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1. Bedaquiline and clofazimine resistance in Mycobacterium tuberculosis: an in-vitro and in-silico data analysis

Author: Sonnenkalb, L, Carter, JJ, Spitaleri, A, Iqbal, Z, Hunt, M, Malone, KM, Utpatel, C, Cirillo, DM, Rodrigues, C, Nilgiriwala, KS, Fowler, PW, Merker, M, Niemann, S, Barilar, I, Battaglia, S, Borroni, E, Brandao, AP, Brankin, A, Cabibbe, AM, Carter, J, Claxton, P, Clifton, DA, Cohen, T, Coronel, J, Crook, DW, Dreyer, V, Earle, SG, Escuyer, V, Ferrazoli, L, Fu Gao, G, Gardy, J, Gharbia, S, Ghisi, KT, Ghodousi, A, Gibertoni Cruz, AL, Grandjean, L, Grazian, C, Groenheit, R, Guthrie, JL, He, W, Hoffmann, H, Hoosdally, SJ, Ismail, NA, Jarrett, L, Joseph, L, Jou, R, Kambli, P, Khot, R, Knaggs, J, Koch, A, Kohlerschmidt, D, Kouchaki, S, Lachapelle, AS, Lalvani, A, Grandjean Lapierre, S, Laurenson, IF, Letcher, B, Lin, W-H, Liu, C, Liu, D, Mandal, A, Mansjö, M, Matias, D, Meintjes, G, De Freitas Mendes, F, Mihalic, M, Millard, J, Miotto, P, Mistry, N, Moore, D, Musser, KA, Ngcamu, D, Hoang, NN, Nimmo, C, Okozi, N, Oliveira, RS, Omar, SV, Paton, N, Peto, TEA, Watanabe Pinhata, JM, Plesnik, S, Puyen, ZM, Rabodoarivelo, MS, Rakotosamimanana, N, Rancoita, PMV, Rathod, P, Rodger, G, Rodwell, TC, Roohi, E, Santos-Lazaro, D, Shah, S, Kohl, TA, Smith, G, Solano, W, Supply, P, Surve, U, Tahseen, S, Thuong, NTT, Thwaites, G, Todt, K, Trovato, A, Van Rie, A, Vijay, S, Walker, TM, Walker, SA, Warren, R, Werngren, J, Wijkander, M, Wilkinson, RJ, Wilson, DJ, Wintringer, P, Yu, XX, Yang, Y, Zhao, Y, Yao, S-Y, Zhu, B, and Consortium, Comprehensive Resistance Prediction for Tuberculosis: an International
Subjects: Microbiology (medical), Infectious Diseases, Virology, Microbiology
Abstract: Background Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data. Methods For this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations. Findings We discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5–MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand. Interpretation Our findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments. Funding Leibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions.
Published: 2023
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2. Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution.

Author: Fröberg, G., Maurer, F.P., Chryssanthou, E., Fernström, L., Benmansour, H., Boarbi, S., Mengshoel, A.T., Keller, P.M., Viveiros, M., Machado, D., Fitzgibbon, M.M., Mok, S., Werngren, J., Cirillo, D.M., Alcaide, F., Hyyryläinen, H.L., Aubry, A., Andres, S., Nadarajan, D., Svensson, E., Turnidge, J., Giske, C.G., Kahlmeter, G., Cambau, E., Ingen, J. van, Schön, T., Fröberg, G., Maurer, F.P., Chryssanthou, E., Fernström, L., Benmansour, H., Boarbi, S., Mengshoel, A.T., Keller, P.M., Viveiros, M., Machado, D., Fitzgibbon, M.M., Mok, S., Werngren, J., Cirillo, D.M., Alcaide, F., Hyyryläinen, H.L., Aubry, A., Andres, S., Nadarajan, D., Svensson, E., Turnidge, J., Giske, C.G., Kahlmeter, G., Cambau, E., Ingen, J. van, and Schön, T.
Abstract: 01 juni 2023, Item does not contain fulltext, OBJECTIVE: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. METHODS: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. RESULTS: The clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. CONCLUSION: As a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.
Published: 2023

3. Pretomanid-resistant tuberculosis.

Author: Koehler, N., Andres, S., Merker, M., Dreyer, V., John, A., Kuhns, M., Krieger, D., Choong, E., Verougstraete, N., Wiesch, P.A. Zur, Wicha, S.G., König, C., Kalsdorf, B., Sanchez Carballo, P.M., Schaub, D., Werngren, J., Schön, T., Peloquin, C.A., Schönfeld, N., Verstraete, A.G., Decosterd, L.A., Aarnoutse, R.E., Niemann, S., Maurer, F.P., Lange, C de, Koehler, N., Andres, S., Merker, M., Dreyer, V., John, A., Kuhns, M., Krieger, D., Choong, E., Verougstraete, N., Wiesch, P.A. Zur, Wicha, S.G., König, C., Kalsdorf, B., Sanchez Carballo, P.M., Schaub, D., Werngren, J., Schön, T., Peloquin, C.A., Schönfeld, N., Verstraete, A.G., Decosterd, L.A., Aarnoutse, R.E., Niemann, S., Maurer, F.P., and Lange, C de
Abstract: Item does not contain fulltext
Published: 2023

4. A crowd of BashTheBug volunteers reproducibly and accurately measure the minimum inhibitory concentrations of 13 antitubercular drugs from photographs of 96-well broth microdilution plates

Author: Fowler, P.W., Wright, C., Spiers, H., Zhu, T., Baeten, E.M.L., Hoosdally, S.W., Cruz, A.L.G., Roohi, A., Kouchaki, S., Walker, T.M., Peto, T.E.A., Miller, G., Lintott, C., Clifton, D., Crook, D.W., Walker, A.S., Barilar, I., Battaglia, S., Borroni, E., Brandao, A.P., Brankin, A., Cabibbe, A.M., Carter, J., Chetty, D., Cirillo, D.M., Claxton, P., Clifton, D.A., Cohen, T., Coronel, Jorge, Dreyer, V., Earle, S.G., Escuyer, V., Ferrazoli, L., Gao, G.F., Gardy, J., Gharbia, S., Ghisi, K.T., Ghodousi, A., Grandjean, Louis, Grazian, C., Groenheit, R., Guthrie, J.L., He, W., Hoffmann, H., Hoosdally, S.J., Martinhunt, M., Iqbal, Z., Ismail, N.A., Jarrett, L., Joseph, L., Jou, R., Kambli, P., Khot, R., Knaggs, J., Koch, A., Kohlerschmidt, D., Lachapelle, A.S., Lalvani, A., Lapierre, S.G., Laurenson, I.F., Letcher, B., Lin, W.-H., Liu, C., Liu, D., Malone, K.M., Mandal, A., Mansjõ, M., Matias, D., Meintjes, G., Mendes, F.D.F., Merker, M., Mihalic, M., Millard, J., Miotto, P., Mistry, N., Moore, David Alexander James, Musser, K.A., Ngcamu, D., Nhung, H.N., Niemann, S., Nilgiriwala, K.S., Nimmo, C., O’Donnell, M., Okozi, N., Oliveira, R.S., Omar, S.V., Paton, N., Pinhata, J.M.W., Plesnik, S., Puyen, Z.M., Rabodoarivelo, M.S., Rakotosamimanana, N., Rancoita, P.M.V., Rathod, P., Robinson, E., Rodger, G., Rodrigues, C., Rodwell, T.C., Santos-Lazaro, D., Shah, S., Kohl, T.A., Smith, G., Solano, Walter, Spitaleri, A., Supply, P., Steyn, A.J.C., Surve, U., Tahseen, S., Thuong, N.T.T., Thwaites, G., Todt, K., Trovato, A., Utpatel, C., Van Rie, A., Vijay, S., Warren, R., Werngren, J., Wijkander, M., Wilkinson, R.J., Wilson, D.J., Wintringer, P., Xiao, Y.-X., Yang, Y., Yanlin, Z., Yao, S.-Y., Zhu, B., The Zooniverse Volunteer Community, The CRyPTIC Consortium, Community, The Zooniverse Volunteer, Consortium, The CRyPTIC, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), and Wellcome Trust
Subjects: Volunteers, Model organisms, infectious disease, [SDV]Life Sciences [q-bio], Immunology, Antitubercular Agents, Infectious Disease, Microbial Sensitivity Tests, Zooniverse Volunteer Community, 0601 Biochemistry and Cell Biology, antibiotics, General Biochemistry, Genetics and Molecular Biology, Imaging, minimum inhibitory concentrations, antitubercular drugs, citizen science, M. tuberculosis, Humans, clinical microbiology, Human Biology & Physiology, General Immunology and Microbiology, CRyPTIC Consortium, Prevention, General Neuroscience, FOS: Clinical medicine, microbiology, BashTheBug, Mycobacterium tuberculosis, General Medicine, microdilution plates, Emerging Infectious Diseases, Infectious Diseases, Good Health and Well Being, tuberculosis, 5.1 Pharmaceuticals, photographs, Antimicrobial Resistance, Biochemistry and Cell Biology, Development of treatments and therapeutic interventions, Infection
Abstract: Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally, this is done by culturing a clinical sample and then exposing aliquots to a panel of antibiotics, each being present at a pre-determined concentration, thereby determining if the sample isresistant or susceptible to each sample. The minimum inhibitory concentration (MIC) of a drug is the lowestconcentration that inhibits growth and is a more useful quantity but requires each sample to be tested at a range ofconcentrations for each drug. Using 96-well broth micro dilution plates with each well containing a lyophilised pre-determined amount of an antibiotic is a convenient and cost-effective way to measure the MICs of several drugs at once for a clinical sample. Although accurate, this is still an expensive and slow process that requires highly-skilled and experienced laboratory scientists. Here we show that, through the BashTheBug project hosted on the Zooniverse citizen science platform, a crowd of volunteers can reproducibly and accurately determine the MICs for 13 drugs and that simply taking the median or mode of 11-17 independent classifications is sufficient. There is therefore a potential role for crowds to support (but not supplant) the role of experts in antibiotic susceptibility testing.Tuberculosis is a bacterial respiratory infection that kills about 1.4 million people worldwide each year. While antibiotics can cure the condition, the bacterium responsible for this disease
Published: 2022
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5. Epidemiological cut-off values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of

