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Objectives: Linezolid treatment has a high risk of toxicity and adverse drug reactions (ADR) are frequent. Few studies have investigated risk factors of major ADRs separately, therefore, we aimed to evaluate major ADRs including peripheral neuropathy in relation to risk factors and drug concentration levels of linezolid in a high-resource setting for multidrug-resistant tuberculosis (MDR-TB)., Methods: We conducted a retrospective cohort study including participants treated with a linezolid-containing MDR-TB regimen in Sweden 1992-2018. Data was collected from medical records. ADRs were classified according to Common Terminology Criteria for Adverse Events (version 5.0)., Results: Of all participants (n = 132), 43.2% were female and the median age 28 y. The median linezolid treatment was 6.5 months (IQR 3.0-12.7) with a median daily dose of 9.6 mg/kg/d. Any ADR was seen in 58.3% (n = 77) of participants, with 35.6% having peripheral neuropathy (n = 47), 27.3% anaemia (n = 36), 22.0% leukopenia (n = 36) while 6.1% (n = 8) had optic neuritis. The median time for peripheral neuropathy was 3.6 months (IQR 2.1-5.9) and 8.3 months (6.2-10.7) for optic neuritis. A >2.0 mg/L trough concentration (n = 40) was associated with anaemia (P = 0.0038) and thrombocytopenia (P = 0.009) but not with peripheral neuropathy. In multivariable analysis, a dose ≥12 mg/kg/d was associated with time to peripheral neuropathy (HR 2.89, 95% CI 1.08-7.74, P = 0.035), anaemia (HR 6.62, 95% CI 2.22-19.8, P = 0.001) and leukopenia (HR 5.23, 95% CI 1.48-18.5, P = 0.010)., Conclusions: Linezolid ADRs were frequent in a high-resource setting. Structured, regular follow-up for ADRs and adjusting dosing according to body weight followed-up by monitoring of drug concentrations early may reduce toxicity., Competing Interests: Declaration of competing interests JJ is heading the Swedish national advisory board on MDR-TB and other difficult to treat cases and JB, LDF and TS are also members of the advisory board. JW is the scientific secretary in the EUCAST steering committee on antimycobacterial susceptibility testing, which is an unpaid position. JWA has been giving lectures on an infectious diseases conference on therapeutic drug monitoring for linezolid and received an educational fee from Pfizer. JB received a payment fee for lectures on Post-COVID by Astra Zeneca and Novartis. JK, AO, MS, MM, and RG have no conflict of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh ( Rv1854c ) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis . This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration., Competing Interests: D.M.C. is the co-chair of the Working Group of the Stop TB Partnership New Diagnostics and is an unpaid member of EUCAST subcommittee for antimicrobial susceptibility testing of mycobacteria, the CLSI mycobacterial committee, and the WHO Strategic and Technical Advisory Group for diagnostics. C.U.K. is a consultant for Becton Dickinson, the Foundation for Innovative New Diagnostics, the TB Alliance, and the WHO Global TB Programme. C.U.K.'s consulting for Becton Dickinson involves a collaboration with Janssen and Thermo Fisher Scientific. C.U.K. is collaborating with PZA Innovation and is an unpaid advisor to Cepheid and GenoScreen. C.U.K. worked as a consultant for the Stop TB Partnership and the WHO Regional Office for Europe. C.U.K. gave a paid educational talk for Oxford Immunotec. C.U.K. was an unpaid advisor to BioVersys.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a β-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as β-lactams in combination with β-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

