Evolutionary and spatiotemporal analyses reveal multiple introductions and cryptic transmission of SARS-CoV-2 VOC/VOI in Malta

ABSTRACT Genomic surveillance and epidemiology have shed light on the viral diversity driving coronavirus disease 2019 (COVID-19) outbreaks and are important during waves of highly transmissible and immune-escaping variants of interest or of concern (VOCs). We analyzed the epidemiological data of the understudied country of Malta and related the patterns observed with viral genetic sequences obtained through the surveillance system headed by the Mater Dei Hospital and the University of Malta. We reconstructed the evolutionary history and spatiotemporal dynamics of Maltese severe acute respiratory syndrome coronavirus 2 viruses using a phylodynamics framework. Our findings suggest that the number of cases associated with B.1.1.7/Alpha, B.1.617.2.X/Delta, and B.1.1.529.X/Omicron VOCs was nine times higher than those associated with wild-type variants. The positivity rates in Malta remained low to moderate (<10%). A combination of public health interventions appeared to have allowed Malta to mitigate the impact of COVID-19. Our phylodynamic reconstruction traced most of the 173 viral introductions inferred to countries in Northern Europe, which is consistent with flight connectivity patterns. We also observed prolonged periods of cryptic transmission (median = 102 days) until expansion into larger outbreaks. These larger outbreaks were more easily detected by the intermittent genomic surveillance in Malta, characterized by periods of sequencing hiatus. Our study demonstrates that integrating epidemiological and genomic data are crucial for uncovering the COVID-19 dynamics of understudied locations, particularly when genomic surveillance is suboptimal. Accordingly, strengthening the genomic surveillance system in Malta should help in the earlier detection of introductions and minimize viral expansion in the country while informing public health interventions. IMPORTANCE Our study provides insights into the evolution of the coronavirus disease 2019 (COVID-19) pandemic in Malta, a highly connected and understudied country. We combined epidemiological and phylodynamic analyses to analyze trends in the number of new cases, deaths, tests, positivity rates, and evolutionary and dispersal patterns from August 2020 to January 2022. Our reconstructions inferred 173 independent severe acute respiratory syndrome coronavirus 2 introductions into Malta from various global regions. Our study demonstrates that characterizing epidemiological trends coupled with phylodynamic modeling can inform the implementation of public health interventions to help control COVID-19 transmission in the community.