Author: Fowler, P.W., Barilar, I., Battaglia, S., Borroni, E., Brandao, A.P., Brankin, A., Cabibbe, A.M., Carter, J., Cirillo, D.M., Claxton, P., Clifton, D.A., Cohen, T., Coronel, Jorge, Crook, D.W., Dreyer, V., Earle, S.G., Escuyer, V., Ferrazoli, L., Gao, G.F., Gardy, J., Gharbia, S., Ghisi, K.T., Ghodousi, A., Cruz, A.L.G., Grandjean, Louis, Grazian, C., Groenheit, R., Guthrie, J.L., He, W., Hoffmann, H., Hoosdally, S.J., Hunt, M., Iqbal, Z., Ismail, N.A., Jarrett, L., Joseph, L., Jou, R., Kambli, P., Khot, R., Knaggs, J., Koch, A., Kohlerschmidt, D., Kouchaki, S., Lachapelle, A.S., Lalvani, A., Lapierre, S.G., Laurenson, I.F., Letcher, B., Lin, W.-H., Liu, C., Liu, D., Malone, K.M., Mandal, A., Mansjö, M., Matias, D., Meintjes, G., de Freitas Mendes, F., Merker, M., Mihalic, M., Millard, J., Miotto, P., Mistry, N., Moore, David Alexander James, Musser, K.A., Ngcamu, D., Nhung, H.N., Niemann, S., Nilgiriwala, K.S., Nimmo, C., Okozi, N., Oliveira, R.S., Omar, S.V., Paton, N., Peto, T.E.A., Pinhata, J.M.W., Plesnik, S., Puyen, Z.M., Rabodoarivelo, M.S., Rakotosamimanana, N., Rancoita, P.M.V., Rathod, P., Robinson, E., Rodger, G., Rodrigues, C., Rodwell, T.C., Roohi, A., Santos-Lazaro, D., Shah, S., Kohl, T.A., Smith, G., Solano, Walter, Spitaleri, A., Supply, P., Surve, U., Tahseen, S., Thuong, N.T.T., Thwaites, G., Todt, K., Trovato, A., Utpatel, C., Van Rie, A., Vijay, S., Walker, T.M., Walker, A.S., Warren, R., Werngren, J., Wijkander, M., Wilkinson, R.J., Wilson, D.J., Wintringer, P., Xiao, Y.-X., Yang, Y., Yanlin, Z., Yao, S.-Y., Zhu, B., Consoritum, CRyPTIC, Walker, AS, and CRyPTIC Consortium
Subjects: microdilution, Pulmonary and Respiratory Medicine, tuberculosis, Respiratory, M. tuberculosis, Humans, Tuberculosis, Human medicine, infections, Microbial Sensitivity Tests, Mycobacterium tuberculosis, Anti-Bacterial Agents
Abstract: Drug susceptibility testing ofM. tuberculosisis rooted in a binary susceptible/resistant paradigm. While there are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, it is necessary to measure the epidemiological cut-off values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-tuberculosis compounds, including bedaquiline and delamanid, derived from 20 637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to most of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex, and therefore aphenotypicallywild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined agenotypicallywild-type population and measured the MIC of the 99th percentile by direct measurement andviafitting a Gaussian using interval regression. The proposed ECOFF/ECVs were then validated by comparing with the MIC distributions of high-confidence genetic variants that confer resistance and with qualitative drug susceptibility tests obtainedviathe Mycobacterial Growth Indicator Tube (MGIT) system or Microscopic-Observation Drug Susceptibility (MODS) assay. These ECOFF/ECVs will inform and encourage the more widespread adoption of broth microdilution: this is a cheap culture-based method that tests the susceptibility of 12–14 antibiotics on a single 96-well plate and so could help personalise the treatment of tuberculosis.
Published: 2021

6. Evaluation of a biphasic media assay for pyrazinamide drug susceptibility testing of Mycobacterium tuberculosis

Author: Gonzalo, X., Drobniewski, F., Hoffner, S., and Werngren, J.
Published: 2014
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7. What is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex?

Author: Schön, T., Köser, C.U., Werngren, J., Viveiros, M., Georghiou, S., Kahlmeter, G., Giske, C., Maurer, F., Lina, G., Turnidge, J., Ingen, J. van, Jankovic, M., Goletti, D., Cirillo, D.M., Santin, M., Cambau, E., Schön, T., Köser, C.U., Werngren, J., Viveiros, M., Georghiou, S., Kahlmeter, G., Giske, C., Maurer, F., Lina, G., Turnidge, J., Ingen, J. van, Jankovic, M., Goletti, D., Cirillo, D.M., Santin, M., and Cambau, E.
Abstract: Contains fulltext : 229539.pdf (Publisher’s version ) (Closed access)
Published: 2020

8. Multi-Label Random Forest Model for Tuberculosis Drug Resistance Classification and Mutation Ranking

Author: Kouchaki, S, Yang, Y, Lachapelle, A, Walker, T, Walker, SA, Hoosdally, S, Gibertoni Cruz, AL, Carter, J, Grazian, C, Earle, SG, Fowler, P, Iqbal, Z, Hunt, M, Knaggs, J, Smith, GE, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaler, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, Niemann, S, Merker, M, Hoffmann, H, Todt, K, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Thwaites, G, Thuong, TNT, Ngoc, NH, Srinivasan, V, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Liu, C, Ma, A, Zhu, B, Laurenson, I, Claxton, P, Koch, A, Wilkinson, R, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, MH, Lin, WH, Ferrazoli, L, Siqueira de Oliveira, R, Arandjelovic, I, Chaiprasert, A, Comas, I, Roig, CR, Drobniewski, FA, Farhat, MR, Gao, Q, Hee, ROT, Sintchenko, V, Supply, P, van Soolingen, D, Peto, TEA, Crook, D, Clifton, D, Kouchaki, S, Yang, Y, Lachapelle, A, Walker, T, Walker, SA, Hoosdally, S, Gibertoni Cruz, AL, Carter, J, Grazian, C, Earle, SG, Fowler, P, Iqbal, Z, Hunt, M, Knaggs, J, Smith, GE, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaler, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, Niemann, S, Merker, M, Hoffmann, H, Todt, K, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Thwaites, G, Thuong, TNT, Ngoc, NH, Srinivasan, V, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Liu, C, Ma, A, Zhu, B, Laurenson, I, Claxton, P, Koch, A, Wilkinson, R, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, MH, Lin, WH, Ferrazoli, L, Siqueira de Oliveira, R, Arandjelovic, I, Chaiprasert, A, Comas, I, Roig, CR, Drobniewski, FA, Farhat, MR, Gao, Q, Hee, ROT, Sintchenko, V, Supply, P, van Soolingen, D, Peto, TEA, Crook, D, and Clifton, D
Published: 2020

