key: cord-0769446-dzgdb1iv
authors: Chapleau, R. R.; Christian, M.; Connors, B.; Premo, C.; Chao, T.; Rodriguez, J.; Huntsberger, S.; Meyer, J.; Javorina, A.; Reynolds, K.; Riddle, D. S.; Lisanby, M. W.; Starr, C. R.
title: Early Identification of SARS-CoV-2 Emergence in the DoD via Retrospective Analysis of 2019-2020 Upper Respiratory Illness Samples
date: 2021-02-20
journal: nan
DOI: 10.1101/2021.02.18.21251368
sha: 5b47e4da13d2c17250093bb1cb71fe6ca104e646
doc_id: 769446
cord_uid: dzgdb1iv

The first case of non-travel related SARS-COV-2 was detected late February in California, however initially stringent testing criteria and subsequent delay of diagnostic testing made it difficult to identify those who could have acquired it through community spread. Early speculation was that the virus may have been circulating from at least January [1], and environmental sampling suggests that versions of this virus were found months before the first human samples were identified [2]. Here we performed a retrospective study from residual samples collected from a global respiratory surveillance effort to establish a tentative timeline by which this virus was circulating in the US military population. We performed RT-PCR for SARS-COV-2 and compared the dates of these cases to the first cases identified in respective states and counties using the Johns Hopkins COVID tracker website. Twenty-four positive samples were identified out of approximately 7,000 tested. Although we found some early cases in certain regions, we did not see circulation before late February in samples collected both in the US and outside the USA.

We know that the first reported case of SARS-COV-2 was mid-January; however, there has been conjecture that the virus was found in the community before this date.

Here we took samples collection from a global respiratory surveillance program and evaluated for the presence of SARS-COV-2 RNA. The first case we found in the surveillance program was approximately 30-60 days before the first case was identified on military installations via diagnostic testing, however was not earlier than the mid-January reported case in California.

The implementation of new and emerging pathogen detection assays into already established surveillance programs could detect early community spread and possibly reduce spread of pathogen among vulnerable populations.

The first case of non-travel related SARS-COV-2 was detected late February 2020 in California, however the delay of diagnostic testing and initial stringent testing criteria made it difficult to identify those who could have acquired it through community spread. There was speculation by many that the virus may have been circulating at least a month earlier [1] , and environmental sampling has suggested that versions of this virus were found many months before the first for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 https://doi.org/10.1101 https://doi.org/10. /2021 human samples were identified [2] . Here we performed a retrospective study from residual samples collected from a global DoD Respiratory Surveillance Program to establish a tentative timeline by which this virus was circulating in our DoD population. We performed RT-PCR for SARS-COV-2 and compared to the dates of these cases to the first cases identified in respective states and counties using the Johns Hopkins COVID tracker website. Twenty-four positive samples were identified out of approximately 7,000 tested. Although we found some early cases in certain regions, we did not see circulation before late February in samples collected both in the US and outside the USA.

De-identified nasopharyngeal swab (NPS) samples collected from a respiratory surveillance program that tracks influenza globally to aid in the yearly vaccine development. Samples were This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 February 20, 2021. ; https://doi.org/10.1101/2021.02.18.21251368 doi: medRxiv preprint DNA, cat. #10006605). Thermocycling conditions on the ABI 7500 FAST analyzer was a 20minute reverse transcriptase step at 50°C, a 10-minute hot-start activation step at 95°C, and 45 cycles of 95°C for 3 seconds followed by 55°C for 30 seconds.

In accordance with the approved CDC assay methodology, samples were positive for SARS-CoV-2 if the RNase P control passed (Ct < 40) and both primer sets N1 and N2 produced Ct values below 40. In the cases where RNase P did not amplify but both N1 and N2 were positive, we called the sample positive in accordance with the CDC emergency use authorization guidance (revision 03, page 34). Samples where only one of the two markers were detected were "inconclusive" and the test was repeated using the previously extracted RNA. Metadata were stored in a separate computer from the analytical results and the Laboratory Director combined the PCR results with metadata prior to analysis. We used Microsoft Access for data management and descriptive statistics such as number of positives, daily positive hit rate, and earliest detection.

Our study identified 24 positive samples from 7,021 total samples (0.3%). Of these samples, 14

were collected from patients prior to the first COVID-19 case clinically reported at their respective installations ( Table 1 ). The first case identified at Wright Patterson AFB in Ohio was collected a full month before the first laboratory-confirmed case at that installation, and two days before the first laboratory-confirmed case in the Department of Defense (reported 26 February for use under a CC0 license.

This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 February 20, 2021. ; https://doi.org/10.1101/2021.02.18.21251368 doi: medRxiv preprint by US Forces Korea [Defense.gov, 2020] ). We also identified a second case of significant interest 46 days before the first case reported from the installation in North Dakota.

We received samples from installations in three geographic combatant commands (Table 2) This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 https://doi.org/10.1101 https://doi.org/10. /2021 As expected, our paradigm of testing patient residual samples remaining from other molecular testing for upper respiratory resulted in detecting a logarithmically increasing positivity rate This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 https://doi.org/10.1101 https://doi.org/10. /2021 In addition to observing an increase in testing, we detected two peaks the positivity rates. For the week ending 29 February, we detected one positive sample of 333 tests, a rate of 0.30%. The following two weeks remained below 1% positive rate (0.17% and 0.37%, respectively) before increasing to 1.45% with 10 detections of 688 tests for the week ending 21 March, and peaking 

Our results suggest that while the epidemic was present in the US military population earlier than previously reported, the change is minimal. Excluding the outlier from Ellsworth AFB, we identified initial positive cases approximately two weeks prior to the first case reported (average = 11.6 days, range 2 to 27 days prior). Our results are similar to other retrospective testing that focused on PCR-based detection of residual samples [4] [5] [6] . Evaluation of residual specimens using antibody tests is still an on-going effort and may lead to different data as better assays comes available [7] .

Due to early resource limitations in the global molecular testing supply chain, we began testing our early samples using the SYBRgreen detection method. Several of the samples ran out of material prior to establishing consistent supply chain provisions for probe-based detection using for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 February 20, 2021. ; https://doi.org/10.1101/2021.02.18.21251368 doi: medRxiv preprint the CDC assay and Superscript real-time detection. One of these samples was the detected sample from late February.

While we tested more than 7,000 samples, the number of independent test sites is small and heavily favored by one Service. We expected a high proportion of samples to originate from NORTHCOM facilities, as most of the DoD MTFs are located there, however representation overseas is limited with only ten Air Force installations and one Army installation. In a demonstration of collaboration, we did receive samples from six Coast Guard clinics, too.

Additionally, we did not receive any samples from Southern Command and less than 10 samples from Central Command, so the impact of the epidemic in our military members stationed in those regions is unknown. In order to provide adequate surveillance during normal operations and epidemics, the Military Health System, and the Defense Health Agency by extension, must increase the participation of sentinel sites in the Respiratory Surveillance Program. With 475 military hospitals and medical clinics across the globe, the DoD is the only healthcare system that is equipped to monitor worldwide infectious diseases before they enter the homeland. The surveillance network for the DoD is primed to immediately add validated assays for new and emerging infections, before the clinical assays are deployed, to actively track community spread once a virus has been detected by travel or any criteria that are tracked early on in a pandemic by the CDC.

Evidence for Limited Early Spread of COVID-19 Within the United States

DOI

It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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

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