key: cord-1020268-43wco3ql
authors: Aubry, M.; Teiti, I.; Teissier, A.; Richard, V.; Mariteragi-Helle, T.; Chung, K.; Deen, F.; Paoaafaite, T.; Cao-Lormeau, V.-M.
title: Self-collection and pooling of samples as resources-saving strategies for RT-PCR-based SARS-CoV-2 surveillance, the example of travelers in French Polynesia
date: 2021-06-21
journal: nan
DOI: 10.1101/2021.06.17.21254195
sha: 1236ef4831fd3577e2bc8093016bb2ec1b3751d0
doc_id: 1020268
cord_uid: 43wco3ql

In French Polynesia, the first case of SARS-CoV-2 infection was detected on March 10th, 2020, in a resident returning from France. Between March 28th and July 14th, international air traffic was interrupted and local transmission of SARS-CoV-2 was brought under control, with only 62 cases recorded. The main challenge for reopening the air border without requiring travelers to quarantine on arrival was to limit the risk of re-introducing SARS-COV-2. Specific measures were implemented, including the obligation for all travelers to have a negative RT-PCR test for SARS-CoV-2 carried out within 3 days before departure, and to perform another RT-PCR testing 4 days after arrival. Because of limitation in available medical staff, travelers were provided a kit allowing self-collection of oral and nasal swabs. In addition to increase our testing capacity, self-collected samples from up to 10 travelers were pooled before RNA extraction and RT-PCR testing. When a pool tested positive, RNA extraction and RT-PCR were performed on each individual sample. We report here the results of COVID-19 surveillance (COV-CHECK PORINETIA) conducted between July 15th, 2020, and February 15th, 2021, in travelers using self-collection and pooling approaches. We tested 5,982 pools comprising 59,490 individual samples, and detected 273 (0.46%) travelers positive for SARS-CoV-2. A mean difference of 1.17 Ct (CI 95% 0.93 - 1.41) was found between positive individual samples and pools (N=50), probably related to the volume of samples used for RNA extraction (200 {micro}L versus 50 {micro}L, respectively). Retrospective testing of positive samples self-collected from October 20th, 2020, using variants-specific amplification kit and spike gene sequencing, found at least 6 residents infected by the B1.1.7 UK variant. Self-collection and pooling approaches allowed large-scale screening for SARS-CoV-2 using less human, material and financial resources. Moreover, this strategy allowed detecting the introduction of SARS-CoV-2 variants in French Polynesia.

Cases of respiratory infection (coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were first reported in December 2019 in Wuhan City, Hubei Province, China [1] . Because of the disease global expansion, COVID-19 was declared by the World Health Organization as a public health emergency of international concern on January 30 th , 2020 [2] , and was then characterized as a pandemic on March 11 th [3] .

French Polynesia is a French overseas collectivity in the South Pacific with ca 190,000 inhabitants living on the island of Tahiti, and 90,000 inhabitants distributed on 73 other islands scattered on a surface area as wide as Europe [4] . The first case of SARS-CoV-2 infection was detected on March 10 th , 2020, in a resident of Tahiti returning from France [5] .

To stop viral transmission, the population of French Polynesia was confined and international air traffic was interrupted on March 20 th and 28 th , respectively. Only residents or foreigners showing a compelling reason were allowed to enter French Polynesia provided they were quarantined on arrival. Confinement was eased from April 20 th in most islands as no active circulation of SARS-CoV-2 had been detected, then was fully leveraged on May 21 st .

Between March 10 th and June 25 th , among 5,390 patients tested for a suspicion of SARS-CoV-2 infection, a total of 62 were found positive, including 32 imported cases, and no COVID-19 related death was recorded [6] .

