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9 hojas, 4 figuras, 1 tabla. Demographic data of the participants are available in the appendix (pp 40–42). Genome sequences are publicly available on GISAID. All scripts used for the analysis, and beta and delta subtree files are publicly available at https://gitlab.com/tbgenomicsunit/mozcovid. The study protocol (appendix pp 1–35) and clinical questionnaire (appendix pp 36–39) will be available with publication, Background: From the start of the SARS-CoV-2 outbreak, global sequencing efforts have generated an unprecedented amount of genomic data. Nonetheless, unequal sampling between high-income and low-income countries hinders the implementation of genomic surveillance systems at the global and local level. Filling the knowledge gaps of genomic information and understanding pandemic dynamics in low-income countries is essential for public health decision making and to prepare for future pandemics. In this context, we aimed to discover the timing and origin of SARS-CoV-2 variant introductions in Mozambique, taking advantage of pandemic-scale phylogenies. Methods: We did a retrospective, observational study in southern Mozambique. Patients from Manhiça presenting with respiratory symptoms were recruited, and those enrolled in clinical trials were excluded. Data were included from three sources: (1) a prospective hospital-based surveillance study (MozCOVID), recruiting patients living in Manhiça, attending the Manhiça district hospital, and fulfilling the criteria of suspected COVID-19 case according to WHO; (2) symptomatic and asymptomatic individuals with SARS-CoV-2 infection recruited by the National Surveillance system; and (3) sequences from SARS-CoV-2-infected Mozambican cases deposited on the Global Initiative on Sharing Avian Influenza Data database. Positive samples amenable for sequencing were analysed. We used Ultrafast Sample placement on Existing tRees to understand the dynamics of beta and delta waves, using available genomic data. This tool can reconstruct a phylogeny with millions of sequences by efficient sample placement in a tree. We reconstructed a phylogeny (~7·6 million sequences) adding new and publicly available beta and delta sequences. Findings: A total of 5793 patients were recruited between Nov 1, 2020, and Aug 31, 2021. During this time, 133 328 COVID-19 cases were reported in Mozambique. 280 good quality new SARS-CoV-2 sequences were obtained after the inclusion criteria were applied and an additional 652 beta (B.1.351) and delta (B.1.617.2) public sequences were included from Mozambique. We evaluated 373 beta and 559 delta sequences. We identified 187 beta introductions (including 295 sequences), divided in 42 transmission groups and 145 unique introductions, mostly from South Africa, between August, 2020 and July, 2021. For delta, we identified 220 introductions (including 494 sequences), with 49 transmission groups and 171 unique introductions, mostly from the UK, India, and South Africa, between April and November, 2021. Interpretation: The timing and origin of introductions suggests that movement restrictions effectively avoided introductions from non-African countries, but not from surrounding countries. Our results raise questions about the imbalance between the consequences of restrictions and health benefits. This new understanding of pandemic dynamics in Mozambique can be used to inform public health interventions to control the spread of new variants. Funding: European and Developing Countries Clinical Trials, European Research Council, Bill & Melinda Gates Foundation, and Agència de Gestió d'Ajuts Universitaris i de Recerca., This publication was produced by MozCovid which is part of the EDCTP2 programme supported by the EU (grant number RIA2020EF-3005-MozCOVID). The COVID-19 testing was supported by Emory Global Health Institute, University of Emory, through the CHAMPS Program funded by the Bill & Melinda Gates Foundation (under the grant OPP1126780 to Robert Breiman, subcontract SC00003286). This work was also supported by the European Research Council under the EU’s Horizon 2020 Research and Innovation Program grant (101001038; TB-RECONNECT), the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global), the Departament d’Universitats i Recerca de la Generalitat de Catalunya (AGAUR; 2021 SGR 01517), and the Ministerio de Ciencia e Innovación (Spanish Government) Project PID2019–104477RB-I00. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development. ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya (http://cerca.cat/en/suma/). We also acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the Centro de Excelencia Severo Ochoa 2019–2023 Program (CEX2018–000806-S).

