Molecular Epidemiology of Sars-CoV-2 in the Pityusic Islands, a Touristic Worldwide Hotspot in the Western Mediterranean

doi:10.21203/rs.3.rs-2727107/v1

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Molecular Epidemiology of Sars-CoV-2 in the Pityusic Islands, a Touristic Worldwide Hotspot in the Western Mediterranean

https://doi.org/10.21203/rs.3.rs-2727107/v1

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published 23 Oct, 2023

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The public health emergency caused by the Covid-19 outbreak in March 2020 encouraged worldwide initiatives to monitor the genetic diversity and features of the SARS-CoV-2 circulating variants, mainly based on the genomic surveillance. However, due to the impossibility to carry out extensive sequencing in resource-limited
hospitals, other PCR-based strategies could be applied to efficiently monitor the circulating variants without the need to greatly expand the sequencing capacity. In our case, overpassing the technical limitations inherent to a second level hospital, we were able to characterize the weekly distribution of SARS-CoV-2 by the amplification patterns visualization, genotyping, and sequencing of randomly selected samples. All these molecular approaches allowed us to trace the epidemiology of SARS-CoV-2 viruses circulating in Ibiza and Formentera (Balearic Islands, Spain) during the third to the sixth pandemic waves, in which three major lineages that were considered as VoCs (alpha, delta, and omicron), and many other non-VoC variants were detected and tracked.

Health sciences/Health care/Public health/Epidemiology

Biological sciences/Microbiology/Policy and public health in microbiology

Since its detection in December 2019, the virus that causes severe acute respiratory syndrome or coronavirus disease (COVID-19) has naturally accumulated mutations in its genome, leading to the emergence of multiple variants worldwide. Some of them have been classified as variants of concern (VoCs), because they have characteristics that imply the possibility of being more transmissible, causing a more serious disease, and even evading the natural immune and vaccination response [1]. The monitoring and follow-up of these variants have been fundamental to fight against the pandemic, and worldwide sequencing initiatives were advised as the best tool for the acute analyses of the mutations accumulated in the circulating variants and their spreading [2]. However, genomic tools, specifically genome sequencing technologies are not widely available, and even in developed countries the facilities needed for large-scale sequencing are limited [3]. This is the case of most county and regional, small hospitals in Spain. These sanitary facilities depend on the reference hospitals sequencing services, and only can afford the analyses of a small representative number of all the positives detected. Nevertheless, the visualization of the amplification patterns as well as genotyping techniques were able to help significantly in delineating the epidemiology of SARS-CoV-2, providing rapid, cost-effective, and reliable ways to monitor SARS-CoV-2 variants circulating during the outbreak [4; 2].

The Pityusic Islands are placed in the Western Mediterranean and comprise two major islands, Ibiza and Formentera, with a total population of about 160.000 habitants [5]. In this area, two second-level hospitals (Can Misses and Formentera Hospital) provide public health services to the resident and touristic population. Both islands are major tourist destinations worldwide, with an important volume of domestic tourism and principal international connections to the UK and Germany [6]. However, the National lockdown imposed in Spain from 14 March to 4 May and the travel restrictions during years 2020 and 2021 to control COVID-19 dissemination increased the insularity of the area. These actions, together with the imposed social measures and the vaccination started at the beginning of 2021, have shaped the epidemiology of SARS-CoV-2 in our health area.

Some specific PCR assays, by including the S gene among the analyzed viral targets, can identify variants carrying the Δ69–70 deletion, as the VoCs alpha and omicron BA.1 and BA.5 [7]. Therefore, this target failure (Spike Gene Target Failure, SGTF) has been used as a screening test to track these VoCs, overcoming the necessity of sequencing. Moreover, other PCR assays (allele-specific RT-qPCR) were developed to identify the presence of other common mutations, in addition to the Δ69–70, emerging as potential solutions for identifying SARS-CoV-2 variants [8]. We have investigated the proportion of SGTF cases among the samples with a positive result for SARS-CoV-2 as an approximation to know the frequency and permanence of the VoCs alpha and omicron BA.1/BA.5 in the Health Area of Ibiza and Formentera (ASEF), as well as single nucleotide polymorphism genotyping to detect the VoCs delta and omicron BA.2. Moreover, randomly selected positive samples were weekly collected and sent to the reference hospital of the Balearic Islands (Son Espases, Majorca) for complete genome sequencing.