The routine use of whole genome sequencing (WGS) as a reference typing technique for Mycobacterium tuberculosis epidemiology combined with the catalogued and extensive knowledge base of resistance-associated mutations means an initial susceptibility prediction can be derived from all cultured isolates in our laboratories based on WGS data alone. Preliminary work has confirmed, in our low-burden settings, these predictions are for first-line drugs, reproducible, robust with an accuracy similar to phenotypic drug susceptibility testing (pDST) and in many cases able to also predict the level of resistance (MIC). Routine screening for drug resistance by WGS results in approximately 80% of the isolates received being predicted as fully susceptible to the first-line drugs. Parallel testing with both WGS and pDST has demonstrated that routine pDST of genotypically fully susceptible isolates yields minimal additional information. Thus, rather than re-confirming all fully sensitive WGS-based predictions, we suggest that a more efficient use of available mycobacterial culture capacity in our setting is the development of a more extensive and detailed pDST targeted at any mono or multi-drug-resistant isolates identified by WGS screening. Phenotypic susceptibility retains a key role in the determination of an extended susceptibility profile for mono/multi-drugresistant isolates identified by WGS screening. The pDST information collected is also needed to support the development of future catalogues of resistance-associated mutations.

We performed 2 surveys during 2022 to estimate point prevalences of SARS-CoV-2 infection compared with overall seroprevalence in Sweden. Point prevalence was 1.4% in March and 1.5% in September. Estimated seroprevalence was >80%, including among unvaccinated children. Continued SARS-CoV-2 surveillance is necessary for detecting emerging, possibly more pathogenic variants.

Here, we describe a clinical case of pyrazinamide-resistant (PZA-R) tuberculosis (TB) reported as PZA-susceptible (PZA-S) by common molecular diagnostics. Phenotypic susceptibility testing (pDST) indicated PZA-R TB. Targeted Sanger sequencing reported wild-type PncA, indicating PZA-S TB. Whole Genome Sequencing (WGS) by PacBio and IonTorrent both detected deletion of a large portion of pncA, indicating PZA-R. Importantly, both WGS methods showed deletion of part of the primer region targeted by Sanger sequencing. Repeating Sanger sequencing from a culture in presence of PZA returned no result, revealing that 1) two minority susceptible subpopulations had vanished, 2) the PZA-R majority subpopulation harboring the pncA deletion could not be amplified by Sanger primers, and was thus obscured by amplification process. This case demonstrates how a small susceptible subpopulation can entirely obscure majority resistant populations from targeted molecular diagnostics and falsely imply homogenous susceptibility, leading to incorrect diagnosis. To our knowledge, this is the first report of a minority susceptible subpopulation masking a majority resistant population, causing targeted molecular diagnostics to call false susceptibility. The consequence of such genomic events is not limited to PZA. This phenomenon can impact molecular diagnostics' sensitivity whenever the resistance-conferring mutation is not fully within primer-targeted regions. This can be caused by structural changes of genomic context with phenotypic consequence as we report here, or by uncommon mechanisms of resistance. Such false susceptibility calls promote suboptimal treatment and spread of strains that challenge targeted molecular diagnostics. This motivates development of molecular diagnostics unreliant on primer conservation, and impels frequent WGS surveillance for variants that evade prevailing molecular diagnostics., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

High-confidence resistance mutations for new and repurposed anti-TB drugs, such as delamanid (DLM) and pretomanid (Pa), are rare and more data are needed in order to correctly interpret the results generated by genotypic drug susceptibility testing. In this study performed on clinical Mycobacterium tuberculosis complex isolates, we report that in the Swedish strain collection the ddn mutation Trp20Stop is found exclusively among DLM and Pa resistant (Pa MIC >16 mg/L) isolates assigned to lineage 4.5.

Background: In order to estimate the prevalence and understand the spread of SARS-CoV-2 in Sweden, the Public Health Agency of Sweden, with support from the Swedish Armed Forces, conducted a series of point prevalence surveys between March and December 2020., Methods: Sampling material and instructions on how to perform self-sampling of the upper respiratory tract were delivered to the homes of the participants. Samples were analysed by real-time PCR, and the participants completed questionnaires regarding symptoms., Findings: The first survey in the Stockholm region in March 2020 included 707 participants and showed a SARS-CoV-2 prevalence of 2.5%. The following five surveys, performed on a national level, with between 2461 and 2983 participants, showed SARS-CoV-2 prevalences of 0.9% (April), 0.3% (May), 0.0% (August), 0.0% (September), and 0.7% (December). All positive cases who responded to questionnaires reported experiencing symptoms that occurred from 2 weeks before the date of sampling up to and including the date of sampling., Interpretation: None of the individuals shown to be PCR-positive were asymptomatic at the time of sampling or in the 14 days prior to sampling. This is in contrast to many other surveys in which a substantial proportion of positive cases have been reported to be asymptomatic. Our surveys demonstrate a decreasing ratio between notified cases and the observed prevalence throughout the year, in line with increasing testing capacity and the consecutive inclusion of all symptomatic individuals in the case definition for testing., (© 2022. The Author(s).)

