MTBDRplus V2 Research Articles

Trends in rifampicin and isoniazid resistance in patients with presumptive TB.

BACKGROUND: Early diagnosis of drug-resistant TB (DR-TB) is crucial in preventing the spread of the disease in the community. Introduction of upfront decentralised drug susceptibility testing to district-level as part of universal drug susceptibility testing (UDST) policy increased the feasibility of rapid and early testing for drug resistance closer to the patient and has resulted in reduced circumstances for transmission. The introduction of the first-line line-probe assay (FL-LPA), GenoType® MTBDRplus v2, has had an extensive impact on the management of multidrug-resistant TB (MDR-TB) in India.MATERIALS and METHODS: Sputum samples of patients with presumptive TB and DR-TB from selected districts of Tamil Nadu received through National TB Elimination Programme (NTEP) were subjected to FL-LPA as per programme guidelines. In this study, we present trends in genotypic resistance to isoniazid (INH) and rifampicin (RIF) during the 4 years (2016-2019) among these patients. Band patterns were analysed as per the updated GLI (Global Laboratory Initiative) LPA interpretation and reporting guidelines.RESULTS: A total of 26,349 samples were received during the study period. Smear-positive samples (n = 20231) were directly subjected to FL-LPA; smear-negative samples were cultured in liquid media and M. tuberculosis-positive cultures were tested using FL-LPA. A total of 18,441 were MTB-positive on FL-LPA. INH monoresistance, RIF monoresistance and MDR-TB was observed in respectively 8.7%, 1.1% and 3.3% of the samples. There was a decreasing trend in all types of resistance observed particularly after 2017 (P < 0.001). MDR-TB showed a steady decrease from 5.6% to 1.8%. S531L (19.5%) and S315T (61.1%) were the most common mutations identified in the rpoB and katG genes, respectively. The percentage of inhA-c-15t promoter mutation, indicating low-level INH resistance, showed a consistent increase (P < 0.001).CONCLUSION: The impact of the UDST policy on the NTEP may have led to this decreasing trend in RIF and INH resistance observed in the study period. The increase in low-level INH resistance mutation inhA-c-15t may be associated with ethionamide/prothionamide resistance, and this should be taken into account when designing DR-TB regimen.

Genetic structure of drug-resistant Mycobacterium tuberculosis strains in Peru based on haplotypes obtained from a line probe assay

To determine the genetic structure of drug-resistant strains of Mycobacterium tuberculosis that circulated throughout Peru during the years 2011-2015, by using haplotypes obtained from a line probe assay. A total of 6589 samples that were admitted to the Instituto Nacional de Salud for routine diagnosis using the GenoType® MTBDRplus v2 assay were analyzed during the study period. Resistant haplotypes were created by concatenating 21 polymorphic sites of the evaluated genes using the line probe assay; and the association analysis was carried out with phenotypes obtained by the 7H10 agar ratio method. The most frequent mutations were: rpoB S531L (55.4%) and rpoB D516V (18.5%) for rifampicin resistance, and katG S315T (59.5%) and inhA c-15t (25.7%) for isoniazid resistance. We obtained 13 representative haplotypes (87.8% of analyzed samples), 6 corresponded to the multidrug-resistant genotype, 4 to the isoniazid mono-resistant genotype and 3 to the rifampicin mono-resistant genotype. Eighteen regions and the province of Callao showed high haplotype diversity; four showed moderate diversity and two showed low diversity. Most regions showed high haplotype diversity; in addition, most drug-resistant strains of Mycobacterium tuberculosis were concentrated in the cities of Lima and Callao. Likewise, drug-resistant Mycobacterium tuberculosis strains circulating in Peru mainly contain the genetic markers with the highest prevalence worldwide, which are associated with resistance to rifampicin and isoniazid.

Magnitude of Phenotypic and MTBDRplus Line Probe Assay First-Line Anti-Tuberculosis Drug Resistance Among Tuberculosis Patients; Northwest Ethiopia.