Herein, we analyze and evaluate the diversity, incidence, and prevalence of circulating SARS-CoV-2 variants in the Health Area of Ibiza and Formentera (ASEF) in the period covering from January 2021 to July 2022, in which three major lineages that were considered as VoCs (alpha, delta, and omicron), and many other non-VoC variants were detected and tracked.

RT-qPCR detection of SARS-CoV-2 in the Health Area of Ibiza and Formentera

As shown in Table 1, 97,866 respiratory samples were processed for the diagnosis of Covid-19 during the study period (77,919 in 2021 and 19,947 in 2022). The number of SARS-CoV-2 positive samples was 16,946 in total, of which 12,073 were detected during 2021 and 4873 during 2022, representing a mean positivity of 12 and 15%, respectively. The highest rate of positivity in year 2021 took place during January (averaged 23%) and August (averaged 26%), corresponding to the 3rd and 4th -5th pandemic waves, with maximums of 29% and 33% in the fourth week of the year (week 2021-04) and the second of August (week 2021-32), respectively (Fig. 1 and Fig. 2). In 2022, the peak in the positivity rate occurred during the 6th pandemic wave, with a historic maximum value of 48% in the second week of the year (week 2022-02, Fig. 3). The mean positivity during the 6th pandemic wave was around 39%, which represents the maximum positivity record during a wave in our health area since the beginning of the Covid-19 pandemic.

Table 1

Monthly number of PCRs analyzed and mean positivity during the six pandemic periods included in the study.
Pandemic period	Year	Month	Total number of PCRs	Mean Positivity (%)
3rd wave	2021	January	15696	22,9
3rd wave		February	7744	16,8
Inter-wave		March	4838	2,9
		April	3571	4,3
		May	3568	3,0
		June	3609	6,7
4th-5th waves		July	8311	25,9
4th-5th waves		August	6844	19,9
Inter-wave		September	5194	7,3
		October	4481	6,6
		November	4785	8,1
6th wave		December	9278	21,2
	2022	January	8015	39,2
		February	3638	16,7
After-wave		March	2376	10,1
		April	1674	8,7
		May	1903	10,8
		June	1447	18,7
		July	894	29,5

Incidence and prevalence of VoC alpha by SGTF amplification pattern detection

The visualization of the RT-qPCR patterns obtained in respiratory samples after the diagnostic procedure for the detection of SARS-CoV-2 allowed detecting the presence of the VoC alpha (SGFT + pattern) in ASEF the last days of December 2020, coinciding with the onset of the third pandemic wave. From its first detection, both the mean global positivity and the frequency of the SGTF pattern showed a clear increasing trend, reaching at the end of January (week 2021-04) maximums of 28% and 35%, respectively (Fig. 1). The maximum peak of detection of VoC alpha was at the end of April 2021, when it represented up to 87% of all the positives detected in ASEF. During February (week 2021-07), the positivity rate started to decrease until reaching values around 6% at the end of June (week 2021-24, Fig. 1), when the 4th and 5th pandemic waves began. At that time, the detection of the SGTF pattern fell to 4%, while the positivity rate increased up to 33%. The weekly number of analyzed samples, the percentage of positivity and the frequency of the SGTF pattern (indicative of alpha variant detection) during this period are shown in Fig. 1.

