With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P <0.05 for rifampicin or isoniazid or streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (κ) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (κ, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (κ, 0.97) while targeted sequencing yielded lower agreement (κ range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (κ range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be more useful when accurate ethambutol susceptibility results are warranted.

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10 Mycobacterium tuberculosis samples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay. rpoB sequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of the rpoB gene. Multidrug-resistant tuberculosis (MDR-TB) (defined as infection with a Mycobacterium tuberculosis strain resistant to at least the two most effective, rifampin and isoniazid, anti-TB drugs) is prevalent throughout the world, difficult to treat, and associated with higher rates of clinical failure and disease relapse (1, 2). The rapid and accurate laboratory diagnosis of MDR-TB is crucial for effective treatment, which will also limit the transmission of MDR-TB (2, 3). The resistance of M. tuberculosis to rifampin (RMP) in nearly 97% of isolates is due to mutations in an 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene (4). Other RMP-resistant isolates contain mutations in either the N-terminal or cluster II region of the rpoB gene, or the resistance is due to other mechanisms (2, 4). Resistance to RMP is a key determinant in treatment failure and also correlates well with MDR-TB, since Ͼ85% of RMP-resistant M. tuberculosis isolates worldwide are also resistant to isoniazid (INH) (2-4). Molecular assays detect mutations in the RRDR of the rpoB gene for the rapid detection of RMP-resistant M. tuberculosis in clinical specimens and culture isolates (2, 3). The World Health Organization (WHO)-approved tests include two line probe assays, the INNO-LiPA Rif. TB (detecting resistance to RMP only) and the GenoType MTBDRplus (detecting resistance to RMP and INH), as well as the real-time PCR-based automated Xpert MTB/RIF (Xpert) assay (detecting resistance to RMP only) (3, 5). However, these tests are not specific, as silent mutations in the rpoB gene occasionally lead to the detection of false-positive RMP resistance (6-9). The current WHO recommendations are to use the Xpert assay as the initial diagnostic test and start treatment for MDR-TB if an RMP resistance result is expected, or, if unexpected, to repeat Xpert assay testing on another sputum sample, particularly in settings in which the prevalence of RMP-resistant TB is Ͻ15% (10). For those settings, treatment for MDR-TB should be initiated when the Xpert assay repeatedly detects RMP resistance. Treatment should be optimized by following susceptibility testing with other first-line and second-line drugs and confirmatory testing for RMP resistance by phenotypic or other genotypic methods; any discordant RMP susceptibility results can be resolved by sequencing of the rpoB gene (10).Phenotypic drug susceptibility testing ...

BackgroundAccurate drug susceptibility testing (DST) of Mycobacterium tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for diagnosis and management of multidrug-resistant tuberculosis (MDR-TB). Resistance of M. tuberculosis to rifampicin is mainly due to mutations in hot-spot region of rpoB gene (HSR-rpoB). The prevalence of disputed (generally missed by rapid phenotypic DST methods) rpoB mutations, which mainly include L511P, D516Y, H526N, H526L, H526S, and L533P in HSR-rpoB and I572F in cluster II region of rpoB gene, is largely unknown. This study determined the occurrence of all disputed mutations in HSR-rpoB and at rpoB codon 572 in M. tuberculosis strains phenotypically susceptible to rifampicin in Kuwait.MethodsA total of 242 M. tuberculosis isolates phenotypically susceptible to rifampicin were used. The DST against first-line drugs was performed by Mycobacteria growth indicator tube (MGIT) 960 system. Mutations in HSR-rpoB (and katG codon 315 and inhA-regulatory region for isoniazid resistance) were detected by GenoType MDBDRplus assay. The I572F mutation in cluster II region of rpoB was detected by developing a multiplex allele-specific (MAS)-PCR assay. Results were confirmed by PCR-sequencing of respective loci. Molecular detection of resistance for ethambutol and pyrazinamide and fingerprinting by spoligotyping were also performed for isolates with an rpoB mutation.ResultsAmong 242 rifampicin-susceptible isolates, 0 of 130 pansusceptible/monodrug-resistant isolates but 4 of 112 polydrug-resistant isolates contained a disputed rpoB mutation. All 4 isolates were also resistant to isoniazid and molecular screening identified additional resistance to pyrazinamide and ethambutol in one isolate each. In final analysis, 2 of 4 isolates were resistant to all 4 first-line drugs. Spoligotyping showed that the isolates belonged to different M. tuberculosis lineages.ConclusionsFour of 242 (1.7%) rifampicin-susceptible M. tuberculosis isolates contained a disputed rpoB mutation including 2 isolates resistant to all four first-line drugs. The occurrence of a disputed rpoB mutation in polydrug-resistant M. tuberculosis isolates resistant at least to isoniazid (MDR-TB) suggests that polydrug-resistant strains should be checked for genotypic rifampicin resistance for optimal patient management since the failure/relapse rates are nearly same in isolates with a canonical or disputed rpoB mutation.

