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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 . Mycobacterium abscessus is considered an emerging pathogen, given its prevalence in patients with pulmonary diseases, such as cystic fibrosis. Here, authors perform a genomic analysis on sequential isolates obtained from patients with persistent infections of M. abscessus .

Estimates 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.

Background During the pandemic, whole genome sequencing was critical to characterize SARS-CoV-2 for surveillance, clinical and therapeutical purposes. However, low viral loads in specimens often led to suboptimal sequencing, making lineage assignment and phylogenetic analysis difficult. We propose an alternative approach to sequencing these specimens that involves sequencing in triplicate and concatenation of the reads obtained using bioinformatics. This proposal is based on the hypothesis that the uncovered regions in each replicate differ and that concatenation would compensate for these gaps and recover a larger percentage of the sequenced genome. Results Whole genome sequencing was performed in triplicate on 30 samples with Ct > 32 and the benefit of replicate read concatenation was assessed. After concatenation: i) 28% of samples reached the standard quality coverage threshold (> 90% genome covered > 30x); ii) 39% of samples did not reach the coverage quality thresholds but coverage improved by more than 40%; and iii) SARS-CoV-2 lineage assignment was possible in 68.7% of samples where it had been impaired. Conclusions Concatenation of reads from replicate sequencing reactions provides a simple way to access hidden information in the large proportion of SARS-CoV-2-positive specimens eliminated from analysis in standard sequencing schemes. This approach will enhance our potential to rule out involvement in outbreaks, to characterize reinfections and to identify lineages of concern for surveillance or therapeutical purposes.

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. 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.

Hundred and ninety-six travellers with negative-COVID-19-tests prior to departure tested positive, on arrival at Madrid (April/June 2021), from a total of 45 211 travellers tested (0.43%). Viral loads (Ct: 20.3) were higher compared to the general population (Ct: 27.09). Our data reveal weaknesses in pre-departure testing and alert about high-viral-load-SARS-CoV-2 carriers in intercontinental flights.

We present clinical, genomic and epidemiological data on the first 106 cases with the SARS-CoV-2 B.1.1.7 variant in Madrid. Even from the start, the increase of this variant was due to transmission events within the community, some causing extensive clusters, rather than further imports. Most cases developed non-severe disease.

Whole-genome sequencing (WGS) enhances precision in predicting antimicrobial resistance and tracking (MTB) transmission. Due to MTB's slow-growing nature, genomic results are delayed; however, few efforts have sought to accelerate them by performing WGS directly on respiratory specimens. Most culture-free efforts have focused on accelerating resistance prediction. The present study provides further evidence to the only preceding study aiming to accelerate precise delineation of transmission, coupling culture-free WGS to a surveillance programme. Our study is distinguished from its predecessor by being the first to apply flexible nanopore sequencing to further accelerate the process. A total of 71 sputa were selected, in which we applied only a procedure to deplete human DNA, thus avoiding costly and cumbersome capture-bait alternatives. Optimal results (>90% genome covered, mean coverage >45× and >70% genome covered >20×) were obtained from 33.8% of cases, allowing the assignment to transmission clusters close to diagnosis of every new case. A further 12.6% of samples yielded suboptimal results (15.5%-90.92% at >10×), which were exploited through a rescue pipeline. This approach was based on identifying informative SNPs acting as markers for relevant transmission clusters in our population. The pipeline enabled pre-allocation of new cases to pre-existing clusters and, in some cases, precise genomic relationships with the preceding cases in the cluster. In summary, this study demonstrates that epidemiologically valuable information can be obtained directly from sputum in approximately half the samples analysed. It represents a new advancement in the pursuit of faster comparative genomics, with epidemiological purposes, at diagnosis.

Healthcare-associated infections (HAIs) are a significant concern worldwide due to their impact on patient safety and healthcare costs. Klebsiella spp., particularly Klebsiella pneumoniae and Klebsiella oxytoca, are frequently implicated in HAIs and often exhibit multidrug resistance mechanisms, posing challenges for infection control. In this study, we evaluated Fourier-transform Infrared (FT-IR) spectroscopy as a rapid method for characterizing a nosocomial outbreak caused by VIM-1-producing K. oxytoca. A total of 47 isolates, including outbreak strains and controls, were collected from Hospital Universitario Gregorio Marañón, Spain and the University Hospital Basel, Switzerland. FT-IR spectroscopy was employed for bacterial typing, offering rapid and accurate results compared to conventional methods like pulsed-field gel electrophoresis (PFGE) and correlating with whole-genome sequencing (WGS) results. The FT-IR spectra analysis revealed distinct clusters corresponding to outbreak strains, suggesting a common origin. Subsequent WGS analysis identified Klebsiella michiganensis as the causative agent of the outbreak, challenging the initial assumption based on FT-IR results. However, both FT-IR and WGS methods showed high concordance, with an Adjusted Rand index (AR) of 0.882 and an Adjusted Wallace coefficient (AW) of 0.937, indicating the reliability of FT-IR in outbreak characterization. Furthermore, FT-IR spectra visualization highlighted discriminatory features between outbreak and non-outbreak isolates, facilitating rapid screening in case and outbreak is suspected. In conclusion, FT-IR spectroscopy offers a rapid and cost-effective alternative to traditional typing methods, enabling timely intervention and effective management of nosocomial outbreaks. Its integration with WGS enhances the accuracy of outbreak investigations, demonstrating its utility in clinical microbiology and infection control practices.