key: cord-0942100-olc00glo
authors: Franco, D.; Gonzalez, C.; Abrego, L. E.; Carrera, J. P.; Diaz, Y.; Caisedo, Y.; Moreno, A.; Chavarria, O.; Gondola, J.; Castillo, M.; Valdespino, E.; Gaitan, M.; Martinez-Mandiche, J.; Hayer, L.; Gonzalez, P.; Lange, C.; Molto, Y.; Mojica, D.; Ramos, R.; Mastelari, M.; Cerezo, L.; Moreno, L.; Donnelly, C. A.; Faria, N. R.; Pascale, J. M.; Lopez-Verges, S.; Martinez, A. A.
title: Early transmission dynamics, spread, and genomic characterization of SARS-CoV-2 in Panama.
date: 2020-08-04
journal: nan
DOI: 10.1101/2020.07.31.20160929
sha: 41f62f07e43809ef2ca5a72b2eb00de07a0ba9f6
doc_id: 942100
cord_uid: olc00glo

Background With more than 50000 accumulated cases, Panama has one of the highest incidences of SARS-CoV-2 in Central America, despite the fast implementation of disease control strategies. We investigated the early transmission patterns of the virus and the outcomes of mitigation measures in the country. Methods We collected information from epidemiological surveillance, including contact tracing, and genetic data from SARS-CoV-2 whole genomes, of the first five weeks of the outbreak. These data were used to estimate the exponential growth rate, doubling time and the time-varying effective reproductive number (Rt) using date of symptom onset in a Bayesian framework. The time of most recent ancestor for the introduced and circulating lineages was estimated by Bayesian analysis. Findings A total of 4210 subjects were SARS-CoV-2 positive during the period evaluated, of them we sequenced 313 cases, detecting the circulation of 10 SARS-CoV-2 lineages. Whole genomes analysis identified the local transmission of one cryptic lineage as early as 2 weeks before it was detected by surveillance systems. Analysis of transmission dynamics showed that lockdown reduced Rt and increased the doubling time, however, these measures did not stop the circulation of this lineage in the country. Interpretation These results demonstrate the value of epidemiological modeling and genome surveillance to assess mitigation strategies. At the same time, an active search for cryptic transmission clusters is crucial to interrupt local transmission of SARS-CoV-2 in a region.

The protocol EC-CNBI-202-04-46 was approved by the National Committee on Bioethics of Research of Panama to use de-identified epidemiological data to analyze SARS-CoV-2 transmission and spread, as well as to sequence the complete genome of SARS-CoV-2 from Gorgas Memorial Institute of Health Studies (GMI)'s confirmed cases.

The surveillance program for COVID-19 was implemented by the Panama Ministry of Health (MoH) on January 20th, 2020. Suspected cases were actively sought at international airports using the WHO/PAHO case definitions and recommendations 16 . In this first stage, suspected cases were defined as presenting Influenza Like Illness (ILI) and severe acute respiratory infections (SARI) symptoms and signs, as well as coming from China.

Later, on March 4 th , the list of countries was expanded to include Italy, Iran, South Korea, and Japan. Travelers from countries with confirmed circulation of SARS-CoV-2 were isolated at home or hotel-quarantine for 14 days and monitored for onset of symptoms. Patients that met the criteria of suspected cases were then confirmed via laboratory diagnosis. For confirmed cases, contact tracing was performed by the National Epidemiology Department of the MoH, and cases and contacts were placed under quarantine. Clinical evaluation and temperature measurement for travelers and contacts were done every day; if symptoms developed, nasopharyngeal or oropharyngeal swabs were taken by the rapid response teams of MoH and sent to the GMI for laboratory diagnosis. Details of COVID-19 surveillance system are described in the Supplementary Information.

Nasopharyngeal and oropharyngeal swabs samples were submitted to the GMI for laboratory confirmation since January 23rd and to additional laboratories from the National COVID-19 Laboratory network since March 16th.

Molecular diagnosis was performed first using a generic coronavirus RT-PCR and then specific Real Time RT-PCR for SARS-CoV-2 17 as described in the Supplementary Information.

Epidemiological data on suspected cases and their contacts were recorded by physicians when nasopharyngeal and oropharyngeal swabs were taken, using a standardized epidemiological form. Data entry was independently undertaken by the National COVID-19 Laboratory network, and then checked by the National Department of Epidemiology of MoH to confirm the accuracy of the information. A dataset of daily incidence based on date of symptom onset was created. The first 61 days of COVID-19 epidemic in Panama from February 15 to April 16 were analyzed. More details are given in the Supplementary Information.