T he emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the onset of the coronavirus disease 2019 (COVID- 19) pandemic in late 2019, has resulted in an unprecedented public health response globally (1).Real-time genomic surveillance of circulating SARS-CoV-2 in different parts of the world has been critical for monitoring the evolution of the virus (2,3).Phylogenetic analyses of SARS-CoV-2 genomes have identified emerging variants over the course of the pandemic with shifting dominance over time (4)(5)(6).Some of these variants have been classified as variants of interest (VOIs) and variants of concern (VOCs) based on their potential for rapid spread, ability to cause severe disease, capacity to evade detection, and natural-/vaccine-related immunity, as well as their ability to evade therapeutics (7).Monitoring of VOCs/VOIs at the regional/country scale is key for an adequate public response in the context of each territory's epidemiological dynamics, even at small-scale levels such as institutions and campuses (8).
Despite being one of the smallest countries in Europe, with a population of just over half a million people, Malta's high population density and proximity to Italy (a country with a significant number of cases early on in the pandemic) put Malta at high risk of SARS-CoV-2 transmission (9).The country's population also has a large prevalence of comorbidities, with nearly one-quarter of the population having hypertension (10) and one-third classified as obese (11); additionally, one-fifth of the population is elderly, which represents a high-risk group (12).Notably, Malta has ties to the tourism industry, specifically medical tourism.These connections are particularly strong with the United Kingdom, a country where the B.1.1.7/AlphaVOC ultimately emerged (13,14).In late February 2020, Malta implemented precautionary measures to prevent the spread of COVID-19 to the island nation, including the use of thermal cameras at the Malta International Airport as well as at the harbors at Port of Valletta and Marsa.Arriving travelers with respiratory symptoms were screened for COVID-19 at Mater Dei Hospital, Malta's only general hospital.
Malta reported its first COVID-19 case on 7 March 2020, from a family arriving in the country from Rome, Italy (9).With the outbreak rapidly growing, the country imposed a mandatory quarantine on five key populations: (i) travelers entering the country; (ii) citizens who had contact with international travelers; (iii) citizens over 65 years old; (iv) citizens with chronic conditions/illnesses; and (v) pregnant women.Between 9 and 11 March, bans on air and sea travel to Italy, Germany, France, Spain, and Switzerland were imposed.Travelers returning from these countries who refused to comply with mandatory quarantine rules were fined.Other public health interventions at this time included closing schools, universities, childcare centers, and day centers for the elderly.Political and religious activities, including weddings, were curtailed, and soccer matches were either postponed or played without crowds.Certain activities, such as hunting, continued to be allowed, and venues like bars, restaurants, and nightclubs remained open.However, these venues were closed within a week.By the end of March 2020, non-essential retail and non-essential services would be shut down, and organized group gatherings would be banned.Compliance from the general population with the measures set in place by public health authorities and the government allowed Malta to control its epidemic successfully (15).Such precautionary measures enabled Malta to handle the first wave relatively easily compared to other countries where the virus was more challenging to control and contain (14).
By 1 May 2020, the viral reproductive number was under 0, allowing the relaxation of some measures.Despite this, a second, more severe wave of the virus began in the summer of 2020 (14), resulting in spikes in cases in mid-October.Multiple measures were quickly reinstated, including early closures of bars and entertainment centers, mandatory facemask requirements in all public spaces, and social distancing guidelines.The outbreak rapidly grew to a cumulative 12,665 confirmed positive cases in December 2020, when the first variant of concern was classified as B.1.1.7/AlphaVOC (16).Malta responded to this and additional surges by reinstating similar intervention strategies.
Malta became the first country in the European Union to offer vaccines for SARS-CoV-2 to its entire population in May 2021.The Pfizer-BioNTech vaccine was the first COVID-19 vaccine to be authorized for emergency use in Malta, and it began being administered in December 2020.The vaccine required two doses, given 3 weeks apart.The Moderna vaccine was authorized for emergency use in Malta in January 2021 and also required two doses, given 4 weeks apart.A third vaccine, manufactured by AstraZeneca, was authorized for emergency use in Malta in February 2021.It required two doses, given 12 weeks apart.By the end of May 2021, over 70% of the Maltese population had been identified as fully vaccinated.At the time, this was the estimated threshold to achieve herd immunity, which thereby made Malta the first nation to meet the milestone.Since the onset of the pandemic, Malta has instituted public health measures conducive to handling large waves of cases.As a result, the healthcare system and hospitals in the country were relatively less burdened than those in other countries.To date, Malta has one of the highest rates of vaccination in the world and serves as a leader in pandemic control and strategies to return to a new normalcy (17).
Prompted by an escalation in the daily number of positive cases in summer 2020, 671 SARS-CoV-2 samples collected between August 2020 and January 2022 were sequenced by the Molecular Diagnostics-Infectious Diseases at the Mater Dei Hospital to identify and trace circulating variants.In this study, we analyzed the viral sequences generated in this study in addition to all SARS-CoV-2 genomes from Malta available at the Global Initiative on Sharing All Influenza Data database (GISAID; https://www.gisaid.org)(18,19) and leveraged a comprehensive approach involving epidemiological and viral genomic data to reconstruct the timelines of viral introductions, their geographic origins, and the contribution of detected introductions to the epidemic evolution of SARS-CoV-2 in Malta (20).

Sample collection, RNA extraction, and real-time PCR
We collected 1,289,627 nasopharyngeal samples during the period between January 2020 and January 2022 that were tested at the Molecular Diagnostics (Infectious Diseases) Laboratory at Mater Dei Hospital, Malta, using reverse transcription real-time PCR (RT-qPCR).Nucleic acid extraction was performed with the MagMax Pathogen kit (Thermofisher) on the KingFisher Flex extraction platform according to the manufactur er's instructions.SARS-CoV-2 detection by RT-qPCR was done using a CE IVD kit (Certest Viasure), targeting the N and ORF1ab genes.Between August 2020 and January 2022, samples with a positive RT-qPCR with a cycle threshold of <30 were considered suitable for sequencing.

