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Centers for Disease Control and Prevention guidelines consider SARS-CoV-2 reinfection when sequential COVID-19 episodes occur >90 days apart. However, genomic diversity acquired over recent COVID-19 waves could mean previous infection provides insufficient cross-protection. We used genomic analysis to assess the percentage of early reinfections in a sample of 26 patients with 2 COVID-19 episodes separated by 20-45 days. Among sampled patients, 11 (42%) had reinfections involving different SARS-CoV-2 variants or subvariants. Another 4 cases were probable reinfections; 3 involved different strains from the same lineage or sublineage. Host genomic analysis confirmed the 2 sequential specimens belonged to the same patient. Among all reinfections, 36.4% involved non-Omicron, then Omicron lineages. Early reinfections showed no specific clinical patterns; 45% were among unvaccinated or incompletely vaccinated persons, 27% were among persons <18 years of age, and 64% of patients had no risk factors. Time between sequential positive SARS-CoV-2 PCRs to consider reinfection should be re-evaluated. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypassed the standard approach of applying systematic mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and used a strain-specific polymerase chain reaction (PCR) that allowed identifying a cluster involving six cases (C1). The second scheme followed the canonical MIRU-VNTR path coupled with a whole-genomic amplification step, by which a second unsuspected overlapping cluster (C2), was detected in the same prison. These findings justified the implementation of a surveillance programme adapted to local resources based on a tailored multiplex allele-specific oligonucleotide (ASO)-PCR targeting C1 and C2. Presence of the C2 strain at a different prison was determined. ASO-PCR was applied extensively and alerted to the active circulation of one of the strains within and beyond prisons. Our study shows that alternative methodological strategies may provide useful data in settings with lack of resources for performing systematic standard molecular epidemiology programmes and/or with suboptimal material for analysis. [ABSTRACT FROM AUTHOR]

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]

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analysed 90 sequential MDR Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR‐TB that recurred in 2018 (1–10 years, 2–10 isolates per patient). Three long‐term predominant strains were responsible for 63% of all MDR‐TB recurrences. Most of the remaining patients were infected by strains different from each other. Reactivation/persistence of the same strain caused all but one recurrence, which was due to a reinfection with a predominant strain. One of the prevalent strains showed marked stability in the recurrences, while in another strain higher SNP‐based diversity was observed. Comparisons of intra‐ versus inter‐patient SNP distances identified two possible reinfections with closely related variants circulating in the community. Our results show a complex scenario of MDR‐TB infections in settings with predominant MDR Mtb strains. [ABSTRACT FROM AUTHOR]

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.)

The transition from MIRU-VNTR-based epidemiology studies in tuberculosis (TB) to genomic epidemiology has transformed how we track transmission. However, short-read sequencing is poor at analyzing repetitive regions such as the MIRU-VNTR loci. This causes a gap between the new genomic data and the large amount of information stored in historical databases. Long-read sequencing could bridge this knowledge gap by allowing analysis of repetitive regions. However, the feasibility of extracting MIRU-VNTRs from long reads and linking them to historical data has not been evaluated. In our study, an in silico arm, consisting of inference of MIRU patterns from long-read sequences (using MIRUReader program), was compared with an experimental arm, involving standard amplification and fragment sizing. We analyzed overall performance on 39 isolates from South Africa and confirmed reproducibility in a sample enriched with 62 clustered cases from Spain. Finally, we ran 25 consecutive incident cases, demonstrating the feasibility of correctly assigning new clustered/orphan cases by linking data inferred from genomic analysis to MIRU-VNTR databases. Of the 3,024 loci analyzed, only 11 discrepancies (0.36%) were found between the two arms: three attributed to experimental error and eight to misassigned alleles from long-read sequencing. A second round of analysis of these discrepancies resulted in agreement between the experimental and in silico arms in all but one locus. Adjusting the MIRUReader program code allowed us to flag potential in silico misassignments due to suboptimal coverage or unfixed double alleles. Our study indicates that long-read sequencing could help address potential chronological and geographical gaps arising from the transition from molecular to genomic epidemiology of tuberculosis., Importance: The transition from molecular epidemiology in tuberculosis (TB), based on the analysis of repetitive regions (VNTR-based genotyping), to genomic epidemiology transforms in the precision with which we track transmission. However, short-read sequencing, the most common method for performing genomic analysis, is poor at analyzing repetitive regions. This means that we face a gap between the new genomic data and the large amount of information stored in historical databases, which is also an obstacle to cross-national surveillance involving settings where only molecular data are available. Long-read sequencing could help bridge this knowledge gap by allowing analysis of repetitive regions. Our study demonstrates that MIRU-VNTR patterns can be successfully inferred from long-read sequences, allowing the correct assignment of new cases as clustered/orphan by linking new data extracted from genomic analysis to historical MIRU-VNTR databases. Our data may provide a starting point for bridging the knowledge gap between the molecular and genomic eras in tuberculosis epidemiology., Competing Interests: The authors declare no conflict of interest.

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.

Genotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spanish-born individuals. In one case, two Mycobacterium tuberculosis clonal variants were identified. We reanalyzed the cluster, supported by the characterization of multiple cultured isolates and respiratory specimens, whole-genome sequencing, and epidemiological case interviews. Our findings showed that the cluster, which was initially thought to have restarted activity with just a single case harboring a small degree of within-host diversity, was in fact currently growing due to coincidental reactivation of past exposures, with clonal diversity transmitted throughout the cluster. In one case, within-host diversity was amplified, probably due to prolonged diagnostic delay. IMPORTANCE The precise study of the dynamics of tuberculosis transmission in socio-epidemiologically complex scenarios may require more thorough analysis than the standard molecular epidemiology strategies. Our study illustrates the epidemiological and bacteriological complexity present in a transmission cluster in a challenging epidemiological setting with a high proportion of migrant cases. The combination of whole-genome sequencing, refined and refocused epidemiological interviews, and in-depth analysis of the bacterial composition of sputa and cultured isolates was crucial in order to correctly reinterpret the true nature of this cluster. Our global approach allowed us to reinterpret correctly the unnoticed epidemiological and bacteriological complexity involved in the Mycobacterium tuberculosis transmission event under study, which had been overlooked by the usual molecular epidemiology approaches.