Mozambique is considered a high multidrug-resistant (MDR-TB) country by the World Health Organization (WHO) (3.7% new MDR-TB cases). The last national WHO survey was performed in 2008. Nowadays, South Africa and Eswatini are reporting an increase in MDR-TB cases heightened, in Eswatini, by the spread of a MDR-TB strain harbouring therpoB_I491F mutation not detected by XpertUltra/RIF. Due to the concerning MDR-TB situation in its neighbouring countries, it is crucial to review the current resistance status in Mozambique. Our aim is to depict the current prevalence of drug-resistance to first and second-line drugs and track its trends since 2008. We analysed 312Mycobacterium tuberculosisgenomes from a population-based survey conducted in Manhiça in 2018 and looked for drug-resistance mutations included in the recently published WHO catalogue and compared with the prevalences reported in 2014 and 2008. We found that the overall resistance increased in 2014 (15.9%) and 2018 (14.4%) was slightly higher than the one reported in 2008. Although, new multidrug/rifampicin resistance cases remained consistent with the 2008 prevalence study (3.5%) indicating that MDR-TB is not spreading as rapidly as in neighbouring countries. Importantly, we detected a high isoniazid (INH) prevalence not associated with MDR-TB (4.2%, 5.5% and 7.6% in 2008, 2014 and 2018, p-value=0.03) suggesting that a sizeable number of cases are INH-resistant before starting treatment and not detected by XpertUltra/RIF. Fortunately, no mutations associated with second-line drug-resistance were found in the dataset. Our results show a stable drug-resistance situation in Manhiça with the need to monitor isoniazid resistance, and highlight the potential of WGS to be used in national surveys to expand our knowledge of drug-resistance prevalence throughout all Mozambican provinces.

15 páginas, 5 figuras, 1 tabla, Outbreak strains of Mycobacterium tuberculosis are promising candidates as targets in the search for intrinsic determinants of transmissibility, as they are responsible for many cases with sustained transmission; however, the use of low-resolution typing methods and restricted geographical investigations represent flaws in assessing the success of long-lived outbreak strains. We can now address the nature of outbreak strains by combining large genomic data sets and phylodynamic approaches. We retrospectively sequenced the whole genome of representative samples assigned to an outbreak circulating in the Canary Islands (the GC strain) since 1993, which accounts for ~20% of local tuberculosis cases. We selected a panel of specific single nucleotide polymorphism (SNP) markers for an in-silico search for additional outbreak-related sequences within publicly available tuberculosis genomic data. Using this information, we inferred the origin, spread, and epidemiological parameters of the GC strain. Our approach allowed us to accurately trace the historical and more recent dispersion of the GC strain. We provide evidence of a highly successful nature within the Canarian archipelago but limited expansion abroad. Estimation of epidemiological parameters from genomic data disagree with a distinctive biology of the GC strain. With the increasing availability of genomic data allowing for the accurate inference of strain spread and critical epidemiological parameters, we can now revisit the link between Mycobacterium tuberculosis genotypes and transmission, as is routinely carried out for SARS-CoV-2 variants of concern. We demonstrate that social determinants rather than intrinsically higher bacterial transmissibility better explain the success of the GC strain. Importantly, our approach can be used to trace and characterize strains of interest worldwide. IMPORTANCE Infectious disease outbreaks represent a significant problem for public health. Tracing outbreak expansion and understanding the main factors behind emergence and persistence remain critical to effective disease control. Our study allows researchers and public health authorities to use Whole-Genome Sequencing-based methods to trace outbreaks, and shows how available epidemiological information helps to evaluate the factors underpinning outbreak persistence. Taking advantage of all the freely available information placed in public repositories, researchers can accurately establish the expansion of an outbreak beyond original boundaries, and determine the potential risk of a strain to inform health authorities which, in turn, can define target strategies to mitigate expansion and persistence. Finally, we show the need to evaluate strain transmissibility in different geographic contexts to unequivocally associate spread to local or pathogenic factors, an important lesson taken from genomic surveillance of SARS-CoV-2., This project has been funded by the European Research Council (101001038-TBRECONNECT), the Ministerio de Economía, Industria y Competitividad (PID2019-104477RB-I00), and the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global) to I.C. This project has been funded by the Instituto de Salud Carlos III (FIS18/0336) and the Gobierno de Aragón/Fondo Social Europeo “Construyendo Europa desde Aragón” to SS

M. brumaeis a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain.M. brumaeis not pathogenic and its in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of theM. brumaetype strain ATCC 51384T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains.M. brumaegenome is composed of a circular chromosome with a high GC content of 69.2 % and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci.M. brumaehas shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although immunogenic potential ofM. brumaewas proved to be as high asMycobacterium bovisBCG, the only mycobacteria licensed to treat cancer, the genomic content ofM. tuberculosisT cell and B cell antigens inM. brumaeis considerably lower than those ofM. bovisBCG. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.

