Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy

Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients' disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences

Microevolution, reinfection and highly complex genomic diversity in patients with sequential isolates of Mycobacterium abscessus

Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.This work was supported by the Instituto de Salud Carlos III [AC16/00057, FIS15/01554, PI21/01823, PI19/00331, FI20/00129, PI21/01738], co-financed by European Regional Development Funds of the European Commission: “A way of making Europe”; a Miguel Servet Contract (ISCIII) CPII20/00001 to LPL. FI22/00145 contract from a PFIS (ISCIII) to SBS and Ministerio de Ciencia (MCIN/AEI/10.13039/501100011033, grant PID2020-112865RB-I00).Peer reviewe

Epidemiological, clinical and genomic snapshot of the first 100 B.1.1.7 SARS-CoV-2 cases in Madrid

A new SARS-CoV-2 variant, B.1.1.7, emerged in September in the UK, and is responsible for 76.6% of COVID-19 cases.1 This variant has also been reported in another 45 countries, 17 of them European.2,3 B.1.1.7 is considered to have higher transmissibility.4 It carries an unusually high number of specific mutations/deletions, 18, mostly non-synonymous and eight concentrate in the S gene,5 including several which might have relevant functional roles. The 69/70 deletion may be associated to immune response evasion6 and the N501Y substitution increases the affinity to the ACE2 receptor.7 These findings have raised the alarm of having to face a new variant with the potential to accelerate the spread of the pandemic. A recent report finds a realistic possibility that B.1.1.7 is associated with an increased risk of death.This work was supported by Instituto de Salud Carlos III (Ref COV20/00140: SeqCOVID—Consorcio para la epidemiología genómica de SARS-CoV-2 en España) and by Consejo Superior de Investigaciones Científicas (CSIC) (PTI Salud Global). LPL holds a Miguel Servet Contract CP15/00075).Peer reviewe

Proper assignation of reactivation in a COVID-19 recurrence initially interpreted as a reinfection

A 77-year-old-male (Case R) who had had a previous diagnosis of mild COVID-19 episode, was hospitalized 35 days later. On Day 23 post-admission, he developed a second COVID-19 episode, now severe, and finally died. Initially, Case R COVID-19 recurrence was interpreted as a reinfection due to the exposure to a SARS-CoV-2 RT-PCR-positive room-mate. However, whole-genome-sequencing indicated that case R recurrence corresponded to a reactivation of the strain involved in his first episode. Case R reactivation had major consequences, leading to a more severe episode, and causing a subsequent transmission to another two hospitalized patients, one of them with fatal outcome.Peer reviewe

Systematic Genomic and Clinical Analysis of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfections and Recurrences Involving the Same Strain

10 páginas, 2 figuras, 3 tablasEstimates of the burden of severe acute respiratory syndrome coronavirus 2 reinfections are limited by the scarcity of population-level studies incorporating genomic support. We conducted a systematic study of reinfections in Madrid, Spain, supported by genomic viral analysis and host genetic analysis, to cleanse laboratory errors and to discriminate between reinfections and recurrences involving the same strain. Among the 41,195 cases diagnosed (March 2020-March 2021), 93 (0.23%) had 2 positive reverse transcription PCR tests (55-346 days apart). After eliminating cases with specimens not stored, of suboptimal sequence quality, or belonging to different persons, we obtained valid data from 22 cases. Of those, 4 (0.01%) cases were recurrences involving the same strain; case-patients were 39-93 years of age, and 3 were immunosuppressed. Eighteen (0.04%) cases were reinfections; patients were 19-84 years of age, and most had no relevant clinical history. The second episode was more severe in 8 cases.This work was supported by the Instituto de Salud Carlos III (Ref COV20/00140: SeqCOVID—Consorcio para la epidemiología genómica de SARS-CoV-2 en España) and by Consejo Superior de Investigaciones Científicas (CSIC) (PTI Salud Global). L.P.L. is the recipient of a Miguel Servet Research contract (CPII20/00001) from the Instituto de Salud Carlos III.Peer reviewe

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2

Publisher Copyright: © 2021 O'Toole Á et al.Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.Peer reviewe

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.</ns3:p