Incidence and prevalence of VoC delta by genotyping analyses

We could implement the mutation panel for VoC delta genotyping in mid-August 2021, so during some period we had evidence of the increasing proportion of the VoC delta through the analyses of the sequencing data obtained from the randomly selected sequences. The sequencing results weekly obtained from SARS-CoV-2 positive samples (see below) revealed that this VoC emerged in ASEF in late May 2021 (week 2021-22), becoming rapidly the predominant one throughout the 4th and 5th pandemic waves. As shown in Table 2, the genotyping panel showed that most of the samples (96%) were affiliated to VoC delta (the remaining 4% were not amplified, Cts > 32). In fact, until the end of the year, it was by far the predominant circulating variant in our health area (Fig. 2). During this period, 1,319 positive samples were genotyped, of which 57 were not amplified (Cts > 32) and 1,246 were confidently affiliated to VoC delta (Table 2). Thereafter, and coinciding with the 6th pandemic wave, we only could afford the genotyping of the positives obtained one day per week (385 in total). One hundred and thirteen samples were affiliated to VoC omicron BA.1 by direct visualization of the amplification patterns (see below). The rest were genotyped (272 in total), of which 37 were not amplified (Cts > 32), and 235 were affiliated to VoC delta, corresponding to 96% of the total positive samples (Table 2).

From the second week of December (week 2021-50) to the end of the study period there was an almost complete displacement of the VoC delta by the VoC omicron, which became the predominant VoC throughout year 2022 (Fig. 3).

Incidence and prevalence of VoC omicron sub variants by SGTF amplification pattern detection and genotyping

The visualization of the RT-qPCR patterns allowed detecting the presence of the VoC omicron BA.1 (SGFT + pattern) in ASEF at the beginning of December 2021 (week 2021-49), when the mean positivity began to increase, and the sixth wave started in Spain. Both, the mean global positivity and the frequency of the SGTF + pattern showed an increasing tendency along January 2022, reaching values during the first week of the year of 48% and 94%, respectively (Fig. 3). The genotyping panel used for the VoC delta discrimination was also useful to confidently discriminate the VoC omicron BA.2, the “stealth” omicron, which emerged in our health area in January 2022. As shown in Table 2, 751 positive samples were genotyped, obtaining 50 samples affiliated to VoC delta (7%) and 626 to VoC omicron BA.2 (83%), whereas 75 samples (10% of the total) could not be amplified (Cts > 32).

Seven weeks after the onset of the 6th wave, the positivity rate began to decline, reaching a value of around 7% on early March (week 2022-13). However, the frequency detection of VoC omicron did not change, remaining close to 100% during the rest of the analyzed period. In contrast, the proportions of the different sub variants within omicron lineage were highly variable along the time, starting with a progressive replacement of BA.1 by BA.2, which became the dominant sub variant from February to May/June 2022 (see below). Sub-variants BA.3 and BA.4 were detected in very low proportions along the year, being BA.5 the one that progressively replaced BA.2 in the last period (see below). The number of genotyped samples and the frequencies of VoCs omicron and delta from early December 2021 (week 2021-49) to July 2022 (week 2022-30) are detailed in Table 2.

SARS-CoV-2 genetic diversity in ASEF as revealed by sequencing. Phylogenetic analyses.