Genomic studies of the

Africa accounts for 1.5% of the global coronavirus disease 2019 (COVID-19) cases and 2.7% of deaths, but this low incidence has been partly attributed to the limited testing capacity in most countries. In addition, the population in many African countries is at high risk of infection with endemic infectious diseases such as malaria. Our aim is to determine the prevalence and circulation of SARS-CoV-2 variants, and the frequency of co-infection with the malaria parasite. We conducted serological tests and microscopy examinations on 998 volunteers of different ages and sexes in a random and stratified population sample in Burkina-Faso. In addition, nasopharyngeal samples were taken for RT-qPCR of SARS-COV-2 and for whole viral genome sequencing. Our results show a 3.2% and a 2.5% of SARS-CoV-2 seroprevalence and PCR positivity; and 22% of malaria incidence, over the sampling period, with marked differences linked to age. Importantly, we found 2 cases of confirmed co-infection and 8 cases of suspected co-infection mostly in children and teenagers. Finally, we report the genome sequences of 13 SARS-CoV-2 isolates circulating in Burkina Faso at the time of analysis, assigned to lineages: A.19, A.21, B.1.1.404, B.1.1.118, B.1 and grouped into clades; 19B, 20A and 20B. This is the first population-based study about SARS-CoV-2 and malaria in Burkina Faso during the first wave of the pandemic, providing a relevant estimation of the real prevalence of SARS-CoV-2 and variants circulating in this Sub-Saharan African country. Besides, it highlights the low frequency of co-infection with malaria in African communities., This research work received funding from by the European Commission NextGenerationEU (Regulation EU 2020/2094) and grant 202020E159 through CSIC Global Health Platform (PTI Salud Global).

Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application. Here, we generate a population-based dataset from Valencia region and compare it with available datasets from different TB-burden settings to reveal transmission dynamics heterogeneity and its public health implications. We sequenced the whole genome of 785

12 páginas, 4 figuras, 1 tabla, Efforts to eradicate tuberculosis are hampered by the rise and spread of antibiotic resistance. Several large-scale projects have aimed to specifically link clinical mutations to resistance phenotypes, but they were limited in both their explanatory and predictive powers. Here, we combine functional genomics and phylogenetic associations using clinical strain genomes to decipher the architecture of isoniazid resistance and search for new resistance determinants. This approach has allowed us to confirm the main target route of the antibiotic, determine the clinical relevance of redox metabolism as an isoniazid resistance mechanism and identify novel candidate genes harboring resistance mutations in strains with previously unexplained isoniazid resistance. This approach can be useful for characterizing how the tuberculosis bacilli acquire resistance to new antibiotics and how to forestall them., This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programs 638553 (TB-ACCELERATE), SAF2016-77346-R from Ministerio de Economía y Competitividad (Spanish Government) and AICO/2018/113 from Generalitat Valenciana (to I.C.). M.M.-M. is recipient of a FPI grant from the Ministerio de Economía y Competitividad (Spanish Government, code BES-2017-079656). V.F. was recipient of a post-doctoral research grant from the Ministerio de Economía y Competitividad (Spanish Government, code FPDI-2013-18757). Action cofinanced by the European Union through the Operational Program of European Regional Development Fund (ERDF) of Valencia Region (Spain) 2014-2020.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants., This work was funded by the Instituto de Salud Carlos III project COV20/00140 and COV20/00437, Spanish National Research Council project CSIC-COV19-021 and CSIC-COVID19-082, and the Generalitat Valenciana (SEJI/2019/011 and Covid_19-SCI). Action co-financed by the European Union through the Operational Program of the European Regional Development Fund (ERDF) of the Valencian Community 2014-2020. M.C. and R.G. are supported by Ramon y Cajal program from Ministerio de Ciencia.

11 páginas, 5 figuras, 1 tabla, Polyclonal infections occur when at least two unrelated strains of the same pathogen are detected in an individual. This has been linked to worse clinical outcomes in tuberculosis, as undetected strains with different antibiotic resistance profiles can lead to treatment failure. Here, we examine the amount of polyclonal infections in sputum and surgical resections from patients with tuberculosis in the country of Georgia. For this purpose, we sequence and analyse the genomes of Mycobacterium tuberculosis isolated from the samples, acquired through an observational clinical study (NCT02715271). Access to the lung enhanced the detection of multiple strains (40% of surgery cases) as opposed to just using a sputum sample (0-5% in the general population). We show that polyclonal infections often involve genetically distant strains and can be associated with reversion of the patient's drug susceptibility profile over time. In addition, we find different patterns of genetic diversity within lesions and across patients, including mutational signatures known to be associated with oxidative damage; this suggests that reactive oxygen species may be acting as a selective pressure in the granuloma environment. Our results support the idea that the magnitude of polyclonal infections in high-burden tuberculosis settings is underestimated when only testing sputum samples., The authors were supported by projects SAF2016-77346-R and PID2019-104477RB-I00 awarded to IC and the grant BES-2017-079656 awarded to MM by the Spanish Ministry of Economy and Competitiveness and Ministry of Science, the ERC project 638553-TBACCELERATE awarded to IC, Spanish Government-FEDER Funds through CV contract CPII18/00031 and grant PI16/01511, and Generalitat Valencia Grant to I.C. (code PROMETEO/2020/012). The grant providers played no part in study design, data collection, and analysis, or the preparation of the manuscript.

