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Background: Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains. Method: In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media. Results: For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = ∞ [95% confidence interval (CI) 1.9–∞], P = 0.004 for MDR, OR = ∞ [95% CI 3.3–∞], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6–710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0–26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98). Conclusion: Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates.

Background: Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains. Method: In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media. Results: For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = 8 [95% confidence interval (CI) 1.9-8], P = 0.004 for MDR, OR = 8 [95% CI 3.3-8], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6-710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0-26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98). Conclusion: Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates. [ABSTRACT FROM AUTHOR]

Abstract Background Multidrug-resistant Mycobacterium tuberculosis (Mtb) is a significant global public health threat. Genotypic resistance prediction from Mtb DNA sequences offers an alternative to laboratory-based drug-susceptibility testing. User-friendly and accurate resistance prediction tools are needed to enable public health and clinical practitioners to rapidly diagnose resistance and inform treatment regimens. Results We present Translational Genomics platform for Tuberculosis (GenTB), a free and open web-based application to predict antibiotic resistance from next-generation sequence data. The user can choose between two potential predictors, a Random Forest (RF) classifier and a Wide and Deep Neural Network (WDNN) to predict phenotypic resistance to 13 and 10 anti-tuberculosis drugs, respectively. We benchmark GenTB’s predictive performance along with leading TB resistance prediction tools (Mykrobe and TB-Profiler) using a ground truth dataset of 20,408 isolates with laboratory-based drug susceptibility data. All four tools reliably predicted resistance to first-line tuberculosis drugs but had varying performance for second-line drugs. The mean sensitivities for GenTB-RF and GenTB-WDNN across the nine shared drugs were 77.6% (95% CI 76.6–78.5%) and 75.4% (95% CI 74.5–76.4%), respectively, and marginally higher than the sensitivities of TB-Profiler at 74.4% (95% CI 73.4–75.3%) and Mykrobe at 71.9% (95% CI 70.9–72.9%). The higher sensitivities were at an expense of ≤ 1.5% lower specificity: Mykrobe 97.6% (95% CI 97.5–97.7%), TB-Profiler 96.9% (95% CI 96.7 to 97.0%), GenTB-WDNN 96.2% (95% CI 96.0 to 96.4%), and GenTB-RF 96.1% (95% CI 96.0 to 96.3%). Averaged across the four tools, genotypic resistance sensitivity was 11% and 9% lower for isoniazid and rifampicin respectively, on isolates sequenced at low depth (< 10× across 95% of the genome) emphasizing the need to quality control input sequence data before prediction. We discuss differences between tools in reporting results to the user including variants underlying the resistance calls and any novel or indeterminate variants Conclusions GenTB is an easy-to-use online tool to rapidly and accurately predict resistance to anti-tuberculosis drugs. GenTB can be accessed online at https://gentb.hms.harvard.edu , and the source code is available at https://github.com/farhat-lab/gentb-site .

Background: Although phenotypic drug susceptibility testing (DST) of Mycobacterium tuberculosis (Mtb) takes up to 6–8 weeks, little is known about how drug susceptibility is affected during this period. Methods: We performed a prospective cohort study to investigate the development of drug resistance (DR) during turnaround time (TAT), including 359 pulmonary tuberculosis (PTB) patients with a baseline DST result of an Mtb isolate collected at TB diagnosis and a follow-up DST result of an Mtb isolate collected when baseline DST result was available between 2013 and 2018. Whole-genome sequencing (WGS) was used to differentiate between acquired drug resistance, exogenous reinfection, and mixed infection. Results: Among the studied patients, during TAT for DST, 116 (32.3%) developed DR to four first-line drugs (rifampicin, isoniazid, pyrazinamide, ethambutol). Among 116 pairs of isolates included for WGS, 21 pairs were classified as acquired drug resistance with single nucleotide polymorphisms (SNPs) differences less than 12. Four pairs with an intermediate SNPs differences displayed minor differences in related genotypes and were assessed as mixed infection. The remaining 91 pairs had high SNPs differences consistent with exogenous reinfection. Conclusions: The exogenous reinfection of drug-resistant strains played a vital role in the development of DR of Mtb isolates during TAT for DST, highlighting the need for both rapid DST methods and improved infection control.