Public health response
We used the data in https://github.com/COVID19-Malta/COVID19-Dataand information on the Maltese government response (21) to shed light on and contextualize the policies implemented in Malta with the reconstructed evolutionary and transmission dynamics.We analyzed the stringency index over time, which is a composite measure based on nine response indicators, including school closures, workplace closures, and travel bans.These indicators include the following: (i) containment policies (C1-C8): these indica tors record information on various containment policies, such as school closures and movement restrictions; (ii) economic policies (E1-E2): these indicators record informa tion on economic policies, particularly income support to citizens; and (iii) health system policies (H1-H3, H6-H8): these indicators record information on health system policies, including the COVID-19 testing regime and vaccination policies.The composite stringency index records the strictness of lockdown-style policies that primarily restrict people's behavior and is scaled to a value from 0 to 100 (100 = strictest).

SARS-CoV-2 sequencing
Amplicon sequencing was performed on 671 samples using the AmpliSeq for Illumina SARS-CoV-2 panel.This panel consists of 247 amplicons targeting >99% coverage of the SARS-CoV-2 genome, including potential variants of concern.Sequencing libraries were purified with the Ampure XP magnetic beads (Beckman Coulter, USA), and their concentration and quality were measured with the Quantus Fluorometer (Promega) and Tapestation (Agilent, USA) devices, respectively.Each library was diluted to 2 nM, pooled, denatured, diluted to 8 pM, and sequenced on the Illumina MiSeq platform using the V2 2× 300 bp kit.

Variant calling and assembly of consensus sequences
Data analysis of the sequencing reads was done by using the bioinformatics pipeline INSaFlu (22).Briefly, the Fastq files were subjected to the following steps: read quality analysis, variant detection and consensus genome construction, coverage analysis.This process resulted in 659 SARS-CoV-2 Maltese sequences generated in this study and uploaded to the GISAID (https://www.gisaid.org/).
The final data set contained 3,540 sequences, of which 666 were collected in Malta (663 new sequences), 55 from Italy, 441 from Eastern Europe, 482 from Northern Europe, 490 from Southern Europe, 423 from Western Europe, 30 from Northern Africa, 189 from Africa, 189 from Asia, 149 from North America, 53 from Oceania, and 147 from South America.

Phylogenetic analysis
A maximum-likelihood tree was inferred with IQ-TREE v2.1.4using the GTR + G + I nucleotide substitution model (23).We assessed the root-to-tip divergence using TempEst (24) and excluded outlier sequences whose genetic divergence and sampling date were incongruent.
The estimation of time-scaled phylogenies, spatial ancestral reconstruction, and estimation of transition rates between regions was performed with TreeTime (25).For the temporal inference, the oldest reroot option was used, and the joint maximum-likelihood tree was reconstructed.Reconstructing the dates at the nodes allowed estimation of the lag between the date of a viral introduction and the first collection date of the sequenced sample in a cluster and thus derive the period of undetected viral circulation or cryptic transmission (26).
The "mugration" package extension of TreeTime was then used to map discrete regions to tips and infer the locations for internal nodes under a GTR model.The regions described above were used as attributes.