9. GenomegaMap: within-species genome-wide d_N/d_S estimation from over 10,000 genomes

Author: Wilson, D, Crook, DW, Peto, TEA, Walker, AS, Hoosdally, SJ, Gibertoni Cruz, AL, Carter, J, Grazian, C, Earle, SG, Kouchaki, S, Lachapelle, A, Yang, Y, Clifton, DA, Fowler, PW, Iqbal, Z, Hunt, M, Knaggs, J, Smith, EG, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaleri, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, NIemann, S, Kohl, TA, Merker, M, Hoffman, H, Todt, K, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Thwaites, G, Thoung, TNT, Ngoc, NH, Srinivasan, V, Walker, TM, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Liu, C, Ma, A, Zhu, B, Laurenson, I, Claxton, P, Koch, A, Wilkinson, R, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, MH, Lin, WH, Ferrazoli, L, Siqueira de Oliveira, R, Arandjelovic, I, Chaipresert, A, Comas, I, Roig, CJ, Drobniewski, FA, Farhat, MR, Gao, Q, Hee, ROT, Sintchenko, V, Wilson, D, Crook, DW, Peto, TEA, Walker, AS, Hoosdally, SJ, Gibertoni Cruz, AL, Carter, J, Grazian, C, Earle, SG, Kouchaki, S, Lachapelle, A, Yang, Y, Clifton, DA, Fowler, PW, Iqbal, Z, Hunt, M, Knaggs, J, Smith, EG, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaleri, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, NIemann, S, Kohl, TA, Merker, M, Hoffman, H, Todt, K, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Thwaites, G, Thoung, TNT, Ngoc, NH, Srinivasan, V, Walker, TM, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Liu, C, Ma, A, Zhu, B, Laurenson, I, Claxton, P, Koch, A, Wilkinson, R, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, MH, Lin, WH, Ferrazoli, L, Siqueira de Oliveira, R, Arandjelovic, I, Chaipresert, A, Comas, I, Roig, CJ, Drobniewski, FA, Farhat, MR, Gao, Q, Hee, ROT, and Sintchenko, V
Abstract: The dN/dS ratio provides evidence of adaptation or functional constraint in protein-coding genes by quantifying the relative excess or deficit of amino acid-replacing versus silent nucleotide variation. Inexpensive sequencing promises a better understanding of parameters such as dN/dS, but analysing very large datasets poses a major statistical challenge. Here I introduce genomegaMap for estimating within-species genome-wide variation in dN/dS, and I apply it to 3,979 genes across 10,209 tuberculosis genomes to characterize the selection pressures shaping this global pathogen. GenomegaMap is a phylogeny-free method that addresses two major problems with existing approaches: (i) it is fast no matter how large the sample size and (ii) it is robust to recombination, which causes phylogenetic methods to report artefactual signals of adaptation. GenomegaMap uses population genetics theory to approximate the distribution of allele frequencies under general, parent-dependent mutation models. Coalescent simulations show that substitution parameters are well-estimated even when genomegaMap’s simplifying assumption of independence among sites is violated. I demonstrate the ability of genomegaMap to detect genuine signatures of selection at antimicrobial resistance-conferring substitutions in M. tuberculosis and describe a novel signature of selection in the cold-shock DEAD-box protein A gene deaD/csdA. The genomegaMap approach helps accelerate the exploitation of big data for gaining new insights into evolution within species.
Published: 2020

10. Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

Author: Schön, T. (Thomas), Werngren, J. (Jim), Machado, D. (Diana), Borroni, E. (Emanuele), Wijkander, M. (Maria), Lina, G. (Gérard), Mouton, J.W. (Johan), Matuschek, E. (Erika), Kahlmeter, G., Giske, C.G. (Christian G.), Santin, M. (Miguel), Cirillo, D.M. (Daniela Maria), Viveiros, M. (Miguel), Cambau, E. (Emmanuelle), Schön, T. (Thomas), Werngren, J. (Jim), Machado, D. (Diana), Borroni, E. (Emanuele), Wijkander, M. (Maria), Lina, G. (Gérard), Mouton, J.W. (Johan), Matuschek, E. (Erika), Kahlmeter, G., Giske, C.G. (Christian G.), Santin, M. (Miguel), Cirillo, D.M. (Daniela Maria), Viveiros, M. (Miguel), and Cambau, E. (Emmanuelle)
Abstract: Scope: Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC. Methods: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. Recommendations: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 105 CFU/mL suspension, obtained from a 10−2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 103 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03–0.12 for isoniazid, 0.12–0.5 for levofloxacin and 0.25–1 mg/L for amikacin. Conclusions: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods
Published: 2020
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11. Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

Author: Schön, T, Werngren, J, Machado, D, Borroni, E, Wijkander, M, Lina, G, Mouton, Johan, Matuschek, E, Kahlmeter, G, Giske, C, Santin, M, Cirillo, DM, Viveiros, M, Cambau, E, Schön, T, Werngren, J, Machado, D, Borroni, E, Wijkander, M, Lina, G, Mouton, Johan, Matuschek, E, Kahlmeter, G, Giske, C, Santin, M, Cirillo, DM, Viveiros, M, and Cambau, E
Published: 2020

12. Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data

Author: Ängeby, K. A., Jureen, P., Giske, C. G., Chryssanthou, E., Sturegård, E., Nordvall, M., Johansson, A. G., Werngren, J., Kahlmeter, G., Hoffner, S. E., and Schön, T.
Published: 2010
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13. DeepAMR for predicting co-occurrent resistance of Mycobacterium tuberculosis

Author: Yang, Y, Walker, TM, Walker, AS, Wilson, DJ, Peto, TEA, Crook, DW, Shamout, F, Zhu, T, Clifton, DA, Arandjelovic, I, Comas, I, Farhat, MR, Gao, Q, Sintchenko, V, Van Soolingen, D, Hoosdally, S, Cruz, ALG, Carter, J, Grazian, C, Earle, SG, Kouchaki, S, Fowler, PW, Iqbal, Z, Hunt, M, Smith, EG, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaleri, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, Niemann, S, Kohl, T, Merker, M, Hoffmann, H, Molodtsov, N, Plesnik, S, Ismail, N, Omar, SV, Thwaites, G, Thuong, NTT, Nhung, HN, Srinivasan, V, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Ma, A, Liu, C, Zhu, B, Laurenson, I, Claxton, P, Koch, A, Wilkinson, R, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, M-H, Lin, W-H, Ferrazoli, L, De Oliveira, RS, and Wellcome Trust
Subjects: DRUG-RESISTANCE, Technology, Biochemistry & Molecular Biology, Science & Technology, INFORMATION, MUTATIONS, CRyPTIC Consortium, Bioinformatics, Statistics & Probability, SUSCEPTIBILITY, 06 Biological Sciences, GENE, Biochemical Research Methods, CLASSIFICATION, DIMENSIONALITY REDUCTION, Biotechnology & Applied Microbiology, Physical Sciences, Computer Science, Computer Science, Interdisciplinary Applications, Mathematical & Computational Biology, 08 Information and Computing Sciences, Life Sciences & Biomedicine, Mathematics, 01 Mathematical Sciences
Abstract: Motivation Resistance co-occurrence within first-line anti-tuberculosis (TB) drugs is a common phenomenon. Existing methods based on genetic data analysis of Mycobacterium tuberculosis (MTB) have been able to predict resistance of MTB to individual drugs, but have not considered the resistance co-occurrence and cannot capture latent structure of genomic data that corresponds to lineages. Results We used a large cohort of TB patients from 16 countries across six continents where whole-genome sequences for each isolate and associated phenotype to anti-TB drugs were obtained using drug susceptibility testing recommended by the World Health Organization. We then proposed an end-to-end multi-task model with deep denoising auto-encoder (DeepAMR) for multiple drug classification and developed DeepAMR_cluster, a clustering variant based on DeepAMR, for learning clusters in latent space of the data. The results showed that DeepAMR outperformed baseline model and four machine learning models with mean AUROC from 94.4% to 98.7% for predicting resistance to four first-line drugs [i.e. isoniazid (INH), ethambutol (EMB), rifampicin (RIF), pyrazinamide (PZA)], multi-drug resistant TB (MDR-TB) and pan-susceptible TB (PANS-TB: MTB that is susceptible to all four first-line anti-TB drugs). In the case of INH, EMB, PZA and MDR-TB, DeepAMR achieved its best mean sensitivity of 94.3%, 91.5%, 87.3% and 96.3%, respectively. While in the case of RIF and PANS-TB, it generated 94.2% and 92.2% sensitivity, which were lower than baseline model by 0.7% and 1.9%, respectively. t-SNE visualization shows that DeepAMR_cluster captures lineage-related clusters in the latent space. Availability and implementation The details of source code are provided at http://www.robots.ox.ac.uk/∼davidc/code.php.
Published: 2019