The COVID-19 pandemic has shaken the world since the beginning of 2020. Spain is among the European countries with the highest incidence of the disease during the first pandemic wave. We established a multidisciplinar consortium to monitor and study the evolution of the epidemic, with the aim of contributing to decision making and stopping rapid spreading across the country. We present the results for 2170 sequences from the first wave of the SARS-Cov-2 epidemic in Spain and representing 12% of diagnosed cases until 14th March. This effort allows us to document at least 500 initial introductions, between early February-March from multiple international sources. Importantly, we document the early raise of two dominant genetic variants in Spain (Spanish Epidemic Clades), named SEC7 and SEC8, likely amplified by superspreading events. In sharp contrast to other non-Asian countries those two variants were closely related to the initial variants of SARS-CoV-2 described in Asia and represented 40% of the genome sequences analyzed. The two dominant SECs were widely spread across the country compared to other genetic variants with SEC8 reaching a 60% prevalence just before the lockdown. Employing Bayesian phylodynamic analysis, we inferred a reduction in the effective reproductive number of these two SECs from around 2.5 to below 0.5 after the implementation of strict public-health interventions in mid March. The effects of lockdown on the genetic variants of the virus are reflected in the general replacement of preexisting SECs by a new variant at the beginning of the summer season. Our results reveal a significant difference in the genetic makeup of the epidemic in Spain and support the effectiveness of lockdown measures in controlling virus spread even for the most successful genetic variants. Finally, earlier control of SEC7 and particularly SEC8 might have reduced the incidence and impact of COVID-19 in our country., This work was funded by the Instituto de Salud Carlos III project COV20/00140, Spanish 593 National Research Council project CSIC-COV19-021 and ERC StG 638553 to IC, and BFU2017- 594 89594R to FGC. MC is supported by Ramón y Cajal program from Ministerio de Ciencia and 595 grants RTI2018-094399-A-I00 and SEJI/2019/011. 596 We gratefully acknowledge Hospital Universitari Vall d'Hebron, Instituto de Salud Carlos 597 III, IrsiCaixa AIDS Research Lab and all the international researchers and institutions that 598 submitted sequenced SARS-CoV-2 genomes to the GISAID’s EpiCov™ Database, as an 599 important part of our analyses have been made possible by the share of their work.

10 páginas, 3 figuras, 3 tablas. Material suplementario en: https://advances.sciencemag.org/content/suppl/2020/02/10/6.7.eaay4453.DC1, The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution., This work was supported by Plan Nacional de I + D + i 2013–2016 and the Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—cofinanced by the European Development Regional Fund “A Way to Achieve Europe”—by Operative Program Intelligent Growth 2014-2020 and grants FIS PI17/00159 from the Instituto de Salud Carlos III and SAF2015-72793-EXP from the Spanish Ministry of Science and Competitiveness (MINECO)-FEDER. E.C.-S. was the recipient of a PFIS predoctoral research fellowship (FI18/00036) cofinanced by the Instituto de Salud Carlos III and the European Social Fund. I.C., A.C.-O., and M.T.-P. are funded by projects of the 581 European Research Council (ERC) (638553-TB-ACCELERATE) and Ministerio de Economía y Competitividad research grant SAF2016-77346-R.

9 páginas, 3 figuras, Background Direct whole-genome sequencing of Mycobacterium tuberculosis from clinical specimens will be a major breakthrough in tuberculosis diagnosis and control. To date, direct whole-genome sequencing has never been used in genomic epidemiology, and its accuracy in transmission inference remains unknown. We investigated the technical challenges imposed by direct whole-genome sequencing, and used it to infer transmission clusters and predict drug resistance. Methods Using an optimised workflow, we did direct whole-genome sequencing for 37 clinical specimens from 23 tuberculosis patients. Nine sputum samples from nine patients who were infected with different non-tuberculous mycobacteria and culture-negative for tuberculosis were used as controls in the qPCR assays and pre-sequencing runs. Additionally, 780 clinical isolates in the region of Comunidad Valenciana (Spain) were whole-genome sequenced between Jan 1, 2014, and Dec 31, 2016. We analysed the genomic variants to build a tuberculosis transmission network for the region, including the clinical specimens, and to predict drug susceptibility profiles. Findings After sequencing 37 clinical specimens, 28 specimens (22 [85%] of 26 smear-positive and six [55%] of 11 smear-negative) met the quality criteria for downstream analysis. All 28 clinical specimens clustered with their matching culture isolates, with a median distance of 0 single nucleotide polymorphisms. Of the 28 clinical specimens, 16 (57%) were accurately assigned to ten transmission clusters in the region, and 12 (43%) were unique cases. Transmission inferences and drug-susceptibility predictions from direct whole-genome sequencing data were concordant with sequences from corresponding cultures and phenotypic drug-susceptibility testing. Complete genomic analysis, within a week of specimen receipt, cost €217 per sample (excluding personnel costs). Interpretation Direct whole-genome sequencing could be used to accurately delineate transmission clusters of tuberculosis and conduct culture-independent surveillance. Compared with conventional approaches, direct wholegenome sequencing allows researchers to do real-time genomic epidemiology and drug resistance surveillance in settings where culture and drug susceptibility testing are not available., This work was supported by projects of the European Research Council (638553-TB-ACCELERATE), Ministerio de Economía y Competitividad, and Ministerio de Ciencia, Innovación y Universidades (Spanish Government; SAF2013-43521-R, SAF2016-77346-R, and SAF2017-92345-EXP [to IC], and BES-2014-071066 [to GAG])