Early transmission dynamics of COVID-19 in Panama were evaluated estimating the daily growth rate, doubling time, basic and effective reproductive numbers. We estimated the basic reproductive number (R 0 ) using the time series of confirmed cases, an early R 0 with Likelihood-based estimation using a branching process: This estimation follows Poisson likelihood standards 18 . A serial interval mean of 4·7 days and standard deviation of 2·9 was used for the estimation 6 . The time-varying effective reproductive number (R t ) was estimated in a Bayesian framework since the cumulative number of cases reached 25 cases as previous decribed 6 , serial . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint interval with 95 % credible intervals (95% Cl), using the EpiEstim packaged implemented in R 18 . Details are provided in Supplementary Information.

Reverse transcription and multiplex PCR were performed according to ARTIC Network protocol (https://artic.network/), with modifications. Amplicons generated were pooled and prepared for Illumina

Sequencing with the Nextera XT library preparation protocol. All samples were sequenced with MiSeq V2 reagent kits for 500 cycles. Reads obtained were aligned with bowtie-2 and consensus genome was generated with Quasitools-Hydra. Details are provided in Supplementary Information.

To evaluate virus lineage distribution in Panama, lineages were classified using Pangolin 14 

Reports from the WHO, the Organization for Economic Co-operation and Development (OECD) and official communications from the ministries of health were used to obtain data on control and mitigation strategies at the country level to reconstruct a timeline representing the strategies adopted by Latin American countries. Only when no official information was found, press articles from local newspapers were consulted, as described in Supplementary Information.

Epidemiological surveillance began on January 23 and, by April 16, a total of 18 559 suspected cases of COVID-19 had been investigated. Of these, 4 210 were confirmed as SARS-CoV-2 infections at that date. On March 7th, the National Surveillance system detected a fatal case in which the SARS-CoV-2 infection was confirmed on March 9th. This case was not associated with previous travel history outside the country. The first case detected by active surveillance at international airports, came from Spain, had mild disease and was confirmed on March 9th. Most imported cases were associated with travel from Europe, North America, and one from China.

Epidemiological investigation confirmed that the date of onset of symptoms of one local case in our study goes back as early as February 15 ( Figure 1A ). Most local cases had mild disease ( Figure 1B ). By April 16, a total of 341 hospitalized cases (77 were hospitalized at diagnosis confirmation) and 116 fatalities (31 were dead at the diagnosis confirmation) were reported in Panama ( Figure 1C and 1D). The highest proportion of confirmed cases were observed in the age group 20-59 years (Supplementary Figure 1A) . A higher proportion of females were tested while more males were positive (Supplementary Figure 1B) . A rapid growth rate of 0·13 cases/per day ( Figure 2A ) and a short doubling time was also observed during the early stages of the epidemic, with an increasing doubling time over the analyzed interval ( Figure 2B ). We estimated an early epidemic potential of transmission of SARS-CoV-2 in Panama of R 0 = 2·22 (95% CI, 2·08 -2·37).

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint Panama detected the first SARS-CoV-2 infection one month after Brazil 15 , being the 11 th country with confirmed cases in Latin America (Supplementary Figure 2) . School closures were implemented the day after first case confirmation, social distance measures six days later, and stay-at-home 24-hour curfew 14 days later.

Compared to other countries in the Latin American region, Panama was among those that rapidly implemented control strategies after its first case (Supplementary Figure 2) . The effective reproduction number since the accumulated 25 cases was R t = 3·01 (95% Crl: 2·54 -3·52) and rapidly dropped over the course of 17 days to Rt= 1·08 (95% Crl: 1·00 -1·17) ( Figure 2C, Supplementary Table 1 ). Until April 16, Panama was the country from Central America with the highest cumulative incidence of cases and fatality (Supplementary Figure 3) .

To characterize the distribution of genetic lineages in Panama, we generated 313 SARS-CoV-2 sequences, which represents 7.4 % of the total confirmed cases by April 15 th (Supplementary Figure 4A Figure 4A ). These sequences, indicated a substantial local transmission as they corresponded to 54·3% of the sequenced cases in the country.