14. GenomegaMap: within-species genome-widedN/dSestimation from over 10,000 genomes

Author: Wilson, DJ, Crook, DW, Peto, TEA, Walker, AS, Hoosdally, SJ, Gibertoni Cruz, AL, Carter, J, Grazian, C, Earle, SG, Kouchaki, S, Lachapelle, A, Yang, Y, Clifton, DA, Fowler, PW, Iqbal, Z, Hunt, M, Knaggs, J, Smith, EG, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaleri, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, Niemann, S, Kohl, TA, Merker, M, Hoffmann, H, Todt, K, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Thwaites, G, Thuong, TNT, Ngoc, NH, Srinivasan, V, Walker, TM, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Liu, C, Ma, A, Zhu, B, Laurenson, I, Claxton, P, Koch, A, Wilkinson, R, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, M-H, Lin, W-H, Ferrazoli, L, De Oliveira, RS, Arandjelovic, I, Chaiprasert, A, Comas, I, Roig, CJ, Drobniewski, FA, Farhat, MR, Gao, Q, Hee, ROT, Sintchenko, V, Supply, P, Van Soolingen, D, University of Oxford, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), D.J.W. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (grant no. 101237/Z/13/B) and is a Big Data Institute Robertson Fellow. The CRyPTIC Consortium was supported by grants from the Bill and Melinda Gates Foundation (OPP1133541) and a Wellcome Trust/Newton Fund-MRC Collaborative Award (200205/Z/15/Z). F.A.D. was supported by the Imperial Biomedical Research Centre., Members of the CRyPTIC Consortium : Derrick W. Crook, Timothy E.A. Peto, A. Sarah Walker, Sarah J. Hoosdally, Ana L. Gibertoni Cruz, Joshua Carter, Clara Grazian, Sarah G. Earle, Samaneh Kouchaki, Alexander Lachapelle, Yang Yang, David A. Clifton, and Philip W. Fowler, University of Oxford, Zamin Iqbal, Martin Hunt, and Jeffrey Knaggs, European Bioinformatics Institute, E. Grace Smith, Priti Rathod, Lisa Jarrett, and Daniela Matias, Public Health England, Birmingham, Daniela M. Cirillo, Emanuele Borroni, Simone Battaglia, Arash Ghodousi, Andrea Spitaleri, and Andrea Cabibbe, Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Milan, Sabira Tahseen, National Tuberculosis Control Program Pakistan, Islamabad, Kayzad Nilgiriwala and Sanchi Shah, The Foundation for Medical Research, Mumbai, Camilla Rodrigues, Priti Kambli, Utkarsha Surve, and Rukhsar Khot, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, Stefan Niemann, Thomas A. Kohl, and Matthias Merker, Research Center Borstel, Harald Hoffmann, Katharina Todt, and Sara Plesnik, Institute of Microbiology & Laboratory Medicine, IML Red, Gauting, Nazir Ismail, Shaheed Vally Omar, and Lavania Joseph, National Institute for Communicable Diseases, Johannesburg, Guy Thwaites, Thuong Nguyen Thuy Thuong, Nhung Hoang Ngoc, Vijay Srinivasan, and Timothy M. Walker, Oxford University Clinical Research Unit, Ho Chi Minh City, David Moore, Jorge Coronel and Walter Solano, London School of Hygiene and Tropical Medicine and Universidad Peruana Cayetano Heredá, Lima, George F. Gao, Guangxue He, Yanlin Zhao, and Chunfa Liu, China CDC, Beijing, Aijing Ma, Shenzhen Third People’s Hospital, Shenzhen, Baoli Zhu, Institute of Microbiology, CAS, Beijing, Ian Laurenson and Pauline Claxton, Scottish Mycobacteria Reference Laboratory, Edinburgh, Anastasia Koch, Robert Wilkinson, University of Cape Town, Ajit Lalvani, Imperial College London, James Posey, CDC Atlanta, Jennifer Gardy, University of British Columbia, Jim Werngren, Public Health Agency of Sweden, Nicholas Paton, National University of Singapore, Ruwen Jou, Mei-Hua Wu, Wan-Hsuan Lin, CDC Taiwan, Lucilaine Ferrazoli, Rosangela Siqueira de Oliveira, Institute Adolfo Lutz, São Paulo. Authors contributing to the CRyPTIC Consortium are (in alphabetical order): Irena Arandjelovic (Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia), Angkana Chaiprasert (Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand), Iñaki Comas (Instituto de Biomedicina de Valencia [IBV-CSIC], Calle Jaime Roig, Valencia, Spain, FISABIO Public Health, Valencia, Spain, CIBER in Epidemiology and Public Health, Madrid, Spain), Francis A. Drobniewski (Imperial College, London, UK), Maha R. Farhat (Harvard Medical School, Boston, USA), Qian Gao (Shanghai Medical College, Fudan University, Shanghai, China), Rick Ong Twee Hee (Saw Swee Hock School of Public Health, National University of Singapore, Singapore), Vitali Sintchenko (Centre for Infectious Diseases and Microbiology—Public Health, University of Sydney, Sydney, Australia), Philip Supply (Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204—CIIL—Centre d’Infection et d’Immunité de Lille, F-59000 Lille, France), and Dick van Soolingen (National Institute for Public Health and the Environment [RIVM], Bilthoven, The Netherlands)., Supply, Philip, Consortium, CRyPTIC, University of Oxford [Oxford], Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Royal Society (UK), Bill & Melinda Gates Foundation, Newton Fund, Comas, Iñaki [0000-0001-5504-9408], and Comas, Iñaki
Subjects: Natural selection, [SDV]Life Sciences [q-bio], Population genetics, adaptation, Computational biology, Biology, AcademicSubjects/SCI01180, 0601 Biochemistry and Cell Biology, Genome, Coalescent theory, DEAD-box RNA Helicases, Big data, 03 medical and health sciences, 0603 Evolutionary Biology, big data, Parent-dependent mutation, Genetics, dN/dS, Adaptation, Selection, Genetic, Molecular Biology, Allele frequency, Ecology, Evolution, Behavior and Systematics, Silent Mutation, Selection (genetic algorithm), 030304 developmental biology, Evolutionary Biology, 0604 Genetics, 0303 health sciences, Models, Genetic, Phylogenetic tree, 030306 microbiology, AcademicSubjects/SCI01130, natural selection, Mycobacterium tuberculosis, Recombination, Resources, recombination, 3. Good health, [SDV] Life Sciences [q-bio], Genetic Techniques, Mutation (genetic algorithm), parent-dependent mutation, Genome, Bacterial
Abstract: 11 págs, 4 figuras y fórmulas matemáticas. Material suplementario en: http://dx.doi.org/10.1093/molbev/msaa069, The dN/dS ratio provides evidence of adaptation or functional constraint in protein-coding genes by quantifying the relative excess or deficit of amino acid-replacing versus silent nucleotide variation. Inexpensive sequencing promises a better understanding of parameters, such as dN/dS, but analyzing very large data sets poses a major statistical challenge. Here, I introduce genomegaMap for estimating within-species genome-wide variation in dN/dS, and I apply it to 3,979 genes across 10,209 tuberculosis genomes to characterize the selection pressures shaping this global pathogen. GenomegaMap is a phylogeny-free method that addresses two major problems with existing approaches: 1) It is fast no matter how large the sample size and 2) it is robust to recombination, which causes phylogenetic methods to report artefactual signals of adaptation. GenomegaMap uses population genetics theory to approximate the distribution of allele frequencies under general, parent-dependent mutation models. Coalescent simulations show that substitution parameters are well estimated even when genomegaMap's simplifying assumption of independence among sites is violated. I demonstrate the ability of genomegaMap to detect genuine signatures of selection at antimicrobial resistance-conferring substitutions in Mycobacterium tuberculosis and describe a novel signature of selection in the cold-shock DEAD-box protein A gene deaD/csdA. The genomegaMap approach helps accelerate the exploitation of big data for gaining new insights into evolution within species., D.J.W. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (grant no. 101237/Z/13/B) and is a Big Data Institute Robertson Fellow. The CRyPTIC Consortium was supported by grants from the Bill and Melinda Gates Foundation (OPP1133541) and a Wellcome Trust/Newton Fund-MRC Collaborative Award (200205/Z/15/Z). F.A.D. was supported by the Imperial Biomedical Research Centre
Published: 2019
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15. Application of machine learning techniques to tuberculosis drug resistance analysis

