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Estimates of the burden of SARS-CoV-2 reinfections continue to be crucial for assessing new SARS-CoV-2 variants with immune escape potential (1). Genomic analysis of SARS-CoV-2 strains involved in sequential COVID-19 [...]

ABSTRACT During the COVID-19 pandemic, several SARS-CoV-2 variants of concern (VOCs) of particular relevance emerged. Early detection of VOCs entering a country is essential to control spread. The alert triggered by the first suspected case of the Omicron variant in Spain in a traveler arriving from South Africa in November 2021 provided a unique opportunity to evaluate four different methodological strategies tailored to rapid identification of Omicron. The different approaches were designed to respond to the different technical resources available in different settings. First, we used melting probes in RT-PCR to determine the presence of four Omicron signatures (K417N, E484A, P681H, and absence of L452R): three probes showed deviations in temperature (Tm) values relative to the reference codons (E484K-15.8°C, P681H-5.2°C, and L452R-7.2°C) and one maintained the reference value (K417N). The deviation in Tm of P681H suggested the presence of the characteristic Omicron N679K mutation in the probe hybridization region; these data pointed to the presence of Omicron alleles. Second, the presence of 29 of the 33 characteristic single nucleotide polymorphisms (SNPs) in the Omicron variant S-gene was identified by Sanger sequencing of nine amplicons. The final two strategies involved identification of 47 of the 50 non-synonymous and indel mutations attributed to Omicron by rapid nanopore whole genome sequencing (WGS) and by Illumina WGS technology. These strategies enabled us to pre-assign the first Omicron case in Spain with high certainty 2 h after receipt of RNA and to confirm it genomically 3 h later, so that the Public Health authorities could be rapidly notified. IMPORTANCE The study presents different experimental alternatives to identify new variants of concern (VOCs) of SARS-CoV-2 entering a certain population. Early detection of a new VOC is crucial for surveillance and control of spread. The objective is to provide laboratories with tools adapted to their resource capabilities that offer a sufficient level of resolution to rule out, confirm, or pre-assign the presence of a suspected VOC. The study describes four different techniques that were applied simultaneously to the first suspected Omicron case in Spain, highlighting the level of resolution and response time achieved in each case. These techniques are based on the detection of mutations in the S-gene of the virus that can easily adapt to potential emerging variants. The results of the study allow any laboratory to prepare for new alerts of SARS-CoV-2 VOCs.

ABSTRACT 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. IMPORTANCE 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.

IntroductionSince the COVID-19 outbreak, specific mRNA-based anti-SARS-CoV-2 vaccines have been developed and distributed worldwide. Because this is the first time that mRNA vaccines have been used, there are several questions regarding their capacity to confer immunity and the durability of the specific anti-SARS-CoV-2 response. Therefore, the objective of this study was to recruit a large cohort of healthcare workers from the Gregorio Marañón Hospital vaccinated with the mRNA-1273 or BNT126b2 vaccines and to follow-up on IgG anti-RBD levels at 8 months post-vaccination.MethodsWe recruited 4,970 volunteers and measured IgG anti-RBD antibodies on days 30 and 240 post-vaccination.ResultsWe observed that both vaccines induced high levels of antibodies on day 30, while a drastic wane was observed on day 240, where mRNA-1273 vaccinated induced higher levels than BNT162b2. Stratifying by vaccine type, age, gender, and comorbidities, we identified that older mRNA-1273-vaccinated volunteers had higher antibody levels than the younger volunteers, contrary to what was observed in the BNT162b2-vaccinated volunteers.DiscussionIn conclusion, we observed that mRNA-1273 has a higher capacity to induce a humoral response than BNT162b2 and that age is a factor in the specific response.

ABSTRACT The SARS-CoV-2 variant of concern (VOC) Delta (B.617.2 lineage) displaced the predominant VOC Alpha (B.1.1.7 lineage) in the United Kingdom. In Madrid, recent start of the decline of predominant VOC Alpha suggested an equivalent phenomenon. However, 11 different variants, none overrepresented in frequency, occupied progressively over a period of 7 weeks the niche previously dominated by VOC Alpha. Only after these 7 weeks, VOC Delta started to emerge. Viral competition due to the entry of VOC Delta is not the major force driving the start of VOC Alpha decline in Madrid. IMPORTANCE Our data indicate that the dynamics of SARS-CoV-2 VOCs turnover in our setting differ from those proposed for other countries. A systematic genomic analysis, updated on a weekly basis, of representative randomly selected samples of SARS-CoV-2 circulating variants allowed us to define a lapse of 7 weeks between the start of VOC Alpha decline and the final emergence of VOC Delta. During this period, VOC Alpha showed a sustained decline, while 11 VOCs, variants of interest (VOIs), and other identified variants, none overrepresented, occupied the niche left by VOC Alpha. Only after these 7 weeks, emergence of VOC Delta occurred, indicating that viral competition involving VOC Delta was not the exclusive direct driving force behind the starting of VOC Alpha decline.

ABSTRACT 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.

A study published in the Journal of Medical Virology examined the emergence of resistance mutations in COVID-19 patients receiving the antiviral drug nirmatrelvir/ritonavir. The study analyzed 60 patients who received the drug and found that none of the known resistance mutations were identified in the patients. However, low-frequency substitutions and fixed mutations were observed in some patients, particularly those who were immunosuppressed. The study suggests that the emergence of resistance mutations to nirmatrelvir/ritonavir is currently rare. [Extracted from the article]

Keywords: SARS-CoV-2; COVID-19; travellers; high viral load EN SARS-CoV-2 COVID-19 travellers high viral load 1 4 4 06/08/22 20220401 NES 220401 International air travel has been a key factor in the global spread of SARS-CoV-2. The unexpectedly common finding of travellers with SARS-CoV-2 on arrival in our study, despite the compulsory negative test result prior to departure, was even more alarming when we analysed SARS-CoV-2 viral loads. [Extracted from the article]

Background: Klebsiella michiganensis , a member of the Klebsiella oxytoca complex, is an emerging nosocomial pathogen known to frequently carry plasmids with antibiotic-resistance genes, including carbapenemases. Using genomics, this study redefined an outbreak alert of K. michiganensis carrying a blaVIM carbapenemase in a pediatric ward in a Spanish hospital., Methods: A total of 31 isolates of Verona integron-encoded metallo-β-lactamase (VIM)-carbapenemase K. oxytoca from suspected outbreak cases and unrelated controls from 2015 to 2022 were analyzed. Whole-genome sequencing (both short and long reads) was applied to determine phylogenetic relationships based on single-nucleotide polymorphisms (SNPs) and identify plasmids and antimicrobial resistance genes., Results: The sequences from 12 isolates identified in 2021 showed pairwise SNP distances ranging from 0 to 16 SNPs, confirming the outbreak. Examination of isolates before and after the study period revealed 7 additional cases, 2 in 2020 and 5 in 2022. The outbreak comprised 18 isolates from 17 patients in 3 different pediatric wards, together with 1 environmental sample. In all outbreak isolates, the blaVIM-1 gene was located within a gene cassette carried by a class 1 integron on an IncFIB(pQil) plasmid. A genomic network based on SNPs revealed 5 unsampled intermediate nodes, suggesting additional subclones that may have involved healthcare staff, patient relatives or environmental reservoirs. Blood and rectal isolates obtained from the same patient were positioned on separate branches of the network, making a direct evolutionary pathway between them unlikely., Conclusions: Our study redefined the full extent of this K. michiganensis -VIM outbreak and highlights the critical importance of genomic analysis in accurately understanding outbreaks in healthcare settings., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)