scholarly journals A Diagnostic Algorithm To Investigate Pyrazinamide and Ethambutol Resistance in Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Low-Incidence Setting

2018 ◽  
Vol 63 (2) ◽  
pp. e01798-18 ◽  
Author(s):  
Söenke Andres ◽  
Matthias I. Gröschel ◽  
Doris Hillemann ◽  
Matthias Merker ◽  
Stefan Niemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sanger Sequencing ◽  
Rational Design ◽  
Clinical Decision Making ◽  
Susceptibility Testing ◽  
Diagnostic Algorithm ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Content Type ◽  
Phenotypic Resistance

ABSTRACT Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol.

scholarly journals Evaluation of the Abbott RealTi m e MTB and RealTi m e MTB INH/RIF Assays for Direct Detection of Mycobacterium tuberculosis Complex and Resistance Markers in Respiratory and Extrapulmonary Specimens

2016 ◽  
Vol 54 (12) ◽  
pp. 3022-3027 ◽  
Author(s):  
Sabine Hofmann-Thiel ◽  
Nikolay Molodtsov ◽  
Uladzimir Antonenka ◽  
Harald Hoffmann
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Nucleic Acid Amplification ◽  
Content Type ◽  
Clinical Specimens ◽  
Different Types ◽  
Resistance Markers ◽  
Smear Negative

The Abbott RealTi m e MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTi m e MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDR plus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

scholarly journals Susceptibility Testing of Extensively Drug-Resistant and Pre-Extensively Drug-Resistant Mycobacterium tuberculosis against Levofloxacin, Linezolid, and Amoxicillin-Clavulanate

2013 ◽  
Vol 57 (6) ◽  
pp. 2522-2525 ◽  
Author(s):  
Imran Ahmed ◽  
Kauser Jabeen ◽  
Raunaq Inayat ◽  
Rumina Hasan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Critical Concentration ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Fluoroquinolone Resistance ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Amoxicillin Clavulanate

ABSTRACTPakistan is a high-burden country for tuberculosis (TB). The emergence and increasing incidence of extensively drug-resistant (XDR) TB has been reported in Pakistan. Similarly, the prevalence of multidrug-resistant TB infections with fluoroquinolone resistance (pre-XDR) is also increasing. To treat these infections, local drug susceptibility patterns of alternate antituberculosis agents, including levofloxacin (LVX), linezolid (LZD), and amoxicillin-clavulanate (AMC), is urgently needed. The aim of this study was to determine the susceptibility frequencies of drug-resistant (DR)Mycobacterium tuberculosisagainst LVX, LZD, and AMC. All susceptibilities were determined on Middlebrook 7H10 agar. A critical concentration was used for LVX (1 μg/ml), whereas MICs were determined for LZD and AMC.M. tuberculosisH37Rv was used as a control strain. A total of 102M. tuberculosisisolates (XDR,n= 59; pre-XDR,n= 43) were tested. Resistance to LVX was observed in 91.2% (93/102). Using an MIC value of 0.5 μg/ml as a cutoff, resistance to LZD (MIC ≥ 1 μg/ml) was noted in 5.9% (6/102). Although the sensitivity breakpoints are not established for AMC, the MIC values were high (>16 μg/ml) in 97.1% (99/102). Our results demonstrate that LZD may be effective for the treatment of XDR and pre-XDR cases from Pakistan. High resistance rates against LVX in our study suggest the use of this drug with caution for DR-TB cases from this area. Drug susceptibility testing against LVX and AMC may be helpful in complicated and difficult-to-manage cases.

scholarly journals Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature

2016 ◽  
Vol 60 (8) ◽  
pp. 4956-4960 ◽  
Author(s):  
Alice L. den Hertog ◽  
Sandra Menting ◽  
Richard Pfeltz ◽  
Matthew Warns ◽  
Salman H. Siddiqi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Low Temperature ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Acidic Ph ◽  
Content Type ◽  
The Past ◽  
Neutral Ph

ABSTRACTFor the past decades, an acidic pH has been used to renderMycobacterium tuberculosissusceptible to pyrazinamide forin vitrotesting. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.

scholarly journals Validation of the FluoroType MTBDR Assay for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates

2018 ◽  
Vol 56 (6) ◽  
pp. e00072-18 ◽  
Author(s):  
Doris Hillemann ◽  
Carsten Haasis ◽  
Sönke Andres ◽  
Tobias Behn ◽  
Katharina Kranzer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sanger Sequencing ◽  
High Sensitivity ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Molecular Diagnostic ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Specificity And Sensitivity ◽  
Genotype Mtbdrplus

ABSTRACT For Mycobacterium tuberculosis complex (MTBC), the rapid and accurate diagnosis of drug resistance is crucial to ensure early initiation of appropriate therapy. Recently, a new molecular diagnostic test, the FluoroType MTBDR, aimed at detecting rifampin and isoniazid resistance has become available. This study aimed to evaluate the FluoroType MTBDR in comparison to phenotypic drug susceptibility testing (DST) using M. tuberculosis complex isolates. MTBC isolates underwent phenotypic DST and were tested using the FluoroType MTBDR and Genotype MTBDRplus. Sanger sequencing of the key regions of rpoB, katG, inhA, and aphC was performed for isolates with discordant phenotypic and molecular results. Furthermore, isolates with specific wild-type bands missing in the Genotype MTBDRplus, indicating the presence of a mutation, were investigated by Sanger sequencing. Specificity and sensitivity, defined as the proportions of isolates correctly determined as susceptible and resistant by the FluoroType MTBDR compared to phenotypic DST, were calculated. A total of 180 culture isolates were included; phenotypic DST showed 85 isolates susceptible to isoniazid and rifampin, 7 with isoniazid monoresistance, 7 with rifampin monoresistance, and 81 with multidrug resistance. The specificity of the FluoroType MTBDR was 100% (95% confidence interval [CI], 96.0 to 100%) for both rifampin and isoniazid. The sensitivity was 91.7% (95% CI, 83.6 to 96.6%) for isoniazid and 98.9% (95% CI, 93.8 to 100.0%) for rifampin. The FluoroType MTBDR has a high sensitivity and specificity for the detection of rifampin and isoniazid resistance when using culture isolates.

scholarly journals Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

2013 ◽  
Vol 58 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Jongseok Lee ◽  
Derek T. Armstrong ◽  
Willy Ssengooba ◽  
Jeong-ae Park ◽  
Yeuni Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Critical Concentration ◽  
Microtiter Plate ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
Content Type ◽  
The Republic ◽  
Phenotypic Methods

ABSTRACTForMycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB forM. tuberculosisdrug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archivedM. tuberculosisisolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results forM. tuberculosissusceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

scholarly journals Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

2011 ◽  
Vol 55 (5) ◽  
pp. 2032-2041 ◽  
Author(s):  
Patricia J. Campbell ◽  
Glenn P. Morlock ◽  
R. David Sikes ◽  
Tracy L. Dalton ◽  
Beverly Metchock ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
First Line ◽  
Content Type ◽  
Line Drug

ABSTRACTThe emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced:rpoB(for resistance to RIF),katGandinhA(INH),pncA(PZA),embB(EMB),gyrA(CIP and OFX), andrrs,eis, andtlyA(KAN, AMK, and CAP). A total of 314 clinicalMycobacterium tuberculosiscomplex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% forrpoB, 85.4% and 100% forkatG, 16.5% and 100% forinhA, 90.6% and 100% forkatGandinhAtogether, 84.6% and 85.8% forpncA, and 78.6% and 93.1% forembB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in theM. tuberculosiscomplex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

scholarly journals Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Paras Jain ◽  
Spencer Garing ◽  
Deepshikha Verma ◽  
Rajagopalan Saranathan ◽  
Nicholas Clute-Reinig ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Limit Of Detection ◽  
Reference Method ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Metabolic Inhibitors ◽  
Health Crisis ◽  
Content Type

ABSTRACT Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis. We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

scholarly journals Occurrence ofrpoBMutations in Isoniazid-Resistant but Rifampin-Susceptible Mycobacterium tuberculosis Isolates from Germany

2013 ◽  
Vol 58 (1) ◽  
pp. 590-592 ◽  
Author(s):  
Sönke Andres ◽  
Doris Hillemann ◽  
Sabine Rüsch-Gerdes ◽  
Elvira Richter
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Content Type ◽  
Indicator Tube ◽  
Lowenstein Jensen ◽  
Proportion Method ◽  
Rifampin Resistance ◽  
Growth Indicator