Whole genome sequencing (WGS) can provide insight into drug-resistance, transmission chains and the identification of outbreaks, but data analysis remains an obstacle to its routine clinical use. Although several drug-resistance prediction tools have appeared, until now no website integrates drug-resistance prediction with strain genetic relationships and species identification of nontuberculous mycobacteria (NTM). We have established a free, function-rich, user-friendly online platform for MTB WGS data analysis (SAM-TB, http://samtb.szmbzx.com) that integrates drug-resistance prediction for 17 antituberculosis drugs, detection of variants, analysis of genetic relationships and NTM species identification. The accuracy of SAM-TB in predicting drug-resistance was assessed using 3177 sequenced clinical isolates with results of phenotypic drug-susceptibility tests (pDST). Compared to pDST, the sensitivity of SAM-TB for detecting multidrug-resistant tuberculosis was 93.9% [95% confidence interval (CI) 92.6–95.1%] with specificity of 96.2% (95% CI 95.2–97.1%). SAM-TB also analyzes the genetic relationships between multiple strains by reconstructing phylogenetic trees and calculating pairwise single nucleotide polymorphism (SNP) distances to identify genomic clusters. The incorporated mlstverse software identifies NTM species with an accuracy of 98.2% and Kraken2 software can detect mixed MTB and NTM samples. SAM-TB also has the capacity to share both sequence data and analysis between users. SAM-TB is a multifunctional integrated website that uses WGS raw data to accurately predict antituberculosis drug-resistance profiles, analyze genetic relationships between multiple strains and identify NTM species and mixed samples containing both NTM and MTB. SAM-TB is a useful tool for guiding both treatment and epidemiological investigation. [ABSTRACT FROM AUTHOR]

Background: Multidrug-resistant Mycobacterium tuberculosis (Mtb) is a significant global public health threat. Genotypic resistance prediction from Mtb DNA sequences offers an alternative to laboratory-based drug-susceptibility testing. User-friendly and accurate resistance prediction tools are needed to enable public health and clinical practitioners to rapidly diagnose resistance and inform treatment regimens. Results: We present Translational Genomics platform for Tuberculosis (GenTB), a free and open web-based application to predict antibiotic resistance from next-generation sequence data. The user can choose between two potential predictors, a Random Forest (RF) classifier and a Wide and Deep Neural Network (WDNN) to predict phenotypic resistance to 13 and 10 anti-tuberculosis drugs, respectively. We benchmark GenTB's predictive performance along with leading TB resistance prediction tools (Mykrobe and TB-Profiler) using a ground truth dataset of 20,408 isolates with laboratory-based drug susceptibility data. All four tools reliably predicted resistance to first-line tuberculosis drugs but had varying performance for second-line drugs. The mean sensitivities for GenTB-RF and GenTB-WDNN across the nine shared drugs were 77.6% (95% CI 76.6–78.5%) and 75.4% (95% CI 74.5–76.4%), respectively, and marginally higher than the sensitivities of TB-Profiler at 74.4% (95% CI 73.4–75.3%) and Mykrobe at 71.9% (95% CI 70.9–72.9%). The higher sensitivities were at an expense of ≤ 1.5% lower specificity: Mykrobe 97.6% (95% CI 97.5–97.7%), TB-Profiler 96.9% (95% CI 96.7 to 97.0%), GenTB-WDNN 96.2% (95% CI 96.0 to 96.4%), and GenTB-RF 96.1% (95% CI 96.0 to 96.3%). Averaged across the four tools, genotypic resistance sensitivity was 11% and 9% lower for isoniazid and rifampicin respectively, on isolates sequenced at low depth (< 10× across 95% of the genome) emphasizing the need to quality control input sequence data before prediction. We discuss differences between tools in reporting results to the user including variants underlying the resistance calls and any novel or indeterminate variants Conclusions: GenTB is an easy-to-use online tool to rapidly and accurately predict resistance to anti-tuberculosis drugs. GenTB can be accessed online at https://gentb.hms.harvard.edu, and the source code is available at https://github.com/farhat-lab/gentb-site. [ABSTRACT FROM AUTHOR]