Mixed cytomegalovirus (CMV) infections are associated with delayed viral clearance in solid organ transplant recipients. We investigated whether this could be extrapolated to allogeneic stem cell transplant (allo-SCT) recipients. A total of 48 plasma specimens, obtained during 29 episodes of active CMV infection in 25 non-consecutive allo-SCT patients, were analysed. Baseline blood specimens, drawn shortly prior to the inception of pre-emptive antiviral therapy (pre-treatment specimen; n=29), as well as follow-up samples obtained either after the initiation of antiviral therapy (post-treatment specimen; n=15) or during recurrent episodes (n=4) were analysed. Plasma CMV DNA loads were quantified by real-time PCR and the CMV genotyping was performed by ultra-deep sequencing of hypervariable regions in the genes coding for glycoproteins N (gN) and O (gO). A trend towards higher CMV DNA peak loads, longer CMV DNAemia episode durations and slower CMV DNAemia decay rates was observed for episodes with mixed CMV genotype populations compared to those caused by single CMV variants, although the differences did not reach statistical significance. The length of the treatment course required to clear DNAemia was significantly longer in these mixed episodes (P=0.002). Significant changes in the number or frequency of CMV gN or gO genetic variants were documented following the initiation of antiviral therapy or in recurrent episodes. CMV diversity may have a major impact on the kinetics of CMV DNAemia clearance during the treatment of active CMV infection episodes in allo-SCT recipients.

5 páginas, 2 figuras. AVAILABILITY AND IMPLEMENTATION: The database of non-tuberculous mycobacteria assemblies can be accessed at: 10.5281/zenodo.3374377. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online: http://dx.doi.org/10.1093/bioinformatics/btz729, MOTIVATION: Tuberculosis (TB) remains one of the main causes of death worldwide. The long and cumbersome process of culturing Mycobacterium tuberculosis complex (MTBC) bacteria has encouraged the development of specific molecular tools for detecting the pathogen. Most of these tools aim to become novel TB diagnostics, and big efforts and resources are invested in their development, looking for the endorsement of the main public health agencies. Surprisingly, no study has been conducted where the vast amount of genomic data available is used to identify the best MTBC diagnostic markers. RESULTS: In this work, we used large-scale comparative genomics to identify 40 MTBC-specific loci. We assessed their genetic diversity and physiological features to select 30 that are good targets for diagnostic purposes. Some of these markers could be used to assess the physiological status of the bacilli. Remarkably, none of the most used MTBC markers is in our catalog. Illustrating the translational potential of our work, we develop a specific qPCR assay for quantification and identification of MTBC DNA. Our rational design of targeted molecular assays for TB could be used in many other fields of clinical and basic research., This work was supported by projects of the European Research Council (ERC) (638553-TB-ACCELERATE), Ministerio de Economía y Competitividad and Ministerio de Ciencia, Innovación y Universidades (Spanish Government), SAF2013-43521-R, SAF2016-77346-R and SAF2017–92345–EXP (to I.C.), BES-2014-071066 (to G.A.G.), FPU 13/00913 (to A.C.O.).

12 páginas, 6 figuras, 4 tablas, The Mycobacterium tuberculosis complex (MTBC) comprises the species that causes tuberculosis (TB) which affects 10 million people every year. A robust classification of species, lineages, and sub-lineages is important to explore associations with drug resistance, epidemiological patterns or clinical outcomes. We present a rapid and easy-to-follow methodology to classify clinical TB samples into the main MTBC clades. Approaches are based on the identification of lineage and sub-lineage diagnostic SNP using a real-time PCR high resolution melting assay and classic Sanger sequencing from low-concentrated, low quality DNA. Thus, suitable for implementation in middle and low-income countries. Once we validated our molecular procedures, we characterized a total of 491 biological samples from human and cattle hosts, representing countries with different TB burden. Overall, we managed to genotype ~95% of all samples despite coming from unpurified and low-concentrated DNA. Our approach also allowed us to detect zoonotic cases in eight human samples from Nigeria. To conclude, the molecular techniques we have developed, are accurate, discriminative and reproducible. Furthermore, it costs less than other classic typing methods, resulting in an affordable alternative method in TB laboratories., This work was supported by European Research Council (grant, 638553-TB-ACCELERATE) and the Ministerio de Economía y Competitividad grants (SAF2013-43521-R and SAF2016-77346-R) both to I.C.

