The molecular epidemiology of SARS-CoV-2 in the Pityusic Islands shows multiple introductions and fast replacements of variants in a touristic worldwide hot spot

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 RT-qPCR amplification patterns visualization, single nucleotide polymorphism 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 (January 2021–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.

The Pityusics are placed in the Western Mediterranean and comprise two major islands, Ibiza and Formentera, with a total population of about 160.000 habitants 1 .Both islands are major tourist destinations worldwide, with an important volume of domestic tourism and principal international connections to the UK and Germany 2 .However, the National lockdown imposed in Spain from 14 March to 4 May 2020 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, affected the epidemiology of SARS-CoV-2 in our health area.The first analyses of the SARS-CoV-2 genomic epidemiology in the Balearic Islands were conducted with the samples taken during the three first pandemic waves, concluding that the Balearics constituted a unique setting in which the course of the pandemic was influenced by a complex interplay between insularity, severe social restrictions, and tourism travels 3 .In this work, we delved into the incidence and prevalence of the main SARS-CoV-2 circulating variants during a prolonged period of time (from the third to the sixth pandemic waves) in two of the main Balearic tourist destinations.
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 4 .The monitoring and follow-up of these variants have been

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 only 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. 1B).During this period, 1319 positive samples were genotyped, of which 57 were not amplified (Cts > 32) and 1246 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. 1C).

Incidence and prevalence of VOC Omicron subvariants 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. 1C).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 declined, 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 subvariants within Omicron lineage were highly variable along the time, starting with a progressive replacement of BA.1 by BA.2, which became the dominant subvariant from February to May/June 2022 (see below).Subvariants 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 accession codes of the sequences deposited in Gisaid (gisaid.org)can be found in Supplementary Table S1.Only 27 almost complete genome sequences, marked in bold in Supplementary Table S1, could not be deposited in Gisaid because did not fulfill the length requirements of this database.However, these sequences were used with confidence for the lineage assignment with the pangolin tool since all of them could be considered as high quality sequences with high coverage level (96% of the reference sequence).All genomes are publicly available at the repository of Discovery Environment (Cyverse) through the following link: https:// data.cyver se.org/ davan on/ iplant/ home/ tomeu/ Viver_ et_ al_ 2023/ GENOM ES_ COVID.zip.
Vol:.( 1234567890 2A, and concordantly with RT-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.1.221was detected in 10% of the analyzed samples, distantly followed by lineage B.1.177(2%), VOC Gamma (1%), 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.1.221were 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. 2A).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. 3) 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. 2A 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. 2B).BA.1 was progressively replaced, first by BA.2 and finally by BA.5, that were the dominant subvariants from the middle March to June (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. 2B and 3).