A Cannas,1 O Butera,1 G Gualano,2 MP Parracino,3 C Venditti,1 A Mazzarelli,1 F Palmieri,2 E Girardi,3,* A Di Caro1,* 1Microbiology and Bio-Repository Unit, National Institute for Infectious Diseases “L. Spallanzani” IRCCS, Rome, Italy; 2Respiratory Infectious Diseases Unit, National Institute for Infectious Diseases “L. Spallanzani” IRCCS, Rome, Italy; 3Clinical Epidemiology Unit, National Institute for Infectious Diseases “L. Spallanzani” IRCCS, Rome, Italy*These authors contributed equally to this workCorrespondence: A CannasNational Institute for Infectious Diseases “L. Spallanzani” IRCCS, Via Portuense 292, Rome 00149, ItalyTel +39 06 5517 0905Email angela.cannas@inmi.itPurpose: Multidrug-resistant tuberculosis (MDR-TB) is a major burden to public health in low incidence countries in Europe. The aim of this study was to attempt to have a better insight into the trends of MDR-TB in the metropolitan area of Rome, within the Italian and the foreign-born population, based on microbiological and demographic data.Patients and methods: We performed a prospective study, collecting microbiological data based on phenotypic drug-resistant testing (DST) of TB strains consecutively isolated in a referral hospital in Rome, the capital city of a low TB incidence country, over a 6-year period, and correlated them to the geographical origin of patients. This study was carried out in a referral hospital for patients with drug-resistant TB from the whole region.Results: Drug-resistance data from 926 patients with a microbiological diagnosis of TB from 2011 to 2016 show a 5.5% rate of MDR-TB, mostly occurring in patients born in a single East European country, that has a high incidence of MDR-TB. The strains isolated from these patients frequently carry additional resistances, leading to an increased risk of developing extensively drug-resistant (XDR) TB.Conclusion: In the great metropolitan area of Rome, MDR-TB more frequently occurs in patients who were born in a single country from Eastern Europe known to have high rates of MDR-TB and long-time residents in Italy. Recent immigrants from non-European countries do not appear to contribute to the rates of MDR-TB reported in this article. This knowledge of local TB trends could help improve the measures of surveillance and prevention of disease.Keywords: tuberculosis, drug-susceptibility testing, MDR-TB, XDR-TB

Abstract Background Mycobacterium tuberculosis resistance to anti-tuberculosis drugs is a major threat to global public health. Whole genome sequencing (WGS) is rapidly gaining traction as a diagnostic tool for clinical tuberculosis settings. To support this informatically, previous work led to the development of the widely used TBProfiler webtool, which predicts resistance to 14 drugs from WGS data. However, for accurate and rapid high throughput of samples in clinical or epidemiological settings, there is a need for a stand-alone tool and the ability to analyse data across multiple WGS platforms, including Oxford Nanopore MinION. Results We present a new command line version of the TBProfiler webserver, which includes hetero-resistance calling and will facilitate the batch processing of samples. The TBProfiler database has been expanded to incorporate 178 new markers across 16 anti-tuberculosis drugs. The predictive performance of the mutation library has been assessed using > 17,000 clinical isolates with WGS and laboratory-based drug susceptibility testing (DST) data. An integrated MinION analysis pipeline was assessed by performing WGS on 34 replicates across 3 multi-drug resistant isolates with known resistance mutations. TBProfiler accuracy varied by individual drug. Assuming DST as the gold standard, sensitivities for detecting multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) were 94% (95%CI 93–95%) and 83% (95%CI 79–87%) with specificities of 98% (95%CI 98–99%) and 96% (95%CI 95–97%) respectively. Using MinION data, only one resistance mutation was missed by TBProfiler, involving an insertion in the tlyA gene coding for capreomycin resistance. When compared to alternative platforms (e.g. Mykrobe predictor TB, the CRyPTIC library), TBProfiler demonstrated superior predictive performance across first- and second-line drugs. Conclusions The new version of TBProfiler can rapidly and accurately predict anti-TB drug resistance profiles across large numbers of samples with WGS data. The computing architecture allows for the ability to modify the core bioinformatic pipelines and outputs, including the analysis of WGS data sourced from portable technologies. TBProfiler has the potential to be integrated into the point of care and WGS diagnostic environments, including in resource-poor settings.

Background: Rifampicin resistant tuberculosis (RR-TB) was frequently detected in Suriname after the introduction of Xpert MTB/RIF in 2012. Subsequent phenotypic drug-susceptibility testing (DST) was not conclusive at that moment, while RR-TB patients treated with first-line tuberculostatics had good treatment outcome. In our study, we analysed this interesting observation. Methods: We collected demographic and clinical characteristics and treatment outcome of TB patients from May 2012-December 2018 and performed a univariate and multivariate analysis to assess possible associations with resistance to rifampicin. Secondly, we conducted whole genome sequencing on all available Mycobacterium tuberculosis isolates that had a rifampicin resistance in the Xpert MTB/RIF test and performed phenotypic DST on selected isolates. Findings: RR-TB was detected in 59 (9.6%) patients confirmed by Xpert. These patients were treated with rifampicin-containing regimens in most (88%) of the cases. In all 32 samples examined, a D435Y mutation in the rpoB gene was identified; only one isolate revealed an additional isoniazid mutation. Phenotypic DST indicated low-level rifampicin resistance. In multivariate analysis, the Creole ethnicity was a factor associated with rifampicin resistance (aOR 3.5; 95%CI 1.9–6.4). The treatment success rate for patients with RR-TB (78.0%) was comparable to the treatment outcome in non-RR-TB patients 77.8%. Interpretation: This study confirms a low-level rifampicin mono-resistance in TB patients of Suriname. These patients could benefit from a first-line regimen with high dose rifampicin (or rifabutin), rather than from the lengthy treatment regimens for rifampicin-resistant and multi-drug resistant TB, a concept of stratified medicine also advocated for the treatment of TB. Funding: None.

