The performance of Xpert MTB/RIF and MTBDRplus within a Programmatic setting at TB Laboratory in Rio de Janeiro, Brazil.

BACKGROUND: Few studies in routine settings have confirmed the high accuracy of the Xpert MTB/RIF assay for detecting rifampicin resistance (RR) and the first-line probe assay (FL-LPA) for detecting both RR and isoniazid resistance (INHR). METHODS: The performance of Xpert MTB/RIF and MTBDRplus VER 2.0 LPA was evaluated in 180 Mycobacterium tuberculosis samples collected from January 2018 to December 2019 in Rio de Janeiro, Brazil. The results were compared with those from BACTEC MGIT 960 culture and drug susceptibility testing (DST). Whole-genome sequencing was performed on the samples with discordant results. RESULTS: The Xpert MTB/RIF assay showed a sensitivity (Se) of 93.3% and a specificity (Sp) of 97.6%, detecting RR. The performance of FL-LPA to identify RIF and INH resistance was, respectively, (Se) 100% and 83.3% and (Sp) 98.8% and 100%. Among 18 clinical isolates with INHR detected by FL-LPA, mutations in the katG gene were observed in 100% of samples, of which only two (11.1%) had mutations in both katG and inhA genes. Overall, the discordant results were identified in 9 (5%) samples. Among the four Xpert RIF-resistant and DST-sensitive, two harbored mutations in rpoB Leu430Pro. Among the four FL-LPA-sensitive and DST-resistant, one had a mutation in inhA 17G>T. FL-LPA showed high accuracy in detecting RR and INHR. CONCLUSIONS: The MTBDRplus test demonstrated excellent performance in detecting RR, and INHR in clinical isolates under routine conditions at a reference laboratory in Rio de Janeiro, Brazil. Incorporating both tests can improve drug-resistant tuberculosis treatment outcomes and monitor the INHR incidence.

The performance of Xpert MTB/RIF and MTBDRplus within a Programmatic setting at TB Laboratory in Rio de Janeiro, Brazil

ABSTRACT Background: Few studies in routine settings have confirmed the high accuracy of the Xpert MTB/RIF assay for detecting rifampicin resistance (RR) and the first-line probe assay (FL-LPA) for detecting both RR and isoniazid resistance (INHR). Methods: The performance of Xpert MTB/RIF and MTBDRplus VER 2.0 LPA was evaluated in 180 Mycobacterium tuberculosis samples collected from January 2018 to December 2019 in Rio de Janeiro, Brazil. The results were compared with those from BACTEC MGIT 960 culture and drug susceptibility testing (DST). Whole-genome sequencing was performed on the samples with discordant results. Results: The Xpert MTB/RIF assay showed a sensitivity (Se) of 93.3% and a specificity (Sp) of 97.6%, detecting RR. The performance of FL-LPA to identify RIF and INH resistance was, respectively, (Se) 100% and 83.3% and (Sp) 98.8% and 100%. Among 18 clinical isolates with INHR detected by FL-LPA, mutations in the katG gene were observed in 100% of samples, of which only two (11.1%) had mutations in both katG and inhA genes. Overall, the discordant results were identified in 9 (5%) samples. Among the four Xpert RIF-resistant and DST-sensitive, two harbored mutations in rpoB Leu430Pro. Among the four FL-LPA-sensitive and DST-resistant, one had a mutation in inhA 17G>T. FL-LPA showed high accuracy in detecting RR and INHR. Conclusions: The MTBDRplus test demonstrated excellent performance in detecting RR, and INHR in clinical isolates under routine conditions at a reference laboratory in Rio de Janeiro, Brazil. Incorporating both tests can improve drug-resistant tuberculosis treatment outcomes and monitor the INHR incidence.

Whole-genome sequencing as a tool for studying the microevolution of drug-resistant serial Mycobacterium tuberculosis isolates.