Discussion
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 11 .First cases in Balearic Islands were detected by the end of March 2020, when initiatives to monitor the genetic diversity and features of the circulating variants, mainly based on the genomic surveillance, were established by the Spanish Health System 12 .
The first study addressing the evolution of SARS-CoV-2 infections in the Balearic Islands concluded that its relatively small size and the organization of health and epidemiological surveillance systems made the official accounts of reported cases more reliable than in other areas 2 .However, due to the impossibility to carry out extensive genomic sequencing in most of the islander hospital facilities, the monitoring and tracking of the circulating variants had to rely in other PCR-based strategies, such as the detection of the SGTF genomic signature and/or high-throughput genotyping methods.
The SGTF genomic signature for the detection of the Alpha and Omicron variants (B.1.1.529,BA.1, BA.4 and BA.5) provide a very rapid and accurate proxy for their identification.In fact, at the peak of their corresponding wave, the positive predictive value of the SGTF was 98% for Alpha and 100% for Omicron 13 .On the other hand, the genotyping RT-qPCR methods have showed increased sensitivity over sequence-based approaches, allowing for their quantitation, and providing readily interpretable results within hours.However, designing assays for VOCs is a process requiring continuous adaptation and validation since emerging VOCs may possess unique mutations that necessitate the development of new assays 5 .Specifically, TaqMan SARS-CoV-2 mutation panel molecular genotyping assays allow the detection and differentiation of the most common variants that have been used for surveillance and epidemic control and prevention: B.1.1.529(Omicron), B.1.617.2 (Delta), B.1.1.7 (Alpha), B.1.526(Iota), B.1.351(Beta), P.1 (Gamma), P.2 (Zeta), B.1.617.1 (Kappa), and B.1.427/B.1.429(Epsilon) 14 .
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.Specifically, the dynamics of the SARS-CoV-2 variants in our health area was characterized mainly by the amplification patterns visualization and SNP genotyping, whereas sequencing was only performed in weekly, randomly selected samples.www.nature.com/scientificreports/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, namely the VOCs Beta and Gamma, although their prevalence remained low.The replacement of these 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 total number of 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 that period.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. 2A).From its first detection in late May 2021, it rapidly increased in prevalence, out-competing pre-existing lineages and becoming the most common variant (almost the totality of the cases detected from July to late November), as occurred in many other Spanish locations and in other countries 15,16 .The emerging of VOC Delta 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 Pytiusics 17,18 .However, nationwide records of COVID-19 hospitalizations in Spain indicated that the mass vaccination prevented a fourth wave of hospitalization after Eastern holidays 19 .
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 20 .The selective advantage of Omicron over Delta may be explained again by increased transmissibility and by higher immune escape 21 .
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 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 uniformly highlight that changes in neutralization activity were not translated into complete vaccine failure with symptomatic illness 22,23 .In fact, vaccine protection remains clinically evident based on the decrease in the rate of COVID-19 hospitalizations in Spain since vaccination started 19 .
Summarizing, the SARS-CoV-2 epidemics in ASEF was characterized by four differentiated phases: (i) in a first stage, a progressive replacement of non-VOC variants by VOC Alpha during the first half of year 2021 and a total replacement of VOC Alpha by VOC Delta during the second half was observed; (ii) in a second stage, a fast replacement of VOC Delta by VOC Omicron BA.1 during December 2021 and early January 2022 was detected, (iii) during the third one, it was detected a rapid replacement of Omicron BA.1 by Omicron BA.2 (from March to June 2022), and lastly, a fast replacement of BA.2 by BA.5, from mid-2022 to the end of the studied period.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 24 .
The relative geographical isolation of the Balearic Islands and their central role as a hub for international tourism along with the extension of vaccination, affected the COVID-19 pandemic in the Health Area of Ibiza and Formentera 3 , outlining the introduction, spread and extinction of different lineages and VOCs through time.The comparative study of the epidemiology of SARS-CoV-2 in different Spanish regions across the six epidemic waves reported that leading European tourist destinations, as Balearics, could explain the high diversity of circulating variants observed as compared with others worldwide 25 .
As SARS-CoV-2 became 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 years 2022 and 2023, novel variants of SARS-CoV-2 could continuously threaten public

Figure 2 .
Figure 2. Sequencing results obtained the randomly, weekly selected positive SARS-CoV-2 samples in years 2021 (A) and year 2022, from January to the end of July (B).

Figure 3 .
Figure 3. Phylogenetic reconstruction of a collection of 917 complete sequenced SARS-CoV-2 genomes.All SARS-CoV-2 variants have been represented in different colours in the internal ring, as indicated in the legend.In the external ring, grey gradient colours indicates the sampling dates (in months) for year 2020, gradient colour from black to brown for samples collected in 2021, and brown to red for samples obtained in 2022.Bar indicates 0.1% sequence divergence.
During year 2021, up to 543 of the generated consensus sequences (99.4%) were suitable for Pangolin lineage assignment.As shown in Fig.

Table 2 .
Affiliation of the genotyped samples.NA Not amplified.