ABSTRACTFour out of 143 phenotypically isoniazid-resistant but rifampin-susceptibleMycobacterium tuberculosisstrains that were isolated from patients in Germany in 2011 had mutations in the rifampin resistance-determining region ofrpoB. After performing drug susceptibility testing (DST) with two methods, the proportion method on Löwenstein-Jensen medium and using the Bactec 960 Mycobacteria Growth Indicator Tube system, we conclude that the two methods are equally reliable for phenotypic DST and MIC determination.

scholarly journals Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Bin Liang ◽  
Yaoju Tan ◽  
Zi Li ◽  
Xueshan Tian ◽  
Chen Du ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Melting Curve ◽  
Digital Pcr ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Melting Curve Analysis ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Highly Sensitive Detection

ABSTRACT Detection of heteroresistance of Mycobacterium tuberculosis remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less-abundant mutants through selective clamping of the wild type in mixed populations. The singleplex DeepMelt assay detected 0.01% katG S315T in 105 M. tuberculosis genomes/μl. The multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 104 M. tuberculosis genomes/μl. The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 precharacterized clinical isolates. Using the 1% proportion method as the gold standard, the sensitivity was found to be increased from 93.6% (176/188, 95% confidence interval [CI] = 89.2 to 96.3%) to 95.7% (180/188, 95% CI = 91.8 to 97.8%) compared to the MeltPro TB/INH assay. Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3% (20/24, 95% CI = 64.2 to 93.3%) to 91.7% (22/24, 95% CI = 74.2 to 97.7%). In both cases, the specificity remained nearly unchanged. All heteroresistant samples newly identified by the DeepMelt TB/INH assay were confirmed by DNA sequencing and even partially by digital PCR. The DeepMelt assay may fill the gap between current genotypic and phenotypic drug susceptibility testing for detecting drug-resistant tuberculosis patients.

scholarly journals Role of Disputed Mutations in the rpoB Gene in Interpretation of Automated Liquid MGIT Culture Results for Rifampin Susceptibility Testing of Mycobacterium tuberculosis

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Paolo Miotto ◽  
Andrea M. Cabibbe ◽  
Emanuele Borroni ◽  
Massimo Degano ◽  
Daniela M. Cirillo
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Silico ◽  
Susceptibility Testing ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Missense Mutations ◽  
Content Type ◽  
Low Level ◽  
Clinical Resistance

ABSTRACT Low-level rifampin resistance associated with specific rpoB mutations (referred as “disputed”) in Mycobacterium tuberculosis is easily missed by some phenotypic methods. To understand the mechanism by which some mutations are systematically missed by MGIT phenotypic testing, we performed an in silico analysis of their effect on the structural interaction between the RpoB protein and rifampin. We also characterized 24 representative clinical isolates by determining MICs on 7H10 agar and testing them by an extended MGIT protocol. We analyzed 2,097 line probe assays, and 156 (7.4%) cases showed a hybridization pattern referred to here as “no wild type + no mutation.” Isolates harboring “disputed” mutations (L430P, D435Y, H445C/L/N/S, and L452P) tested susceptible in MGIT, with prevalence ranging from 15 to 57% (overall, 16 out of 55 isolates [29%]). Our in silico analysis did not highlight any difference between “disputed” and “undisputed” substitutions, indicating that all rpoB missense mutations affect the rifampin binding site. MIC testing showed that “undisputed” mutations are associated with higher MIC values (≥20 mg/liter) compared to “disputed” mutations (4 to >20 mg/liter). Whereas “undisputed” mutations didn't show any delay (Δ) in time to positivity of the test tube compared to the control tube on extended MGIT protocol, “disputed” mutations showed a mean Δ of 7.2 days (95% confidence interval [CI], 4.2 to 10.2 days; P < 0.05), providing evidence that mutations conferring low-level resistance are associated with a delay in growth on MGIT. Considering the proved relevance of L430P, D435Y, H445C/L/N, and L452P mutations in determining clinical resistance, genotypic drug susceptibility testing (DST) should be used to replace phenotypic results (MGIT) when such mutations are found.

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