Concordance between Molecular and Phenotypic Testing of Mycobacterium tuberculosis Complex Isolates for Resistance to Rifampin and Isoniazid in the United States

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Overdiagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by the molecular detection of drug resistance (MDDR) for mutations associated with resistance to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculosis drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC from September 2009 to February 2011. We compared CDC''s results with those from the submitting public health laboratories (PHL). Concordances between molecular and phenotypic testing at CDC were 97.4% for RMP and 92.5% for INH resistance. Concordances between CDC''s molecular testing and PHL DST results were 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to the absence of known INH resistance mutations in isolates found to be INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for the diagnosis of MDR TB.     

Rapid diagnosis of extensively drug-resistant tuberculosis by use of a reverse line blot hybridization assay

Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.     

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.     

The management of tuberculosis: epidemiology, resistance and monitoring

This PhD thesis is based on 5 studies conducted in the period 2006-2010 during my employment at the International Reference Laboratory of Mycobacteriology, Statens Serum Institut. The overall aim was to assess tuberculosis (TB) treatment in Denmark with specific focus on the risk of relapse of TB disease, and to analyse treatment outcome of patients with multidrug-resistant (MDR) or isoniazid-resistant TB. The project established the need for rapid methods to detect resistance and follow-up of treatment. A rapid method to detect drug resistance was optimised and evaluated for use directly in clinical specimens. The studies were based on data from the Mycobacterial registry in the period 1992-2007, which included the results from microscopy, culture, drug-susceptibility and restriction fragment length polymorphism (RFLP). Information on dates of death/emigration were taken from the CPR-registry and treatment from surveillance data and patient records. The rate of recurrent TB was found to be low in Denmark, during 13.5 years of follow-up. Relapse accounted for 1.3% of the recurrent cases and reinfection was rare, only in 0.5% cases. The relapse hazard increased up to four years after diagnosis. Cavitary disease was associated with relapse as opposed to reinfection and may need prolonged treatment and closer monitoring. The incidence of MDR-TB and isoniazid resistance was confirmed to be low. Successful short- and long-term treatment outcome of MDR-TB and isoniazid-resistant TB was high. High- and low-level isoniazid resistance did not affect treatment outcome. A multiplex PCR hybridization mutation analysis, that simultaneously detects the most frequent rpoB and katG gene mutations conferring rifampin and high-level isoniazid resistance, was optimized for direct use and evaluated in smear-positive specimens as opposed to slow conventional drug-susceptibility testing (DST). The second-generation rifampin and isoniazid resistance mutation assay additionally included detection of mutations within the inhA gene conferring low-level isoniazid resistance. This assay was found to be rapid (< 48 h) and easy to perform in isolates and clinical specimens. A high concordance between mutation and conventional DST results was found for rifampin, while results varied for isoniazid . The mutation analysis identified all MDR-TB cases and the majority of isoniazid-resistant cases in Denmark. Standard 6-month multiple anti-TB drug therapy is necessary to treat drug-susceptible TB. Drug-resistant TB often requires therapy adjustments and extended treatment. MDR-TB particularly poses therapeutic challenges. Rapid detection of resistance mutations directly in smear-positive patient specimens may improve MDR-TB patient treatment, although the impact on isoniazid-resistant TB treatment outcome remains to be determined. The mutation assay is a rapid supplement to the gold standard conventional DST in high-income countries such as Denmark, while in low-income countries it can be used for preliminary DST. The assay may be applied to smear-positive samples from patients suspected of treatment failure, recurrent TB, drug-resistant TB exposure or originating from countries with high levels of DR. The new extended mutation assay has proved to be a useful tool, which has now been included in the World Health Organization's policy to combat and prevent new cases of MDR and extensively drug-resistant TB.     

Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens

The new GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) was tested with 125 clinical isolates and directly with 72 smear-positive sputum specimens for its ability to detect rifampin (RMP) and isoniazid (INH) resistance in Mycobacterium tuberculosis complex (MTBC) strains. In total, 106 RMP(r)/INH(r), 10 RMP(s)/INH(r), and 80 RMP(s)/INH(s) MTBC strains were comparatively analyzed with the new and the old MTBDR assays. Besides the detection of mutations within the 81-bp hot spot region of rpoB and katG codon 315, the GenoType MTBDRplus assay is designed to detect mutations in the regulatory region of inhA. The applicability of the new assay directly to specimens was shown, since 71 of 72 results for smear-positive sputa and all 125 results for clinical isolates were interpretable and no discrepancies compared with the results of real-time PCR or DNA sequencing were obtained. In comparison to conventional drug susceptibility testing, both assays were able to identify RMP resistance correctly in 74 of 75 strains (98.7%) and 30 of 31 specimens (96.8%). The misidentification of RMP resistance was obtained for two strains containing rpoB P533L mutations. Compared to the old MTBDR assay, the new GenoType MTBDRplus assay enhanced the rate of detection of INH resistance from 66 (88.0%) to 69 (92.0%) among the 75 INH-resistant strains and 36 (87.8%) to 37 (90.2%) among the 41 specimens containing INH-resistant strains. Thus, the new GenoType MTBDRplus assay represents a reliable and upgraded tool for the detection of INH and RMP resistance in strains or directly from smear-positive specimens.     

Evaluation of two molecular assays for rapid detection of mycobacterium tuberculosis resistance to fluoroquinolones in high-tuberculosis and -multidrug-resistance Settings

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 C→Τ 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-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.     

Use of molecular methods to identify the Mycobacterium tuberculosis complex (MTBC) and other mycobacterial species and to detect rifampin resistance in MTBC isolates following growth detection with the BACTEC MGIT 960 system

A prospective study was organized by using a total of 1,585 consecutive clinical specimens to determine whether biomass obtained from positive growth in the MGIT 960 system could be used directly in AccuProbe DNA hybridization tests, the PCR-based Inno-LiPA Rif.TB (LiPA) assay, and a PCR-based DNA sequencing of the rpoB gene for the rapid identification of the Mycobacterium tuberculosis complex (MTBC) and other mycobacterial species and for the determination of rifampin (RIF) resistance in MTBC strains. The results were compared to routine culture, identification, and susceptibility testing techniques performed on the same samples. The study results revealed that the DNA AccuProbe assay (on the day of growth positivity) readily identified 95.7%, the LiPA assay readily identified 98.6%, and rpoB sequencing readily identified 97.1% of the 70 MTBC isolates from mycobacterial growth indicator tubes (MGIT). In addition, application of the LiPA for the identification and RIF susceptibility testing of the MTBC in growth-positive MGIT resulted in a turnaround time of less than 2 weeks after specimen receipt. Although DNA sequencing of rpoB required a slightly longer (16 days) turnaround time, this method was capable of identifying several species of nontuberculous mycobacteria in addition to identifying MTBC and determining RIF susceptibility or resistance. The molecular methods were also found to rapidly identify RIF-susceptible and -resistant MTBC in two of the three mixed mycobacterial cultures weeks earlier than conventional methods. In conclusion, the biomass obtained in MGIT at the time of growth positivity in the 960 system is sufficient for use in all three molecular tests, and this approach can reduce the turnaround time for reporting results.     

Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n = 104; South Africa, n = 52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n = 5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.     

Mixed Mycobacterium tuberculosis Complex Infections and False-Negative Results for Rifampin Resistance by GeneXpert MTB/RIF Are Associated with Poor Clinical Outcomes

The Xpert MTB/RIF (Xpert) assay is becoming a principal screening tool for diagnosing rifampin-resistant Mycobacterium tuberculosis complex (MTBC) infection. However, little is known about the performance of the Xpert assay in infections with both drug-sensitive and drug-resistant strains (mixed MTBC infections). We assessed the performance of the Xpert assay for detecting rifampin resistance using phenotypic drug sensitivity testing (DST) as the reference standard in 370 patients with microbiologically proven pulmonary tuberculosis. Mixed MTBC infections were identified genetically through 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) analysis. Logistic regression was used to identify the factors associated with poor (defined as treatment failure, default, and death from any cause) or good (defined as cure or successful treatment completion) clinical outcomes. The analytic sensitivity of the Xpert assay for detecting rifampin resistance was assessed in vitro by testing cultures containing different ratios of drug-sensitive and drug-resistant organisms. Rifampin resistance was detected by the Xpert assay in 52 (14.1%) and by phenotypic DST in 55 (14.9%) patients. Mixed MTBC infections were identified in 37 (10.0%) patients. The Xpert assay was 92.7% (95% confidence interval [CI], 82.4% to 97.9%) sensitive for detecting rifampin resistance and 99.7% (95% CI, 98.3% to 99.9%) specific. When restricted to patients with mixed MTBC infections, Xpert sensitivity was 80.0% (95% CI, 56.3 to 94.3%). False-negative Xpert results (adjusted odds ratio [aOR], 6.6; 95% CI,1.2 to 48.2) and mixed MTBC infections (aOR, 6.5; 95% CI, 2.1 to 20.5) were strongly associated with poor clinical outcome. The Xpert assay failed to detect rifampin resistance in vitro when <90% of the organisms in the sample were rifampin resistant. Our study indicates that the Xpert assay has an increased false-negative rate for detecting rifampin resistance with mixed MTBC infections. In hyperendemic settings where mixed infections are common, the Xpert results might need further confirmation.     

Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.     

Use of smear-positive samples to assess the PCR-based genotype MTBDR assay for rapid, direct detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin

Isoniazid (INH) and rifampin (RIF) are two of the most important antituberculosis drugs, and resistance to both of these drugs can often result in treatment failure and fatal clinical outcome. Resistance to these two first-line drugs is most often attributed to mutations in the katG, inhA, and rpoB genes. Historically, the identification and testing of the susceptibility of Mycobacterium tuberculosis complex (MTBC) strains takes weeks to complete. Rapid detection of resistance using the PCR-based Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) has the potential to significantly shorten the turnaround time from specimen receipt to reporting of results of susceptibility testing. Therefore, the aim of the present study was to determine (i) the sensitivity and accuracy of the Genotype MTBDR assay for the detection of MTBC strains and (ii) the ability of the assay to detect the presence of INH and RIF resistance-associated mutations in katG and rpoB from samples taken directly from smear-positive clinical specimens. The results were compared with those obtained with the reference BACTEC 460TB system combined with standard DNA sequencing analysis methods for katG, inhA, and rpoB. A total of 92 drug-resistant and 51 pansusceptible smear-positive specimens were included in the study. The Genotype MTBDR assay accurately and rapidly detected MTBC strains in 94.4% of the 143 specimens and showed a sensitivity of 94.4% for katG and 90.9% for rpoB when used directly on smear-positive specimens. The assay correctly identified INH resistance in 48 (84.2%) of the 57 specimens containing strains with resistance to high levels of INH (0.4 microg/ml) and RIF resistance in 25 (96.2%) of the 26 specimens containing RIF-resistant strains.     

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.     

Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a single multiplex PCR and DNA sequencing

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.     

First evaluation of an improved assay for molecular genetic detection of tuberculosis as well as rifampin and isoniazid resistances

The commercially available line probe assay MTBDRplus 2.0 (Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect Mycobacterium tuberculosis complex (MTBC) and mutations conferring resistance to rifampin (RMP) and isoniazid (INH) directly in smear-negative and smear-positive pulmonary clinical specimens under routine laboratory conditions. A total of 348 samples originating from Moldova, a high-incidence country for tuberculosis (TB), were investigated. Two hundred fifty-seven (73.9%) were smear negative, 12 samples were excluded, and 81 (23.3%) were smear positive. Two DNA extraction methods were applied. Compared to culture and clinical data as the reference standard (adapted from Vadwai V et al., J. Clin. Microbiol. 49:2540-2545, 2011), overall sensitivity and specificity were 87.6 and 99.2%, respectively. One hundred four of the 257 smear-negative samples turned out to be culture positive, and 20 were MTBC culture negative but were positive based on clinical symptoms. The combined sensitivity and specificity in the subgroup of smear-negative samples were calculated to be 79.8 and 99.2%, respectively. MTBDRplus 2.0 detected RMP and INH resistance with sensitivity and specificity of 94.3 and 96.0%, respectively. In conclusion, the MTBDRplus 2.0 assay is a rapid and highly sensitive test for the detection of M. tuberculosis strains from smear-positive and -negative clinical specimens and provides additional information on RMP and INH resistance status, which can easily be included in routine laboratory work flow.     

Detection of resistance to second-line antituberculosis drugs by use of the genotype MTBDRsl assay: a multicenter evaluation and feasibility study

The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement.     

