ABSTRACT
The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.
ABSTRACT
To control the SARS-CoV-2 pandemic, healthcare systems have focused on ramping up their capacity for epidemiological surveillance through viral whole genome sequencing. In this paper, we tested the performance of two protocols of SARS-CoV-2 nucleic acid enrichment, an amplicon enrichment using different versions of the ARTIC primer panel and a hybrid-capture method using KAPA RNA Hypercap. We focused on the challenge of the Omicron variant sequencing, the advantages of automated library preparation and the influence of the bioinformatic analysis in the final consensus sequence. All 94 samples were sequenced using Illumina iSeq 100 and analysed with two bioinformatic pipelines: a custom-made pipeline and an Illumina-owned pipeline. We were unsuccessful in sequencing six samples using the capture enrichment due to low reads. On the other hand, amplicon dropout and mispriming caused the loss of mutation G21987A and the erroneous addition of mutation T15521A respectively using amplicon enrichment. Overall, we found high sequence agreement regardless of method of enrichment, bioinformatic pipeline or the use of automation for library preparation in eight different SARS-CoV-2 variants. Automation and the use of a simple app for bioinformatic analysis can simplify the genotyping process, making it available for more diagnostic facilities and increasing global vigilance.
ABSTRACT
Background: SARS-CoV-2 variant tracking is key to the genomic surveillance of the pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification.Case presentation: A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike aminoacids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation. Conclusions: To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the targets of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.
ABSTRACT
Background: SARS-CoV-2 variation represents a serious challenge to current COVID-19 vaccines. Recent reports suggest that B.1.351 and other variants may escape the neutralization activity of the antibodies generated by current vaccines. Methods: Ninety-nine healthcare workers undertaking BNT162b2 mRNA vaccination were sampled at baseline, on the day of the second dose, and 14 days after the latter. Neutralization activity against SARS-CoV-2 B.1, B.1.1.7 and B.1.351 was investigated using a Vero-E6 model. Results: Eleven of the study participants had prior infection with SARS-CoV-2. Neutralization titers against the B.1 and the B.1.1.7 variants were not statistically different and were significantly higher than titers against the B.1.351 variant across pre-exposed and non-pre-exposed vaccinated individuals ( p <0.01). While all vaccinated individuals presented neutralizing antibodies against B.1 and B 1.1.7 after the second dose, 14% were negative against B.1.351, and 76% had low titers (1/20-1/80). Pre-exposed vaccinated individuals showed higher titers than non-pre-exposed after the first (median titers of 1/387 versus 1/28, respectively) and the second doses (1/995 versus 1/703, respectively). As high as 72% of the pre-exposed vaccinees presented titers >1/80 after a single dose, while only 11% of non-exposed vaccinated individuals had titers >1/80. Conclusions: BNT162b2 mRNA-induced antibodies show a lower in vitro neutralizing activity against B.1.351 variant compared to neutralization against B.1.1.7 or B.1 variants. Interestingly, for individuals pre-exposed to SARS-CoV-2, one dose of BNT162b2 mRNA may be adequate to produce neutralizing antibodies against B.1.1.7 and B.1, while two doses of BNT162b2 mRNA provide optimal neutralizing antibody response against B.1.351 too.
Subject(s)
COVID-19ABSTRACT
A detailed understanding of how and when SARS-CoV-2 transmission occurs is crucial for designing effective prevention measures. Other than contact tracing, genome sequencing provides information to help infer who infected whom. However, the effectiveness of the genomic approach in this context depends on both (high enough) mutation and (low enough) transmission rates. Today, the level of resolution that we can obtain when describing SARS-CoV-2 outbreaks using just genomic information alone remains unclear. In order to answer this question, we sequenced 49 SARS-CoV-2 patient samples from ten local clusters for which partial epidemiological information was available, and inferred transmission history using genomic variants. Importantly, we obtained high-quality genomic data, sequencing each sample twice and using unique barcodes to exclude cross-sample contamination. Phylogenetic and cluster analyses showed that consensus genomes were generally sufficient to discriminate among independent transmission clusters. However, levels of intrahost variation were low, which prevented in most cases the unambiguous identification of direct transmission events. After filtering out recurrent variants across clusters, the genomic data were generally compatible with the epidemiological information but did not support specific transmission events over possible alternatives. We estimated the effective transmission bottleneck size to be 1-2 viral particles for sample pairs whose donor-recipient relationship was likely. Our analyses suggest that intrahost genomic variation in SARS-CoV-2 might be generally limited and that homoplasy and recurrent errors complicate identifying shared intrahost variants. Reliable reconstruction of direct SARS-CoV-2 transmission based solely on genomic data seems hindered by a slow mutation rate, potential convergent events, and technical artifacts. Detailed contact tracing seems essential in most cases to study SARS-CoV-2 transmission at high resolution.