A national point seroprevalence study of SARS-CoV-2 was conducted in Sweden in April-May 2021. In total, 2860 individuals 3 to 90 years old from a probability-based web panel were included. Results showed that an estimated 32.6% of the population in Sweden had detectable levels of antibodies, and among non-vaccinated 20.1% had detectable levels of antibodies. We tested for differences in seroprevalence between age groups and by sex and estimated seroprevalence among previously infected participants by time since reporting., (© 2022. The Author(s).)

Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs :A1401G and rrs :G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter ( eis :G-10A, eis :C-12T, and eis :C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.

BACKGROUND: Distinguishing TB relapse from re-infection is important from a clinical perspective to document transmission patterns. We investigated isolates from patients classified as relapse to understand if these were true relapses or re-infections. We also investigated shifts in drug susceptibility patterns to distinguish acquired drug resistance from re-infection with resistant strains. METHODS: Isolates from pulmonary TB patients from 2009 to 2017 were analysed using whole-genome sequencing (WGS). RESULTS: Of 11 patients reported as relapses, WGS results indicated that 4 were true relapses (single nucleotide polymorphism difference ≤5), 3 were re-infections with new strains, 3 were both relapse and re-infection and 1 was a suspected relapse who was later categorised as treatment failure based on sequencing. Of the 9 patients who went from a fully susceptible to a resistant profile, WGS showed that none had acquired drug resistance; 6 were re-infected with new resistant strains, 1 was probably infected by at least two different genotype strains and 2 were phenotypically misclassified. CONCLUSIONS: WGS was shown to distinguish between relapse and re-infection in an unbiased way. The use of WGS minimises the risk of false classification of treatment failure instead of re-infection. Furthermore, our study showed that strains without major genetic differences can cause re-infection.

Background: Pyrazinamide (PZA) resistance in multidrug-resistant tuberculosis (MDR-TB) is common; yet, it is not clear how it affects interim and treatment outcomes. Although rarely performed, phenotypic drug susceptibility testing (pDST) is used to define PZA resistance, but genotypic DST (gDST) and minimum inhibitory concentration (MIC) could be beneficial. We aimed to assess the impact of PZA gDST and MIC on time to sputum culture conversion (SCC) and treatment outcome in patients with MDR-TB., Methods: Clinical, microbiological, and treatment data were collected in this cohort study for all patients diagnosed with MDR-TB in Sweden from 1992-2014. MIC, pDST, and whole-genome sequencing of the pncA, rpsA, and panD genes were used to define PZA resistance. A Cox regression model was used for statistical analyses., Results: Of 157 patients with MDR-TB, 56.1% (n = 88) had PZA-resistant strains and 49.7% (n = 78) were treated with PZA. In crude and adjusted analysis (hazard ratio [HR], 0.49; 95% conficence interval [CI], .29-.82; P = .007), PZA gDST resistance was associated with a 29-day longer time to SCC. A 2-fold decrease in dilutions of PZA MIC for PZA-susceptible strains showed no association with SCC in crude or adjusted analyses (HR, 0.98; 95% CI, .73-1.31; P = .89). MIC and gDST for PZA were not associated with treatment outcome., Conclusions: In patients with MDR-TB, gDST PZA resistance was associated with a longer time to SCC. Rapid PZA gDST is important to identify patients who may benefit from PZA treatment., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Heteroresistance is defined as the coexistence of both susceptible and resistant bacteria in a bacterial population. Previously published data show that it may occur in 9 to 57% of Mycobacterium tuberculosis isolates for various drugs. Pyrazinamide (PZA) is an important first-line drug used for treatment of both drug-susceptible and PZA-susceptible multidrug-resistant TB. Clinical PZA resistance is defined as a proportion of resistant bacteria in the isolate exceeding 10%, when the drug is no longer considered clinically effective. The ability of traditional drug susceptibility testing techniques to detect PZA heteroresistance has not yet been evaluated. The aim of this study was to compare the capacity of Bactec MGIT 960, Wayne's test, and whole-genome sequencing (WGS) to detect PZA-resistant subpopulations in bacterial suspensions prepared with different proportions of mutant strains. Both Bactec MGIT 960 and WGS were able to detect the critical level of 10% PZA heteroresistance, whereas Wayne's test failed to do so, with the latter falsely reporting highly resistant samples as PZA susceptible. Failure to detect drug-resistant subpopulations may lead to inadvertently weak treatment regimens if ineffective drugs are included, with the risk of treatment failure with the selective growth of resistant subpopulations. We need clinical awareness of heteroresistance as well as evaluation of new diagnostic tools for their capacity to detect heteroresistance in TB.

