Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network.

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network's technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain.

Frequent Emergence of Resistance Mutations Following Complex Intra-Host Genomic Dynamics in SARS-CoV-2 Patients Receiving Sotrovimab.

The emergence of the Omicron variant of SARS-CoV-2 represented a challenge to the treatment of COVID-19 using monoclonal antibodies. Only Sotrovimab maintained partial activity, allowing it to be used in high-risk patients infected with the Omicron variant. However, reports of resistance mutations to Sotrovimab demand efforts to better understand the intra-patient emergence of Sotrovimab resistance. A retrospective genomic analysis was conducted on respiratory samples from immunocompromised patients infected with SARS-CoV-2 who received Sotrovimab at our hospital between December 2021 and August 2022. The study involved 95 sequential specimens from 22 patients (1 to 12 samples/patient; 3 to 107 days post-infusion; threshold cycle [CT] ≤ 32). Resistance mutations (in P337, E340, K356, and R346) were detected in 68% of cases; the shortest time to detection of a resistance mutation was 5 days after Sotrovimab infusion. The dynamics of resistance acquisition were highly complex, with up to 11 distinct amino acid changes in specimens from the same patient. In two patients, the mutation distribution was compartmentalized in respiratory samples from different sources. This is the first study to examine the acquisition of Sotrovimab resistance in the BA.5 lineage, enabling us to determine the lack of genomic or clinical differences between Sotrovimab resistance in BA.5 relative to that in BA.1/2. Across all Omicron lineages, the acquisition of resistance delayed SARS-CoV-2 clearance (40.67 versus 19.5 days). Close, real-time genomic surveillance of patients receiving Sotrovimab should be mandatory to facilitate early therapeutic interventions.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

SARS-CoV-2 superinfection and reinfection with three different strains.

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.

Deciphering the Tangible Spatio-Temporal Spread of a 25-Year Tuberculosis Outbreak Boosted by Social Determinants.

Outbreak strains of Mycobacterium tuberculosis are promising candidates as targets in the search for intrinsic determinants of transmissibility, as they are responsible for many cases with sustained transmission; however, the use of low-resolution typing methods and restricted geographical investigations represent flaws in assessing the success of long-lived outbreak strains. We can now address the nature of outbreak strains by combining large genomic data sets and phylodynamic approaches. We retrospectively sequenced the whole genome of representative samples assigned to an outbreak circulating in the Canary Islands (the GC strain) since 1993, which accounts for ~20% of local tuberculosis cases. We selected a panel of specific single nucleotide polymorphism (SNP) markers for an in-silico search for additional outbreak-related sequences within publicly available tuberculosis genomic data. Using this information, we inferred the origin, spread, and epidemiological parameters of the GC strain. Our approach allowed us to accurately trace the historical and more recent dispersion of the GC strain. We provide evidence of a highly successful nature within the Canarian archipelago but limited expansion abroad. Estimation of epidemiological parameters from genomic data disagree with a distinctive biology of the GC strain. With the increasing availability of genomic data allowing for the accurate inference of strain spread and critical epidemiological parameters, we can now revisit the link between Mycobacterium tuberculosis genotypes and transmission, as is routinely carried out for SARS-CoV-2 variants of concern. We demonstrate that social determinants rather than intrinsically higher bacterial transmissibility better explain the success of the GC strain. Importantly, our approach can be used to trace and characterize strains of interest worldwide. IMPORTANCE Infectious disease outbreaks represent a significant problem for public health. Tracing outbreak expansion and understanding the main factors behind emergence and persistence remain critical to effective disease control. Our study allows researchers and public health authorities to use Whole-Genome Sequencing-based methods to trace outbreaks, and shows how available epidemiological information helps to evaluate the factors underpinning outbreak persistence. Taking advantage of all the freely available information placed in public repositories, researchers can accurately establish the expansion of an outbreak beyond original boundaries, and determine the potential risk of a strain to inform health authorities which, in turn, can define target strategies to mitigate expansion and persistence. Finally, we show the need to evaluate strain transmissibility in different geographic contexts to unequivocally associate spread to local or pathogenic factors, an important lesson taken from genomic surveillance of SARS-CoV-2.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant Primera confirmación de importación y transmisión en España de la variante B.1.1.7 del SARS-CoV-2 recientemente identificada

Introduction A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and methods We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. Results We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. Conclusion The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission.

