We report a corona virus disease (COVID‐19) case with unprecedented viral complexity. In the first severe episode, two different severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) strains ...(superinfection) were identified within a week. Three months after discharge, the patient was readmitted and was infected in a nosocomial outbreak with a different strain, suffering a second milder COVID‐19 episode.
SARS‐CoV‐2 RT‐PCR cycle threshold values from 18,803 cases (2 March–4 October) in Madrid define three stages: (i) initial ten weeks with sustained reduction in viral load (Ct: 23.4–32.3), (ii) ...stability with low viral loads (Ct: 31.9–35.5) in the next nine weeks and (iii) sudden increase with progressive higher viral loads until reaching stability at high levels in the next twelve weeks, coinciding with an increased percentage of positive cases and reduced median age. These data indicate differential virological/epidemiological patterns between the first and second COVID‐19 waves in Madrid.
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.
Introduction
is a member of the
complex (MTBC) not routinely identified to species level. It lacks specific clinical features of presentation and may therefore not be identified as the causative ...agent of tuberculosis. Use of whole genome sequencing (WGS) in the investigation of a family microepidemic of tuberculosis in Almería, Spain, unexpectedly identified the involvement of
.AimWe aimed to evaluate the presence of additional unidentified
cases and to determine the magnitude of this occurrence.MethodsFirst-line characterisation of the MTBC isolates was done by MIRU-VNTR, followed by WGS. Human and animal
isolates were integrated in the analysis.ResultsA comprehensive One Health strategy allowed us to (i) detect other 11
infections in humans in a period of 18 years, (ii) systematically analyse
infections on an epidemiologically related goat farm and (iii) geographically expand the study by including 16
isolates from other provinces. Integrative genomic analysis of 41 human and animal
isolates showed a high diversity of strains. The animal isolates' diversity was compatible with long-term infection, and close genomic relationships existed between isolates from goats on the farm and recent cases of
infection in humans.DiscussionZoonotic circulation of
strains had gone unnoticed for 18 years. Systematic characterisation of MTBC at species level and/or extended investigation of the possible sources of exposure in all tuberculosis cases would minimise the risk of overlooking similar zoonotic events.
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.
SARS-CoV-2 genomic analysis has been key to the provision of valuable data to meet both epidemiological and clinical demands. High-throughput sequencing, generally Illumina-based, has been necessary ...to ensure the widest coverage in global variant tracking. However, a speedier response is needed for nosocomial outbreak analyses and rapid identification of patients infected by emerging VOCs. An alternative based on nanopore sequencing may be better suited to delivering a faster response when required; however, although there are several studies offering side-by-side comparisons of Illumina and nanopore sequencing, evaluations of the usefulness in the hospital routine of the faster availability of data provided by nanopore are still lacking.
We performed a prospective 10-week nanopore-based sequencing in MinION in a routine laboratory setting, including 83 specimens where a faster response time was necessary. The specimens analyzed corresponded to i) international travellers in which lineages were assigned to determine the proper management/special isolation of the patients; ii) nosocomial infections and health-care-worker infections, where SNP-based comparisons were required to rule in/out epidemiological relationships and tailor specific interventions iii) sentinel cases and breakthrough infections to timely report to the Public Health authorities. MinION-based sequencing was compared with the standard procedures, supported on Illumina sequencing; MinION accelerated the delivery of results (anticipating results 1-12 days) and reduced costs per sample by 28€ compared to Illumina, without reducing accuracy in SNP calling.
Parallel integration of Illumina and nanopore sequencing strategies is a suitable solution to ensure both high-throughput and rapid response to cope with accelerating the surveillance demands of SARS-CoV-2 while also maintaining accuracy.
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.
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.
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.
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.
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.
Despite the proven value of applying genomic data for epidemiological purposes, commonly used high-throughput sequencing formats are not adapted to the response times required to intervene and ...finally control outbreaks. In this study, we propose a fast alternative to whole-genome sequencing (WGS) to track relevant microbiological strains: nanopore sequencing of multiple amplicons including strain marker single nucleotide polymorphisms (SNPs). As a proof a concept, we evaluated the performance of our approach to offer a rapid response to the most recent public health global alarm, the monkeypox virus (MPXV) global outbreak. Through a multisequence alignment, a list of 42 SNPs were extracted as signature makers for this outbreak. Twenty primer pairs were designed to amplify in a multiplex PCR the regions including 22 of these SNPs. Amplicon pools were sequenced in a MinION device, and SNPs were called in real time by an in-house bioinformatic pipeline. A total of 120 specimens (95 MPXV-PCR positive, Ct values from 14 to 39) were selected. In 67.37% of the positive subset, all 22 SNPs were called. After excluding low viral load specimens, in 92% of samples ≥11 outbreak SNPs were called. No false positives were observed in any of the 25 negative specimens. The total turnaround time required for this strategy was 5 hours, and the cost per sample was 14 euros. Nanopore sequencing of multiple amplicons harboring signature SNPs escapes the targeting limitations of strain-specific PCRs and offers a powerful alternative to systematic WGS, paving the way to real-time genomic epidemiology and making immediate intervention possible to finally optimize transmission control.
Nanopore sequencing of multiple amplicons harboring signature single nucleotide polymorphisms (SNPs) escapes the targeting limitations of strain-specific PCRs and offers a powerful alternative to systematic whole-genome analysis, paving the way to real-time genomic epidemiology and making immediate intervention possible to finally optimize transmission control.
Reverse transcription-PCRs (RT-PCRs) targeting SARS-CoV-2 variant of concern (VOC) mutations have been developed to simplify their tracking. We evaluated an assay targeting E484K/N501Y to identify ...B.1.351/P1. Whole-genome sequencing (WGS) confirmed only 72 (59.02%) of 122 consecutive RT-PCR P.1/B.1.351 candidates. Prescreening RT-PCRs must target a wider set of mutations, updated from WGS data from emerging variants.
PDF