Treatment of drug-resistant tuberculosis requires extended use of more toxic and less effective drugs and may result in retreatment cases due to failure, abandonment or disease recurrence. It is therefore important to understand the evolutionary process of drug resistance in Mycobacterium tuberculosis. We here in describe the microevolution of drug resistance in serial isolates from six previously treated patients. Drug resistance was initially investigated through phenotypic methods, followed by genotypic approaches. The use of whole-genome sequencing allowed the identification of mutations in the katG, rpsL and rpoB genes associated with drug resistance, including the detection of rare mutations in katG and mixed populations of strains. Molecular docking simulation studies of the impact of observed mutations on isoniazid binding were also performed. Whole-genome sequencing detected 266 single nucleotide polymorphisms between two isolates obtained from one patient, suggesting a case of exogenous reinfection. In conclusion, sequencing technologies can detect rare mutations related to drug resistance, identify subpopulations of resistant strains, and identify diverse populations of strains due to exogenous reinfection, thus improving tuberculosis control by guiding early implementation of appropriate clinical and therapeutic interventions.

Genomic-based surveillance reveals high ongoing transmission of multi-drug-resistant Mycobacterium tuberculosis in Southern Brazil.

Genomic-based surveillance on the occurrence of drug resistance and its transmission dynamics has emerged as a powerful tool for the control of tuberculosis (TB). A whole-genome sequencing approach, phenotypic testing and clinical-epidemiological investigation were used to undertake a retrospective population-based study on drug-resistant (DR)-TB in Rio Grande do Sul, the largest state in Southern Brazil. The analysis included 305 resistant Mycobacterium tuberculosis strains sampled statewide from 2011 to 2014, and covered 75.7% of all DR-TB cases identified in this period. Lineage 4 was found to be predominant (99.3%), with high sublineage-level diversity composed mainly of 4.3.4.2 [Latin American and Mediterranean (LAM)/RD174], 4.3.3 (LAM/RD115) and 4.1.2.1 (Haarlem/RD182) sublineages. Genomic diversity was also reflected in resistance of the variants to first-line drugs. A large number of distinct resistance-conferring mutations, including variants that have not been reported previously in any other setting worldwide, and 22 isoniazid-monoresistant strains with mutations described as disputed in the rpoB gene but causing rifampicin resistance generally missed by automated phenotypic tests as BACTEC MGIT. Using a cut-off of five single nucleotide polymorphisms, the estimated recent transmission rate was 55.1%, with 168 strains grouped into 28 genomic clusters. The most worrying fact concerns multi-drug-resistant (MDR) strains, of which 73.4% were clustered. Different resistance profiles and acquisition of novel mutations intraclusters revealed important amplification of resistance in the region. This study described the diversity of M. tuberculosis strains, the basis of drug resistance, and ongoing transmission dynamics across the largest state in Southern Brazil, stressing the urgent need for MDR-TB transmission control state-wide.

Author Correction: Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil.

Mycobacterium tuberculosis (M.tb), the pathogen responsible for tuberculosis (TB) poses as the major cause of death among infectious diseases. The knowledge about the molecular diversity of M.tb enables the implementation of more effective surveillance and control measures and, nowadays, Whole Genome Sequencing (WGS) holds the potential to produce high-resolution epidemiological data in a high-throughput manner. Florianópolis, the state capital of Santa Catarina (SC) in south Brazil, shows a high TB incidence (46.0/100,000). Here we carried out a WGS-based evaluation of the M.tb strain diversity, drug-resistance and ongoing transmission in the capital metropolitan region. Resistance to isoniazid, rifampicin, streptomycin was identified respectively in 4.0% (n = 6), 2.0% (n = 3) and 1.3% (n = 2) of the 151 studied strains by WGS. Besides, resistance to pyrazinamide and ethambutol was detected in 0.7% (n = 1) and reistance to ethionamide and fluoroquinolone (FQ) in 1.3% (n = 2), while a single (0.7%) multidrug-resistant (MDR) strain was identified. SNP-based typing classified all isolates into M.tb Lineage 4, with high proportion of sublineages LAM (60.3%), T (16.4%) and Haarlem (7.9%). The average core-genome distance between isolates was 420.3 SNPs, with 43.7% of all isolates grouped across 22 genomic clusters thereby showing the presence of important ongoing TB transmission events. Most clusters were geographically distributed across the study setting which highlights the need for an urgent interruption of these large transmission chains. The data conveyed by this study shows the presence of important and uncontrolled TB transmission in the metropolitan area and provides precise data to support TB control measures in this region.

Emergence of multidrug-resistant Mycobacterium tuberculosis of the Beijing lineage in Portugal and Guinea-Bissau: a snapshot of moving clones by whole-genome sequencing.