BackgroundMycobacterium tuberculosis (Mtb) drug resistance is a key challenge in ending TB.ObjectiveThe study aimed to determine anti-TB drug resistance and compare the discordance between phenotypic and genotypic drug-susceptibility testing (DST).MethodsProspective enrollment and sputum collection from patients suspected of active pulmonary TB from May 2018 to December 2019 at the University of Gondar Hospital. Phenotypic DST study for streptomycin, isoniazid, rifampin, and ethambutol was done by MGIT 360 SIRE Kit. Genotypic resistance for isoniazid and rifampin was performed by MTBDRplus v2 line probe assay (LPA) and compared to phenotypic drug resistance.ResultsA total of 376 patients, median age 32 years, and 53.7% male were enrolled. Mtb was isolated from 126 patients. 106/126 (84%) patients were newly diagnosed with TB and 20 patients with prior TB treatment. Seventy (66.0%) were susceptible to all anti‐TB drugs tested. Twenty-five (19.8%) of the isolates were resistant to isoniazid, 12 (9.5%) to rifampicin and six (5%) were multidrug resistant. Among previously treated TB patients, 4 (20.0%) and 5 (25.0%) were mono-resistant and poly-resistant, respectively. The sensitivity and specificity of LPA resistance for isoniazid were 94.4% and 100%, and for rifampin was 75.0% and 100%, respectively.ConclusionThe frequency of mono- and poly-drug resistance among both newly diagnosed and previously treated TB patients was high to the rest of the nation. MTBDRplus showed excellent concordance for isoniazid and rifampin. We concluded that DST should be performed for all patients to improve management and decrease spread of drug-resistant Mtb strains in the community.

Evaluation of genotype MTBDRplus V2 and genotype MTBDRsl V2 for the diagnosis of extrapulmonary tuberculosis in India

Microbiological diagnosis of extra-pulmonary tuberculosis (EPTB) has been one of the most difficult aspects of tuberculosis (TB) management. Availability of better imaging and diagnostic modalities has led to an increase in the number of diagnosed cases. The current upsurge in multidrug-resistant tuberculosis warrants routine testing of EPTB samples for resistance at baseline with shorter turn-around time.A total of 369 EPTB specimens were subjected to Ziehl-Neelsen (ZN) stain, liquid culture (LC) with phenotypic drug susceptibility testing, MTBDRplus V.2 and MTBDRsl V.2. The molecular categorisation of resistant specimens was further reconfirmed with sequencing.The sensitivity and specificity of MTBDRplus V.2 to detect Mycobacterium tuberculosis (MTB) when compared to ZN stain was 97.9% and 89.2%, respectively while it was 73.4% and 83.8%, respectively when compared to LC. Similarly, for MTBDRsl V.2, the sensitivity and specificity for detection of MTB when compared with ZN was 95.6% and 91.9%, respectively and 75% and 89.2%, respectively when compared to LC. In smear-positive specimens, 94% (141/150) and 86% (129/150) valid results were observed in MTBDRplus V.2 and MTBDRsl V.2, respectively.The utilisation of both MTBDRplus V.2 and MTBDRsl V.2 for the diagnosis of smear-positive EPTB specimens would be useful in programmatic management of TB in high-burden settings.

The Performance of the Abbott Real Time MTB RIF/INH Compared to the MTBDRplus V2 for the Identification of MDR-TB Among Isolates.

BackgroundThe GenoType MTBDRplus V2 line-probe assay (LPA) is routinely used in clinical patient management to characterise the susceptibility of Mycobacterium tuberculosis complex to rifampicin (Rif) and isoniazid (INH) directly from sputum and cultured isolates. The laboratory workflow requires skill and three separate areas to minimize contamination and banding pattern interpretation requires experienced laboratory personnel. We explored the use of the RT MTB RIF/INH assay performed on the Abbott m2000 platform as an alternative laboratory platform.MethodsIsolates (n=93) consisting of fully susceptible, Rif- or INH-mono-resistant and multi-drug resistant (MDR) strains were tested on both MTBDRplus v2 and RT MTB RIF/INH assays. Both assays target the rpoB, katG and inhA genes for resistance-detection mutations. Concordance was assessed using percent agreement and the kappa statistic. Those specimens with discordant results were further assessed using Sanger sequencing.ResultsA total of 89% (83/93) of cultured isolates generated successful results on the RT MTB/RIF-INH assay and MTBDRplus assays. Of the 10 discordant results, where sequencing was used as the reference method, the RT MTB RIF/INH assays misclassified six resistance isolates, while the LPA misclassified seven.DiscussionOverall, the RT MTB RIF/INH demonstrated good agreement with the LPA, and a better correlation with sequencing on discrepant isolates specifically with mutations occurring in codon 511 of the rpoB gene. The RT MTB RIF/INH therefore can be used to complement existing laboratory algorithms determining Rif and INH resistance profiles, with less emphasis on manual laboratory processing.

Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú

Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.

Potential Application of Digitally Linked Tuberculosis Diagnostics for Real-Time Surveillance of Drug-Resistant Tuberculosis Transmission: Validation and Analysis of Test Results.

