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Background: Treatment for fluoroquinolone-resistant multidrug-resistant/rifampicin-resistant tuberculosis (pre-XDR TB) often lasts longer than treatment for less resistant strains, yields worse efficacy results, and causes substantial toxicity. The newer anti-tuberculosis drugs, bedaquiline and delamanid, and repurposed drugs clofazimine and linezolid, show great promise for combination in shorter, less-toxic, and effective regimens. To date, there has been no randomized, internally and concurrently controlled trial of a shorter, all-oral regimen comprising these newer and repurposed drugs sufficiently powered to produce results for pre-XDR TB patients., Methods: endTB-Q is a phase III, multi-country, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of a treatment strategy for patients with pre-XDR TB. Study participants are randomized 2:1 to experimental or control arms, respectively. The experimental arm contains bedaquiline, linezolid, clofazimine, and delamanid. The control comprises the contemporaneous WHO standard of care for pre-XDR TB. Experimental arm duration is determined by a composite of smear microscopy and chest radiographic imaging at baseline and re-evaluated at 6 months using sputum culture results: participants with less extensive disease receive 6 months and participants with more extensive disease receive 9 months of treatment. Randomization is stratified by country and by participant extent-of-TB-disease phenotype defined according to screening/baseline characteristics. Study participation lasts up to 104 weeks post randomization. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 324 participants across 2 arms affords at least 80% power to show the non-inferiority, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per-protocol populations., Discussion: This internally controlled study of shortened treatment for pre-XDR TB will provide urgently needed data and evidence for clinical and policy decision-making around the treatment of pre-XDR TB with a four-drug, all-oral, shortened regimen., Trial Registration: ClinicalTrials.Gov NCT03896685. Registered on 1 April 2018; the record was last updated for study protocol version 4.3 on 17 March 2023., (© 2023. The Author(s).)

Background: Fluoroquinolones (FQs) have substantial activity against the Mycobacterium tuberculosis complex (MTBc) by preventing bacterial DNA synthesis through DNA gyrase inhibition. The reference standard for FQ-resistance testing is phenotypic drug-susceptibility testing (pDST) based on growth inhibition of MTBc in drug-containing Mycobacteria Growth Indicator Tube system (MGIT) media at a critical concentration (CC) that differentiates phenotypically wild-type from nonwild-type MTBc and at a clinical breakpoint that identifies strains that will likely still respond to treatment at higher doses. Despite the recent introduction of powerful new TB drugs, highly sensitive detection of clinically defined FQ resistance remains key., Method: In this study, we re-evaluated the current WHO-recommended CCs of Lfx (1.0 mg/ml), Mfx (0.25 mg/ml), Gfx (0.25 μg/ml), and the nowadays, obsolete CC of Ofx (2.0 mg/ml) for MGIT, using 147 MTBc isolates with known gyrA and gyrB sequences including both high-and low-level FQ resistance-conferring mutants. We tested a wide range of drug concentrations covering the current and former/obsolete WHO-recommended CCs for FQs and some intermediate concentrations to challenge the current WHO-recommended CCs., Results: The specificity of all four CCs was 100%. The sensitivities varied: 92.4% for Ofx and Lfx, 85.7% for Mfx, and 83.2% for Gfx. Lowering the CC of Mfx to 0.125 mg/ml would allow to correctly classify all wild-type and mutant isolates while lowering the CC of Gfx to 0.125 mg/ml would still misclassify some gyrA/gyrB mutants as susceptible., Conclusion: Based on our findings, a minimal inhibitory concentration of 0.125 mg/ml on MGIT medium is a more appropriate CC for Mfx and probably also as a surrogate for overall FQ resistance in the MTBc., Competing Interests: None

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., Competing Interests: None

Competing Interests: AVR reports receiving funding from Research Foundation Flanders (Belgium) related to whole-genome sequencing of M tuberculosis. BdJ reports an institutional contract for central laboratory support to the C211 pediatric bedaquiline trial with Janssen and participation in the scientific advisory boards of the Dioraphte foundation and Panacea. PS reports personal consulting fees from Genoscreen. DMC reports receiving grants from European Committee on Antimicrobial Susceptibility Testing (EUCAST) to study epidemiological cut-off values (ECOFFs) in EUCAST-recommenced methods, funding from UNITE4TB to study ECOFF distributions of drugs included in clinical trials, and participating in the TB alliance study on the M tuberculosis lineage 1 distribution. All other authors declare no competing interests.