• One third developed extra drug resistance within turnaround time for DST results. • One fourth of MDR Mtb acquired PZA/EMB resistance within turnaround time for DST. • Exogenous reinfection played a ponderous role in the drug resistance acquisition. Although phenotypic drug susceptibility testing (DST) of Mycobacterium tuberculosis (Mtb) takes up to 6–8 weeks, little is known about how drug susceptibility is affected during this period. We performed a prospective cohort study to investigate the development of drug resistance (DR) during turnaround time (TAT), including 359 pulmonary tuberculosis (PTB) patients with a baseline DST result of an Mtb isolate collected at TB diagnosis and a follow-up DST result of an Mtb isolate collected when baseline DST result was available between 2013 and 2018. Whole-genome sequencing (WGS) was used to differentiate between acquired drug resistance, exogenous reinfection, and mixed infection. Among the studied patients, during TAT for DST, 116 (32.3%) developed DR to four first-line drugs (rifampicin, isoniazid, pyrazinamide, ethambutol). Among 116 pairs of isolates included for WGS, 21 pairs were classified as acquired drug resistance with single nucleotide polymorphisms (SNPs) differences less than 12. Four pairs with an intermediate SNPs differences displayed minor differences in related genotypes and were assessed as mixed infection. The remaining 91 pairs had high SNPs differences consistent with exogenous reinfection. The exogenous reinfection of drug-resistant strains played a vital role in the development of DR of Mtb isolates during TAT for DST, highlighting the need for both rapid DST methods and improved infection control. [ABSTRACT FROM AUTHOR]

Background: Multidrug-resistant tuberculosis (MDR-TB) is a threat to the accomplishments of the World Health Organization's (WHO) End TB Strategy. The treatment of MDR-TB is less effective and more toxic; additional resistance to second-line drugs makes the treatment more difficult. India accounts for one-fourth of the global TB burden. In 2015 alone, 2.8 million cases of TB were diagnosed of which 79,000 cases had MDR/rifampicin-resistant TB (RR-TB). This study was conducted to analyze the baseline susceptibility pattern of MDR/RR-TB isolates against second-line drugs. Methods: A total of 374 culture-positive MDR and rifampicin-resistant M. tuberculosis isolates were tested for susceptibility against capreomycin (CAP), kanamycin, levofloxacin, moxifloxacin, clofazimine, and linezolid. Results: Of the total 374 isolates, 236 (63.10%) strains were susceptible to all drugs, whereas 138 (36.89%) were resistant at least to one of the second-line drugs. One hundred and sixteen (31.01%) strains were identified as preextensively drug-resistant (XDR) (MDR isolates with additional resistance to either fluoroquinolones or second-line injectables) and 22 (5.88%) of the isolates tested were identified as XDR. Conclusion: India is a signatory to the WHO's “The End TB Strategy” with the aim of ending the global TB epidemic; intensified efforts for early detection and treatment of drug-resistant cases from both public and private sectors are required to accelerate the rate at which TB incidence falls and accomplish the desired results.

As the causative bacteria of tuberculosis, Mycobacteriumtuberculosis (M. tb) is aggravated by the emergence of its multidrug-resistant isolates in China. Mutations of six of the most frequently reported resistant genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were detected for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), ofloxacin (OFX), and streptomycin (STR) in this study. The amino acid missense mutations (MMs) and their corresponding single nucleotide polymorphism mutations for all drug-resistant (DR) isolates are described in detail. All isolates were divided into non-extensively drug-resistant (Non-XDR) and preXDR/XDR groups. No statistical differences were detected among MMs and linked MMs (LMs) between the two groups, except for rpsL 88 (p = 0.037). In the preXDR/XDR group, the occurrence of MMs in rpoB, katG, and inhA developed phenotypic resistance and MMs of rpoB 531, katG 315, rpsL 43, and rpsL 88 could develop high levels of DR. It is necessary to carry out epidemiological investigations of DR gene mutations in the local region, and thus provide necessary data to support the design of new technologies for rapid detection of resistant M. tb and the optimization of detection targets.