5 páginas, 2 figuras, Understanding Multidrug-resistant tuberculosis cases not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostics and advance towards personalized TB treatments. Here, we combine WGS with single-colony phenotyping testing to diagnose a MDR strain infecting a patient over nine years. Our investigation revealed the failure of rapid testing assays and genome-based prediction tools to identify the MDR strain. The false negative outcome was caused by uncommon RIF and INH resistance mutations. Despite WGS data helped to personalize treatment, the patient developed XDR-TB, highlighting the importance of coupling new diagnostic methods with appropriate treatment regimens., This work was supported by the European Research Council (638553-TB-ACCELERATE to I. C.) and the Ministerio de Economía y Competitividad (SAF2016-77346-R to I. C.).

Artículo con 20 páginas, 5 figuras, 1 tabla. All the sequence data are deposited in the European Nucleotide Archive under the Bioproject number PRJEB29604 (https://www.ebi.ac.uk/ena/data/view/PRJEB29604) and the accession numbers ERR2099780 (https://www.ebi.ac.uk/ena/data/search?query=ERR2099780) and ERR2099784 (https://www.ebi.ac.uk/ena/data/search?query=ERR2099784)., BACKGROUND: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened. METHODS AND FINDINGS: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region. CONCLUSIONS: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control., IC was supported by European Research Council (638553-TB-ACCELERATE), the Ministerio de Economía y Competitividad (SAF2016-77346-R). CC and YX were supported by the Engineering and Physical Sciences Research Council of the UK (EPSRC EP/K026003/1 (CC) and EPSRC EP/N014529/1 (CC and YX).

Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. In this study, we have employed a large data set of sequences (14,654 sequences from between 25 and 100 clone sequences per analyzed region and per patient) from 67 patients infected with HCV genotype 1 (23 subtype 1a and 44 subtype 1b). For all patients, a sample prior to combined therapy with alpha interferon plus ribavirin was available, whereas for some patients additional samples after 6 or 12 months of treatment were also available. Twenty-seven patients responded to treatment (12 subtype 1a and 15 subtype 1b) and forty patients did not respond to treatment (11 subtype 1a vs. 29 subtype 1b). Two regions of the HCV genome were analyzed, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the envelope 2 glycoprotein and another one including the interferon sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Previously (Cuevas, J.M., Torres-Puente, M., Jiménez-Hernández, N., Bracho, M.A., García-Robles, I., Wrobel, B., Carnicer, F., del Olmo, J., Ortega, E., Moya, A., González-Candelas, F., 2008b. Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin. Plos One 3 (8), e3058), several amino acid positions in both regions analyzed were detected to be under positive selection. Here, we have compared the amino acid composition of each positively selected position between responder and non-responder patients for both subtypes. If we exclude some non-conclusive cases, no clear differences were detected in any case. In conclusion, identifying specific positions as completely discriminatory of treatment response seems to be a difficult task. Our results, in concordance with previous studies, suggest that HCV evasion strategies are more likely based on a global increased variability, which would yield combinations of mutations with an increased resistance, than on the fixation of specific amino acids conferring resistance to antiviral treatment or immune response. In this sense, the particular systemic response from each patient could play an essential role in determining the outcome of the antiviral treatment.

The envelope 2 protein of hepatitis C virus (HCV) presents three hypervariable regions, named HVR1, HVR2 and HVR3, in which the presence of antigenic sites has been described. Genetic variability in these regions may reflect the generation of escape mutants as a consequence of the immune response. Therefore, these regions would tend to accumulate amino acid changes along the infection process, an effect that could be accelerated by antiviral treatments. In this study, we have analyzed the E1–E2 region of 23 HCV patients non-responders to antiviral treatment, 7 of which were infected with subtype 1a, 15 with subtype 1b, and 1 with a new HCV-1 subtype, before and after 6 and/or 12 months of peg-interferon + ribavirin treatment. We have sequenced about 100 clones from each sample, analyzing a total of 4906 sequences. A detailed analysis of the evolutionary forces acting along the genome region studied confirmed the existence of the three hypervariable regions, characterized by significant changes in amino acid composition between samples taken at different times from the same patient and a high number of sites evolving under positive selection. Moreover, for the recently described HVR3, our results suggest that its location could be restricted to residues 434–450, instead of the originally postulated 431–466.

Summary. Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. The high genetic variability of HCV contributes to the chronicity of hepatitis C. Here, we report results from a large-scale sequence analysis of 67 patients infected with HCV genotype 1, 23 with subtype 1a and 44 with subtype 1b. Two regions of the HCV genome were analysed in samples prior to combined therapy with alpha interferon plus ribavirin, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the E2 glycoprotein and another one including the interferon-sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Genetic diversity measures showed a clear tendency to higher genetic variability levels in nonresponder patients to antiviral treatment than in responder patients, although highly disperse values were present within each response group for both subtypes. A more detailed analysis of amino acid composition revealed the presence of several subtype-specific variants in a few positions, but no discriminating positions between responder and nonresponder patients were detected. Our results also revealed that most amino acid positions were highly conserved, especially for subtype 1a. We conclude that the outcome of the antiviral treatment might depend not only on the nature of one or a few independent positions, but more likely on the combination of several positions along the HCV genome. Moreover, the own host’s ability to generate an appropriate systemic response, in combination with the action of antivirals, is also likely to be essential for treatment outcome.