Tourism is an important financial resource for French Polynesia [7] . Consequently it was urgent to reopen the international air traffic, but without imposing a quarantine on arrival so as not to deter tourists from traveling. At the same time, a strategy needed to be found to The traveler had to declare to have tested negative for SARS-CoV-2 by RT-PCR within 3 days prior to departure, certify to present no symptoms of COVID-19 at boarding, agree to comply with all sanitary rules required by the government of French Polynesia (including wearing a mask in public areas, avoiding close contact with people as much as possible, reporting any symptom suggestive of , certify to have a travel insurance for non-French citizens and agree to assume all health costs incurred in French Polynesia, and accept to perform a self-test for SARS-CoV-2 detection using a kit provided upon arrival. Finally, non-resident travelers had to accurately describe their itinerary by indicating arrival and departure dates and flight numbers, and each visited island with the dates of stay as well as the name and contact of the accommodation(s). Data collected on the ETIS platform were stored in accordance with applicable General Data Protection Regulation (GDPR) laws. They could be consulted only by the French Polynesia health authorities and used for the protection of public health and epidemiological research. Once finalized, the traveler received the receipt of the ETIS form at the email address indicated for the registration. Each traveler was identified by a unique ETIS number and a QR code. On the day of departure, the traveler had to present the ETIS receipt and the proof of negative RT-PCR test at check-in to be allowed to board the flight.

On arrival at Tahiti-Faa'a international airport (Tahiti), each traveler aged at least 6 years and staying more than 4 days in French Polynesia received a self-sampling kit consisting of a zipper plastic bag containing 2 swabs (one for the oral sampling and the other for the nasal sampling), a tube with 2-3 ml of viral transport medium (VTM) or universal transport medium (UTM), and a plastic bag approved for the transport of bio-hazardous materials with an absorbent inside. With the kit were also provided in both French and English languages an information notice presenting the protocol implemented for COVID-19 surveillance of travelers, an instruction sheet describing how to proceed to sample self-collection, and the list of the health care centers located on the different islands of French Polynesia where selfcollected samples could be dropped off (S1 File).

The date of completion of the self-collection of samples was indicated on the kit and corresponded to the 4 th day after arrival in French Polynesia, excluding weekends because of the closure of most health care centers (if the 4 th day fell on a Saturday, the test was brought forward to the previous Friday, and if it fell on a Sunday the test was postponed to the following Monday). The choice of the 4 th day for sample self-collection was based on the mean incubation period, ie the delay between the exposure to the virus and the apparition of symptoms of COVID-19, which had been estimated to 5-6 days in previously published studies [16] [17] [18] . Since the first RT-PCR testing had to be performed within 3 days prior to departure, the delay between possible infection and detectable viremia would correspond to 2-6 days (mean 4 days) after arrival in French Polynesia. Each kit was identified by a unique number represented by a barcode affixed to both the zipper plastic bag and the tube containing the transport medium. Prior to giving the kit to the traveler, the kit number was matched to the traveler's ETIS number using an application installed on mobile digital tools (tablet or smartphone) that successively scanned the barcode on the kit and the QR code on the traveler's ETIS receipt. Those data were sent automatically to the server of the ETIS platform to be associated with the traveler's identity. The data allowing the link between the kit number and the identity of the traveler could only be viewed by the physicians of the Surveillance office from the Ministry of Health of French Polynesia. questions.

In order to increase the probability of collection of viral particles at the respiratory tract, the traveler had to collect both nasal and oral samples. Briefly, the traveler had to insert the swab provided for the nasal sampling into each nostril until some resistance was felt, and rub the walls 4 times (the swab provided for the nasal sampling had a large cotton tip to prevent the traveler from pushing it too deep and getting hurt). In addition, the traveler had to insert the swab provided for the oral sampling into the mouth and rub several times inside of cheeks, the top and the bottom of the tongue, the palate, and the lower and upper gums. Then, the traveler had to insert the 2 swabs into the tube containing the transport medium and close it, turn the tube 3 times to mix the samples with the medium, put the tube into the bio-hazard plastic bag and seal it, and finally put the bio-hazard plastic bag back into the zipper plastic bag.

The traveler had to keep the self-collected samples at a temperature between +4°C and +8°C until dropping it off at the Institut Louis Malardé (ILM, Papeete, Tahiti) or one of the health care centers on the list provided with the kit. Travelers staying in a hotel, guesthouse or on a cruise ship participating in the surveillance strategy also had the opportunity to drop off their self-collected samples at the lobby of their accommodation. Then, the accommodation was responsible for forwarding the self-collected samples to the nearest health care center under the temperature conditions recommended during transport. In Tahiti, self-collected samples were collected from health care centers every weekday. In the other islands, self-collected samples dropped off at the health care centers were shipped by boat or plane to Tahiti once to several times a week, depending on the frequency of sea and air rotations (Fig 1) . All selfcollected samples were ultimately delivered to ILM. 