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in Mycobacterium tuberculosis strains with existing resistance to isoniazid and rifampin (i.e., multidrug resistance [MDR]) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotyped pncA and purported alternative PZA-R genes ( panD , rpsA , and clpC1 ) with long-read sequencing of 19 phenotypically PZA-monoresistant isolates collected in Sweden and compared their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDR PZA-R ) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter ( clpC1p -138 ) and the N terminus of ClpC1 (ClpC1 Val63Ala ). Mutations have emerged in both these regions under POA selection in vitro , and the N-terminal region of ClpC1 has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1 Val63Ala mutants spanned 4 Indo-Oceanic sublineages. Indo-Oceanic isolates invariably harbored ClpC1 Val63Ala and were starkly overrepresented (odds ratio [OR] = 22.2, P < 0.00001) among PZA-monoresistant isolates (11/19) compared to MDR PZA-R isolates (5/80). The genetic basis of Indo-Oceanic isolates' overrepresentation in PZA-monoresistant tuberculosis (TB) remains undetermined, but substantial circumstantial evidence suggests that ClpC1 Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development., (Copyright © 2021 Modlin et al.)

In this retrospective study, whole-genome sequencing (WGS) data generated on an Ion Torrent platform was used to predict phenotypic drug resistance profiles for first- and second-line drugs among Swedish clinical Mycobacterium tuberculosis isolates from 2016 to 2018. The accuracy was ∼99% for all first-line drugs and 100% for four second-line drugs. Our analysis supports the introduction of WGS into routine diagnostics, which might, at least in Sweden, replace phenotypic drug susceptibility testing in the future., (Copyright © 2020 American Society for Microbiology.)