Policy and epidemiological trends for SARS-CoV-2 in Malta
The first government response to the COVID-19 pandemic in Malta can be traced to January 2020 (Fig. 1) (21).This early period saw the establishment of the coronavi rus national response team, composed of public health doctors and other healthcare professionals.Within the same month as the first detected case, the first swabbing center was opened to the public, and multiple countries were placed on a red list and banned from traveling to Malta.A "work from home" strategy was also strongly encouraged and promoted.As shown in Fig. 1, the peak of all indices, related to government regulations designed to stop the spread of COVID-19 in 2020, occurred during the months of April and May.During this peak, the stringency index (21) was 87.0, which was tantamount to lockdown with only essential services operating.From the summer of 2020 onward, the response was relatively constant, with a very slight increase across all indexes.The new year of 2021 brought with it a series of vaccination programs, starting with old people's homes and gradually leading to all the populace.No major or new measures were put in place until March 2021, when once again there was the closure of many shops and services deemed non-essential, and traveling between Malta and its sister island of Gozo was banned.These control measures were to endure for most of 2021 until a large portion of the Maltese population was vaccinated, and gradually all services were allowed to resume operation with protective measures in place.
To better understand the dynamics of the SARS-CoV-2 epidemic in Malta, we analyzed trends in the number of new cases, deaths, tests, and positivity rates in Malta from 7 March 2020 through 5 January 2022, using the epidemiological data obtained from OurWorldInData.org ( 27) on 27 April 2022 (Fig. 2).
Although most VOC/VOI lineages in our study have been downgraded to variants being monitored, for clarity on their relative importance throughout the pandemic, we will refer to them by their VOC/VOI status at the time of their circulation.

Variants circulating in Malta
We explored the frequency of sequenced SARS-CoV-2 lineages circulating in Malta (Fig. 4; Fig. S1).The first sequenced sample was collected on 19

Evolutionary and spatiotemporal patterns of SARS-CoV-2 in Malta
In order to assess the origins and estimate the number of SARS-CoV-2 introductions into Malta between August 2020 and January 2022, we performed a phylodynamic analysis integrating all isolates and their respective collection dates and regions (Table S1, available at https://epicov.org/epi3/epi_set/230323pr).
Looking at the viral dynamics in more detail (Fig. 6), we estimated that the earliest introduction of the B.1.1.7/AlphaVOC to Malta traces back to a most recent common ancestor (MRCA) from Northern Europe on 11 November 2020, leading to an outbreak of at least 61 cases.This was followed by 20 more B.1.1.7/AlphaVOC introductions from Northern Europe throughout the study period.We also reconstructed seeding events between November 2020 and January 2021 from other regions, including Africa, Asia, Eastern Europe, and Southern Europe, that caused 14 smaller sequenced outbreaks, each  We inferred that the initial B.1.1.529.X/Omicron VOC introduction into Malta traces back to an MRCA that existed in early August 2021 in Asia.This was followed by 11 introductions from Northern Europe, Southern Europe, Eastern Europe, Africa, or Asia, producing between 1 and 2 sequenced infections each.The largest sequenced outbreak of B.1.1.529.X/Omicron VOC was characterized by a clade of 74 sequences that originated from Northern Europe in early November 2021.Similar to the B.1.1.7/Alphaand B.1.617.2.X/Delta VOC epidemics, the increase in B.1.1.529.X/Omicron VOC cases in Malta appears to have occurred from multiple independent introductions from different geographic regions in a short time frame.
Three independent introductions of the Eta VOI were identified, with two introduc tions from Africa in December and January of 2021 resulting in one to two sequenced infections each.One introduction was dated with an MRCA of 4 February 2020 and originated from Western Europe.
We also reconstructed a single introduction of the Mu VOI into Malta from North America.This clade consisted of only one sequenced infection with an estimated MRCA of 5 April 2021.

Period of cryptic transmission
We investigated the extent of cryptic transmission that led to the detection of variants in Malta.The analyses revealed a median time lag of 102 days (range = 0 and 399 days; The three most prolonged periods of cryptic transmission were associated with introductions from a wild-type lineage, Eta VOI, and P.1/Gamma VOC, ranging from 370 to 399 days.The length of cryptic transmission for these introductions may have been due to the limited genomic background for these lineages.Otherwise, the longest period of cryptic transmission in Malta was observed during the B.1.617.2.X/Delta VOC wave and lasted for 248 days of undetected circulation.
Estimates of the lineage-specific lag time reveal that the median number of days was much lower during the B.1.1.529.X/Omicron VOC wave than during the B.1.1.7/AlphaVOC and B.1.617.2.X/Delta VOC cycles.