The development and implementation of rapid molecular diagnostics for tuberculosis (TB) drug-susceptibility testing is critical to inform treatment of patients and to prevent the emergence and spread of resistance. Optimal trial planning for existing tests and those in development will be critical to rapidly gather the evidence necessary to inform World Health Organization review and to support potential policy recommendations. The evidence necessary includes an assessment of the performance for TB and resistance detection as well as an assessment of the operational characteristics of these platforms. The performance assessment should include analytical studies to confirm the limit of detection and assay ability to detect mutations conferring resistance across globally representative strains. The analytical evaluation is typically followed by multisite clinical evaluation studies to confirm diagnostic performance in sites and populations of intended use. This paper summarizes the considerations for the design of these analytical and clinical studies. [ABSTRACT FROM AUTHOR]

Objective/background: The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs (X52917) and eis (AF144099) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. Methods: Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsl (version 1) assay. Results: The rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40 mg/L, >20 mg/L, and 5–15 mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0 mg/L, 0.625–10 mg/L, and 0.625–2.5 mg/L, respectively. Conclusion: This study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation.

Objective/background: Early initiation of therapy in patients with tuberculosis is imperative for its control. Conventional methods of susceptibility testing such as the proportion method (PM) require visual detection and counting of colonies that takes up to 6 weeks. Rapid and simple phenotypic methods that have been endorsed by the World Health Organization can serve as alternatives. Methods: In this study, we evaluated the colorimetric nitrate reductase assay, which utilizes the detection of nitrate reduction as an indicator of growth much earlier compared with PM (within 7–14 days). The susceptibility of 75 clinical isolates of Mycobacterium tuberculosis to four first-line antitubercular drugs was tested by nitrate reductase assay and compared with the standard PM. In this assay, inoculation was done on both drug-free and drug-containing Löwenstein–Jensen medium containing sodium nitrate. After incubation for 7–14 days, reduction to nitrite was taken as an indicator of growth, which was detected by color change on addition of Griess reagent. Results: Agreement between nitrate reductase assay and PM was 100% for rifampicin, 97.30% for isoniazid, 93.30% for streptomycin, and 98.60% for ethambutol. Cost/isolate with this assay was found to be approximately two times lesser than that of PM. All results were obtained in 7–14 days by nitrate reductase assay, which was significantly rapid compared with 42 days taken for obtaining results by PM. Conclusion: Nitrate reductase assay can be used as a rapid and inexpensive method for drug-susceptibility testing of M. Tuberculosis for first-line antitubercular drugs without compromising accuracy of standard methods.

To say that tuberculosis (TB) has regained a strong foothold in the global human health and wellbeing scenario would be an understatement. Ranking alongside HIV/AIDS as the top reason for mortality due to a single infectious disease, the impact of TB extends far into socio-economic context worldwide. As global efforts led by experts and political bodies converge to mitigate the predicted outcome of growing antimicrobial resistance, the academic community of students, practitioners and researchers have mobilised to develop integrated, inter-disciplinary programmes to bring the plans of the former to fruition. Enabling this crucial requirement for unimpeded dissemination of scientific discovery was the TB Summit 2016, held in London, United Kingdom. This report critically discusses the recent breakthroughs made in diagnostics and treatment while bringing to light the major hurdles in the control of the disease as discussed in the course of the 3-day international event. Conferences and symposia such as these are the breeding grounds for successful local and global collaborations and therefore must be supported to expand the understanding and outreach of basic science research. [ABSTRACT FROM AUTHOR]

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT, NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90%. With most of the assays, sensitivity for ethambutol was low (62-87%) whereas for streptomycin, sensitivity values ranged from 84 to 100%; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff. MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.