Background: Minimum inhibitory concentration (MIC) testing, unlike routine drug susceptibility testing (DST) at a single critical concentration, quantifies drug resistance. The association of MICs and treatment outcome in multidrug-resistant (MDR)-tuberculosis patients is unclear. Therefore, we correlated MICs of first- and second-line tuberculosis drugs with time to sputum culture conversion (tSCC) and treatment outcome in MDR-tuberculosis patients., Methods: Clinical and demographic data of MDR-tuberculosis patients in Sweden, including DST results, were retrieved from medical records from 1992 to 2014. MIC determinations were performed retrospectively for the stored individual Mycobacterium tuberculosis (Mtb) isolates using broth microdilution in Middlebrook 7H9. We fitted Cox proportional hazard models correlating MICs, DST results, and clinical variables to tSCC and treatment outcome., Results: Successful treatment outcome was observed in 83.5% (132/158) of MDR-tuberculosis patients. Increasing MICs of fluoroquinolones, diabetes, and age >40 years were significantly associated with unsuccessful treatment outcome. Patients treated with pyrazinamide (PZA) had a significantly shorter tSCC compared to patients who were not (median difference, 27 days)., Conclusions: Increasing MICs of fluoroquinolones were correlated with unsuccessful treatment outcome in MDR-tuberculosis patients. Further studies, including MIC testing and clinical outcome data to define clinical Mtb breakpoints, are warranted. PZA treatment was associated with shorter tSCC, highlighting the importance of PZA DST., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction: Individualised treatment through therapeutic drug monitoring (TDM) may improve tuberculosis (TB) treatment outcomes but is not routinely implemented. Prospective clinical studies of drug exposure and minimum inhibitory concentrations (MICs) in multidrug-resistant TB (MDR-TB) are scarce. This translational study aims to characterise the area under the concentration-time curve of individual MDR-TB drugs, divided by the MIC for Mycobacterium tuberculosis isolates, to explore associations with markers of treatment progress and to develop useful strategies for clinical implementation of TDM in MDR-TB., Methods and Analysis: Adult patients with pulmonary MDR-TB treated in Xiamen, China, are included. Plasma samples for measure of drug exposure are obtained at 0, 1, 2, 4, 6, 8 and 10 hours after drug intake at week 2 and at 0, 4 and 6 hours during weeks 4 and 8. Sputum samples for evaluating time to culture positivity and MIC determination are collected at days 0, 2 and 7 and at weeks 2, 4, 8 and 12 after treatment initiation. Disease severity are assessed with a clinical scoring tool (TBscore II) and quality of life evaluated using EQ-5D-5L. Drug concentrations of pyrazinamide, ethambutol, levofloxacin, moxifloxacin, cycloserine, prothionamide and para-aminosalicylate are measured by liquid chromatography tandem-mass spectrometry and the levels of amikacin measured by immunoassay. Dried blood spot on filter paper, to facilitate blood sampling for analysis of drug concentrations, is also evaluated. The MICs of the drugs listed above are determined using custom-made broth microdilution plates and MYCOTB plates with Middlebrook 7H9 media. MIC determination of pyrazinamide is performed in BACTEC MGIT 960., Ethics and Dissemination: This study has been approved by the ethical review boards of Karolinska Institutet, Sweden and Fudan University, China. Informed written consent is given by participants. The study results will be submitted to a peer-reviewed journal., Trial Registration Number: NCT02816931; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Therapeutic drug monitoring (TDM) could improve current TB treatment, but few studies have reported pharmacokinetic data together with MICs., Objectives: To investigate plasma concentrations of rifampicin, isoniazid, pyrazinamide and ethambutol along with MICs., Methods: Drug concentrations of rifampicin, isoniazid, pyrazinamide and ethambutol were analysed pre-dose and 2, 4 and 6 h after drug intake at week 2 in 31 TB patients and MICs in BACTEC 960 MGIT were determined at baseline. The highest plasma concentrations at 2, 4 and 6 h post-dose (Chigh) were determined, as well as estimates of Chigh/MIC and area under the concentration-time curve (AUC0-6)/MIC including the corresponding ratios based on calculated free-drug concentrations. This trial was registered at www.clinicaltrials.gov (NCT02042261)., Results: After 2 weeks of treatment, the median Chigh values for rifampicin, isoniazid, pyrazinamide and ethambutol were 10.0, 5.3, 41.1 and 3.3 mg/L respectively. Lower than recommended drug concentrations were detected in 42% of the patients for rifampicin (<8 mg/L), 19% for isoniazid (<3 mg/L), 27% for pyrazinamide (<35 mg/L) and 16% for ethambutol (<2 mg/L). The median Chigh/MIC values for rifampicin, isoniazid, pyrazinamide and ethambutol were 164, 128, 1.3 and 2.5, respectively, whereas the AUC0-6/MIC was 636 (range 156-2759) for rifampicin and 351 (range 72-895) for isoniazid., Conclusions: We report low levels of first-line TB drugs in 16%-42% of patients, in particular for rifampicin. There was a wide distribution of the ratios between drug exposures and MICs. The future use of MIC determinations in TDM is dependent on the development of a reference method and clinically validated pharmacokinetic/pharmacodynamic targets.

