Tracking SARS-CoV-2 introductions in Mozambique using pandemic-scale phylogenies: a retrospective observational study.

BACKGROUND: From the start of the SARS-CoV-2 outbreak, global sequencing efforts have generated an unprecedented amount of genomic data. Nonetheless, unequal sampling between high-income and low-income countries hinders the implementation of genomic surveillance systems at the global and local level. Filling the knowledge gaps of genomic information and understanding pandemic dynamics in low-income countries is essential for public health decision making and to prepare for future pandemics. In this context, we aimed to discover the timing and origin of SARS-CoV-2 variant introductions in Mozambique, taking advantage of pandemic-scale phylogenies. METHODS: We did a retrospective, observational study in southern Mozambique. Patients from Manhiça presenting with respiratory symptoms were recruited, and those enrolled in clinical trials were excluded. Data were included from three sources: (1) a prospective hospital-based surveillance study (MozCOVID), recruiting patients living in Manhiça, attending the Manhiça district hospital, and fulfilling the criteria of suspected COVID-19 case according to WHO; (2) symptomatic and asymptomatic individuals with SARS-CoV-2 infection recruited by the National Surveillance system; and (3) sequences from SARS-CoV-2-infected Mozambican cases deposited on the Global Initiative on Sharing Avian Influenza Data database. Positive samples amenable for sequencing were analysed. We used Ultrafast Sample placement on Existing tRees to understand the dynamics of beta and delta waves, using available genomic data. This tool can reconstruct a phylogeny with millions of sequences by efficient sample placement in a tree. We reconstructed a phylogeny (~7·6 million sequences) adding new and publicly available beta and delta sequences. FINDINGS: A total of 5793 patients were recruited between Nov 1, 2020, and Aug 31, 2021. During this time, 133 328 COVID-19 cases were reported in Mozambique. 280 good quality new SARS-CoV-2 sequences were obtained after the inclusion criteria were applied and an additional 652 beta (B.1.351) and delta (B.1.617.2) public sequences were included from Mozambique. We evaluated 373 beta and 559 delta sequences. We identified 187 beta introductions (including 295 sequences), divided in 42 transmission groups and 145 unique introductions, mostly from South Africa, between August, 2020 and July, 2021. For delta, we identified 220 introductions (including 494 sequences), with 49 transmission groups and 171 unique introductions, mostly from the UK, India, and South Africa, between April and November, 2021. INTERPRETATION: The timing and origin of introductions suggests that movement restrictions effectively avoided introductions from non-African countries, but not from surrounding countries. Our results raise questions about the imbalance between the consequences of restrictions and health benefits. This new understanding of pandemic dynamics in Mozambique can be used to inform public health interventions to control the spread of new variants. FUNDING: European and Developing Countries Clinical Trials, European Research Council, Bill & Melinda Gates Foundation, and Agència de Gestió d'Ajuts Universitaris i de Recerca.

Epidemiological and Genomic Analysis of a Large SARS-CoV-2 Outbreak in a Long-Term Care Facility in Catalonia, Spain.

Limiting outbreaks in long-term care facilities (LTCFs) is a cornerstone strategy to avoid an excess of COVID-19-related morbidity and mortality and to reduce its burden on the health system. We studied a large outbreak that occurred at an LTCF, combining methods of classical and genomic epidemiology analysis. The outbreak lasted for 31 days among residents, with an attack rate of 98% and 57% among residents and staff, respectively. The case fatality rate among residents was 16% (n = 15). Phylogenetic analysis of 59 SARS-CoV-2 isolates revealed the presence of two closely related viral variants in all cases (B.1.177 lineage), revealing a far more complex outbreak than initially thought and suggesting an initial spread driven by staff members. In turn, our results suggest that resident relocations to mitigate viral spread might have increased the risk of infection for staff members, creating secondary chains of transmission that were responsible for prolonging the outbreak. Our results highlight the importance of considering unnoticed chains of transmission early during an outbreak and making an adequate use and interpretation of diagnostic tests. Outbreak containment measures should be carefully tailored to each LTCF. IMPORTANCE The impact of COVID-19 on long-term care facilities (LTCFs) has been disproportionately large due to the high frailty of the residents. Here, we report epidemiological and genomic findings of a large outbreak that occurred at an LTCF, which ultimately affected almost all residents and nearly half of staff members. We found that the outbreak was initially driven by staff members; however, later resident relocation to limit the outbreak resulted in transmission from residents to staff members, evidencing the complexity and different phases of the outbreak. The phylogenetic analysis of SARS-CoV-2 isolates indicated that two closely related variants were responsible for the large outbreak. Our study highlights the importance of combining methods of classical and genomic epidemiology to take appropriate outbreak containment measures in LTCFs.

SARS-CoV-2 Outbreak on a Spanish Mink Farm: Epidemiological, Molecular, and Pathological Studies.

Farmed minks have been reported to be highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may represent a risk to humans. In this study, we describe the first outbreak of SARS-CoV-2 occurred on a mink farm in Spain, between June and July 2020, involving 92,700 animals. The outbreak started shortly after some farm workers became seropositive for SARS-CoV-2. Minks showed no clinical signs compatible with SARS-CoV-2 infection throughout the outbreak. Samples from 98 minks were collected for histopathological, serological, and molecular studies. Twenty out of 98 (20.4%) minks were positive by RT-qPCR and 82 out 92 (89%) seroconverted. This finding may reflect a rapid spread of the virus at the farm with most of the animals overcoming the infection. Additionally, SARS-CoV-2 was detected by RT-qPCR in 30% of brain samples from positive minks. Sequencing analysis showed that the mink sequences were not closely related with the other mink SARS-CoV-2 sequences available, and that this mink outbreak has its probable origin in one of the genetic variants that were prevalent in Spain during the first COVID-19 epidemic wave. Histological studies revealed bronchointerstitial pneumonia in some animals. Immunostaining of viral nucleocapsid was also observed in nasal turbinate tissue. Farmed minks could therefore constitute an important SARS-CoV-2 reservoir, contributing to virus spread among minks and humans. Consequently, continuous surveillance of mink farms is needed.

Spatial and temporal distribution of SARS-CoV-2 diversity circulating in wastewater.

Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.7 lineage and the ability of the technique to anticipate the detection of certain mutations before they are detected in clinical samples. The study proves the usefulness of sewage sequencing to track Variants of Concern that can complement clinical testing to help in decision-making and in the analysis of the evolution of the pandemic.

Evolutionary and Phenotypic Characterization of Two Spike Mutations in European Lineage 20E of SARS-CoV-2.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.

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.