DISCUSSION
Our study provides an understanding of the evolution of the COVID-19 pandemic in Malta by combining epidemiological and phylodynamic analyses.
Public health interventions in Malta, including closures of national borders and non-essential services, appeared to impact the viral transmission dynamics into and within Malta.During periods of strict intervention, the positivity rate and incidence of new cases sharply declined.This is most notable during the early response to the B.1.617.2.X/Delta VOC wave between March and May 2021.After ramping up testing for the SARS-CoV-2 virus in July 2020, Malta consistently had many more tests administered compared to the number of positive cases.This does not imply that viral circulation was being monitored.Similarly observed elsewhere (28,29), the first 2 years of the COVID-19 pandemic in Malta was characterized by a high incidence of cases coinciding with the emergence of new viral variants of concern (30).
Although the first COVID-19 case in Malta was identified on 7 March 2020, the first sequenced case did not occur until more than 5 months later, on 19 August 2020.There was also no genomic surveillance between October 2020 and early February 2021, resulting in a gap in information on the viral diversity circulating in Malta at that time.This is especially concerning as it is one of the most prolonged periods with a high incidence of cases and deaths.Additionally, this period coincides with the introduction of the first described variants of concern and interest, the B.1.1.7/AlphaVOC and the Eta VOI.A similar hiatus in sequencing efforts was observed during August and September 2021, falling within the May-September 2021 months when the majority of TMRCAs were estimated.During these periods, the Mater Dei Hospital and the University of Malta maintained track of variants using the DeepChek-8-plex CoV-2 Genotyping Assay from Advanced Biological Laboratories (data not shown).Although B.1.617.2.X/Delta was the dominant lineage before and after the August 2021 hiatus, the lack of sequenc ing and the genotyping assay employed do not provide insight into other wild-type lineages circulating in Malta during this time.By the time the B.1.1.529.X/Omicron VOC emerged, more active genomic surveillance in Malta had allowed the variant to be quickly identified and traced as it became the dominant lineage within a month.This is consistent with the B.1.1.529.X/Omicron VOC's shorter estimated periods of cryptic transmission.
Epidemiological data provide insight into the patterns of epidemics.However, phylodynamics allows for reconstructing the viral evolutionary history and estimating detailed past events, including the time and geographical origin of viral introductions.We detected 173 independent SARS-CoV-2 introductions into Malta, almost half of which corresponded to the B.1.617.2.X/Delta VOC.This is clearly an underestimate of the real number of viral introductions, as viral sequencing was unavailable during some periods.Overall, our analysis reflects the global viral dynamics observed elsewhere (4,(31)(32)(33)(34)(35), demonstrating that SARS-CoV-2 lineages can spread to a diverse range of international locations, particularly highly transmissible variants of concern (4,6,(36)(37)(38)(39), with the largest and most common introduction to Malta being the result of the B.1.1.7/Alpha(up to March 2021), B.1.617.2.X/Delta (March-October 2021), and B.1.1.529.X/Omicron (October 2021-January 2022) VOCs.We observed peaks in the number of new posi tive cases that coincided with multiple introductions of the B.1.1.7/Alpha,B.1.617.2.X/ Delta, and B.1.1.529.X/Omicron VOCs from locations worldwide within a short period.Geographic proximity to Malta was also a notable factor in viral introductions.About three-quarters of the viral introductions originated from a European region, with the largest being Northern Europe (38.7%),followed by Eastern Europe (15.0%),Southern Europe (11.6%), and Western Europe (9.8%).We also saw several viral introductions from Asia (16.2%), primarily associated with the B.1.617.2.X/Delta VOC first detected in India (37).
Even though Malta ramped up its genomic surveillance since August 2020, sequenc ing remained limited throughout the first 2 years of the pandemic.Many introductions of VOCs, VOIs, and wild-type lineages were missed or undetected due to gaps in the country's genomic surveillance (26).Long periods of cryptic transmission (median 102 days) were associated with larger, more easily detected outbreaks not caught by genomic surveillance at an earlier stage of the transmission dynamics.For some lineages, we identified exceptionally high periods of cryptic transmission, often over 1 year.However, these results are likely overestimated due to gaps in the genomic background data set and limited sequencing of these lineages in Malta.Excluding these, the longest periods of undetected circulation were of the B.1.617.2.X/Delta VOC, associated with the lower levels of genomic surveillance prior to and during the B.1.617.2.X/Delta epidemic in Malta.
Malta has yet to fully employ sequencing information to manage its COVID-19 outbreaks, and it has contributed few SARS-CoV-2 genomic data toward the global pool in growing open-access repositories.Epidemiologically, there were no records of the number of tests in Our World in Data (OWID) in the first 4 months of the pandemic (March-July 2020).
While initially being one of the hardest-hit countries and despite being one of the smallest countries in Europe, Malta found early success in controlling the spread of the virus.However, Malta's success was not covered in as thorough detail in international media compared to the more difficult-to-control epidemic faced by other European countries, such as Italy.Adherence to public health regulations set by authorities enabled Malta to handle the pandemic with greater efficiency and confidence compared to surrounding European and major international countries.Malta, similar to Cyprus and Iceland in terms of population and a relatively small island archipelago in size, bene fits from efficient internal communication, both through official channels and through interpersonal communication; this was probably one of the main factors that contrib uted to the successful public health interventions.Because of the tight community network, interventions at the national government level very easily percolated and were implemented at the community level (40).
This study provides evidence of circulating SARS-CoV-2 wild-type lineages, VOCs, and VOIs in Malta.It also shows that characterization of epidemiological trends coupled with phylodynamic reconstruction is important in tracing SARS-CoV-2 cases, uncovering the viral diversity driving the dynamics, and disentangling the timing and spatiotemporal patterns of viral introductions.This knowledge can inform the implementation of public health interventions to help control COVID-19 transmission in the community.
Full border closures from March 2020 through July 2020 appear to have minimized across-country spread during this period.Cases increased from July 2020 through March 2021, coinciding with the emergence and circulation of the B.1.1.7/AlphaVOC.A relaxation of travel bans in August 2020 might have been one of the drivers of the subsequent upward trend in new cases.From March 2021 through early May 2021, non-essential workplaces were required to close in response to the spike in new cases during this period.These were subsequently downgraded to some required closings in May 2021 and recommended closings for June 2021, leading to a decrease in new cases between April 2021 and June 2021.There was a resurgence of cases in July 2021, concomitant with the widespread circulation of the B.1.617.2.X/Delta VOC.The incidence of new cases declined until a steep increase in November and December 2021, aligning with the timeline of the global emergence of the B.1.1.529.X/Omicron VOC.We observed that high death counts paralleled periods of high incidence with a short 1-2-week delay (Fig. 2).Total counts for new tests are not available prior to mid-July 2020.The testing capacity steadily ramped up between July 2020 and early March 2021.Closings in May and June 2021 resulted in a sharp decline in tests performed.The number of weekly tests increased again between June and September 2021 and post-November 2021 in response to the country's B.1.617.2.X/Delta and B.1.1.529.X/Omicron VOC waves.The positivity rate for new tests followed a similar pattern to that of new cases and tests, spiking during initial VOC waves and eventually declining to consistently low levels.The maximum positivity rates identified were 7.1% during the period July 2020-June 2021 (B.1.1.7/Alphaand P.1/Gamma VOC epidemics), 5.5% during the period July 2021-November 2021 (B.1.617.2.X/Delta VOC epidemic), and 16.2% between December 2021 and early January 2022 (early B.1.1.529.X/Omicron VOC epidemic) (Fig. 2 to 4).