Whole genome sequences were obtained from 916 randomly selected among the positive respiratory samples. The obtained sequences were deposited in Gisaid (gisaid.org) and accession codes can be found in Supplementary Table S1. Only 27 almost complete genome sequences, marked in bold in Supplementary Table S1, could not be deposited. These sequences could be used for the accurate lineage assignment with the pangolin tool but did not fulfil the length requirements of Gisaid database. During year 2021, up to 543 of the generated consensus sequences (99.4%) were suitable for Pangolin lineage assignment. As shown in Fig. 4, and concordantly with qPCR amplification patterns analyses and genotyping, the VoCs alpha and delta were identified as the two major lineages circulating in ASEF in 2021, representing 32% and 50% of the total sequences, respectively. Lineage B.1.221 was detected in 10% of the analyzed samples, distantly followed by lineage B.1.177 (2%), VoC gamma (1%), VoI iota (1%), VoC beta (0.8%), lineage B.1 (0,6%), VoI epsilon (0.6%), VoI lambda, and lineage B.1.165 (0.3% each), being VoI “Variant of Interest”. During the third epidemic wave (week 01-week 05 in 2021), the VoC alpha and lineage B.1.221 were the most dominant variants, appearing at different proportions along this period (averaging 76% and 27%, respectively), although a very small number of other variants were also identified, including VoCs beta and gamma (Fig. 4). The VoC alpha remained the most prevalent variant until mid-June, when the 4-5th epidemic curve began in Spain and the VoC delta emerged in our health area. The phylogenetic analyses (Fig. 6) also showed that VoC delta gradually increased its proportion and displaced completely other variants by middle July (week 2021-28), in concordance with the fact that it was the only variant detected by sequencing and genotyping until the end of the year (Fig. 4 and Table 2). In year 2022, 373 sequences were suitable for Pangolin assessment, being those affiliated to VoC omicron BA.1 the most detected at the beginning of the year (accounting for 82% of the total sequences obtained during the first three weeks, Fig. 5). BA.1 was progressively replaced, first by BA.2 and finally by BA.5, that were the dominant sub variants from the middle March to Juny (week 2022-11 to week 2022-22) and at the end of the study period (from week 2022-23 to week 2022-30), respectively (Figs. 5 and 6).

SARS-CoV-2-related outbreak was declared by the World Health Organization (WHO) as a public health emergency of international concern on 30 January 2020, due to an increase in the number of imported cases worldwide [9]. First cases in Balearic Islands were detected by the end of March 2020, when initiatives to monitor the genetic diversity and features of circulating variants established by the National Health System, mainly based on the genomic surveillance, started to be applied in Balearic Islands [10]. However, due to the impossibility to carry out extensive genomic sequencing in our hospital facilities, other PCR-based strategies have been used to continuously monitor the circulating variants in our health area. Thus, the weekly distribution of SARS-CoV-2 in Ibiza and Formentera islands was characterized by the amplification patterns visualization, genotyping, and/or sequencing of weekly, randomly selected samples. These strategies allowed us to detect the predominant lineages and VoCs in each pandemic period, as well as the emergence and extinction of the different lineages through time.

Overall, the most prevalent variant during the first half of year 2021 was the VoC alpha, which was first detected in ASEF on the 29th of December 2020 and strongly associated with an increase in the incidence. Interestingly, during this period other lineages carrying multiple mutations in the spike and considered as VoCs by the WHO and the European Center for Disease Control (ECDC) emerged in our area, mainly the VoCs beta and gamma, although their prevalence remained low. The replacement of these VoCs and other non-VoC lineages occurred progressively, and VoC alpha did not become majoritarian until March 2021, when it accounted for more than 50% of the positives detected. Of note, the VoC alpha did not trigger a high hospital burden in Spain nor in Balearics, but it likely contributed to high community transmission rates due to its enhanced transmissibility and the increasing social interactions during the first part of year 2021. The restrictive measures established in the Pityusics during 2020 to control the movement of travelers in ports and airports were progressively relaxed during 2021, restoring in part the social interactions, the family and mass gatherings, and the possibility of traveling to and from the islands.

During the second half of year 2021, when the vaccination campaign was in an advanced stage, with about 80% of the ASEF population immunized, the VoC delta was by far the predominant one (see Table 2 and Fig. 4). From its first detection in late May 2021, it rapidly increased in prevalence, out-competing pre-existing lineages and becoming the most common variant in ASEF (virtually 100% of the cases detected from July to late November), as occurred in many other Spanish locations and in other countries [11]. The emerging of VoC delta and sub-lineages (mainly AY.43 and AY.122) was associated with a rapid surge of Covid-19 cases in the area, provoking the fourth and fifth waves and the highest number of positive cases detected during 2021. It is now known that the competitive advantage of the delta variant relied in two main characteristics, its increased transmissibility, with higher infectious virus loads, and its resistance to natural and vaccine-induced immunity, which probably contributed to the rapid and intense transmission in the Pytiuses [12, 13]. However, nationwide records of Covid-19 hospitalizations in Spain indicated that the mass vaccination prevented a fourth wave of hospitalization after Eastern Time [14].