Performance of Cepheid® Xpert MTB/RIF® and TB-Biochip® MDR in two regions of Russia with a high prevalence of drug-resistant tuberculosis

The purpose of this study was to assess the performance of Cepheid® Xpert MTB/RIF® (“Xpert”) and TB-Biochip® MDR (“TB-Biochip”). Sputum specimens from adults with presumptive tuberculosis (TB) were homogenized and split for: (1) direct Xpert and microscopy, and (2) concentration for Xpert, microscopy, culture [Lowenstein–Jensen (LJ) solid media and Mycobacteria Growth Indicator Tube® (MGIT)], indirect drug susceptibility testing (DST) using the absolute concentration method and MGIT, and TB-Biochip. In total, 109 of 238 (45.8 %) specimens were culture-positive for Mycobacterium tuberculosis complex (MTBC), and, of these, 67 isolates were rifampicin resistant (RIF-R) by phenotypic DST and 64/67 (95.5 %) were isoniazid resistant (INH-R). Compared to culture of the same specimen, a single direct Xpert was more sensitive for detecting MTBC [95.3 %, 95 % confidence interval (CI), 90.0–98.3 %] than direct (59.6 %, 95 % CI, 50.2–68.5 %) or concentrated smear (85.3 %, 95 % CI, 77.7–91.1 %) or LJ culture (80.8 %, 95 % CI, 72.4–87.5 %); the specificity was 86.0 % (95 % CI, 78.9–91.3 %). Compared with MGIT DST, Xpert correctly identified 98.2 % (95 % CI, 91.5–99.9 %) of RIF-R and 95.5 % (95 % CI, 85.8–99.2 %) of RIF-susceptible (RIF-S) specimens. In a subset of 104 specimens, the sensitivity of TB-Biochip for MTBC detection compared to culture was 97.3 % (95 % CI, 91.0–99.5 %); the specificity was 78.1 % (95 % CI, 61.5–89.9 %). TB-Biochip correctly identified 100 % (95 % CI, 94.2–100 %) of RIF-R, 94.7 % (95 % CI, 76.7–99.7 %) of RIF-S, 98.2 % (95 % CI, 91.4–99.9 %) of INH-R, and 78.6 % (95 % CI, 52.1–94.2 %) of INH-S specimens compared to MGIT DST. Xpert and Biochip were similar in accuracy for detecting MTBC and RIF resistance compared to conventional culture methods.     

Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases

The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.     

Use of GenoType® MTBDRplus assay to assess drug resistance and mutation patterns of multidrug-resistant tuberculosis isolates in northern India

Purpose: The emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. The aim of this study was to evaluate drug resistance and mutational patterns in clinical isolates MDR-TB by GenoType® MTBDRplus assay. Material and Methods: A total of 350 non-repeated sputum specimens were collected from highly suspected drug-resistant pulmonary tuberculosis (PTB) cases; which were processed by microscopy, culture, differentiation and first line drug susceptibility testing (DST) using BacT/ALERT 3D system. Results: Among a total of 125 mycobacterium tuberculosis complex (MTBC) strains, readable results were obtained from 120 (96%) strains by GenoType® MTBDRplus assay. Only 45 MDR-TB isolates were analysed for the performance, frequency and mutational patterns by GenoType® MTBDRplus assay. The sensitivity of the GenoType® MDRTBplus assay for detecting individual resistance to rifampicin (RIF), isoniazid (INH) and multidrug resistance was found to be 95.8%, 96.3% and 97.7%, respectively. Mutation in codon S531L of the rpoB gene and codon S315T1 of katG genes were dominated in MDR-TB strains, respectively (P < 0.05). Conclusions: The GenoType® MTBDRplus assay is highly sensitive with short turnaround times and a rapid test for the detection of the most common mutations conferring resistance in MDR-TB strains that can readily be included in a routine laboratory workflow.     

Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INH^r, 356 were RIF^r, 292 were MOX^r/OFX^r, 230 were AMK^r, 219 were CAP^r, and 286 were KAN^r. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INH^r. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIF^r. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOX^r/OFX^r. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMK^r/CAP^r but 71% sensitivity for KAN^r. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMK^r and to 91% for detecting KAN^r. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.