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.     

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.     

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 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.     

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.     

Comparison of the characteristics of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates from sputum and lung lesions in chronic tuberculosis patients

Mycobacterium tuberculosis (Mtb) in sputum originates from lung cavities in tuberculosis (TB) patients. But drug susceptibility testing (DST) of sputum Mtb can not be conducted the same as in the lung because mutagenesis of bacilli may be happening in the lung during treatment and result in the possibility of the presence of heterogeneous drug-resistant subpopulations in the different lung lesions. This could be one of the reasons for low cure rates for multi-drug resistant (MDR)-TB. We studied the resected lungs of nine surgery patients with chronic TB. The isolates isolated from the sputum and different lung lesions of each patient were tested for phenotypic DST and genotyped using restriction fragment length polymorphism (RFLP) typing method. Genetic analysis to resistance to first and second line drugs was also performed. Five of nine patients were MDR-TB and three XDR-TB. DST results for ten anti-TB drugs were in accordance among different lung lesions in eight patients. However, only three of these eight patients showed the concordance of DST with sputum. Even though the isolates were heteroresistant, genotyping them by RFLP showed the clonal population in each individual patient. Six of eight followed-up patients achieved successful cure. In conclusion, the heteroresistance between sputum and lung lesions and a clonal population without mixed infection might provide useful information in establishing treatment regimen and surgery decision for MDR- and XDR-TB.     

Multidrug-resistant tuberculosis: rapid detection of resistance to rifampin and high or low levels of isoniazid in clinical specimens and isolates

The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis.     

Tuberculosis drug-resistance in Lisbon,Portugal: a 6-year overview

Multidrug-resistance and extensive drug-resistance pose a serious threat to tuberculosis management in Portugal. The country has high TB incidence rates in comparison with other European Union countries, with the Lisbon Health Region being one of the most affected. In the present study we have analysed a convenience sample of 3025 Mycobacterium tuberculosis clinical isolates, recovered over a 6-year period (2001–2006) in the Lisbon Health Region, regarding drug-resistance both to first-line and second-line drugs. Moreover, 100 of these isolates were also genotyped by 12-loci Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeats (MIRU-VNTR) analysis. We have compared each year and observed the existence of 22 different resistance profiles, with MDR-TB rates ranging between 9.9% and 15.2% and XDR-TB rates, relative to the number of MDR-TB isolates, between 44.3% and 66.1% (excluding 1 year here considered as an outlier). A steady increase in the fraction of MDR-TB isolates resistant to all first-line drugs was also noticed. The genotyping analysis of MDR-TB isolates revealed six clusters, of which three (Lisboa3, Lisboa4 and Q1) were related to XDR-TB. Our results show that active transmission of MDR- and XDR-TB is taking place and that the high prevalence of observed XDR-TB is due to the continued transmission of particular genetic clusters. Enforcement of the implementation of genotyping in diagnostic routines would lead to early detection of resistant cases.     

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.     

Use of Mycobacteriophage Quantitative PCR on MGIT Broths for a Rapid Tuberculosis Antibiogram

Phenotypic culture-based drug susceptibility testing (DST) for Mycobacterium tuberculosis is a valuable tool to identify four to six active drugs for individualized multidrug-resistant (MDR) tuberculosis (TB) regimens. Current culture-based methods are slow, however; therefore, we evaluated a rapid mycobacteriophage-based quantitative PCR (qPCR) assay for use directly on M. tuberculosis-positive MGIT broths. We compared phage qPCRs, using a simple cutoff of 3 for the ΔCq value (where Cq is quantification cycle, and ΔCq is calculated as the Cq of starting phage minus the Cq of TB isolates in drug-containing medium), on 325 clinical M. tuberculosis MGIT broth cultures versus the respective subcultured isolates tested by agar proportion. The median accuracy for the 13 drugs/concentrations tested was 98%, with most discrepancies being false-resistant results. Evaluation of phage qPCR on greater numbers of resistant strains of 393 isolates grown on Löwenstein-Jensen medium showed similar findings, with a median accuracy, sensitivity, and specificity of 97%, 90%, and 99%, respectively. This rapid culture-based DST methodology can be performed for any drug on TB-positive MGIT broths, with a specimen-to-antibiogram turnaround time of approximately 23.9 days, compared with waiting 58.6 days for isolate growth on solid medium followed by agar proportion DST.     