FIG 2
FIG 2 SARS-CoV-2 new cases and deaths in Malta.Frequency of new positive samples (black) and newly recorded deaths (red).Counts were aggregated by collection week for better visualization.Shaded rectangles represent the periods of the universal travel ban (March 2020-July 2020) and non-essential workplace closures (March 2021-May 2021).Green dashed line indicates the date of the most recent sequence in the genomic data set.
August 2020 and belonged to PANGO lineage B.1.Subsequent collection of six samples between 20 August 2020 and 7 September 2020 was all identified as belonging to PANGO lineage B.1.389.Through 5 January 2022, the dominant variants identified were B.1.617.2.X/Delta VOC (n = 368; 55.3%), B.1.1.7/AlphaVOC (n = 144; 21.6%), and B.1.1.529.X/Omicron VOC (n = 87; 13.1%).The lack of information on the viral diversity circulating from October 2020 until early February 2021 is due to the lack of sequencing during those months.The large incidence of cases in mid-February 2021 (Fig. 2) was met with increased genomic surveillance, which permitted detecting the circulation of several VOC/VOI, including the B.1.1.7/AlphaVOC and B.1.525/EtaVOI.From mid-February 2021 through early June 2021, the B.1.1.7/AlphaVOC was the dominant lineage detected, with the B.1.351/BetaVOC only sequenced three times and lower levels of circulation of the P.1/Gamma VOC.The B.1.621/MuVOI was detected through genomic surveillance only once in Malta.The B.1.617.2.X/Delta VOC was first detected in Malta in June 2021 and became the dominant circulating variant through the end of 2021.Despite the peak in positivity rate during July and August 2021 (Fig. 3), the hiatus in genomic surveillance does not allow insight into the viral diversity circulating during that period.The B.1.1.529.X/Omicron VOC was detected in Maltese viral sequences in December 2021, quickly replacing the B.1.617.2.X/Delta VOC as the dominant circulating variant by January 2022.Sequencing counts appear to follow a similar pattern as new cases, with more sequences generated during periods of high incidence.