The VoC omicron was detected in December 2021, becoming dominant across the area in less than one month, significantly increasing SARS-CoV-2 transmission, even when the vaccinated population was around 90%. This VoC accumulated many mutations that conferred a marked advantage over pre-circulating lineages, causing a major upsurge of cases in multiple countries worldwide since November 2021 [15]. The selective advantage of omicron over delta may be explained again by increased transmissibility and by higher immune escape [16].

The introduction of vaccines at the end of 2020, prioritizing the elderly and healthcare workers, and the subsequent steady vaccination by age groups, could have had an effect in favoring the selection of vaccine-scape mutants, although no evidence has been found in favor of this threat. On the contrary, a number of published studies addressing this issue uniformly highlight that changes in neutralization activity are not translated into complete vaccine failure with symptomatic illness [17, 18]. In fact, vaccine protection remains clinically evident based on the decrease in the rate of Covid-19 hospitalizations in Spain since vaccination started [14].

The omicron epidemics in ASEF was characterized by three phases: i) progressive replacement of non-VoC variants by VoC alpha during the first half of year 2021 and total replacement of VoC alpha by VoC delta during the second half of the year; ii) very rapid replacement of VoC delta by omicron BA.1 during December 2021 and early January 2022, iii) rapid replacement of omicron BA.1 from March to Juny 2022 by omicron BA.2, and, iv) again a fast replacement of BA.2 by BA.5, from mid-year to the date of writing this manuscript. As has been extensively reported, the VoC omicron did not suppose a public health concern in our area, since has reduced probability of hospital admission, with shorter period of illness and less infectiousness potential [19].

The present study attends to report the epidemiology of SARS-CoV-2 viruses circulating during the third to the sixth pandemic waves in Ibiza and Formentera (Balearic Islands, Spain), when restrictive measures began to relax, and the vaccination was steadily increased. The relative geographical isolation of the Balearic Islands and their central role as a hub for international tourism along with the extension of vaccination seem to have shaped the Covid-19 pandemic in the Health Area of Ibiza and Formentera, outlining the introduction, spread and extinction of different lineages and VoCs through time.

As SARS-CoV-2 has become endemic in most areas, local governments relaxed the quarantine policies and public health measures for a gradual return to normal life. Although the number of confirmed cases was gradually decreasing during year 2022, novel variants of SARS-CoV-2 continuously threaten public health [20]. In this regard, it is important to highlight that the multiple introductions and fast replacement of VoCs detected during this study reinforce the idea that the quarantine policies will continue require improved variant monitoring systems and virus spread control, in addition to other effective strategies such as vaccination, hygienic habits, social measures, and health education.

Sample collection

For SARS-CoV-2 diagnosis, nasopharyngeal samples were collected in 3ml vials with minitip flocked swabs, designed for the conservation and transport of respiratory pathogens (“KaiBiLi” viral transport medium, Genesis). The vials were transported at 4°C from the different sampling centers to the Can Misses Hospital, individually bagged in order to avoid possible cross contaminations.

We analyzed the daily diagnostic data of SARS-CoV-2 obtained by RT-PCR in the Molecular Biology Laboratory of the Microbiology and Parasitology Service of the Can Misses Hospital, in the period covering from 1st January 2021 to 31st July 2022. During the studied period, our laboratory received samples from all public health centers and services belonging to ASEF, that comprises two hospitals (located in Ibiza and Formentera), 59 health centers, and 104 primary care units that are scattered over the two islands, all of them belonging to the Balearic Public Health System (Ib-Salut) and the Spanish Government Health System (SNS).

One day per week, randomly selected positive samples (1040 in total) were sent to the Microbiology Department of the reference hospital (Son Espases University Hospital) for sequencing, where they were stored frozen at -80ºC until use.