Microscopic Observation Drug Susceptibility Assay for Rapid Diagnosis of Lymph Node Tuberculosis and Detection of Drug Resistance

In this study, 132 patients with lymphadenopathy were investigated. Fifty-two (39.4%) were diagnosed with tuberculosis (TB). The microscopic observation drug susceptibility (MODS) assay provided rapid (13 days), accurate diagnosis (sensitivity, 65.4%) and reliable drug susceptibility testing (DST). Despite its lower sensitivity than that of other methods, its faster results and simultaneous DST are advantageous in resource-poor settings, supporting the incorporation of MODS into diagnostic algorithms for extrapulmonary 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.     

Primary drug resistance to anti-tuberculosis drugs in major towns of Amhara region, Ethiopia

Drug resistance is a major obstacle to effective TB control program performance. In this study, we assessed the prevalence of primary drug resistance in Mycobacterium tuberculosis (Mtb) isolates in Amhara Region, Ethiopia. A total of 112 Mtb isolates from cases with newly diagnosed pulmonary TB were subjected to drug susceptibility testing (DST) in a cross-sectional study. Isolates were tested for sensitivity to isoniazid, rifampicin, ethambutol, and streptomycin using the MGIT 960 protocol. A total of 93 Mtb isolates yielded valid DST results and 28 (30.1%) were resistant to one or more of first line anti-TB drugs. One isolate (1.0%) was multi-drug resistant (MDR), five (5.4%) were classified as poly-resistant and 22 showed single drug resistance to either streptomycin (n = 19) or isoniazid (n = 3). Isolates from HIV-positive patients were more likely to be resistant to at least one of the four anti-TB drugs compared with HIV-negative individuals (odds ratio 2.76, 95% confidence interval 1.06-7.22; p = 0.03). The study showed a high prevalence of primary drug resistance. Even though the prevalence of MDR was low, conditions that can contribute to the development of MDR are increasing. Therefore, regular monitoring of drug resistance and enhanced implementation of TB/HIV collaborative activities in the study region are imperative.     

Evaluation of Five User-Friendly Whole Genome Sequencing Software for Mycobacterium tuberculosis in Clinical Application

BackgroundWhole genome sequencing (WGS) is an increasingly useful tool for tuberculosis (TB) diagnosis and disease management. In this study, we evaluated the utility of user-friendly WGS tools in reporting resistance profiles and identifying lineages of clinical TB isolates from South Korea.MethodsForty clinical samples from TB patients showing discrepancies between their rapid molecular and conventional drug susceptibility tests were used in this study. Among these clinical isolates, 37 strains were successfully evaluated via WGS software, using the GenTB, TB Profiler, PhyResSE, CASTB, and Mykrobe.ResultsMore accurate and faster susceptibility results could be obtained with isoniazid than with rifampin. Using the phenotypic test as the gold standard, the isoniazid concordance rate between phenotypic drug susceptibility test (DST) and WGS (GenTB: 45.9%, TB profiler: 40.5%, PhyResSE: 40.5%, CASTB: 48.6%, and Mykrobe: 43.2%) was much higher than between phenotypic DST and rapid molecular genotypic DST (18.9%) among the 37 strains. In contrast, the rifampin concordance rate between phenotypic DST and WGS and that between phenotypic DST and rapid molecular genotypic DST was similar (81.1–89.2%). We also found novel mutations associated with INH in katG and ahpC gene region, not covered by the line probe assay. In addition, lineage analysis identified 81.1% of these samples as L2 East Asian lineage strains, and 18.9% as L4 Euro-American lineage strains.ConclusionWGS may play a pivotal role in TB diagnosis and the detection of drug resistance, genetic diversity, and transmission dynamics in the near future because of its accuracy, speed, and extensibility.     