The Beijing genotype comprises a highly disseminated strain type that is frequently associated with multidrug resistant (MDR) tuberculosis (TB) and increased transmissibility but, countries such as Portugal and Guinea-Bissau fall outside the regions phylogeographically associated with this specific genotype. Nevertheless, recent data shows that this genotype might be gradually emerging in these two countries as an underlying cause of primary MDR-TB. Here, we describe the emergence of Mycobacterium tuberculosis Beijing strains associated with MDR-TB in Portugal and Guinea-Bissau demonstrating the presence of the well described superclusters 100-32 and 94-32 in Portugal and Guinea-Bissau, respectively. Genome-wide analysis and comparison with a global genomic dataset of M. tuberculosis Beijing strains, revealed the presence of two genomic clusters encompassing isolates from Portugal and Guinea-Bissau, GC1 (n = 121) and GC2 (n = 39), both of which bore SNP signatures compatible with the 100-32/B0/W148 and 94-32/Central Asia Outbreak clades, respectively. Moreover, GC2 encompasses a cross-border cluster between Portugal, Guinea-Bissau and Brazil thus supporting migration-associated introduction of MDR-TB and subsequent clonal expansion at the community-level. The comparison with global Beijing datasets demonstrates the global reach of the disease and its complex dissemination across multiple countries while in parallel there are clear microevolutionary trajectories towards extensively drug resistant TB.

The role of mini-bronchoalveolar lavage fluid in the diagnosis of pulmonary tuberculosis in critically ill patients.

BACKGROUND: The detection of Mycobacterium tuberculosis (MTB) in the intensive care unit (ICU) presents several challenges, mainly associated to the clinical state of the patient. The presence of HIV infection further aggravates this scenario, requiring a reliable collection method, with better performance in the microbiological/molecular techniques to be used. We evaluated the performance of two methods for sample collection, mini bronchoalveolar lavage (Mini-BAL) and endotracheal aspirate (ETA), for diagnosis of pulmonary tuberculosis (PTB) in critically ill patients. METHODS: This prospective study involved 26 HIV positive ICU internalized patients, with presumptive PTB who required mechanical ventilation. Two samples were obtained prospectively from 26 HIV ICU patients with presumptive PTB by Mini-BAL and ETA. The samples were processed for smear microscopy, Löwenstein-Jensen medium and the BACTEC Mycobacteria Growth Indicator Tube 960 system®. We define as confirmed PTB patients with positive MTB culture. Furthermore, all samples obtained through the Mini-BAL were analyzed by Xpert® MTB/RIF. RESULTS: Our results demonstrated that the respiratory samples obtained by Mini-BAL were able to increase MTB detection in critically ill patients with presumptive PTB. The Mini-BAL allowed 30% increased recovery and guaranteed enough sample volume for processing in all methods. In addition, the larger volume of the samples obtained with this technique enabled the Xpert® MTB/RIF molecular test for diagnosis of TB. CONCLUSIONS: The Mini-BAL showed be an acceptable alternative to ETA in this population, since these critically ill and often-immunocompromised patients are more likely to develop complications related to invasive procedures.

First insights into circulating XDR and pre-XDR Mycobacterium tuberculosis in Southern Brazil.

Drug-resistant tuberculosis (DR-TB) is major problem in the fight against TB. Multidrug resistant (MDR) TB patients have a reduced treatment success rates and for, extensively drug-resistant (XDR) TB the cure rate does not exceed 25% in many countries. To evaluate the pre-XDR-TB and XDR-TB prevalence and transmission in Rio Grande do Sul State, in southern Brazil, we performed a retrospective WGS-based analysis of 87 MDR-TB cases, aiming to identify resistance-conferring mutations and its phylogenetic distinctiveness. Using a five SNP threshold for genomic clustering, 60 strains were genomically linked within 10 clusters, including 14 likely transmission events identified by retrospective conventional epidemiological investigation. Moreover, five likely transmission events involved 17 patients deprived of liberty in the same prison establishment. Mutations associated with isoniazid and rifampicin resistance were identified respectively in 97.70% and 98.85% of MDR M.tb strains, more frequently in katG and rpoB genes. In total, we identified eight (9.19%) pre-XDR and four (4.59%) XDR M.tb strains. Resistance to ofloxacin was observed in seven (8.04%) strains, all of them presenting resistance-conferring mutations. Phenotypic resistance from capreomycin and kanamycin was found in seven (8.04%) and four (4.59%) strains respectively, but no classic mutations associated with resistance to these drugs was identified. The results put in evidence a scenario involving multiple phylogenetically distinctive clades associated with pre-XDR and XDR-TB in the largest state of southern Brazil, while stressing the potential of using WGS to predict anti-TB drug resistance and need to halt MDR-TB transmission in the region.

Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

Abstract (1) Background: The Commercial Kit SIRE Nitratase® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study. (2) Methods: To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTECTMMGITTM960 system was used as a gold standard. (3) Results: The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTECTMMGITTM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. (4) Conclusion: Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

The Contribution of Efflux Pumps in Mycobacterium abscessus Complex Resistance to Clarithromycin.

The basis of drug resistance in Mycobacterium abscessus is still poorly understood. Nevertheless, as seen in other microorganisms, the efflux of antimicrobials may also play a role in M. abscessus drug resistance. Here, we investigated the role of efflux pumps in clarithromycin resistance using nine clinical isolates of M. abscessus complex belonging to the T28 erm(41) sequevar responsible for the inducible resistance to clarithromycin. The strains were characterized by drug susceptibility testing in the presence/absence of the efflux inhibitor verapamil and by genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. Efflux pump gene expression was studied by RT-qPCR upon exposure to clarithromycin. Verapamil increased the susceptibility to clarithromycin from 4- to ≥64-fold. The efflux pump genes MAB_3142 and MAB_1409 were found consistently overexpressed. The results obtained demonstrate that the T28 erm(41) polymorphism is not the sole cause of the inducible clarithromycin resistance in M. abscessus subsp. abscessus or bolletii with efflux activity providing a strong contribution to clarithromycin resistance. These data highlight the need for further studies on M. abscessus efflux response to antimicrobial stress in order to implement more effective therapeutic regimens and guidance in the development of new drugs against these bacteria.

A case report of disseminated Mycobacterium colombiense infection in an HIV patient.

Mycobacterium colombiense is a newly recognized member of the Mycobacterium avium complex, and only a few cases of infections caused by this pathogen have been reported. We present an imported case of disseminated disease caused by M. colombiense in an HIV patient in early February 2017.

In silico and in vitro evaluation of tetrahydropyridine compounds as efflux inhibitors in Mycobacterium abscessus.

Herein, we evaluated tetrahydropyridine (THP) compounds (NUNM) as antimicrobials and inhibitors of the efflux mechanism in M. abscessus. subsp. abscessus. The modulation factor (MF) of efflux inhibitors was calculated from the minimum inhibitory concentrations (MICs) of amikacin (AMI), ciprofloxacin (CIP) and clarithromycin (CLA) in the absence and presence of subinhibitory concentrations of the NUNM compounds and canonical inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and verapamil (VP). The kinetics of the intracellular accumulation of the fluorimetric substrate ethidium bromide (EtBr) was evaluated and calculated by the relative final fluorescence (RFF). In addition, molecular modeling simulations for the MmpL5 and Tap efflux transporters with ligands (CLA, NUNM, CCCP, VP and EtBr) were performed to better understand the efflux mechanism. We highlight the NUNM01 compound because it reduced the MICs of AMI, CIP and CLA by 4-, 4- and 16-fold, respectively, had the highest effect on EtBr accumulation (RFF = 3.1) and showed a significant in silico affinity for the evaluated proteins in docking simulations. Based on the analyses performed in vitro and in silico, we propose that NUNM01 is a potential pharmacophore candidate for the development of a therapeutic adjuvant for M. abscessus infections.

Genetic Diversity and Molecular Epidemiology of Mycobacterium tuberculosis in Roraima State, Brazil.