Nucleic acid extractions and amplification of viral genes

After vigorous vortexing of the vials, 200µl of homogenized samples were used for extractions. To obtain the genetic material, extraction was performed with MagMax™-96 Viral RNA Isolation Kit (Applied Biosystems) and the KingFisher automatic extractor (Thermofisher), following the protocols indicated by the manufacturers. Once the process was finished, the viral purified nucleic acids were eluted in 53µl of the elution buffer provided in the kit.

Subsequently, the amplification of three viral gene fragments was performed with 10 µl of the extracted nucleic acids with the "TaqPath™ COVID-19 CE-IVD RT-PCR" diagnostic kit from Applied Biosystems in a QuantStudio 5 thermocycler (Thermofisher), as recommended by the manufacturer. The RT-PCR assay is aimed at the amplification of three specific regions of the SARS-CoV-2 genome contained in the ORF1ab, N and S genes. The thermal protocol consists in 40 amplification cycles (3 s at 95°C, 30 s 60°C), preceded by 2 min at 25°C (UNG incubation) and 10 min at 53°C (retrotranscription). Data were analyzed with the QuantStudio Design and Analysis Software v2.4 and the COVID-19 Interpretive Software (Thermofisher).

Detection of VoCs alpha and omicron BA.1/BA.5 by amplification patterns visualization

Depending on the amplification pattern obtained after diagnostic PCR, positive samples were classified into two groups: those with the failure in the amplification of the S gene fragment (SFGT + samples, corresponding to VoCs alpha and omicron BA.1/BA.5, depending of the period of time), and those with the typical amplification pattern in which the S gene is normally amplified (SFGT-, corresponding to other variants).

Detection of VoCs delta and omicron BA.2 by genotyping analyses

In the positive samples with the typical amplification pattern (SGFT-), we could detect the delta and omicron BA.2 variants by using the Applied Biosystems TaqMan SARS-CoV-2 Mutation Panel assay L452R combined with TaqPath 1-Step RT-qPCR Master Mix, following the manufacturers´ instructions. Genotyping was performed with a QuantStudio5 Real-Time PCR Instrument. Data were analyzed with the QuantStudio Design and Analysis Software v2.4 with the Genotyping Analysis Module. During the 6th pandemic wave, from December 2021 to late January 2022, we could only afford the genotyping of the positives obtained one day per week, as the positivity rate increases considerably, and the high quantity of samples were not manageable in our facilities. During these two months, the positive samples obtained in days 9, 13, 21, and 28 of December 2021 (representing weeks 48–52 2021) and days 4, 11, 18 and 25 of January 2022 (weeks 1–4 2022) were selected for genotyping. Thereafter, and comprising the period from February to the end of Juny, all the positive samples with SFGT- pattern obtained were genotyped.

Genomic library preparation and sequencing

Genomic library preparation was conducted following the SeqCovid Consortium protocols [9]. Briefly, RNA was first retro-transcribed into cDNA and SARS-CoV-2 complete genome amplification was conducted in two parallel multiplex PCR, accordingly to the openly available protocol developed by the ARTIC network [21] and using the V3, V4 y V4.1 multiplex primers scheme. Resulting amplicon pools were then combined and cleaned using AMPure beads (Beckman Coulter), and 50 ng were used to prepare the Illumina sequencing libraries (Illumina DNA Prep kit, Illumina Inc., San Diego, CA) according to the manufacturer’s protocol and with 5 cycles for indexing PCR (Nextera DNA CD Indexes, Illumina). Finally, indexed genomic libraries were pooled in equimolar amounts and loaded onto a MiSeq v3 cartridge (2x250 cycles).