Little is known about the potential effects of insertions and deletions (indels) on the evolutionary dynamics of hepatitis C virus (HCV). In fact, the consequences of indels on antiviral treatment response are a field of investigation completely unexplored. Here, an extensive sequencing project was undertaken by cloning and sequencing serum samples from 25 patients infected with HCV subtype 1a and 48 patients with subtype 1b. For 23 patients, samples obtained after treatment with alpha interferon plus ribavirin were also available. Two genome fragments containing the hypervariable regions in the envelope 2 glycoprotein and the PKR-BD domain in NS5A were sequenced, yielding almost 16 000 sequences. Our results show that insertions are quite rare, but they are often present in biologically relevant domains of the HCV genome. Moreover, their frequency distributions between different time samples reflect the quasispecies dynamics of HCV populations. Deletions seem to be subject to negative selection.

The assumption of a molecular clock for dating events from sequence information is often frustrated by the presence of heterogeneity among evolutionary rates due, among other factors, to positively selected sites. In this work, our goal is to explore methods to estimate infection dates from sequence analysis. One such method, based on site stripping for clock detection, was proposed to unravel the clocklike molecular evolution in sequences showing high variability of evolutionary rates and in the presence of positive selection. Other alternatives imply accommodating heterogeneity in evolutionary rates at various levels, without eliminating any information from the data. Here we present the analysis of a data set of hepatitis C virus (HCV) sequences from 24 patients infected by a single individual with known dates of infection. We first used a simple criterion of relative substitution rate for site removal prior to a regression analysis. Time was regressed on maximum likelihood pairwise evolutionary distances between the sequences sampled from the source individual and infected patients. We show that it is indeed the fastest evolving sites that disturb the molecular clock and that these sites correspond to positively selected codons. The high computational efficiency of the regression analysis allowed us to compare the site-stripping scheme with random removal of sites. We demonstrate that removing the fast-evolving sites significantly increases the accuracy of estimation of infection times based on a single substitution rate. However, the time-of-infection estimations improved substantially when a more sophisticated and computationally demanding Bayesian method was used. This method was used with the same data set but keeping all the sequence positions in the analysis. Consequently, despite the distortion introduced by positive selection on evolutionary rates, it is possible to obtain quite accurate estimates of infection dates, a result of especial relevance for molecular epidemiology studies.

Hepatitis C virus (HCV) presents several regions involved potentially in evading antiviral treatment and host immune system. Two regions, known as PKR-BD and V3 domains, have been proposed to be involved in resistance to interferon. Additionally, hypervariable regions in the envelope E2 glycoprotein are also good candidates to participate in evasion from the immune system. In this study, we have used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples obtained just before initiation of therapy and after 6 or/and 12 months of treatment were available. A range of 25-100 clones per patient, genome region and time sample were obtained. The predominant amino acid sequences for each time sample and patient were determined. Next, the sequences of the PKR-BD and V3 domains and the hypervariable regions from different time samples were compared for each patient. The highest levels of variability were detected at the three hypervariable regions of the E2 protein and,to a lower extent, at the V3 domain of the NS5A protein. However, no clear patterns of adaptation to the host immune system or to antiviral treatment were detected. In summary, although high levels of variability are correlated to viral adaptive response, antiviral treatment does not seem to promote convergent adaptive changes. Consequently, other regions must be involved in evasion strategies likely based on a combination of multiple mechanisms, in which pools of changes along the HCV genome could confer viruses the ability to overcome strong selective pressures. J. Med. Virol. 81:650656, 2009. (C) 2009 Wiley-Liss, Inc.

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective pressures. cuevast@uv.es; mtorresp@uv.es; njimenez@uv.es; bracho@uv.es; garciai@uv.es; wrobel@uv.es; moya@uv.es; gonzalef@uv.es

Summary. Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres-Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large-scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha-interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region.

The aim of this study was to analyze proliferative verrucous leukoplakia (PVL) and oral squamous cell carcinoma (OSCC) for the possible presence of Epstein-Barr virus (EBV). We studied three groups: Sub-Group 1 was composed of 10 patients with PVL, (6 of whom had developed OSCC); Sub-Group 2 comprised 5 patients with OSCC but no preceding PVL; and Sub-Group 3 were 5 controls with clinically normal oral mucosa. Oral biopsies from all cases were examined for Epstein-Barr virus (EBV) by nested PCR. EBV was detected in 60% of Sub-Group 1 patients (PVL ) and in 40% of Sub-Group 2 (OSCC), but in 0% of Sub-Group 3 (controls).