Background: The emergence of resistance to anti-tuberculosis drugs is a serious and growing threat to public health. Next-generation sequencing is rapidly gaining traction as a diagnostic tool for investigating drug resistance in to aid treatment decisions. However, there are few little data regarding the precision Mycobacterium tuberculosis of such sequencing for assigning resistance profiles. Methods: We investigated two sequencing platforms (Illumina MiSeq, Ion Torrent PGM™) and two rapid analytic pipelines (,) using a well characterised reference strain (H37Rv) and clinical isolates from TBProfiler Mykrobe predictor patients with tuberculosis resistant to up to 13 drugs. Results were compared to phenotypic drug susceptibility testing. To assess analytical robustness individual DNA samples were subjected to repeated sequencing. Results: The MiSeq and Ion PGM systems accurately predicted drug-resistance profiles and there was high reproducibility between biological and technical sample replicates. Estimated variant error rates were low (MiSeq 1 per 77 kbp, Ion PGM 1 per 41 kbp) and genomic coverage high (MiSeq 51-fold, Ion PGM 53-fold). MiSeq provided superior coverage in GC-rich regions, which translated into incremental detection of putative genotypic drug-specific resistance, including for resistance to para-aminosalicylic acid and pyrazinamide. The bioinformatics pipeline TBProfiler was concordant with reported phenotypic susceptibility for all drugs tested except pyrazinamide and para-aminosalicylic acid, with an overall concordance of 95.3%. When using the concordance with phenotypic testing Mykrobe predictor was 73.6%. Conclusions: We have demonstrated high comparative reproducibility of two sequencing platforms, and high predictive ability of the mutation library and analytical pipeline, when profiling resistance to first- and TBProfiler second-line anti-tuberculosis drugs. However, platform-specific variability in coverage of some genome regions may have implications for predicting resistance to specific drugs. These findings may have implications for future clinical practice and thus deserve further scrutiny, set within larger studies and using updated mutation libraries. [ABSTRACT FROM AUTHOR]

Objective/background Early initiation of therapy in patients with tuberculosis is imperative for its control. Conventional methods of susceptibility testing such as the proportion method (PM) require visual detection and counting of colonies that takes up to 6 weeks. Rapid and simple phenotypic methods that have been endorsed by the World Health Organization can serve as alternatives. Methods In this study, we evaluated the colorimetric nitrate reductase assay, which utilizes the detection of nitrate reduction as an indicator of growth much earlier compared with PM (within 7–14 days). The susceptibility of 75 clinical isolates of Mycobacterium tuberculosis to four first-line antitubercular drugs was tested by nitrate reductase assay and compared with the standard PM. In this assay, inoculation was done on both drug-free and drug-containing Löwenstein–Jensen medium containing sodium nitrate. After incubation for 7–14 days, reduction to nitrite was taken as an indicator of growth, which was detected by color change on addition of Griess reagent. Results Agreement between nitrate reductase assay and PM was 100% for rifampicin, 97.30% for isoniazid, 93.30% for streptomycin, and 98.60% for ethambutol. Cost/isolate with this assay was found to be approximately two times lesser than that of PM. All results were obtained in 7–14 days by nitrate reductase assay, which was significantly rapid compared with 42 days taken for obtaining results by PM. Conclusion Nitrate reductase assay can be used as a rapid and inexpensive method for drug-susceptibility testing of M. tuberculosis for first-line antitubercular drugs without compromising accuracy of standard methods. [ABSTRACT FROM AUTHOR]

Objective/background The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs ( X52917 ) and eis ( AF144099 ) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. Methods Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDR sl (version 1) assay. Results The rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T ( n = 3), G-10C ( n = 3), G-10A ( n = 3), and C-12T ( n = 2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40 mg/L, >20 mg/L, and 5–15 mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0 mg/L, 0.625–10 mg/L, and 0.625–2.5 mg/L, respectively. Conclusion This study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation. [ABSTRACT FROM AUTHOR]

Background: Early detection for tuberculosis (TB) and rifampicin-resistant TB (RRTB) is crucial for proper control of this disease. WHO recommended the use of the GeneXpert assay at district level to cover these two public health demands? A study evaluated the diagnostic impact of the GeneXpert assay in detecting TB and RRTB. Odd results were observed in this study in the form of discordance between the GeneXpert assay and the conventional culture and drug-susceptibility testing (DST). Aim of the study To assess the molecular diagnostic validity of the GeneXpert assay when results do not match phenotypic results given by DST. Methods: Pulmonary TB patients with recently detected sputum positive for acid-fast bacilli (AFB) were recruited from random geographical clusters (18 out of 36 primary healthcare districts in the middle five governorates in Iraq) during a 1-year period (November 2013–October 2014). Sputum samples from all enrolled patients were sent for GeneXpert assay testing, culture, and DST. Genotype mycobacterium (GM) from Hain Lifescience (Nehren, Germany) was used to detect non-tuberculosis mycobacteria (NTM) whenever suspected. Those with discordant results regarding the status of RRTB between GeneXpert assay and DST were retested with the line probe assay (LPA). Simple frequency distribution was used to describe study results. Results: Four-hundred ten patients were enrolled, all of whom were culture positive. Only two patients were found negative for TB on GeneXpert assay who were then diagnosed as NTM by LPA (GM). Out of the 408 patients, discordance between GeneXpert and DST regarding the status of rifampicin susceptibility was observed in 17 cases (4%). Nine patients were RR on GeneXpert but rifampicin susceptible (RS) on DST. LPA agreed with GeneXpert assay for all nine cases. Eight patients were RS on GeneXpert but RR on DST. Here, LPA disagreed with GeneXpert assay only in one patient who was found to be RR by LPA. Conclusion: GeneXpert assay is a valid molecular test for TB and RRTB regardless of its discordance with conventional culture and DST.