W ith 10 million active disease cases, 558,000 cases of rifampin-resistant/multidrugresistant tuberculosis (MDR-TB), and 1.6 million deaths recorded worldwide in 2017, TB is a leading cause of death from a single infectious agent (1). Early detection of Mycobacterium tuberculosis in clinical specimens, its susceptibility to anti-TB drugs (particularly, rifampin), and effective treatment are essential for global TB control efforts (1). Although culture is the gold standard for definitive diagnosis of active TB, molecular methods are now preferred due to their speed and ability to simultaneously detect resistance to rifampin with/without additional resistance to isoniazid for rapid diagnosis of TB and MDR-TB. The GeneXpert MTB/RIF Ultra (Xpert) assay rapidly detects M. tuberculosis and its resistance to rifampin (a surrogate marker for MDR-TB) in pulmonary and extrapulmonary specimens (2, 3). BBD Max MDR-TB (BBD Max) is a new test for the detection of M. tuberculosis and its resistance to rifampin and isoniazid (MDR-TB). Kuwait is a low-TB-incidence (23 cases/100,000 population) country, and ϳ1% of M. tuberculosis isolates are MDR-TB (4). This study tested the Xpert and BBD Max assays in a head-to-head comparison for the rapid detection of M. tuberculosis/MDR-TB using culture and clinical diagnosis of active TB disease as a reference in Kuwait.Fifty-one pulmonary and 30 extrapulmonary (cavitary fluids, n ϭ 24; fine needle aspirate/pus, n ϭ 6) specimens collected from 81 consecutive patients presenting with TB-like symptoms were used. Samples were processed for Ziehl-Neelsen staining and culture in the MGIT 960 system (MGIT) (5). The Xpert (Cepheid) and BBD Max (Becton and Dickinson) assays were performed according to the manufacturers' instructions.M. tuberculosis H37Rv and a well-characterized MDR-TB strain used as a control yielded expected results. Fourteen specimens were culture positive, including nine specimens that were smear positive for acid-fast bacilli. Of 14 culture-positive specimens, 12 were positive for M. tuberculosis and one negative for M. tuberculosis by both the Xpert and BBD Max assays. One specimen contained nontuberculous mycobacteria. Of 67 smear-negative and culture-negative specimens, eight samples were M. tuberculosis positive by the Xpert assay only, one sample was positive by the BBD Max assay only, and 58 samples were M. tuberculosis negative by both tests. Six of eight Xpertpositive pulmonary specimens were obtained from patients with a clinical diagnosis of TB who responded to anti-TB treatment.All M. tuberculosis-positive specimens (n ϭ 13) were rifampin susceptible by the MGIT and Xpert assays, while 11 specimens were rifampin susceptible by the BBD Max assay, and two specimens yielded indeterminate results. Although two of 13 M. tuberculosis isolates were isoniazid resistant by the MGIT assay, the BBD Max assay detected resistance in only one specimen, while 11 specimens were isoniazid susceptible, and one specimen yielded an indeterminate result.

Objective: Early detection and effective treatment are essential for global tuberculosis control. This study evaluated the performance of GeneXpert MTB/RIF (Xpert) and ProbeTec ET (PTec-ET) assays in diagnosing extrapulmonary tuberculosis (EPTB) in Kuwait. Materials and Methods: Nonrespiratory clinical specimens (n=3995) collected from 3995 patients suspected to have EPTB were tested. These included cavitary fluids (n=2054), fine needle aspirate (FNA)/pus/tissue biopsy (n=1461), urine (n=302), cerebrospinal fluid (CSF, n=118), and others (n=60). All specimens were processed for acid-fast bacilli (AFB), culture in MGIT 960 system and nucleic acid detection by Xpert and PTec-ET according to manufacturer’s instructions. Results: Of 3995 specimens, 95 were AFB-positive, 403 were culture-positive and an additional 86 samples had histopathology suggestive of TB. Using culture as reference, the sensitivity and specificity values were 88.33% and 97.3% for Xpert and 72.95% and 97.80% for PTec-ET, respectively. Although performance of both tests was comparable in AFB-positive samples, Xpert detected significantly more cases in culture-positive samples. Among culture-negative samples, Xpert detected 18 more cases including 16 with histopathological evidence of TB. Lowest positivity was detected for both tests in cavitary fluids. Xpert performed better than PTec-ET in culture-positive FNA/pus/tissue biopsy and CSF samples. Conclusions: Although performance of both tests was suboptimal for AFB-negative/culture-positive samples, Xpert performed better than PTec-ET and also detected more cases of AFB-negative/culture-nagative/histopathology-positive samples. PTec-ET was positive in three while Xpert was positive in all six culture-positive CSF specimens for rapid diagnosis of TB meningitis. Xpert was thus superior to PTec-ET or smear microscopy in rapidly diagnosing EPTB.