INTRODUCTION: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. METHODS: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population. RESULTS: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions CONCLUSION: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission Primeras importaciones de las variantes SARS-CoV-2 P.1 y P.2 de Brasil a Madrid y transmisión comunitaria

Introduction SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. Methods Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population. Results In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions Conclusion Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

Post-COVID-19 syndrome. SARS-CoV-2 RNA detection in plasma, stool, and urine in patients with persistent symptoms after COVID-19.

BACKGROUND: There is a paucity of knowledge on the long-term outcome in patients diagnosed with COVID-19. We describe a cohort of patients with a constellation of symptoms occurring four weeks after diagnosis causing different degrees of reduced functional capacity. Although different hypothesis have been proposed to explain this condition like persistent immune activation or immunological dysfunction, to date, no physiopathological mechanism has been identified. Consequently, there are no therapeutic options besides symptomatic treatment and rehabilitation. METHODS: We evaluated patients with symptoms that persisted for at least 4 weeks after COVID-19. Epidemiological and clinical data were collected. Blood tests, including inflammatory markers, were conducted, and imaging studies made if deemed necessary. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) in plasma, stool, and urine were performed. Patients were offered antiviral treatment (compassionate use). RESULTS: We evaluated 29 patients who reported fatigue, muscle pain, dyspnea, inappropriate tachycardia, and low-grade fever. Median number of days from COVID-19 to positive RT-PCR in extra-respiratory samples was 55 (39-67). Previous COVID-19 was mild in 55% of the cases. Thirteen patients (45%) had positive plasma RT-PCR results and 51% were positive in at least one RT-PCR sample (plasma, urine, or stool). Functional status was severely reduced in 48% of the subjects. Eighteen patients (62%) received antiviral treatment. Improvement was seen in most patients (p = 0.000) and patients in the treatment group achieved better outcomes with significant differences (p = 0.01). CONCLUSIONS: In a cohort of COVID-19 patients with persistent symptoms, 45% of them have detectable plasma SARS-CoV-2 RNA. Our results indicate possible systemic viral persistence in these patients, who may benefit of antiviral treatment strategies.

Nosocomial Transmission of SARS-CoV-2 Involving Vaccinated Health Care Workers.

COVID-19 vaccination has proven to be effective at preventing symptomatic disease but there are scarce data to fully understand whether vaccinated individuals can still behave as SARS-CoV-2 transmission vectors. Based on viral genome sequencing and detailed epidemiological interviews, we report a nosocomial transmission event involving two vaccinated health care-workers (HCWs) and four patients, one of them with fatal outcome. Strict transmission control measures, as during the prevaccination period, must be kept between HCWs and HCWs-patients in nosocomial settings. IMPORTANCE COVID-19 vaccination has proven to be effective at preventing symptomatic disease. Although some transmission events involving vaccinated cases have also been reported, scarce information is still available to fully understand whether vaccinated individuals may still behave as vectors in SARS-CoV-2 transmission events. Here, we report a SARS-CoV-2 nosocomial transmission event, supported on whole genome sequencing, in early March 2021 involving two vaccinated HCWs and four patients in our institution. Strict transmission control measures between HCWs and HCWs - patients in nosocomial settings must not be relaxed, and should be kept as strictly as during the prevaccination period.

Different dynamics of mean SARS-CoV-2 RT-PCR Ct values between the first and second COVID-19 waves in the Madrid population.

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.

SARS-CoV-2 B.1.1.7 Decline Is Not Driven by the Introduction of a More Successful Variant.

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.

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.

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Complete Analysis of the Epidemiological Scenario around a SARS-CoV-2 Reinfection: Previous Infection Events and Subsequent Transmission.