Objectives: The implementation of rapid and reliable drug susceptibilities diagnosis is fundamental for effective treatment of multidrug-resistant tuberculosis(MDR-TB). The present study aimed to assess the diagnostic performance of the 2nd-version GenoType MTBDRsl kit as well as the impact of its implementation on the turnaround time in a multi-center Chinese study., Methods: Totally 353 MDR-TB patient specimens were consecutively tested. The 2nd-version GenoType MTBDRsl assay, drug susceptibility testing with the MGIT 960 system, and sequencing were performed and compared., Results: MTBDRsl testing identified the major genotypes associated with fluoroquinolones resistance, predominated by gyrA MUT3B (Asp94Asn and Asp94Tyr, 26.5%) and MUT3C (Asp94Gly, 19.5%). The genotypes associated with resistance to 2nd-line injectable drugs(SLIDs) were rrsMUT1(A1401G, 64.9%) and absence of WT1(C1402T, 10.5%). The sensitivities for detection of resistance to fluoroquinolones, SLIDs, and their combination (extensively drug resistance, XDR) were 80.5%, 80.7% and 73.5% and specificities were 100.0%, 99.3% and 99.1%, respectively. Implementation of this test significantly reduced the turnaround time between sample collection and result reporting from 45 to 3 days, a reduction by 93.3% (p, 0.001)., Conclusion: With a favorable diagnostic performance and short turnaround time, the 2nd-version GenoType MTBDRsl assay proves its value for early diagnosis of resistance to 2nd-line drugs as well as of XDR-TB in China., (Copyright © 2018 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Background: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis., Methods: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing., Findings: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87-94) for rpoB (rifampicin resistance), 86% (74-93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39-68) for pncA (pyrazinamide resistance), 85% (77-91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81-92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing., Interpretation: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation., Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development., (© 2018 World Health Organization; licensee Elsevier. This is an Open Access article published under the CC BY 3.0 IGO license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In any use of this article, there should be no suggestion that WHO endorses any specific organisation, products or services. The use of the WHO logo is not permitted. This notice should be preserved along with the article's original URL.)

This study aimed to validate the epidemiological significance and temporal stability of Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) typing in a genetically and geographically diverse set of clinical isolates from patients diagnosed with pulmonary tuberculosis in China. Between 2010 and 2013, a total of 982 Mycobacterium tuberculosis isolates were collected from four population-based investigations in China. Apart from the currently applied 24-locus MIRU-VNTR, six additional hypervariable loci were analyzed in order to validate the MIRU-VNTR combinations in terms of their epidemiological links, clustering time span, and paired geographic distance. In vitro temporal stability was analyzed for both individual MIRU-VNTR loci, and for several combinations of loci. In the present study, four MIRU-VNTR combinations, including the hypervariable loci 3820, 3232, 2163a, and 4120, were evaluated. All of these combinations obtained a Hunter-Gaston discriminatory index (HGDI) value over 0.9900 with a reduced clustering proportion (from 32.0% to 25.6%). By comparing epidemiological links, clustering time span, and paired geographic distance, we found that the performances of the four MIRU-VNTR combinations were comparable to the insertion sequence 6110 restriction fragment length polymorphism (IS 6110 -RFLP), and significantly better than that of 24-locus MIRU-VNTR genotyping alone. The proportion of temporally stable loci ranged from 90.5% to 92.5% within the combined MIRU-VNTR genotyping, which is higher than IS 6110 -RFLP (85.4%). By adding four hypervariable loci to the standard 24-locus MIRU-VNTR genotyping, we obtained a high discriminatory power, stability and epidemiological significance. This algorithm could therefore be used to improve tuberculosis transmission surveillance and outbreak investigation in China.

Background: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis., Method: Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA., Results: When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO., Conclusion: In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.

Pyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDR PZA-S ) tuberculosis (TB). pncA gene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDR PZA-S TB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-type pncA gene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy: panD or rpsA mutations identified by whole-genome sequencing (WGS) ( n = 7), heteroresistance ( n = 8), and mixed populations with Mycobacterium avium ( n = 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDR PZA-R clinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymous pncA mutation (Thr87Met), whereas a nonsynonymous rpsA mutation was found in 17 isolates. None of these isolates carried a nonsynonymous panD mutation, while all 15 of the MDR PZA-R isolates harbored a nonsynonymous pncA mutation. Our findings indicate that it is necessary to consider the occurrence of panD mutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymous rpsA mutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role of rpsA in PZA resistance., (Copyright © 2017 American Society for Microbiology.)