Author: Kouchaki, S, Yang, YY, Walker, TM, Walker, AS, Wilson, DJ, Peto, TEA, Crook, DW, Clifton, DA, Hoosdally, SJ, Gibertoni Cruz, AL, Carter, J, Grazian, C, Fowler, PW, Iqbal, Z, Hunt, M, Smith, EG, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaleri, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, Niemann, S, Kohl, T, Merker, M, Hoffmann, H, Molodtsov, N, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Marubini, E, Thwaites, G, Thuong, TNT, Ngoc, NH, Srinivasan, V, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Ma, A, Liu, C, Zhu, B, Laurenson, I, Claxton, P, Wilkinson, RJ, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, MH, Lin, WH, Ferrazoli, L, De Oliveira, RS, Arandjelovic, I, Comas, I, Drobniewski, F, Gao, Q, Sintchenko, V, Supply, P, Van Soolingen, D, Kouchaki, S, Yang, YY, Walker, TM, Walker, AS, Wilson, DJ, Peto, TEA, Crook, DW, Clifton, DA, Hoosdally, SJ, Gibertoni Cruz, AL, Carter, J, Grazian, C, Fowler, PW, Iqbal, Z, Hunt, M, Smith, EG, Rathod, P, Jarrett, L, Matias, D, Cirillo, DM, Borroni, E, Battaglia, S, Ghodousi, A, Spitaleri, A, Cabibbe, A, Tahseen, S, Nilgiriwala, K, Shah, S, Rodrigues, C, Kambli, P, Surve, U, Khot, R, Niemann, S, Kohl, T, Merker, M, Hoffmann, H, Molodtsov, N, Plesnik, S, Ismail, N, Omar, SV, Joseph, L, Marubini, E, Thwaites, G, Thuong, TNT, Ngoc, NH, Srinivasan, V, Moore, D, Coronel, J, Solano, W, Gao, GF, He, G, Zhao, Y, Ma, A, Liu, C, Zhu, B, Laurenson, I, Claxton, P, Wilkinson, RJ, Lalvani, A, Posey, J, Gardy, J, Werngren, J, Paton, N, Jou, R, Wu, MH, Lin, WH, Ferrazoli, L, De Oliveira, RS, Arandjelovic, I, Comas, I, Drobniewski, F, Gao, Q, Sintchenko, V, Supply, P, and Van Soolingen, D
Abstract: Motivation: Timely identification of Mycobacterium tuberculosis (MTB) resistance to existing drugs is vital to decrease mortality and prevent the amplification of existing antibiotic resistance. Machine learning methods have been widely applied for timely predicting resistance of MTB given a specific drug and identifying resistance markers. However, they have been not validated on a large cohort of MTB samples from multi-centers across the world in terms of resistance prediction and resistance marker identification. Several machine learning classifiers and linear dimension reduction techniques were developed and compared for a cohort of 13 402 isolates collected from 16 countries across 6 continents and tested 11 drugs. Results: Compared to conventional molecular diagnostic test, area under curve of the best machine learning classifier increased for all drugs especially by 23.11%, 15.22% and 10.14% for pyrazinamide, ciprofloxacin and ofloxacin, respectively (P < 0.01). Logistic regression and gradient tree boosting found to perform better than other techniques. Moreover, logistic regression/gradient tree boosting with a sparse principal component analysis/non-negative matrix factorization step compared with the classifier alone enhanced the best performance in terms of F1-score by 12.54%, 4.61%, 7.45% and 9.58% for amikacin, moxifloxacin, ofloxacin and capreomycin, respectively, as well increasing area under curve for amikacin and capreomycin. Results provided a comprehensive comparison of various techniques and confirmed the application of machine learning for better prediction of the large diverse tuberculosis data. Furthermore, mutation ranking showed the possibility of finding new resistance/susceptible markers.
Published: 2019

16. Evaluation of the BacT/ALERT 3D system for recovery and drug susceptibility testing of Mycobacterium tuberculosis

Author: Ängeby, K. A. K., Werngren, J., Toro, J. C., Hedström, G., Petrini, B., and Hoffner, S. E.
Published: 2003

17. DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS: FROM PHENOTYPIC MIC-ANALYSIS TO WGS FOR ROUTINE DRUG SUSCEPTIBILITY TESTING

Author: Werngren, J., primary
Published: 2019
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18. Proficiency of drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide: the Swedish experience

Author: Hoffner, S., Ängeby, K., Sturegård, E., Jönsson, B., Johansson, A., Sellin, M., and Werngren, J.
Published: 2013
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19. Emergence and genetic characteristics of pyrazinamide resistant tuberculosis in China, a multi-center study

Author: Li, D, Hu, Y, Werngren, J, Mansjö, M, Zheng, X, Drobniewski, F, Hoffner, S, Xu, B, and Wellcome Trust
Subjects: 1108 Medical Microbiology, 1115 Pharmacology And Pharmaceutical Sciences, Microbiology, 0605 Microbiology
Abstract: Background: The aim of this study was to investigate the rates 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. Method: A population-based multi-center study of pulmonary TB cases was carried out from 2011 to 2013 in four Chinese district/counties with different geographic and socio-economic features. Testing for multidrug-resistant tuberculosis (MDR-TB) and susceptibility to PZA were determined by the proportion method on Löwenstein-Jensen medium and BACTEC MGIT 960 respectively. Mutations in the pncA gene were identified by sequencing. Results: Among 878 culture positive cases, 147(16.7%) were resistant to PZA, with a significantly higher proportion among MDR isolates compared to the drug sensitive isolates (30.2% VS 7.7%, p500mg/L) were observed mainly in three regions of the pncA gene (codon 51-76, codon 130-142 and codon 163-180). Patients with prior treatment history had a significantly higher risk for PZA mono-resistance (OR:2.86,95%CI:1.363-6.015) and MDR PZA resistance (OR:6.47, 95%CI:3.186-13.15), while the additional factors associated with MDR-PZA resistance were the patient's age (OR:1.02, 95%CI:1.003-1.042), lung cavity (OR:2.64, 95%CI:1.296-5.391). Conclusions: These findings suggest that it is a priority to identify PZA resistance in MDR-TB and a rapid molecular diagnostic test based on pncA mutations in the Chinese settings where MDR-TB prevalence is high should be developed.
Published: 2016

20. Protecting Pyrazinamide, a Priority for Improving Outcomes in Multidrug-Resistant Tuberculosis Treatment

Author: Anthony, R. M., primary, Cynamon, M., additional, Hoffner, S., additional, Werngren, J., additional, den Hertog, A. L., additional, and van Soolingen, D., additional
Published: 2017
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21. Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10

Author: Sturegård, E., Ängeby, K. A., Werngren, J., Jureen, P., Kronvall, G., Giske, C. G., Kahlmeter, Gunnar, Schön, T., Sturegård, E., Ängeby, K. A., Werngren, J., Jureen, P., Kronvall, G., Giske, C. G., Kahlmeter, Gunnar, and Schön, T.
Abstract: The MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first-and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within +/- one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification. Clinical Microbiology and Infection (C) 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Published: 2015
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22. Evaluation of loop mediated isothermal amplification for diagnosis of Mycobacterium tuberculosis complex in clinical samples

Author: Kohan, L., Shahhosseiny, M. H., Razavi, M. R., kazem parivar, Moslemi, E., and Werngren, J.
Subjects: Loop mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), IS6110 gene, Mycobacterium tuberculosis, diagnosis
Abstract: Tuberculosis (TB) remains an important global public health problem. The lack of rapid and accurate diagnostic testing is an important impediment to global tuberculosis control. Loop mediated isothermal amplification (LAMP) is a rapid method for nucleic acid amplification. In this study, we assessed the performance of an in-house LAMP assay for the detection of tuberculosis. Six oligonucleotide primers specific for Mycobacterium tuberculosis complex were designed corresponding to IS6110 gene sequence. Optimization of LAMP reaction was performed. A total of 133 clinical sputum samples and 80 bacterial cultures were studied by LAMP method. Sensitivity of this assay for detection of genomic DNA was 5 fg. This assay successfully detected M. tuberculosis complex not only in the bacterial cultures but also in the clinical sputum samples from patients with TB. The sensitivity of LAMP in culturepositive samples was 100% (60/60) and the specificity in culture-negative samples was 95.9% (70/73, 95% confidence interval 91.3 to 98.7%). Thus, LAMP is a rapid, highly sensitive and specific DNA amplification technique for early diagnosis of TB.Key words: Loop mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), IS6110 gene, Mycobacterium tuberculosis, diagnosis.
Published: 2013