Estimates of the tMRCA of the Panamanian sequences place the median age of A.1, A.3, B.1 between two weeks range before the first reported COVID-19 case, which agrees well with the time of onset of symptoms observed in the data ( Figure 1A , Figure 3A ), however the A.2, had the oldest median tMRCA corresponding to early February ( Figure 3A ).

There is no structured spatial distribution of the lineages across the country, however the locally transmitted A.2 linage was found in all regions. Additionally, Center Panama and West Panama (regions that correspond to Panama City and its suburbs), had a more diverse lineage distribution ( Figure 3B ). Using active contact tracing of positive cases at the beginning of the outbreak, the National Epidemiology Department of MoH defined 30 epidemiological clusters (supplementary table S2), linked to a school, police officers and local outbreaks. Most sequenced cases were not classified inside these epidemiological clusters (unknown category in Figure 3C ) and had a predominance of lineage A.2.

The previously described spike glycoprotein variants D614 and G614 21,22 were co-circulating early in the epidemic among all the regions analyzed, but G614 variant was predominant in 18.8% of the sequences analyzed ( Figure 4B ). Both variants were also distributed among lineages, however D614 was highly predominant in A.1 and A.2. (Figure 4C ).

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint Panama has the most confirmed SARS-CoV-2 infections and associated fatalities in Central America, although control strategies were rapidly implemented at the beginning of the outbreak. The reasons for this pattern are unclear, but they may include, 1) cryptic circulation, 2) rapid early transmission dynamics and 3) actively search of cases and their contacts, combined with strong molecular capacity of testing in the country.

Epidemiological investigation showed early local transmission events as February 23 th , based on reports of symptoms onset of the first detected local case on March 9 th . The potential source of this infection dated back an onset of symptoms on February 15 th . Taken together the epidemiological evidence, along with genetic analysis of SARS-CoV-2, confirm cryptic circulation in Panama with an interval window of introduction between late January and early February.

A high early median transmission potential of SARS-CoV-2-was estimated in R 0 = 2.22 (2.08-2.37), like those early estimated reported in China and Europe 23,24 . In addition, the initial time-variant reproduction number R t =3.01 (2.54-3.51), estimated since the incidence reached 25 cases, also suggest a high early transmission dynamic. However, the R t rapidly dropped after implementation of control strategies.

The genetic analysis of the diverse introductions of lineages and the dynamics of their transmission support these epidemiological observations. At the beginning of the outbreak, we detected the introduction of ten lineages, and from them, five were still co-circulating in the country in mid-April. The numbers of detected lineages could be underestimated because we did not sequence all positive cases, thus there is a possibility of not-detecting uncommon lineages. Most of the lineages associated with imported cases (A.1, A.3, B, B.1, B.2.1) were detected by surveillance of suspected cases and their contacts through active contact tracing. However, this did not happen with the cases associated to the lineage A.2. According to our phylogenetic dating analysis, this lineage circulated in the country at least three weeks before it was first detected. This lineage was detected among the school and the police officers clusters, and most of the cases belonging to these groups had their . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint

The pattern of cryptic transmission in Panama was also observed in other countries which also experienced early and rapid dissemination of SARS-CoV-2. 27, 30 We suspect that the tight classification of cases at the beginning of the surveillance in January, which focused mainly in symptomatic China travelers, precluded the opportunity to detect these early importations from Europe and USA. Indeed, in these regions the virus was already circulating at that time. 23 hospitalized cases and D) fatal cases, with symptom onset from February 13 th to April 13 th . In all four charts, the y axis represents the daily incidence and the x axis represents the date of symptom onset for each reported case.

The blue vertical dashed lines represent the first recorded onset of symptoms and vertical red dashed lines the date of first confirmed case by the surveillance system.

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint study. Distribution of S protein variants (D614 or G614) B) in different regions of the country, and C) among lineages.

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20160929 doi: medRxiv preprint

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Thanks to Gorgas' administration, general services for their support and extra-work during the COVID19 response. Thanks to all the Health institutions from MINSA, CSS and private hospitals. AAM, JMP, SLV, LEA, JMP are members of the Sistema Nacional de Investigación (SNI) del SENACYT, Panamá.

Laboratory surveillance was done using the Panama National COVID19 response MoH funds and private donations, Genome surveillance was partially funded by SENACYT COVID19-43 grant and SNI from AAM, SLV, JMP, and LEA.