Lineage assignment and spike mutation surveillance

Two different bioinformatic approaches were used for whole genome sequence analysis: an open source pipeline based on IVAR (https://gitlab.com/fisabio-ngs/sars-cov2-mapping) and the DRAGEN COVID Lineage App available version (Illumina®). Pipelines, map quality- and primer- trimmed viral reads to the hCoV-19/Wuhan/WIV04/2019 reference sequence genome (MN908947.3/ NC_045512.2), result in the generation of consensus whole genome sequences.

Consensus sequences covering at least 75% of the reference sequence and with a median coverage > 100 reads were considered for lineage assignment. For this purpose, the Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) tool was used (https://github.com/cov-lineages/pangolin), employing the latest version and the most updated lineage database available at the time of assignment. Detection of additional spike amino acid substitutions was conducted by using the Basic Local Alignment Search Tool (BLAST) and the spike protein of the hCoV-19/Wuhan/WIV04/2019 strain as reference.

Phylogenetic analyses

The 916 complete sequenced SARS-CoV-2 genomes were aligned using the MUSCLE tool v3.8.31 [22]. The phylogenetic analyses and tree reconstruction were carried out using the neighbour-joining algorithm implemented in the ARB software (v6.0.6) using the Jukes-Cantor correction [23]. The obtained phylogenetic tree was annotated and edited using the on-line tool “Interactive Tree of Life (iTol)”, available at https://itol.embl.de [24].

Ethics statement

This work has followed the ethical guidelines included in the Declaration of Helsinki. Informed consent was not required as this study was performed in the context of the SARS-CoV-2 surveillance system in Balearics, as a part of the Spanish Surveillance Network (https://www.seqcovid.csic.es). Hospital Universitari Son Espases is our reference hospital in which the sequencing was performed following standarized and approved protocols validated by the Instituto de Salud Carlos III of Spain (www.isciii.es).

ACKNOWLEDGEMENTS

We gratefully acknowledge the technical and facultative staff of the Microbiology Services of Can Misses Hospital and Son Espases Universitary Hospital.

AUTHOR CONTRIBUTIONS

TV analyzed the sequencing results, made the phylogenetic analyses, wrote the results section and critically revised the rest of the manuscript. CLC, PFR, and GT collected the data, contributed data and analysis tools. CMP, DLS and GJG performed the PCR experiments and contributed with laboratory protocols and analysis tools. DLS made the bibliographic searching and formating. ALL conceived the study, analyzed and plotted the PCR and genotyping results, made the figures and wrote the manuscript. All authors critically reviewed the manuscript.

DATA AVAILABILITY

The datasets obtained in this study can be found in Gisaid database (gisaid.org), under the accession codes listed in Supplementary Table S1.

FUNDING

This work has been supported by the Instituto de Salud Carlos III of Spain through the project COV20/00140: Addressing unknowns of COVID-19 transmission and infection combining pathogen genomics and epidemiology to inform public health interventions and European Union HERA Incubator program through grant ECDC/HERA/2021/024 ECD.12241. CL-C was supported by a Juan Rodés contract (JR19/00003) from Instituto de Salud Carlos III.

CONFLICT OF INTEREST

The authors declare that this research was conducted without any commercial relationships that could be construed as potential conflict of interest.

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Molecular Epidemiology of Sars-CoV-2 in the Pityusic Islands, a Touristic Worldwide Hotspot in the Western Mediterranean

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Journal Publication

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Abstract

Figures

Introduction

Results

RT-qPCR detection of SARS-CoV-2 in the Health Area of Ibiza and Formentera

Incidence and prevalence of VoC alpha by SGTF amplification pattern detection

Incidence and prevalence of VoC delta by genotyping analyses

Incidence and prevalence of VoC omicron sub variants by SGTF amplification pattern detection and genotyping

Discussion

Methods

Sample collection

Nucleic acid extractions and amplification of viral genes

Detection of VoCs alpha and omicron BA.1/BA.5 by amplification patterns visualization

Detection of VoCs delta and omicron BA.2 by genotyping analyses

Genomic library preparation and sequencing

Lineage assignment and spike mutation surveillance

Phylogenetic analyses

Ethics statement

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1