National border areas are special places for the spread of Mycobacterium tuberculosis (MTB). These regions concentrate vulnerable populations and constant population movements. Understanding the dynamics of the transmission of MTB is fundamental to propose control measures and to monitor drug resistance. We conducted a population-based prospective study of tuberculosis (TB) to evaluate molecular characteristics of MTB isolates circulating in Roraima, a state on the border of Venezuela and Guyana. Eighty isolates were genotyped by IS6110-RFLP (restriction fragment length polymorphism), spoligotyping, and 24-locus mycobacterial interspersed repetitive unit-variable number of repeats tandem (MIRU-VNTR). Drug susceptibility tests were performed by using the proportion method and GeneXpert® MTB/RIF (Cepheid, Sunnyvale, CA). Isolates showing a phenotypic resistance profile were submitted to polymerase chain reaction (PCR) and sequencing. Spoligotyping showed 40 distinct patterns with a high prevalence of Latin-American and Mediterranean (LAM), Haarlem (H), and the "ill-defined" T clades. Mycobacterial interspersed repetitive unit -VNTR and IS6110-RFLP showed clustering rates of 21.3% and 30%, respectively. Drug resistance was detected in 11 (15.1%) isolates, and all were found to have primary resistance; among these, six (8.2%) isolates were streptomycin mono-resistant, four (5.4%) isoniazid mono-resistant, and one (1.3%) multidrug resistant. This is the first study on the molecular epidemiology and drug resistance profile of MTB from Roraima. Herein, we describe high diversity of genetic profiles circulating in this region that may be driven by the introduction of new strain types because of large population flow in this region. In summary, our results showed that analyses of these circulating strains can contribute to a better understanding of TB epidemiology in the northern Brazilian border and be useful to establish public health policies on TB prevention.

Clonal expansion across the seas as seen through CPLP-TB database: A joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countries.

Tuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analysed the largest CPLP M. tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space. To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends.

Antimicrobial and Efflux Inhibitor Activity of Usnic Acid Against Mycobacterium abscessus.

New drugs are needed to treat infections with antimicrobial-resistant Mycobacterium abscessus; therefore, we evaluated usnic acid as an antimicrobial agent and efflux inhibitor (EI) against M. abscessus. Usnic acid showed antimicrobial activity, and synergistically, the EI verapamil increased this activity. In addition, when we evaluated the interaction of antimicrobials with usnic acid, the increase of their activity was observed. Finally, usnic acid showed an efflux inhibitory effect between the classical EIs verapamil and carbonyl cyanide m-chlorophenylhydrazine. In conclusion, usnic acid showed both antimicrobial and EI activity, indicating that this natural compound may be a promising scaffold for new drugs against this difficult-to-treat microorganism.

Activity of Rifabutin and Hemi-synthetic Derivatives Against Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus causes a wide range of clinical diseases that are difficult to treat. This microorganism is resistant not only to the classical antituberculosis agents but also to most of the antimicrobials that are currently available, resulting in limited therapeutic options and treatment failure. This scenario stresses the need to search for new drugs with activity against M. abscessus. OBJECTIVE: To evaluate in vitro the antimycobacterial activity and cytotoxicity of rifabutin (RFB 1) and ten derivatives (2-11) against M. abscessus ATCC 19977. METHOD: The minimum inhibitory concentration (MIC) of the molecules was determined by the microdilution broth method according to the guideline described in CLSI. The toxicity evaluation was carried in 96-well microplates, using the cell line J774A.1 (ATCC TIB-67). RESULT: From the eleven molecules tested, RFB 1 and RFB 4 were the compounds showing higher activities against M. abscessus, with MICs of 0.9 and 1.0 µM, respectively. The R1 and R2 moieties seem to have deciding influence over the final activity. Furthermore, N-oxide derivatives 9, 10, and 11 were also active against M. abscessus, with MICs of 7.2 µM, 1.8 µM and 3.8 µM, respectively. An explanatory hypothesis for the better activities of compounds RFB 1, RFB 4, RFB 10 and RFB 11 considers the likely hydrogen bonding between ligands and receptor, balancing the global flexibility and interaction energies. RFB 1 and its most effective derivatives were found to be not toxic. CONCLUSION: Besides RFB 1, its derivatives 4, 10 and 11 show potential for clinical development in the M. abscessus treatment.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

BACKGROUND: To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE: To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD: 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS: With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION: Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

In vitro and in silico analysis of the efficiency of tetrahydropyridines as drug efflux inhibitors in Escherichia coli.

The objectives of this study were to evaluate tetrahydropyridine derivatives as efflux inhibitors and to understand the mechanism of action of the compounds by in silico studies. Minimum inhibitory concentration (MIC) determination, fluorometric methods and docking simulations were performed. The compounds NUNL02, NUNL09 and NUNL10 inhibited efflux, and NUNL02 is very likely a substrate of the transporter protein AcrB. Docking studies suggested that the mechanism of action could be by competition with substrate for binding sites and protein residues. We showed for the first time the potential of tetrahydropyridines as efflux inhibitors and highlighted compound NUNL02 as an AcrB-specific inhibitor. Docking studies suggested that competition is the putative mechanism of action of these compounds.