In order to increase testing capacity for SARS-CoV-2 detection at ILM, we assessed the use of pooled samples from different travelers before viral RNA extraction and RT-PCR steps. A previous study had shown that 8 to 32 individual samples could be pooled without compromising the benefit of the pooling strategy for a SARS-CoV-2 prevalence ranging from 2 to 0.1 % [19] . Since travelers entering French Polynesia had a negative SARS-CoV-2 RT-. CC-BY-NC-ND 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 June 21, 2021. ;  PCR test dating less than 3 days before departure, the expected prevalence among this population was low.

In order to assess the use of a pool size of 10 individual samples, we performed 10-fold serial dilutions of a nasopharyngeal sample collected from a patient tested RT-PCR positive for SARS-CoV-2 in French Polynesia in March 2020 (Fig 2) . Then, we mixed 50 µL of each 10fold dilution with 50 µL of 9 nasopharyngeal samples collected from patients tested RT-PCR negative for SARS-CoV-2, with a final volume of 500 µL per pool. We subsequently performed viral RNA extraction and RT-PCR. Germany) [19] . Reverse transcription was carried out at 55 °C for 5 min, followed by the initial denaturation and polymerase activation steps at 95 °C for 5 min, 45 cycles of . CC-BY-NC-ND 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 June 21, 2021. ;  amplification at 95 °C for 5 sec, 60 °C for 15 sec and 72 °C for 5 sec, and a final cooling step at 40 °C for 30 sec.

In the second protocol, 5 µL of each extracted RNA were added to 15 µL of reaction mixture 

Upon receipt at ILM, the barcode of each self-collected sample was scanned, and up to 10 self-collected samples were randomly grouped. A new barcode assigned to the group of selfcollected samples was scanned and stuck on a tube containing 2 mL of NucliSENS lysis buffer (BioMérieux, France). Under a class II biological safety cabinet, the tubes containing the transport medium and the 2 swabs used for nasal and oral samples self-collection were removed from the bio-hazard and zipper plastic bags, and vortexed for 30 sec. Then, 50 μL of transport medium were taken from each of the grouped tubes and transferred into the pooling tube containing the lysis buffer. The pooling tube was subsequently vortexed for 10 sec and incubated at room temperature for at least 10 min before the extraction step.

Viral RNA extraction and duplex RT-PCR were performed from pooled self-collected samples as described above, using LightMix® Modular SARS and Wuhan CoV E gene kit and LightMix® Modular EAV RNA Extraction Control kit. If a pool tested positive, 200 µL of each self-collected sample were individually subjected to viral RNA extraction, by adding 50 . CC-BY-NC-ND 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 June 21, 2021. µL of magnetic silica and 10 µL of internal positive extraction control. The presence of SARS-CoV-2 RNA was confirmed by the detection of specific RdRP gene using LightMix® Modular Wuhan CoV RdRP-gene kit, with similar conditions of RT-PCR as described above.

Barcode numbers corresponding to individual self-collected samples tested positive were sent to the Surveillance office from the Ministry of Health. Using the data provided on the ETIS platform, travelers were contacted and immediately isolated. A naso-pharyngeal swab was collected from each traveler by a health worker and tested by RT-PCR to confirm SARS-CoV-2 infection. CC-BY-NC-ND 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 June 21, 2021. USA), using primers provided by the Laboratory for Urgent Response to Biological Threats (CIBU) at the Institut Pasteur (Paris, France), and reagents from the Big Dye Terminator V3.1 kit (Applied Biosystems, USA). Partial sequences were cleaned and assembled using the Sequencher 4.10 software (Gene Codes Corporation, USA). The final sequence was uploaded on the GISAID CoVsurver (available at https://www.gisaid.org/epiflu-applications/covsurvermutations-app/) that automatically determines nucleotide/amino acid mutations and deletions compared to the reference sequence hCoV-19/Wuhan/WIV04/2019.