The first descriptions of reinfection by SARS-CoV-2 have been recently reported. However, these studies focus exclusively on the reinfected case, without considering the epidemiological context of the event. Our objectives were to perform a complete analysis of the sequential infections and community transmission events around a SARS-CoV-2 reinfection, including the infection events preceding it, the exposure, and subsequent transmissions. Our analysis was supported by host genetics, viral whole-genome sequencing, phylogenomic viral population analysis, and refined epidemiological data obtained from interviews with the involved subjects. The reinfection involved a 53-year-old woman with asthma (Case A), with a first COVID-19 episode in April 2020 and a much more severe second episode 4-1/2 months later, with SARS-CoV-2 seroconversion in August, that required hospital admission. An extended genomic analysis allowed us to demonstrate that the strain involved in Case A's reinfection was circulating in the epidemiological context of Case A and was also transmitted subsequently from Case A to her family context. The reinfection was also supported by a phylogenetic analysis, including 348 strains from Madrid, which revealed that the strain involved in the reinfection was circulating by the time Case A suffered the second episode, August-September 2020, but absent at the time range corresponding to Case A's first episode. IMPORTANCE We present the first complete analysis of the epidemiological scenario around a reinfection by SARS-CoV-2, more severe than the first episode, including three cases preceding the reinfection, the reinfected case per se, and the subsequent transmission to another seven cases.

Overlapping of Independent SARS-CoV-2 Nosocomial Transmissions in a Complex Outbreak.

SARS-CoV-2 nosocomial outbreaks in the first COVID-19 wave were likely associated with a shortage of personal protective equipment and scarce indications on control measures. Having covered these limitations, updates on current SARS-CoV-2 nosocomial outbreaks are required. We carried out an in-depth analysis of a 27-day nosocomial outbreak in a gastroenterology ward in our hospital, potentially involving 15 patients and 3 health care workers. Patients had stayed in one of three neighboring rooms in the ward. The severity of the infections in six of the cases and a high fatality rate made the clinicians suspect the possible involvement of a single virulent strain persisting in those rooms. Whole-genome sequencing (WGS) of the strains from 12 patients and 1 health care worker revealed an unexpected complexity. Five different SARS-CoV-2 strains were identified, two infecting a single patient each, ruling out their relationship with the outbreak; the remaining three strains were involved in three independent, overlapping, limited transmission clusters with three, three, and five cases. Whole-genome sequencing was key to understand the complexity of this outbreak. IMPORTANCE We report a complex epidemiological scenario of a nosocomial COVID-19 outbreak in the second wave, based on WGS analysis. Initially, standard epidemiological findings led to the assumption of a homogeneous outbreak caused by a single SARS-CoV-2 strain. The discriminatory power of WGS offered a strikingly different perspective consisting of five introductions of different strains, with only half of them causing secondary cases in three independent overlapping clusters. Our study exemplifies how complex the SARS-CoV-2 transmission in the nosocomial setting during the second COVID-19 wave occurred and leads to extending the analysis of outbreaks beyond the initial epidemiological assumptions.

Different Within-Host Viral Evolution Dynamics in Severely Immunosuppressed Cases with Persistent SARS-CoV-2.

A successful Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variant, B.1.1.7, has recently been reported in the UK, causing global alarm. Most likely, the new variant emerged in a persistently infected patient, justifying a special focus on these cases. Our aim in this study was to explore certain clinical profiles involving severe immunosuppression that may help explain the prolonged persistence of viable viruses. We present three severely immunosuppressed cases (A, B, and C) with a history of lymphoma and prolonged SARS-CoV-2 shedding (2, 4, and 6 months), two of whom finally died. Whole-genome sequencing of 9 and 10 specimens from Cases A and B revealed extensive within-patient acquisition of diversity, 12 and 28 new single nucleotide polymorphisms, respectively, which suggests ongoing SARS-CoV-2 replication. This diversity was not observed for Case C after analysing 5 sequential nasopharyngeal specimens and one plasma specimen, and was only observed in one bronchoaspirate specimen, although viral viability was still considered based on constant low Ct values throughout the disease and recovery of the virus in cell cultures. The acquired viral diversity in Cases A and B followed different dynamics. For Case A, new single nucleotide polymorphisms were quickly fixed (13-15 days) after emerging as minority variants, while for Case B, higher diversity was observed at a slower emergence: fixation pace (1-2 months). Slower SARS-CoV-2 evolutionary pace was observed for Case A following the administration of hyperimmune plasma. This work adds knowledge on SARS-CoV-2 prolonged shedding in severely immunocompromised patients and demonstrates viral viability, noteworthy acquired intra-patient diversity, and different SARS-CoV-2 evolutionary dynamics in persistent cases.