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare in vivo. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naive), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially relevant mechanism generating genetic variation in HCV and with important implications for the treatment of this infection.

The degree of variability of the interferon sensitivity determining region (ISDR) in the hepatitis C virus (HCV) genome has been postulated to predict the response to interferon therapy, mainly in patients infected with subtype 1b, although this prediction has been the subject of a long controversy. This prediction has been tested by analyzing a cohort of 67 Spanish patients infected with HCV genotype 1, 23 of which were infected with subtype 1a and 44 with subtype 1b. A sample previous to therapy with α-interferon plus ribavirin was obtained and several clones (between 25 and 96) including the ISDR were sequenced from each patient. A significant correlation between mutations at the ISDR and response to treatment for subtype 1b patients, but not for those infected with subtype 1a, has been detected. Although the results suggest that the same relationship holds true for subtype 1a, lack of statistical power because of the small sample size of this subtype prevented firmer conclusions. However, identical ISDR sequences were found in responder and non-responder patients, suggesting that the stability of the ISDR sequence can occasionally help HCV to evade interferon therapy, although this is not a sufficient condition. More complex interactions, including the ISDR or not, are likely to exist and govern the HCV response to interferon treatment. J. Med. Virol. 80:247–253, 2008. © 2007 Wiley-Liss, Inc.

We have conducted a large sequence study of the E1–E2 and NS5A regions of the HCV, subtypes 1a and b, both in patients previously treated with interferon, and untreated patients, who later responded, or not, to a combination therapy based on interferon plus ribavirin. We have examined the role played by the number of positively selected sites on disease progression and its relationship with several variables such as patients’ age, sex and their risk of acquiring the disease. We have detected three groups of patients that respond or not to combination therapy: responders of intermediate age, older non-responders and young non-responders, they possess an increasing average number of positively selected sites in the E1–E2 region, respectively. We conclude that the host's genetic factors play an important role in whether the disease is contained or becomes chronic.

Hepatitis C virus (HCV) infection affects about 3 % of the human population. Phylogenetic analyses have grouped its variants into six major genotypes, which have a star-like distribution and several minor subtypes. The most abundant genotype in Europe is the so-called genotype 1, with two prevalent subtypes, 1a and 1b. In order to explain the higher prevalence of subtype 1b over 1a, a large-scale sequence analysis (100 virus clones) has been carried out over 25 patients of both subtypes in two regions of the HCV genome: one comprising hypervariable region 1 and another including the interferon sensitivity-determining region. Neither polymorphism analysis nor molecular variance analysis (attending to intra- and intersubtype differences, age, sex and previous history of antiviral treatment) was able to show any particular difference between subtypes that might account for their different prevalence. Only the demographic history of the populations carrying both subtypes and analysis of molecular variance (AMOVA) for risk practice suggested that the route of transmission may be the most important factor to explain the observed difference.

Purpose To analyze proliferative verrucous leukoplakia (PVL) for the presence of human papillomavirus (HPV) in different stages of the disease. Materials and Methods We studied 13 patients with PVL. In 10 patients (76.9%), a lesional biopsy was taken and frozen at −40°C. Four patients were instructed to mouth rinse with sterile sera. The biopsy and rinse samples were analyzed for HPV by PCR. Results We did not detect HPV infection in the PVL tissue or in the oral rinse of any of the 13 patients in any stage of the disease analyzed, neither in oral squamous cell carcinoma nor in the simple hyperkeratosis. Conclusion There was no association between PVL and HPV infection in our patients.

Tuberculosis remains one of the main causes of death worldwide. The long and cumbersome process of culturingMycobacterium tuberculosiscomplex (MTBC) bacteria has encouraged the development of specific molecular tools for detecting the pathogen. Most of these tools aim to become novel tuberculosis diagnostics, and big efforts and resources are invested in their development, looking for the endorsement of the main public health agencies. Surprisingly, no study had been conducted where the vast amount of genomic data available is used to identify the best MTBC diagnostic markers. In this work, we use large-scale comparative genomics to provide a catalog of 30 characterized loci that are unique to the MTBC. Some of these genes could be targeted to assess the physiological status of the bacilli. Remarkably, none of the conventional MTBC markers is in our catalog. In addition, we develop a qPCR assay to accurately quantify MTBC DNA in clinical samples.

15 páginas, 6 figuras, 7 tablas., A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis, identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study's main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results., This project has been funded by the European Research Council (638553-TB-ACCELERATE, 101001038-TB-RECONNECT), the Ministerio de Economía, Indústria y Competividad (SAF2016-77346-R, PID2019-104477RB-I00) and the Conselleria d'Educació, Investigació, Cultura i Esport (PROMETEO 2020/012).