Pyrazinamide (PZA) is included in the 2016 World Health Organization multidrug-resistant tuberculosis treatment guidelines and is a key component of most ongoing clinical trials investigating novel antibiotic combinations. PZA resistance is associated with worse tuberculosis treatment outcomes. Unfortunately, for such an important drug, phenotypic susceptibility testing is extremely challenging. The exacting bacterial growth conditions required to induce susceptibility to the drug reduce the accuracy of the susceptibility assay, even in experienced laboratories, and widespread testing is not performed. This situation is unacceptable for such a valuable and important drug. A more complete understanding of the mechanism of action of PZA would be expected to lead to improvements in this situation. Although the exact mechanism of action of PZA is not known yet, it is widely accepted that PZA is a prodrug requiring transformation to pyrazinoic acid, the active form, by the mycobacterial enzyme encoded by the pncA gene. Most clinical resistance indeed appears to be a result of a diverse range of mutations in this gene and sequencing of the pncA gene has been shown to have excellent predictive power for PZA resistance. The wider availably of pncA sequencing in combination with databases of the phenotypic implications of these mutations has helped make genetic testing for PZA resistance a practical proposition. For the past decades, it has been generally accepted that an extracellular low pH is required for PZA activity but work in our laboratory [1] and others [2] has recently challenged this assumption. Alternative bacterial stresses, apart from a reduced pH of the growth media (such as reduced temperature), can also induce a PZA-susceptible phenotype. The characterization of spontaneous in vitro-resistant pyrazinoic acid mutants selected under neutral pH conditions suggests a key role for the pantothenate/coenzyme A biosynthetic pathway. This has profound implications for the mechanism of action of PZA as well as potentially the bacterial population against which PZA is active in the host. These findings will be discussed as well as their implications for further research and the future of PZA susceptibility testing., (Copyright © 2016.)