BackgroundTuberculosis (TB) is the highest-mortality infectious disease in the world and the main cause of death related to antimicrobial resistance, yet its surveillance is still paper-based. Rifampicin-resistant TB (RR-TB) is an urgent public health crisis. The World Health Organization has, since 2010, endorsed a series of rapid diagnostic tests (RDTs) that enable rapid detection of drug-resistant strains and produce large volumes of data. In parallel, most high-burden countries have adopted connectivity solutions that allow linking of diagnostics, real-time capture, and shared repository of these test results. However, these connected diagnostics and readily available test results are not used to their full capacity, as we have yet to capitalize on fully understanding the relationship between test results and specific rpoB mutations to elucidate its potential application to real-time surveillance.ObjectiveWe aimed to validate and analyze RDT data in detail, and propose the potential use of connected diagnostics and associated test results for real-time evaluation of RR-TB transmission.MethodsWe selected 107 RR-TB strains harboring 34 unique rpoB mutations, including 30 within the rifampicin resistance–determining region (RRDR), from the Belgian Coordinated Collections of Microorganisms, Antwerp, Belgium. We subjected these strains to Xpert MTB/RIF, GenoType MTBDRplus v2.0, and Genoscholar NTM + MDRTB II, the results of which were validated against the strains’ available rpoB gene sequences. We determined the reproducibility of the results, analyzed and visualized the probe reactions, and proposed these for potential use in evaluating transmission.ResultsThe RDT probe reactions detected most RRDR mutations tested, although we found a few critical discrepancies between observed results and manufacturers’ claims. Based on published frequencies of probe reactions and RRDR mutations, we found specific probe reactions with high potential use in transmission studies: Xpert MTB/RIF probes A, Bdelayed, C, and Edelayed; Genotype MTBDRplus v2.0 WT2, WT5, and WT6; and Genoscholar NTM + MDRTB II S1 and S3. Inspection of probe reactions of disputed mutations may potentially resolve discordance between genotypic and phenotypic test results.ConclusionsWe propose a novel approach for potential real-time detection of RR-TB transmission through fully using digitally linked TB diagnostics and shared repository of test results. To our knowledge, this is the first pragmatic and scalable work in response to the consensus of world-renowned TB experts in 2016 on the potential of diagnostic connectivity to accelerate efforts to eliminate TB. This is evidenced by the ability of our proposed approach to facilitate comparison of probe reactions between different RDTs used in the same setting. Integrating this proposed approach as a plug-in module to a connectivity platform will increase usefulness of connected TB diagnostics for RR-TB outbreak detection through real-time investigation of suspected RR-TB transmission cases based on epidemiologic linking.

Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays

ObjectivesNeither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. MethodsWe designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. ResultsThe technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. ConclusionsWe propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.

Comparison of Two Molecular Assays For Detecting Smear Negative Pulmonary Tuberculosis

ObjectiveTo compare the performance of MTBDRplus V2 and Xpert MTB/RIF for detecting smear negative pulmonary tuberculosis (PTB). MethodsClinical PTB suspects were enrolled consecutively in Anhui Chest Hospital and Xi'an Chest Hospital from January to December in 2014. The sputum samples of smear negative PTB suspects were collected and decontaminated. The sediment was used to conduct MTBDRplus V2, Xpert MTB/RIF and drug susceptibility test (DST). All the samples with discrepant drug susceptibility result between molecular methods and phenotypic method were confirmed by DNA sequencing. ResultsA total of 1973 cases were enrolled in this study. The detection rates of Mycobacterium tuberculosis complex (MTBC) by MTBDRplus V2 and Xpert MTB/RIF were 27.67% and 27.98%, respectively. When setting MGIT culture result as a gold standard, the sensitivity and specificity of MTBDRplus V2 were 86.74% and 93.84%, and the sensitivity and specificity of Xpert MTB/RIF were 86.55% and 93.43%, respectively. For the detection of the resistance to rifampin, the sensitivity and specificity of MTBDRplus V2 were 94.34% and 96.62%, and the sensitivity and specificity of Xpert MTB/RIF were 88.68% and 95.96%, respectively. For the detection of the resistance to isoniazid, the sensitivity and specificity of MTBDRplus V2 were 77.38% and 98.02%, respectively. ConclusionMTBDRplus V2 and Xpert MTB/RIF can be used to detect MTBC in smear negative samples with satisfactory performance.

Multi-center Study to Assess the Non-inferiority of Nipro NTM + MDRTB and Hain Genotype Mtbdrplus V2 Line Probe Assays Compared to Hain Genotype Mtbdrplus V1

Multi-center Study to Assess the Non-inferiority of Nipro NTM + MDRTB and Hain Genotype Mtbdrplus V2 Line Probe Assays Compared to Hain Genotype Mtbdrplus V1

The Diagnostic Performance of the GenoType MTBDR plus Version 2 Line Probe Assay Is Equivalent to That of the Xpert MTB/RIF Assay

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.