23. Pyrazinamide resistance in Swedish multidrug-resistant tuberculosis 2003–2013

Author: Mansjö, M., primary, Werngren, J., additional, and Hoffner, S., additional
Published: 2015
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24. Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10

Author: Sturegård, E., primary, Ängeby, K.A., additional, Werngren, J., additional, Juréen, P., additional, Kronvall, G., additional, Giske, C.G., additional, Kahlmeter, G., additional, and Schön, T., additional
Published: 2015
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25. A 24-well plate assay for simultaneous testing of first and second line drugs against Mycobacterium tuberculosis in a high endemic setting

Author: Wedajo, W., Schön, Thomas, Bedru, A., Kiros, T., Hailu, E., Mebrahtu, T., Yamuah, L., Angeby, K., Werngren, J., Onyebujoh, P., Dagne, K., Aseffa, A., Wedajo, W., Schön, Thomas, Bedru, A., Kiros, T., Hailu, E., Mebrahtu, T., Yamuah, L., Angeby, K., Werngren, J., Onyebujoh, P., Dagne, K., and Aseffa, A.
Abstract: Background: Early detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas. Results: Baseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks. Conclusions: The 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful
Published: 2014
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26. Proficiency of drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide: the Swedish experience

Author: Hoffner, S, Angeby, K, Sturegard, E, Jonsson, B, Johansson, A, Sellin, M, Werngren, J, Hoffner, S, Angeby, K, Sturegard, E, Jonsson, B, Johansson, A, Sellin, M, and Werngren, J
Abstract: BACKGROUND: Pyrazinamide (PZA) is a key drug in the treatment of tuberculosis (TB), including multidrug-resistant TB. Drug susceptibility testing (DST) of Mycobacterium tuberculosis against PZA is not included in the World Health Organizations yearly proficiency testing. There is an increasing need to establish quality control of PZA DST. less thanbrgreater than less thanbrgreater thanOBJECTIVE: To evaluate the performance of PZA DST and to introduce a quality assurance system for the test in Sweden. less thanbrgreater than less thanbrgreater thanMETHOD: Panels with PZA-susceptible and -resistant isolates were used in three rounds of proficiency testing in all five Swedish clinical TB laboratories and our reference laboratory. All laboratories used the MGIT 960 system. Minimum inhibitory concentrations (MICs) were determined and the pncA gene was sequenced to further characterise the 52 panel strains. less thanbrgreater than less thanbrgreater thanRESULTS: Good agreement was seen between the phenotypic PZA DST and pncA sequence data, and MIC determination confirmed high levels of resistance. However, in contrast to other drugs, for which correct proficiency test results were observed, specificity problems occurred for PZA DST in some laboratories. less thanbrgreater than less thanbrgreater thanCONCLUSIONS: In Sweden, using panel testing, differences were seen in the proficiency of TB laboratories in correctly identifying PZA susceptibility. Improved results were noted in the third round; PZA has therefore been included in yearly proficiency testing., Funding Agencies|European Union|223681
Published: 2013
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27. Resistance to First-Line Anti-TB Drugs is Associated with Reduced Nitric Oxide Susceptibility in Mycobacterium tuberculosis

Author: Idh, Jonna, Mekonnen, M., Abate, E., Wedajo, W., Werngren, J., Ängeby, K., Lerm, Maria, Elias, D., Sundqvist, Tommy, Aseffa, A., Stendahl, Olle, Schön, T., Idh, Jonna, Mekonnen, M., Abate, E., Wedajo, W., Werngren, J., Ängeby, K., Lerm, Maria, Elias, D., Sundqvist, Tommy, Aseffa, A., Stendahl, Olle, and Schön, T.
Abstract: Background and objective: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug resistance and clinical outcome is not known. Methods: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. Results: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters, but a tendency towards lower rate of weight gain after two months of treatment. Conclusion: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions.
Published: 2012
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28. Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data

Author: Ängeby, Kristian A., Jureen, P., Giske, C. G., Chryssanthou, E., Sturegård, E., Nordvall, M., Johansson, A. G., Werngren, J., Kahlmeter, Gunnar, Hoffner, S. E., Schön, T., Ängeby, Kristian A., Jureen, P., Giske, C. G., Chryssanthou, E., Sturegård, E., Nordvall, M., Johansson, A. G., Werngren, J., Kahlmeter, Gunnar, Hoffner, S. E., and Schön, T.
Abstract: OBJECTIVES: To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. METHODS: A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. RESULTS: The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. CONCLUSIONS: We propose S (susceptible)
Published: 2010
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29. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections

Author: Juréen, P., Ängeby, K., Sturegård, E., Chryssanthou, E., Giske, C. G., Werngren, J., Nordvall, M., Johansson, A., Kahlmeter, Gunnar, Hoffner, S., Schön, T., Juréen, P., Ängeby, K., Sturegård, E., Chryssanthou, E., Giske, C. G., Werngren, J., Nordvall, M., Johansson, A., Kahlmeter, Gunnar, Hoffner, S., and Schön, T.
Abstract: The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.
Published: 2010
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30. Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions and pncA Gene Sequencing

Author: Werngren, J., primary, Sturegård, E., additional, Juréen, P., additional, Ängeby, K., additional, Hoffner, S., additional, and Schön, T., additional
Published: 2012
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31. Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis

Author: Engstrom, A., primary, Perskvist, N., additional, Werngren, J., additional, Hoffner, S. E., additional, and Jureen, P., additional
Published: 2011
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32. Multicentre laboratory validation of the colorimetric redox indicator (CRI) assay for the rapid detection of extensively drug-resistant (XDR) Mycobacterium tuberculosis

Author: Martin, A., primary, Paasch, F., additional, Docx, S., additional, Fissette, K., additional, Imperiale, B., additional, Ribon, W., additional, Gonzalez, L. A., additional, Werngren, J., additional, Engstrom, A., additional, Skenders, G., additional, Jureen, P., additional, Hoffner, S., additional, Del Portillo, P., additional, Morcillo, N., additional, and Palomino, J. C., additional
Published: 2011
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33. Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections

Author: Juréen, P., primary, Ängeby, K., additional, Sturegård, E., additional, Chryssanthou, E., additional, Giske, C. G., additional, Werngren, J., additional, Nordvall, M., additional, Johansson, A., additional, Kahlmeter, G., additional, Hoffner, S., additional, and Schön, T., additional
Published: 2010
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34. Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis

Author: Schon, T., primary, Jureen, P., additional, Giske, C. G., additional, Chryssanthou, E., additional, Sturegard, E., additional, Werngren, J., additional, Kahlmeter, G., additional, Hoffner, S. E., additional, and Angeby, K. A., additional
Published: 2009
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35. Concise Communication Evaluation of the BacT/ALERT 3D system for recovery and drug susceptibility testing of Mycobacterium tuberculosis.