Evaluation of TBc identification immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in samples from broth cultures

Tuberculosis (TB) is a disease of major public health concern worldwide, especially in developing countries. In addition, the human immunodeficiency virus (HIV) epidemic has increased the incidence of infection with nontuberculous mycobacteria (NTM). Rapid, accurate, and simple methods for differentiation of Mycobacterium tuberculosis complex (MTBC) isolates from NTM is greatly needed for successful control of the TB epidemic. This study was done to evaluate the clinical performance of the BD MGIT TBc identification test (TBc ID) for rapid identification of MTBC in samples from broth cultures. A total of 229 Ziehl-Neelsen (ZN) stain-positive MGIT cultures were tested using the TBc ID test, and the results were compared with those of the AccuProbe MTBC identification test (GenProbe, San Diego, CA). The agreement between the TBc ID test and the AccuProbe assay was 96% (kappa = 0.92; confidence interval [CI] = 0.869 to 0.971). The sensitivity, specificity, and negative and positive predictive values of the TBc ID test compared to the AccuProbe assay were 100%, 92.4%, 100%, and 92.2%, respectively. After additional molecular testing, the agreement between the two methods increased to 97.8% (kappa = 0.96; CI = 0.917 to 0.994), and the specificity and positive predictive value increased to 95.6% and 95.7%, respectively. The TBc ID test is a simple, sensitive, and specific test for identification of MTBC in samples from acid-fast bacillus (AFB) smear-positive cultures. The TBc ID test could be a good alternative to the AccuProbe test in TB diagnostic laboratories.     

Comparison of Three Molecular Assays for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is an emerging problem of great importance to public health, with higher mortality rates than drug-sensitive TB, particularly in immunocompromised patients. MDR-TB patients require treatment with more-toxic second-line drugs and remain infectious for longer than patients infected with drug-sensitive strains, incurring higher costs due to prolonged hospitalization. It is estimated that 90% of United Kingdom rifampin-resistant isolates are also resistant to isoniazid, making rifampin resistance a useful surrogate marker for multidrug resistance and indicating that second- and third-line drugs to which these isolates are susceptible are urgently required. Resistance in approximately 95% of rifampin-resistant isolates is due to mutations in a 69-bp region of the rpoB gene, making this a good target for molecular genotypic diagnostic methods. Two molecular assays, INNO-LiPA Rif.TB (Innogenetics, Zwijndrecht, Belgium) and MisMatch Detect II (Ambion, Austin, Tex.), were performed on primary specimens and cultures to predict rifampin resistance, and these methods were compared with the resistance ratio method. A third method, the phenotypic PhaB assay, was also evaluated in comparison to cultures in parallel with the genotypic assays. In an initial evaluation 16 of 16, 15 of 16, and 16 of 16 rifampin-resistant cultures (100, 93.8, and 100%, respectively), were correctly identified by line probe assay (LiPA), mismatch assay, and PhaB assay, respectively. Subsequently 38 sputa and bronchealveolar lavage specimens and 21 isolates were received from clinicians for molecular analysis. For the 38 primary specimens the LiPA and mismatch assay correlated with culture and subsequent identification and susceptibility tests in 36 and 38 specimens (94.7 and 100%), respectively. For the 21 isolates submitted by clinicians, both assays correlated 100% with routine testing.     

Evaluation of direct detection of Mycobacterium tuberculosis rifampin resistance by a nitrate reductase assay applied to sputum samples in Cotonou, Benin

The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1+ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates.     

Experimental platform utilising melting curve technology for detection of mutations in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates

Tuberculosis (TB) remains one of the most deadly infections with approximately a quarter of cases not being identified and/or treated mainly due to a lack of resources. Rapid detection of TB or drug-resistant TB enables timely adequate treatment and is a cornerstone of effective TB management. We evaluated the analytical performance of a single-tube assay for multidrug-resistant TB (MDR-TB) on an experimental platform utilising RT-PCR and melting curve analysis that could potentially be operated as a point-of-care (PoC) test in resource-constrained settings with a high burden of TB. Firstly, we developed and evaluated the prototype MDR-TB assay using specimens extracted from well-characterised TB isolates with a variety of distinct rifampicin and isoniazid resistance conferring mutations and nontuberculous Mycobacteria (NTM) strains. Secondly, we validated the experimental platform using 98 clinical sputum samples from pulmonary TB patients collected in high MDR-TB settings. The sensitivity of the platform for TB detection in clinical specimens was 75% for smear-negative and 92.6% for smear-positive sputum samples. The sensitivity of detection for rifampicin and isoniazid resistance was 88.9 and 96.0% and specificity was 87.5 and 100%, respectively. Observed limitations in sensitivity and specificity could be resolved by adjusting the sample preparation methodology and melting curve recognition algorithm. Overall technology could be considered a promising PoC methodology especially in resource-constrained settings based on its combined accuracy, convenience, simplicity, speed, and cost characteristics.     

