacterium tuberculosis

The MTBDRsl assay (Hain Lifescience GmbH, Germany) is a new line probe assay for the detection of extensively drug-resistant tuberculosis (XDR TB). The test simultaneously detects resistance to ethambutol, aminoglycosides/cyclic peptides, and fluoroquinolones through detection of mutations in the relevant genes. The assay format is identical to the MTBDR Hain assay. The assay was evaluated for the detection of second-line-drug resistance in Vietnamese isolates using two sample sets from the microbiology department of Pham Ngoc Thach Hospital, Ho Chi Minh City, Viet Nam, with existing conventional phenotypic drug susceptibility results for second-line drugs: 41 consecutive fluoroquinolone-resistant isolates and 21 consecutive multidrug-resistant but fluoroquinolone-sensitive isolates. The sensitivity for detection of fluoroquinolone resistance was 75.6% (31/41) (95% confidence interval 95% CI], 59.7% to 87.6%), and for kanamycin resistance, the sensitivity was 100% (5/5) (95% CI, 47.8% to 100%). The sensitivity of the test for detection of ethambutol resistance was low, consistent with previous reports, at 64.2% (34/53) (95% CI, 49.8% to 76.9%). The specificity of the test was 100% for all three drugs. These data suggest that the MTBDRsl assay is a rapid, specific test for the detection of XDR TB but should not be used exclusively to “rule out” second-line-drug resistance. Further operational evaluation is required and should be integrated with evaluations of the MTBDR test.The World Health Organization (WHO) has estimated that 5% of all tuberculosis (TB) cases globally are now multidrug-resistant tuberculosis (MDRTB) (resistance to at least rifampin RIF] and isoniazid INH]), based on data acquired since 2000 from more than 100 countries (14). Every year, an estimated 490,000 new cases of MDRTB occur, causing more than 130,000 deaths (14). In 2006, the documentation of a rapidly fatal TB outbreak among hospitalized HIV patients in Kwa Zulu Natal, South Africa (5) led to the definition of extensively drug-resistant tuberculosis (XDR TB) as TB resistant to a fluoroquinolone and injectable second-line drug (amikacin, capreomycin, or kanamycin) in addition to isoniazid and rifampin. XDR TB has subsequently been reported from over 50 countries by WHO (14). It is likely that the majority of XDR TB cases worldwide remain undetected due to the lack of second-line-drug testing in most high-burden settings. There are an estimated 40,000 new cases of extensively drug-resistant tuberculosis each year (15).The recognition of XDR TB worldwide has made timely identification of XDR TB cases to achieve effective disease management and to prevent their spread a priority (3, 8).Significant challenges exist; although standard protocols exist for second-line-drug susceptibility testing, strong evidence is lacking on many factors, such as the reproducibility and reliability of results, applicability of MIC to clinical outcomes, and intermethod variability. Proficiency testing for second-line-drug susceptibility testing has only recently been integrated into the supranational reference laboratory panel in an effort to improve standardization of second-line drugs across the WHO reference laboratory network.Conventional drug resistance testing takes more than 2 weeks to return a result even after a positive culture has been isolated. Rapid commercial liquid-based culture systems, such as Bactec MGIT 960 testing (Becton Dickinson), for second-line drugs are not yet formally FDA/WHO approved but are reported to be accurate, widely used in developed settings, and reduce turnaround times to approximately 8 days (7). In recent years, many second-line-drug susceptibility testing methods have been developed. The most rapid results are achieved by direct testing of patient specimens by molecular methods; however, in addition to the high cost of such tests, the sensitivity remains suboptimal, and rigorous contamination control is required to maintain accuracy. The majority of high-burden settings currently lack the resources to implement such tests effectively. Two commercial DNA strip assays, INNO-LiPA RifTB (Innogenetics, Zwijndrecht, Belgium) and MTBDRplus (Hain Lifescience GmbH, Germany), targeting the rpoB plus katG and inhA genes have been extensively evaluated for use with Mycobacterium tuberculosis culture and directly on sputum to identify MDR TB cases (2, 10). The Foundation for Innovative Diagnostics (FIND) demonstration projects in South Africa of the GenoType MTBDRplus assay resulted in the recommendation of commercial line probe assays for use in high-burden settings by WHO (16). This assay is based on a multiplex PCR in combination with reverse hybridization. Either the absence of wild-type bands or the appearance of bands targeting specific mutations indicates the presence of a resistant strain. MDR TB cases can be detected within 1 or 2 days of sputum sampling using this assay.In order to rapidly detect second-line-drug resistance, the MTBDRsl test (Hain Lifescience GmbH, Germany) has been developed. This assay can detect mutations in gyrA, rrs, and embB genes, detecting resistance to the fluoroquinolones (FQ), aminoglycosides/cyclic peptides, and ethambutol (EMB), respectively, with a single assay. A previous evaluation study in Germany showed that this assay has a high accuracy for FQ and amikacin-capreomycin resistance testing in clinical strains and sputum samples (6). EMB detection was specific (100%), but its sensitivity (69.2%) was low.The MTBDRsl assay contains 22 probes, including 16 probes for gene mutation detection and 6 probes for the control of the test procedure. The six control probes include a conjugate control (CC), an amplification control (AC), a Mycobacterium tuberculosis complex control (TUB), and three locus probes (gyrA, rrs, and embB) for gene amplification control. The remaining probes detect FQ resistance (gyrA), amikacin/capreomycin resistance (rrs), and ethambutol resistance (embB). The probes contained in the assay do not detect all mutations in these genes but are targeted to the most commonly occurring mutations.The aim of the present study was to determine the accuracy of this assay for detection of FQ, kanamycin, and ethambutol resistance against conventional phenotypic testing as the gold standard on Vietnamese isolates of M. tuberculosis.