Background: Treatment of multidrug- and rifampin-resistant tuberculosis (MDR/RR-TB) is expensive, labour-intensive, and associated with substantial adverse events and poor outcomes. While most MDR/RR-TB patients do not receive treatment, many who do are treated for 18 months or more. A shorter all-oral regimen is currently recommended for only a sub-set of MDR/RR-TB. Its use is only conditionally recommended because of very low-quality evidence underpinning the recommendation. Novel combinations of newer and repurposed drugs bring hope in the fight against MDR/RR-TB, but their use has not been optimized in all-oral, shorter regimens. This has greatly limited their impact on the burden of disease. There is, therefore, dire need for high-quality evidence on the performance of new, shortened, injectable-sparing regimens for MDR-TB which can be adapted to individual patients and different settings., Methods: endTB is a phase III, pragmatic, multi-country, adaptive, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of shorter treatment regimens containing new drugs for patients with fluoroquinolone-susceptible, rifampin-resistant tuberculosis. Study participants are randomized to either the control arm, based on the current standard of care for MDR/RR-TB, or to one of five 39-week multi-drug regimens containing newly approved and repurposed drugs. Study participation in all arms lasts at least 73 and up to 104 weeks post-randomization. Randomization is response-adapted using interim Bayesian analysis of efficacy endpoints. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 750 patients across 6 arms affords at least 80% power to detect the non-inferiority of at least 1 (and up to 3) experimental regimens, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per protocol populations., Discussion: The lack of a safe and effective regimen that can be used in all patients is a major obstacle to delivering appropriate treatment to all patients with active MDR/RR-TB. Identifying multiple shorter, safe, and effective regimens has the potential to greatly reduce the burden of this deadly disease worldwide., Trial Registration: ClinicalTrials.gov Identifier NCT02754765. Registered on 28 April 2016; the record was last updated for study protocol version 3.3, on 27 August 2019., (© 2021. The Author(s).)

The objective of this study was to assess the sensitivity of the Omni Klentaq-LA DNA polymerase for detecting Aleutian mink disease virus (AMDV) in mink blood and tissues by PCR without DNA extraction. The presence of AMDV DNA was directly tested by Klentaq in the plasma, serum, whole blood, and spleen homogenates of 188 mink 4 and 16 months after inoculation with the virus. Samples from bone marrow, small intestine, liver, lungs, kidneys, and lymph nodes of 20 of the same mink were also tested by Klentaq. DNA was extracted from paired samples of plasma and the aforesaid tissues by a commercial nucleic acid extraction kit (Dynabeads Silane) and tested by PCR. Compared with the extracted DNA, Klentaq detected a significantly greater number of samples in the whole blood, serum, plasma, spleen, and small intestine. It was concluded that Klentaq is a preferred system for directly detecting AMDV DNA in mink blood and tissues. The lower success rate of extracted DNA compared with Klentaq could be the result of DNA losses during the extraction process. This is an important factor in chronically infected mink, which have a low AMDV copy number in the bloodstream. Direct AMDV detection also reduces the cost of PCR amplification and lowers the risk of sample contamination.

There is a growing interest among mink ranchers to select their stock for tolerance to the Aleutian mink disease virus (AMDV). Enzyme-linked immunosorbent assays (ELISA) are used to identify mink which have low anti-AMDV antibody titres and are expected to tolerate the AMDV infection. The objective of this study was to calculate the accuracy of three ELISA systems which were performed on blood or serum of AMDV-inoculated American mink (Neovison vison) at five laboratories in Canada, USA, Finland, the Netherlands and Denmark. The accuracy was determined by comparing the ELISA results with antibody titres measured by the counter-immunoelectrophoresis (CIEP) using 10 two-fold serial dilutions of the plasma. Antibody titres of 880 black mink which were inoculated with a spleen homogenate from a naturally infected mink were measured between 16 and 176 weeks post-inoculation. Each ELISA result from every laboratory covered a wide range of antibody titres and the Spearman's rank correlation coefficients between CIEP and ELISA results from different laboratories varied between 0.41 and 0.83, indicating a low to moderate accuracy of ELISA systems for ranking mink by antibody titre. The recombinant VP2-based ELISA used in the Netherlands and Finland ranked the mink by antibody titres more accurately than did the AMDV-G-based ELISA platforms developed in Denmark and the USA, suggesting that the source of antigen was one of the factors affecting the accuracy of ELISA results. It was concluded that the ELISA systems, particularly those based on AMDV-G antigen, require further refinement to improve their accuracy for ranking mink by antibody titre., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Spleen samples from 14 mink that were trapped in 4 counties of Nova Scotia were tested for the presence of the Aleutian mink disease virus (AMDV) by polymerase chain reaction. Viral DNA was not detected in samples from Kings County (n = 2), but was detected in all the mink sampled from Colchester (n = 2) and Halifax (n = 6) counties, and 3 of 4 mink from Yarmouth County. The high level of AMDV-infected mink in Colchester and Halifax counties may pose a serious threat to the captive mink and wild animal populations. Because treatment of infected free-ranging mink is not an option, AMDV control strategies for the captive mink should be primarily focused on bio-security to protect clean ranches.