Prevalence,associated factors,outcomes and transmission of extensively drug-resistant tuberculosis among multidrug-resistant tuberculosis patients in São Paulo,Brazil: a cross-sectional study

ObjectivesTo describe the prevalence, associated factors, treatment outcomes and transmission of extensively drug-resistant (XDR) tuberculosis (TB) in the state of São Paulo, Brazil, for 2011 to 2013.MethodsDrug susceptibility testing to first- and second-line drugs was performed by BACTEC MGIT 960 and molecular typing, by IS6110 restriction fragment length polymorphism. Clinical, epidemiologic and demographic data were obtained from surveillance information systems for TB. Patients were divided into three groups: multidrug resistant (MDR) TB (resistance to at least isoniazid and rifampicin), pre–XDR-TB (MDR-TB resistant to a fluoroquinolone or to at least one of the second-line injectable drugs) and XDR-TB (MDR-TB resistant to a fluoroquinolone and to at least one of the second-line injectables).ResultsAmong the 313 MDR-TB patients identified, the prevalence of XDR-TB and pre–XDR-TB was 10.2% (n = 32) and 19.2% (n = 60), respectively. Compared to MDR-TB patients, XDR-TB patients were more likely to be female (odds ratio (OR) = 2.74, 95% confidence interval (CI), 1.29–5.83), have a history of TB (OR = 5.16; 95% CI, 1.52–17.51) and present higher death rates (OR= 3.74; 95% CI 1.70–8.25). XDR-TB transmission was observed in households, between neighbours and between a patient and a healthcare worker in a hospital.ConclusionsThe prevalence of XDR-TB in the state of São Paulo is close to that estimated globally. Most of the XDR-TB patients were treated previously for TB and presented the lowest successful outcome rates. Because transmission of XDR-TB occurred, it is important that timely diagnosis of drug resistance is performed.     

Diagnostic Value of GeneChip for Detection of Resistant Mycobacterium tuberculosis in Patients with Differing Treatment Histories

The increasing burden of drug-resistant tuberculosis (TB) poses an escalating threat to national TB control programs. To assist appropriate treatment for TB patients, accurate and rapid detection of drug resistance is critical. The GeneChip test is a novel molecular tool for the diagnosis of TB drug resistance. Performance-related data on GeneChip are limited, and evaluation in new and previously treated TB cases has never been performed. We evaluated the diagnostic performance of GeneChip in detecting resistance to rifampin (RMP) and isoniazid (INH) and in detecting multidrug-resistant tuberculosis (MDR-TB) in comparison with standard drug susceptibility testing (DST) and compared the results in a group of previously treated and newly detected TB patients in an urban area in southeastern China. One thousand one hundred seventy-three (83.8%) new cases and 227 (16.2%) previously treated cases were collected between January 2011 and September 2013. The GeneChip showed a specificity of 97.8% and a sensitivity of 94.8% for detection of RMP resistance and 97.3% and 70.9%, respectively, for INH resistance in new cases. For previously treated cases, the overall sensitivity, specificity, and agreement rate are 94.6%, 91.3%, and 92.1%, respectively, for detection of RMP resistance and 69.7%, 95.4%, and 86.8%, respectively, for INH resistance. The sensitivity and specificity of MDR-TB were 81.8% and 99.0% in new cases and 77.8% and 93.4% in previously treated cases, respectively. The GeneChip system provides a simple, rapid, reliable, and accurate clinical assay for the detection of TB drug resistance, and it is a potentially important diagnostic tool in a high-prevalence area.