Author: Angeby, K. A. K., Werngren, J., Toro, J. C., Hedström, G., Petrini, B., and Hoffner, S. E.
Subjects: *TUBERCULOSIS, *CLINICAL drug trials, *RIFAMPIN, *ISONIAZID
Abstract: We evaluated the BacT/ALERT 3D system for recovery and drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). Of 2659 clinical specimens, MTB was detected in 92 using BacT/ALERT, compared to 94 using Löwenstein–Jensen culture. Detection time was 25% shorter with BacT/ALERT. Sensitivities were 92%, 96%, 78% and 100% for resistance to rifampicin, isoniazid, streptomycin and ethambutol, respectively, while specificity was 100% for all antibiotics, when BacT/ALERT was compared with the BACTEC 460 method on 50 MTB isolates. The BacT/ALERT system is fully automated and creates no radioactive waste. It seems to be a valid alternative for primary isolation, but further evaluation is needed regarding DST. [ABSTRACT FROM AUTHOR]
Published: 2003
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36. Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosisagainst Pyrazinamide Using Wild-Type MIC Distributions and pncAGene Sequencing

Author: Werngren, J., Sturegård, E., Juréen, P., Ängeby, K., Hoffner, S., and Schön, T.
Abstract: ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistant Mycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates of Mycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinical M. tuberculosisisolates and compared the results to pncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and no pncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances between pncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated to pncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.
Published: 2012
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37. Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosisInfections

Author: Jure´en, P., A¨ngeby, K., Sturega°rd, E., Chryssanthou, E., Giske, C. G., Werngren, J., Nordvall, M., Johansson, A., Kahlmeter, G., Hoffner, S., and Scho¨n, T.
Abstract: ABSTRACTThe aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.
Published: 2010
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38. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study

Author: Yulia Rodionova, Sabine Rüsch-Gerdes, Paolo Miotto, Jim Werngren, Daiva Bakonyte, Petras Stakenas, Sven Hoffner, Francis Drobniewski, Nicola Casali, Mikael Mansjö, Ewa Augustynowicz-Kopeć, Alessandro Ambrosi, Stefan Niemann, Andrea M. Cabibbe, Silke Feuerriegel, Massimo Degano, Daniela Maria Cirillo, Edita Pimkina, Miotto, P, Cabibbe, Am, Feuerriegel, Casali, N, Drobniewski, F, Rodionova, Y, Bakonyte, D, Stakenas, P, Pimkina, E, Augustynowicz Kopeć, E, Degano, M, Ambrosi, Alessandro, Hoffner, S, Mansjö, M, Werngren, J, Rüsch Gerdes, S, Niemann, S, and Cirillo, D. M.
Subjects: ACCURACY, Antitubercular Agents, SUSCEPTIBILITY, Biology, Microbiology, Amidohydrolases, Mycobacterium tuberculosis, chemistry.chemical_compound, PNCA, Pyrazinoic acid, MIRU-VNTRPLUS, Virology, Drug Resistance, Bacterial, medicine, Humans, Tuberculosis, ASSAY, ANTITUBERCULOSIS DRUGS, Gene, Phylogeny, Genetics, COMPLEX, Phylogenetic tree, MUTATIONS, Genetic Variation, Gold standard (test), Sequence Analysis, DNA, CHEMOTHERAPY, Pyrazinamide, biology.organism_classification, Phenotype, QR1-502, 3. Good health, chemistry, PncA, Mutation, SYSTEM, medicine.drug, Research Article
Abstract: 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 (PZAr). 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 PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr 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 PZAr; 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.
Published: 2014

39. Increased risk of adverse drug reactions by higher linezolid dose per weight in multidrug-resistant tuberculosis.

Author: Kuhlin J, Davies Forsman L, Osman A, Skagerberg M, Jonsson J, Groenheit R, Mansjö M, Werngren J, Alffenaar JW, Schön T, and Bruchfeld J
Subjects: Humans, Female, Male, Retrospective Studies, Adult, Sweden epidemiology, Risk Factors, Young Adult, Drug-Related Side Effects and Adverse Reactions epidemiology, Middle Aged, Anemia chemically induced, Leukopenia chemically induced, Linezolid adverse effects, Linezolid administration & dosage, Tuberculosis, Multidrug-Resistant drug therapy, Antitubercular Agents adverse effects, Antitubercular Agents administration & dosage, Peripheral Nervous System Diseases chemically induced
Abstract: 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.)
Published: 2024
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40. Loss-of-function mutations in ndh do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in Mycobacterium tuberculosis .

Author: Pandey S, Vilchèze C, Werngren J, Bainomugisa A, Mansjö M, Groenheit R, Miotto P, Cirillo DM, Coulter C, Baulard AR, Schön T, Jacobs WR Jr, Djaout K, and Köser CU
Subjects: Humans, Isoniazid pharmacology, Ethionamide pharmacology, Antitubercular Agents pharmacology, Bacterial Proteins genetics, Mutation, Mycobacterium tuberculosis genetics, Tuberculosis, Multidrug-Resistant microbiology
Abstract: 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.
Published: 2024
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41. In vitro activity of new combinations of β-lactam and β-lactamase inhibitors against the Mycobacterium tuberculosis complex.

Author: Economou Lundeberg E, Andersson V, Wijkander M, Groenheit R, Mansjö M, Werngren J, Cortes T, Barilar I, Niemann S, Merker M, Köser CU, and Davies Forsman L
Abstract: 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.
Published: 2023
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42. The Relative Positioning of Genotyping and Phenotyping for Tuberculosis Resistance Screening in Two EU National Reference Laboratories in 2023.

Author: Anthony R, Groenheit R, Mansjö M, de Zwaan R, and Werngren J
Abstract: 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.
Published: 2023
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43. Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution.

Author: Fröberg G, Maurer FP, Chryssanthou E, Fernström L, Benmansour H, Boarbi S, Mengshoel AT, Keller PM, Viveiros M, Machado D, Fitzgibbon MM, Mok S, Werngren J, Cirillo DM, Alcaide F, Hyyryläinen HL, Aubry A, Andres S, Nadarajan D, Svensson E, Turnidge J, Giske CG, Kahlmeter G, Cambau E, van Ingen J, and Schön T
Subjects: Humans, Mycobacterium avium Complex, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Nontuberculous Mycobacteria, Amikacin pharmacology, Moxifloxacin pharmacology, Linezolid pharmacology, Microbial Sensitivity Tests, Drug Resistance, Bacterial, Macrolides pharmacology, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium avium-intracellulare Infection microbiology, Mycobacterium tuberculosis, Mycobacterium Infections, Nontuberculous drug therapy
Abstract: Objective: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints., Methods: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains., Results: The clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges., Conclusion: As a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
Published: 2023
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44. Population Pharmacokinetics and Dose Evaluation of Cycloserine among Patients with Multidrug-Resistant Tuberculosis under Standardized Treatment Regimens.

Author: Zhu Y, Zhu L, Davies Forsman L, Paues J, Werngren J, Niward K, Schön T, Bruchfeld J, Xiong H, Alffenaar JW, and Hu Y
Subjects: Humans, Antitubercular Agents pharmacokinetics, Prospective Studies, Microbial Sensitivity Tests, Cycloserine therapeutic use, Cycloserine pharmacokinetics, Tuberculosis, Multidrug-Resistant drug therapy
Abstract: Although cycloserine is a recommended drug for the treatment of multidrug-resistant tuberculosis (MDR-TB) according to World Health Organization (WHO), few studies have reported on pharmacokinetics (PK) and/or pharmacodynamics (PD) data of cycloserine in patients with standardized MDR-TB treatment. This study aimed to estimate the population PK parameters for cycloserine and to identify clinically relevant PK/PD thresholds, as well as to evaluate the current recommended dosage. Data from a large cohort with full PK curves was used to develop a population PK model. This model was used to estimate drug exposure in patients with MDR-TB from a multicentre prospective study in China. The classification and regression tree was used to identify the clinically relevant PK/PD thresholds. Probability of target attainment was analyzed to evaluate the currently recommended dosing strategy. Cycloserine was best described by a two-compartment disposition model. A percentage of time concentration above MICs (T >MIC ) of 30% and a ratio of area under drug concentration-time curve (AUC 0-24h ) over MIC of 36 were the valid predictors for 6-month sputum culture conversion and final treatment outcome. Simulations showed that with WHO-recommended doses (500 mg and 750 mg for patients weighing <45 kg and ≥45 kg), the probability of target attainment exceeded 90% at MIC ≤16 mg/L in MGIT for both T >MIC of 30% and AUC 0-24h /MIC of 36. New clinically relevant PK/PD thresholds for cycloserine were identified in patients with standardized MDR-TB treatment. WHO-recommended doses were considered adequate for the MGIT MIC distribution in our cohort of Chinese patients with MDR-TB., Competing Interests: The authors declare no conflict of interest.
Published: 2023
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45. Pretomanid-resistant tuberculosis.