Extensively drug-resistant (XDR)-TB, defined as TB with resistance to at least isoniazid, rifampin, a fluoroquinolone and either amikacin, kanamycin or capreomycin, is a stark setback for global TB control. Overburdened public-health systems with inadequate resources for case detection and management and high HIV coinfection rates in many regions have contributed to the emergence of XDR-TB. Patients with XDR-TB have poor outcomes, prolonged infectious periods and limited treatment options. To prevent an epidemic of untreatable XDR-TB, improvements in XDR-TB surveillance, increased laboratory capacity for rapid detection of drug-resistant strains, better infection control and the development of new therapeutics are urgently needed. [ABSTRACT FROM AUTHOR]

Extensively-Drug Resistant Tuberculosis (XDR-TB) is emerging as an even more ominous treat. XDR-TB is defined as TB that is resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin and amikacin), in addition to isoniazid and rifampicin. This makes XDR-TB treatment extremely complicated, if not impossible, in general treatment. In a 2006 XDR-TB outbreak in KwaZulu-Natal, South Africa, 52 of 53 people who contracted the disease died within months, It is estimated that 70% of XDR-TB patients die within a month of diagnosis. The most recent drug-resistance surveillance data issued by the WHO estimates that an average of roughly 5 percent of Multi-drug resistant tuberculosis (MDR-TB) cases are XDR-TB. Estimating the incidence of XDR-TB is extremely difficult because most laboratories are ill-equipped to detect and diagnose. To avoid the emergence of more resistant strains that may lead to almost untreatable disease, we must focus our efforts on the right management of drug resistance tuberculosis. Bull Chiang Mai Assoc Med Sci 2015; 48(1): 18-28. Doi: 10.14456/jams.2015.5

As the causative bacteria of tuberculosis, Mycobacteriumtuberculosis (M. tb) is aggravated by the emergence of its multidrug-resistant isolates in China. Mutations of six of the most frequently reported resistant genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were detected for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), ofloxacin (OFX), and streptomycin (STR) in this study. The amino acid missense mutations (MMs) and their corresponding single nucleotide polymorphism mutations for all drug-resistant (DR) isolates are described in detail. All isolates were divided into non-extensively drug-resistant (Non-XDR) and preXDR/XDR groups. No statistical differences were detected among MMs and linked MMs (LMs) between the two groups, except for rpsL 88 (p = 0.037). In the preXDR/XDR group, the occurrence of MMs in rpoB, katG, and inhA developed phenotypic resistance and MMs of rpoB 531, katG 315, rpsL 43, and rpsL 88 could develop high levels of DR. It is necessary to carry out epidemiological investigations of DR gene mutations in the local region, and thus provide necessary data to support the design of new technologies for rapid detection of resistant M. tb and the optimization of detection targets. [ABSTRACT FROM AUTHOR]

Objectives Kanamycin is an important second-line drug used to treat multidrug-resistant (MDR) tuberculosis (TB). Molecular analysis of the rrs gene seems to be not enough to identify every case of kanamycin resistance. In the present study we evaluated the incidence of eis mutations in kanamycin-resistant Mycobacterium tuberculosis isolates. Methods We analysed 70 MDR M. tuberculosis clinical isolates. All isolates were screened for rrs and eis mutations using single-strand conformation polymorphism and sequencing. Phenotypic drug susceptibility testing was performed using Bactec MGIT 960 and the absolute concentration method on Lowenstein–Jensen medium. Results eis mutations were found in 10 isolates. The most prevalent mutations were the A1401G substitution in the rrs gene and the C14T substitution in the eis promoter region. Conclusions Our study shows that the eis promoter region is a useful molecular marker of kanamycin resistance in the Moscow region. Complex analysis of rrs and eis mutations will significantly reduce the time to diagnose kanamycin resistance in TB patients, compared with phenotypic drug resistance testing. [ABSTRACT FROM PUBLISHER]