FIG 3
FIG 3 SARS-CoV-2 new tests in Malta.Counts of newly collected samples (black) and the positivity rate for these new samples (blue).Sample counts were aggregated by collection week for better visualization.Shaded rectangles represent the periods of the universal travel ban (March 2020-July 2020) and non-essential workplace closures (March 2021-May 2021).The green dashed line indicates the date of the most recent sequence in the genomic data set.The number of new tests collected and the positivity rate are not available after June 2022.Total counts of newly collected samples and positivity rates are not available after 22 June 2022.

FIG 4
FIG 4 Frequency of PANGO lineages circulating in Malta between August 2020 and January 2022.The stacked area chart reflects the proportion of collected sequences and their respective PANGO lineages, clustered by week.VOI/VOC lineages are identified, and all other lineages are grouped.No sequences were collected during the months of October 2020 to February 2021 and August to September 2021.Fig. S1 depicts the counts of lineages circulating in Malta over time.

FIG 5 9 FIG 6
FIG 5 The flow of SARS-CoV-2 lineages into Malta.Left: time-resolved maximum-likelihood tree containing high-quality near-complete genome sequences from Malta obtained in this study (n = 659; red dots) analyzed against a background of global sequences.Right: Sankey plot depicts the reconstructed origin location and PANGO lineage of the Maltese sequences.The number between square brackets depicts the number of sequences attributed to the specific reconstructed origin location (left side) or PANGO lineage (right side).

Fig. 7 )
between the MRCA and the earliest collected genome for each introduction.In particular, the MRCA of most clades (n = 105; 60.7%) occurred between 1 May 2021 and 30 September 2021, aligning with the global surge in B.1.617.2.X/Delta VOC cases.The temporal reconstruction estimated that the earliest and largest introduction of the B.1.1.7/AlphaVOC into Malta had a lag to detection of 103 days.The largest introduction of the B.1.617.2.X/Delta VOC into Malta was estimated to have occurred as early as 1 April 2021 from Asia but remained undetected for 88 days.The first introduction of the B.1.1.529.X/Omicron VOC into Malta occurred as early as 4 August 2021 from Northern Europe and remained undetected for 154 days.However, all other B.1.1.529.X/Omi cron VOC introductions, including the largest with 74 sequenced infections, remained undetected for 81 days or fewer.

FIG 7
FIG 7 Cryptic transmission of SARS-CoV-2 in Malta revealed by genomic epidemiology.Top: violin plots represent the date of sample collection of the earliest genome in a Maltese clade (blue) and the TMRCA (green) of an introduction.Middle: violin and box plots depict the time lag between the introduction and the first surveilled genome.Bottom: box plots depict the time lag between introduction and the first surveilled genome for each PANGO lineage.