ABSTRACT
Background: SARS-CoV-2 variation represents a serious challenge to current COVID-19 vaccines. Recent reports suggest that B.1.351 and P1/P2 variants may escape the neutralization activity of the antibodies generated by BNT162b2 mRNA vaccine.Methods: Ninety-nine healthcare workers undertaking BNT162b2 mRNA vaccination were sampled at baseline, on the day of the second dose, and 14 days after the latter. Neutralization activity against SARS-CoV-2 B.1, B.1.1.7 and B.1.351 was investigated using a Vero-E6 model.Results: Eleven of the study participants had prior infection with SARS-CoV-2. Neutralization titers against the B.1 and the B.1.1.7 variants were not statistically different and were significantly higher than titers against the B.1.351 variant across pre-exposed and non-pre-exposed vaccinated individuals (p<0.01). While all vaccinated individuals presented neutralizing antibodies against B.1 and B 1.1.7 after the second dose, 14% were negative against B.1.351, and 76% had low titers (1/20-1/80). Pre-exposed vaccinated individuals showed higher titers than non-pre-exposed after the first (median titers of 1/387 versus 1/28, respectively) and the second doses (1/995 versus 1/703, respectively). As high as 72% of the pre-exposed vaccinees presented titers >1/80 after a single dose, while only 11% of non-exposed vaccinated individuals had titers >1/80.Conclusions: BNT162b2 mRNA-induced antibodies show a lower in vitro neutralizing activity against B.1.351 variant compared to neutralization against B.1.1.7 or B.1 variants. Interestingly, for individuals pre-exposed to SARS-CoV-2, one dose of BNT162b2 mRNA may be adequate to produce neutralizing antibodies against B.1.1.7 and B.1, while two doses of BNT162b2 mRNA provide optimal neutralizing antibody response against B.1.351 too.Funding Statement: None to declare.Declaration of Interests: None to declare.Ethics Approval Statement: The protocol was approved by the Ethics Committee of the Hospital Universitario Clínico San Cecilio (HUSC 0670-N-21). All participants provided informed consent.
Subject(s)
COVID-19ABSTRACT
Background: Workers and residents in Care Homes are considered at special risk for the acquisition of SARS-CoV-2 infection, due to the infectivity and high mortality rate in the case of residents, compared to other containment areas. The role of presymptomatic people in transmission has been shown to be important and the early detection of these people is critical for the control of new outbreaks. Pooling strategies have proven to preserve SARS-CoV-2 testing resources.The aims of the present study, based in our local experience, were (a) to describe SARS-CoV-2 prevalence in institutionalized people in Galicia (Spain) during the Coronavirus pandemic and (b) to evaluate the expected performance of a pooling strategy using RT-PCR for the next rounds of screening of institutionalized people. Methods: A total of 25,386 Nasopharyngeal swab samples from the total of the residents and workers at Care Homes in Galicia (March to May 2020) were individually tested using RT-PCR. Prevalence and quantification cycle (Cq) value distribution of positives was calculated. Besides, 26 pools of 20 samples and 14 pools of 5 samples were tested using RT-PCR as well (1 positive/pool). Pooling proof of concept was performed in two populations with 1.7% and 2% prevalence. Results: Distribution of SARS-CoV-2 infection at Care Homes was uneven (0-60%). As the virus circulation global rate was low in our area (3.32%), the number of people at risk of acquiring the infection continues to be very high. In this work, we have successfully demonstrated that pooling of different groups of samples at low prevalence clusters, can be done with a small average delay on Cq values (5 and 2.85 cycles for pools of 20 and 5 samples, respectively). Conclusions: A new screening system with guaranteed protection is required for small clusters, previously covered with individual testing. Our proposal for Care Homes, once prevalence zero is achieved, would include successive rounds of testing using a pooling solution for transmission control preserving testing resources. Scale-up of this method may be of utility to confront larger clusters to avoid the viral circulation and keeping them operative.
Subject(s)
COVID-19 , Coronavirus InfectionsABSTRACT
SARS-CoV-2 genetic material is detectable in the faeces of a considerable part of COVID-19 cases and hence, in the urban wastewater. This fact was confirmed early during the spread of the COVID-19 pandemic and prompted several studies that proposed monitoring its incidence by wastewater. This paper studies the fate of SARS-CoV-2 genetic material in wastewater treatment plants using RT-qPCR with a two-fold goal: i) to check the safety of the water effluent and also of the sludge produced and ii) based on the understanding of the virus particles fate, to identify the most suitable spots for detecting the incidence of COVID-19 and monitor its evolution. On the grounds of the affinity of enveloped virus towards biosolids, we hypothesized that the sludge line acts as a concentrator of SARS-CoV-2 genetic material. Sampling several spots in primary, secondary and sludge treatment at the Ourense (Spain) WWTP showed that, in effect, most of SARS-CoV-2 particles cannot be detected in the water effluent as they are retained by the sludge line. We identified the sludge thickener as a suitable spot for detecting SARS-CoV-2 particles thanks to its higher solids concentration (more virus particles) and longer residence time (less sensitive to dilution caused by precipitation). Although more studies will be needed for confirmation, these results contribute to clarify the role of WWTPs in COVID-19 mitigation.