Background: Rifampicin resistant tuberculosis (RR-TB) was frequently detected in Suriname after the introduction of Xpert MTB/RIF in 2012. Subsequent phenotypic drug-susceptibility testing (DST) was not conclusive at that moment, while RR-TB patients treated with first-line tuberculostatics had good treatment outcome. In our study, we analysed this interesting observation., Methods: We collected demographic and clinical characteristics and treatment outcome of TB patients from May 2012-December 2018 and performed a univariate and multivariate analysis to assess possible associations with resistance to rifampicin. Secondly, we conducted whole genome sequencing on all available Mycobacterium tuberculosis isolates that had a rifampicin resistance in the Xpert MTB/RIF test and performed phenotypic DST on selected isolates., Findings: RR-TB was detected in 59 (9.6%) patients confirmed by Xpert. These patients were treated with rifampicin-containing regimens in most (88%) of the cases. In all 32 samples examined, a D435Y mutation in the rpoB gene was identified; only one isolate revealed an additional isoniazid mutation. Phenotypic DST indicated low-level rifampicin resistance. In multivariate analysis, the Creole ethnicity was a factor associated with rifampicin resistance (aOR 3.5; 95%CI 1.9-6.4). The treatment success rate for patients with RR-TB (78.0%) was comparable to the treatment outcome in non-RR-TB patients 77.8%., Interpretation: This study confirms a low-level rifampicin mono-resistance in TB patients of Suriname. These patients could benefit from a first-line regimen with high dose rifampicin (or rifabutin), rather than from the lengthy treatment regimens for rifampicin-resistant and multi-drug resistant TB, a concept of stratified medicine also advocated for the treatment of TB., Funding: None., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier Ltd.)

Firstly, this study demonstrated that natural product-inspired coumarin-based nitrofuranyl calanolides (NFCs) can form the Rv2466c-mycothiol (MSH)-NFC (RvMN) ternary complex via NFC binding to W21, N51, and Y61 of Rv2466c and be specifically reduced by Rv2466c, which is accompanied by the generation of a high level of fluorescence. Additionally, the results unveiled that the acetylated cysteine-glucosamine (AcCys-GlcN) motif of MSH is sufficient to interact with Rv2466c and adopt the active conformation that is essential for fully reducing NFCs. Further clinical translational investigation in this Article indicated that the novel fluorescent NFC probe can serve as a much needed high-throughput and low-cost detection method for detection of living Mycobacterium tuberculosis ( Mtb) and can precisely determine MIC values for a full range of available drugs. This method can greatly facilitate the development of phenotypic drug-susceptibility testing (pDST) that will allow the point-of-care treatment of tuberculosis (TB) within a week after diagnosis.

Background Early detection for tuberculosis (TB) and rifampicin-resistant TB (RRTB) is crucial for proper control of this disease. WHO recommended the use of the GeneXpert assay at district level to cover these two public health demands? A study evaluated the diagnostic impact of the GeneXpert assay in detecting TB and RRTB. Odd results were observed in this study in the form of discordance between the GeneXpert assay and the conventional culture and drug-susceptibility testing (DST). Aim of the study To assess the molecular diagnostic validity of the GeneXpert assay when results do not match phenotypic results given by DST. Methods Pulmonary TB patients with recently detected sputum positive for acid-fast bacilli (AFB) were recruited from random geographical clusters (18 out of 36 primary healthcare districts in the middle five governorates in Iraq) during a 1-year period (November 2013–October 2014). Sputum samples from all enrolled patients were sent for GeneXpert assay testing, culture, and DST. Genotype mycobacterium (GM) from Hain Lifescience (Nehren, Germany) was used to detect non-tuberculosis mycobacteria (NTM) whenever suspected. Those with discordant results regarding the status of RRTB between GeneXpert assay and DST were retested with the line probe assay (LPA). Simple frequency distribution was used to describe study results. Results Four-hundred ten patients were enrolled, all of whom were culture positive. Only two patients were found negative for TB on GeneXpert assay who were then diagnosed as NTM by LPA (GM). Out of the 408 patients, discordance between GeneXpert and DST regarding the status of rifampicin susceptibility was observed in 17 cases (4%). Nine patients were RR on GeneXpert but rifampicin susceptible (RS) on DST. LPA agreed with GeneXpert assay for all nine cases. Eight patients were RS on GeneXpert but RR on DST. Here, LPA disagreed with GeneXpert assay only in one patient who was found to be RR by LPA. Conclusion GeneXpert assay is a valid molecular test for TB and RRTB regardless of its discordance with conventional culture and DST. [ABSTRACT FROM AUTHOR]