Author: Koehler N, Andres S, Merker M, Dreyer V, John A, Kuhns M, Krieger D, Choong E, Verougstraete N, Zur Wiesch PA, Wicha SG, König C, Kalsdorf B, Sanchez Carballo PM, Schaub D, Werngren J, Schön T, Peloquin CA, Schönfeld N, Verstraete AG, Decosterd LA, Aarnoutse R, Niemann S, Maurer FP, and Lange C
Subjects: Humans, Antitubercular Agents pharmacology, Antitubercular Agents therapeutic use, Tuberculosis drug therapy, Nitroimidazoles, Tuberculosis, Multidrug-Resistant drug therapy
Abstract: Competing Interests: Declaration of Competing Interest C.K. received personal fees from INFECTOPHARM outside the scope of this work; C.K. received speakers honoraria from GILEAD and SHIONOGI outside the scope of this work; B.K. received personal fees from INSMED outside the scope of this work; S.N. received grants from German Center for Infection Research, Excellence Cluster Precision Medicine in Chronic Inflammation EXC 2167, and Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG), during the conduct of the study; S.N. received consultation fees from ILLUMINA outside the scope of this work; C.L. received consulting fees from INSMED outside the scope of this work; C.L. received speakers honoraria from INSMED, GILEAD, and JANSSEN outside the scope of this work; C.L. is a participant on Data Safety Boards of Medicines sans Frontiers outside the scope of this work; all other authors have nothing to disclose.
Published: 2023
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46. Pyrazinamide-resistant Tuberculosis Obscured From Common Targeted Molecular Diagnostics.

Author: Modlin SJ, Mansjö M, Werngren J, Ejike CM, Hoffner SE, and Valafar F
Subjects: Humans, Pyrazinamide pharmacology, Pyrazinamide therapeutic use, Antitubercular Agents pharmacology, Antitubercular Agents therapeutic use, Pathology, Molecular, Amidohydrolases genetics, Amidohydrolases therapeutic use, Microbial Sensitivity Tests, Mutation, Mycobacterium tuberculosis genetics, Tuberculosis, Multidrug-Resistant diagnosis, Tuberculosis, Multidrug-Resistant drug therapy, Tuberculosis, Multidrug-Resistant genetics
Abstract: 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.)
Published: 2023
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47. Population pharmacokinetics and model-based dosing evaluation of bedaquiline in multidrug-resistant tuberculosis patients.

Author: Shao G, Bao Z, Davies Forsman L, Paues J, Werngren J, Niward K, Schön T, Bruchfeld J, Alffenaar JW, and Hu Y
Abstract: Aims: Bedaquiline is now recommended to all patients in the treatment of multidrug-resistant tuberculosis (MDR-TB) using standard dosing regimens. As the ability to measure blood drug concentrations is very limited, little is known about drug exposure and treatment outcome. Thus, this study aimed to model the population pharmacokinetics as well as to evaluate the currently recommended dosage. Methodology: A bedaquiline population pharmacokinetic (PK) model was developed based on samples collected from the development cohort before and 1, 2, 3, 4, 5, 6, 8, 12, 18, and 24 h after drug intake on week 2 and week 4 of treatment. In a prospective validation cohort of patients with MDR-TB, treated with bedaquiline-containing standardized regimen, drug exposure was assessed using the developed population PK model and thresholds were identified by relating to 2-month and 6-month sputum culture conversion and final treatment outcome using classification and regression tree analysis. In an exploratory analysis by the probability of target attainment (PTA) analysis, we evaluated the recommended dosage at different MIC levels by Middlebrook 7H11 agar dilution (7H11). Results: Bedaquiline pharmacokinetic data from 55 patients with MDR-TB were best described by a three-compartment model with dual zero-order input. Body weight was a covariate of the clearance and the central volume of distribution, albumin was a covariate of the clearance. In the validation cohort, we enrolled 159 patients with MDR-TB. The 7H11 MIC mode (range) of bedaquiline was 0.06 mg (0.008-0.25 mg/L). The study participants with AUC 0-24h /MIC above 175.5 had a higher probability of culture conversion after 2-month treatment (adjusted relative risk, aRR:16.4; 95%CI: 5.3-50.4). Similarly, those with AUC 0-24h /MIC above 118.2 had a higher probability of culture conversion after 6-month treatment (aRR:20.1; 95%CI: 2.9-139.4), and those with AUC 0-24h /MIC above 74.6 had a higher probability of successful treatment outcome (aRR:9.7; 95%CI: 1.5-64.8). Based on the identified thresholds, simulations showed that the WHO recommended dosage (400 mg once daily for 14 days followed by 200 mg thrice weekly) resulted in PTA >90% for the majority of isolates (94%; MICs ≤0.125 mg/L). Conclusion: We established a population PK model for bedaquiline in patients with MDR-TB in China. Based on the thresholds and MIC distribution derived in a clinical study, the recommended dosage of bedaquiline is sufficient for the treatment of MDR-TB., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EMS declared a past co-authorship with one of the authors JA to the handling Editor., (Copyright © 2023 Shao, Bao, Davies Forsman, Paues, Werngren, Niward, Schön, Bruchfeld, Alffenaar and Hu.)
Published: 2023
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48. Population pharmacokinetics and dose evaluations of linezolid in the treatment of multidrug-resistant tuberculosis.

Author: Zhang H, He Y, Davies Forsman L, Paues J, Werngren J, Niward K, Schön T, Bruchfeld J, Alffenaar JW, and Hu Y
Abstract: Background: The pharmacokinetic/pharmacodynamics (PK/PD) target derived from the hollow-fiber system model for linezolid for treatment of the multidrug-resistant tuberculosis (MDR-TB) requires clinical validation. Therefore, this study aimed to develop a population PK model for linezolid when administered as part of a standardized treatment regimen, to identify the PK/PD threshold associated with successful treatment outcomes and to evaluate currently recommended linezolid doses. Method: This prospective multi-center cohort study of participants with laboratory-confirmed MDR-TB was conducted in five TB designated hospitals. The population PK model for linezolid was built using nonlinear mixed-effects modeling using data from 168 participants. Boosted classification and regression tree analyses (CART) were used to identify the ratio of 0- to 24-h area under the concentration-time curve (AUC 0-24h ) to the minimal inhibitory concentration (MIC) threshold using the BACTEC MGIT 960 method associated with successful treatment outcome and validated in multivariate analysis using data from a different and prospective cohort of 159 participants with MDR-TB. Furthermore, based on the identified thresholds, the recommended doses were evaluated by the probability of target attainment (PTA) analysis. Result: Linezolid plasma concentrations (1008 samples) from 168 subjects treated with linezolid, were best described by a 2-compartment model with first-order absorption and elimination. An AUC 0-24h /MIC > 125 was identified as a threshold for successful treatment outcome. Median time to sputum culture conversion between the group with AUC 0-24h /MIC above and below 125 was 2 versus 24 months; adjusted hazard ratio (aHR), 21.7; 95% confidence interval (CI), (6.4, 72.8). The boosted CART-derived threshold and its relevance to the final treatment outcome was comparable to the previously suggested target of AUC 0-24h /MIC (119) using MGIT MICs in a hollow fiber infection model. Based on the threshold from the present study, at a standard linezolid dose of 600 mg daily, PTA was simulated to achieve 100% at MGIT MICs of ≤ .25 mg which included the majority (81.1%) of isolates in the study. Conclusion: We validated an AUC 0-24h /MIC threshold which may serve as a target for dose adjustment to improve efficacy of linezolid in a bedaquiline-containing treatment. Linezolid exposures with the WHO-recommended dose (600 mg daily) was sufficient for all the M. tb isolates with MIC ≤ .25 mg/L., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, He, Davies Forsman, Paues, Werngren, Niward, Schön, Bruchfeld, Alffenaar and Hu.)
Published: 2023
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49. The ddn Trp20Stop Mutation and Its Association with Lineage 4.5 and Resistance to Delamanid and Pretomanid in Mycobacterium tuberculosis.

Author: Mansjö M, Karlsson Lindsjö O, Grönfors Seeth C, Groenheit R, and Werngren J
Subjects: Humans, Antitubercular Agents pharmacology, Antitubercular Agents therapeutic use, Microbial Sensitivity Tests, Mutation genetics, Drug Resistance, Multiple, Bacterial genetics, Mycobacterium tuberculosis, Nitroimidazoles pharmacology, Tuberculosis, Multidrug-Resistant drug therapy
Abstract: 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.
Published: 2022
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50. Distribution of Common and Rare Genetic Markers of Second-Line-Injectable-Drug Resistance in Mycobacterium tuberculosis Revealed by a Genome-Wide Association Study.

Author: Conkle-Gutierrez D, Kim C, Ramirez-Busby SM, Modlin SJ, Mansjö M, Werngren J, Rigouts L, Hoffner SE, and Valafar F
Subjects: Amikacin pharmacology, Antitubercular Agents pharmacology, Capreomycin pharmacology, Genetic Markers, Genome-Wide Association Study, Kanamycin pharmacology, Microbial Sensitivity Tests, Mutation, Drug Resistance, Multiple, Bacterial genetics, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis genetics
Abstract: 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.
Published: 2022
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