Data were analyzed with GraphPad Prism 7.04. The paired t test was used to compare RT-PCR results between individual and pooled samples. P values < 0.05 were considered to be significant.

The COV-CHECK PORINETIA surveillance strategy was implemented by the government of 

To assess the impact of pooling samples on SARS-CoV-2 detection sensitivity, we compared RT-PCR results (Ct values) obtained following viral RNA extraction from 50 µL of 10-fold serial dilutions of a positive nasopharyngeal sample, and from 500 µL of pools containing 50 µL of each diluted sample with 50 µL of 9 negative nasopharyngeal samples. Because of the . CC-BY-NC-ND 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 June 21, 2021. ; https://doi.org/10.1101/2021.06.17.21254195 doi: medRxiv preprint limited number of positive nasopharyngeal samples available at the time the assessment was performed and the short delay before reopening the air border, only one sample could be tested.

Reagents from 2 different RT-PCR kits (SuperScript™ III Platinum™ One-Step qRT-PCR Kit or iTaq Universal Probes One-Step kit) were used to detect the SARS-CoV-2 E and RdRP genes. Whatever the RT-PCR kit used and the gene detected, Ct values were not significantly different between individual and pooled samples, with p-values ranging from 0.051 to 0.699 (Table 1) . 

From July 15 th , 2020, to February 15 th , 2021, we tested 5,982 pools comprising 59,490

individual self-collected samples, and found 238 pools (3.98%) positive for SARS-CoV-2 E gene (S1 Table) . RNA extraction and RT-PCR subsequently performed on individual samples confirmed that 273 travelers were positive for SARS-CoV-2 specific RdRP gene detection.

. CC-BY-NC-ND 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. Table 2) . The 2 remaining travelers tested positive using variants-specific amplification kit were closely related to the 2 other travelers for whom sequencing of the S gene revealed the presence of mutations specific to the B.1.1.7 UK variant.

. CC-BY-NC-ND 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 June 21, 2021. ; 

Since the emergence of SARS-CoV-2 in China in December 2019, the virus has been circulating worldwide [21] . Several countries, including French Polynesia, rapidly closed their borders to prevent the introduction of new COVID-19 cases [5] . In contrast to other countries in the Pacific which have maintained their borders closed, French Polynesia reopened international air traffic from July 15 th , 2020, while COVID-free at that time [6, 22] , in order to revive tourism-related economic activity.

The requirement to have a negative RT-PCR test for SARS-CoV-2 prior to departure to French Polynesia had been in place since April 2020 for residents returning from abroad.

They also had to observe a period of isolation after arrival and perform another RT-PCR test before their discharge. Despite these control measures, 2 travelers tested positive at the end of their period of isolation on June 26 th , 2020 [23], suggesting that they had been infected during . CC-BY-NC-ND 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 June 21, 2021. ; https://doi.org/10.1101/2021.06.17.21254195 doi: medRxiv preprint the last days preceding their departure from Metropolitan France. These observations supported the idea that in addition to the pre-departure RT-PCR test, a second post-arrival test was needed to detect travelers who may have been infected just before departure.

The COV-CHECK PORINETIA surveillance strategy, consisting in testing the samples self- The COV-CHECK PORINETIA surveillance strategy had some limitations. Indeed, of the 61,397 travelers who were given a self-test kit on arrival, 1,907 (3.11%) did not return their self-collected samples, despite reminders. In addition, since self-collection was not supervised by a health worker, it was impossible to check whether the travelers had correctly collected their samples by strictly following the instructions supplied with the kit. Finally, the pooling . CC-BY-NC-ND 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. 

Depending on the evolution of the epidemiological context of COVID-19 in French Polynesia, the strategy consisting in pooling samples self-collected by travelers was initially used to detect the introduction of SARS-CoV-2 in a COVID-free area with limited medical staff and material resources, then more recently to prevent the emergence of variants. Our . CC-BY-NC-ND 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 June 21, 2021. ; https://doi.org/10.1101/2021.06.17.21254195 doi: medRxiv preprint results showed that pooling samples had a minor impact on the sensitivity of the RT-PCR test.