Background: Pyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available., Methods: In a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system., Findings: Pyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0-42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0-16·6% for ofloxacin, to 0·5-12·4% for levofloxacin, and 0·9-14·6% for moxifloxacin when tested at 0·5 μg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 μg/mL was low in all countries., Interpretation: Although pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19-63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness., Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development., (Copyright © 2016 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY license. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to investigate the epidemiology of pyrazinamide (PZA) resistance and the associated risk factors as well as to evaluate the pncA gene loci as a marker for PZA resistance in China. A population-based multicenter study of pulmonary tuberculosis (TB) cases was carried out from 2011 to 2013 in four Chinese districts/counties with different geographic and socioeconomic features. Testing for multidrug-resistant tuberculosis (MDR-TB) and susceptibility to PZA was done by the proportion method on Lowenstein-Jensen medium and Bactec MGIT 960, respectively. Mutations in the pncA gene were identified by sequencing. Among 878 culture-positive cases, 147 (16.7%) were resistant to PZA, with a significantly higher proportion among MDR isolates than among the first-line drug-susceptible isolates (30.2% versus 7.7%; P < 0.001). In total, 136 isolates had a nonsynonymous pncA mutation, with a comparable diagnostic performance between Beijing family and non-Beijing family as well as between MDR-TB and first-line drug-susceptible TB. Furthermore, the mutations in isolates with high-level PZA resistance (MIC > 500 mg/liter) were observed mainly in three regions of the pncA gene (codons 51 to 76, codons 130 to 142, and codons 163 to 180). Patients with prior treatment history had a significantly higher risk for PZA monoresistance (odds ratio [OR], 2.86; 95% confidence interval [CI], 1.363 to 6.015) and MDR PZA resistance (OR, 6.47; 95% CI, 3.186 to 13.15), while the additional factors associated with MDR PZA resistance were the patient's age (OR, 1.02; 95% CI, 1.003 to 1.042), lung cavity (OR, 2.64; 95% CI, 1.296 to 5.391). These findings suggest that it is a priority to identify PZA resistance in MDR-TB and that a rapid molecular diagnostic test based on pncA mutations in the Chinese settings where MDR-TB prevalence is high should be developed., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Our study aims to identify the clinical breakpoints (CBPs) of second-line drugs (SLDs) above which standard therapy fails in order to improve multidrug-resistant tuberculosis (MDR-TB) treatment. MICs of SLDs were determined for M. tuberculosis isolates cultured from 207 MDR-TB patients in a prospective cohort study in China between January 2010 and December 2012. Classification and regression tree (CART) analysis was used to identify the CBPs predictive of treatment outcome. Of the 207 MDR-TB isolates included in the present study, the proportion of isolates above the critical concentration recommended by WHO ranged from 5.3% in pyrazinamide to 62.8% in amikacin. By selecting pyrazinamide as the primary node (CBP, 18.75 mg/liter), 72.1% of sputum culture conversions at month four could be predicted. As for treatment outcome, pyrazinamide (CBP, 37.5 mg/liter) was selected as the primary node to predict 89% of the treatment success, followed by ofloxacin (CBP, 3 mg/liter), improving the predictive capacity of the primary node by 10.6%. Adjusted by identified confounders, the CART-derived pyrazinamide CBP remained the strongest predictor in the model of treatment outcome. Our findings indicate that the critical breakpoints of some second-line drugs and PZA need to be reconsidered in order to better indicate MDR-TB treatment outcome., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: To investigate natural-resistance-associated macrophage protein 1 (NRAMP1), mannose-binding lectin (MBL), vitamin D receptor (VDR) gene polymorphisms and their interaction with susceptibility to pulmonary tuberculosis (PTB) in a Chinese population., Methods: A case-control study was conducted in PTB (n=151), age- and sex- matched healthy controls (HCs) (n=453). Genetic polymorphisms of NRAMP1 (INT4, D543NA and 3'UTR), MBL (HL, PQ, XY and AB) and VDR (FokI and Taq) were analyzed by using PCR-restriction fragment length polymorphism (RFLP) and PCR- single- strand conformation polymorphism (SSCP) techniques. Multifactor dimensionality reduction (MDR) analysis was carried out to assess the effects of the interaction between SNPs., Results: The distribution of NRAMP1- 3'UTR (TGTG/del), MBL- HL (H/L) and FokI (F/f) were significantly different between PTB patients and HCs (p<0.05). HPYA (OR: 1.88; 95% CI: 1.22-2.91), LPXA (OR: 3.17; 95% CI: 1.69- 5.96), LQYA (OR: 3.52; 95%CI: 1.50-8.23) and LPYB (OR: 12.37; 95%CI: 3.75- 40.85) of MBL were risk haplotypes for PTB. The TGTG- H- f (OR: 1.70; 95%CI: 1.10-2.62) and del- H-f (OR: 3.48; 95% CI: 1.45-8.37) of 3'UTR- HL- FokI were also high-risk haplotypes associated with tuberculosis., Conclusions: Our study suggests that genotypes of many polymorphic genes are associated with TB, it is necessary to further explore the mechanism of genotypes and gene-gene interaction in susceptibility to tuberculosis., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment., (Copyright © 2015 Tagliani et al.)

Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients., (Copyright © 2014 Miotto et al.)

During recent years, riboswitches have emerged as potential targets for novel antibacterial substances. In this study, we investigated how one flavin analog, roseoflavin, affected the gene-expression, growth and infectivity of the human bacterial pathogen Listeria monocytogenes to determine the potential of this analog to function as an antibacterial substance. The results indicate that roseoflavin has a profound inhibiting effect on the growth of L. monocytogenes at very low concentrations. Also, expression of the gene located downstream of the FMN riboswitch, a riboflavin transporter, was blocked by the addition of roseoflavin. Base-substitution mutations in the FMN riboswitch allowed the bacteria to grow in the presence of roseoflavin, showing that roseoflavin targeted the FMN riboswitch directly. Surprisingly, we found that roseoflavin stimulated L. monocytogenes virulence gene expression and infection abilities in a mechanism independent of the FMN riboswitch. Our results suggest that roseoflavin can block growth but also enhance Listeria virulence.