In order to facilitate SARS-CoV-2 screening in the population, more and more countries have validated the use of self-collected samples [28] [29] [30] . Therefore, self-collection and pooling approaches should be considered to prevent, or at least limit, the introduction of COVID-19 cases when tourist trade resumes in countries whose borders are currently closed, and for mass population screening in countries with active SARS-CoV-2 circulation.

We acknowledge the staff of the Ministry of Health, the Ministry of Tourism, Tahiti-Faa'a airport, and the Institut Louis Malardé for their involvement in the COV-CHECK PORINETIA surveillance system. We also thank the staff of hotels and guest houses for . CC-BY-NC-ND 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 June 21, 2021. Available from: https://www.fda.gov/news-events/press-announcements/coronavirus-. CC-BY-NC-ND 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 June 21, 2021. . CC-BY-NC-ND 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 June 21, 2021. ; https://doi.org/10.1101/2021.06.17.21254195 doi: medRxiv preprint Supporting information S1 File: Documents provided with the self-sampling kit. S1 Table: . CC-BY-NC-ND 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 June 21, 2021. ; https://doi.org/10.1101/2021.06.17.21254195 doi: medRxiv preprint . CC-BY-NC-ND 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. 

. CC-BY-NC-ND 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 June 21, 2021. 

. CC-BY-NC-ND 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 June 21, 2021. 

Individual n°1 Individual n°2

. CC-BY-NC-ND 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 June 21, 2021. 

. CC-BY-NC-ND 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. This test being carried out as part of a surveillance system, the result will not be communicated to you. However, if a SARS-CoV-2 infection is suspected, you will be contacted by the Health surveillance office of the Direction de la santé.

If during your stay you have at least one of the symptoms of Covid-19 (fever, cough, sore throat, headache, diarrhea, 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 June 21, 2021. 

9. Take a deep breath then cough 5 times in your elbow. 10. Remove the swab for the oral sampling from the wrapper by holding the plastic handle. Do not touch the cotton tip! 11. Insert the cotton tip of the swab into the mouth.

Rub several times inside of both cheeks, the top and the bottom of the tongue, the palate, and finally the lower and upper gums (make sure the cotton is well soaked in saliva). 12. Break the swab at the break point. Do not touch the cotton tip! 13. Put the cotton end of the swab in the tube containing the transport liquid previously used for the nasal sampling. 14. Close the tube by firmly screwing the cap. 15. Turn the tube 3 times to mix the samples impregnated on the cotton with the liquid. 16 . Put the tube in the « biohazard » bag then make sure to close the bag tightly (do not remove the absorbant). 17. Put the « biohazard » bag in the « barcode » bag and seal it (do net remove the barcodes stuck on the bag). Store the bag in the refrigerator (between 4°C and 8°C). 

•Centre médical -Tel: (+689) 40 980 325. Open from Monday to Thursday 7:30am to 3:30pm, Friday from 7:30am to 2:30pm •Infirmerie -Tel: (+689) 40 980 325. Open from Monday to Friday 7:30am to 12:00am. Emergency 24h/24, everyday . CC-BY-NC-ND 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 June 21, 2021. . CC-BY-NC-ND 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 June 21, 2021. . CC-BY-NC-ND 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 June 21, 2021. . CC-BY-NC-ND 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 June 21, 2021. ; https://doi.org/10.1101/2021.06.17.21254195 doi: medRxiv preprint

Novel Coronavirus (2019-nCoV), situation report -1

Novel Coronavirus (2019-nCoV), situation report -11

Coronavirus disease 2019 (COVID-19), situation report -51

Répartition de la population en Polynésie française en 2017

Bulletin épidémiologique hebdomadaire Covid-19 -N°1 -Point en semaine 15 -Polynésie française

Bulletin épidémiologique hebdomadaire Covid-19 -N°8 -Point en semaine 34 -15

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

Incubation period of 2019 novel coronavirus (2019-nCoV) infections among travellers from Wuhan

Household Transmission of SARS-CoV-2

Pool size selection when testing for Severe Acute Respiratory Syndrome Coronavirus 2

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

COVID-19 Weekly Epidemiological Update

Reprise de l'activité touristique et rétablissement des liaisons aériennes à partir du 15 juillet prochain