Transmission of SARS-CoV-2 among marine recruits during quarantine

Study design and participants

To reduce the risk of introducing SARS-CoV-2 into basic training at Marine Corps Recruit Depot, Parris Island, South Carolina, the Marine Corps has established a 14-day supervised quarantine period on a college campus used exclusively for this purpose. Potential recruits were instructed to quarantine at home for 2 weeks immediately before traveling to campus. At the end of the second supervised quarantine on campus, all recruits had to have a negative qPCR result before they could enter Parris Island. Recruits were asked to participate in the COVID-19 Health Action Response for Marines (CHARM) study, which included weekly qPCR testing and blood sampling for IgG antibody assessment.

After potential recruits completed the 14-day home quarantine, they showed up at a local military entrance processing station, where a medical history was taken and a physical examination was performed. If potential recruits were deemed physically and mentally fit for enlistment, they were instructed to always wear masks and maintain a social distance of at least 6 feet while traveling to the quarantine campus. Almost weekly classes of 350 to 450 recruits arrived on campus. The new classes were divided into platoons of 50 to 60 recruits and roommates were assigned regardless of participation in the CHARM study. The overlapping classes were housed in different dormitories and had different lunch times and training times.

During the controlled quarantine, public health measures were applied to suppress transmission of SARS-CoV-2 (Table S1 in the supplementary appendix, available with the full text of this article on NEJM.org). All recruits always wore double-layered cloth masks inside and out, except when they slept or ate; practiced social distancing of at least 6 feet; they were not allowed to leave campus; had no access to personal electronics and other items that could contribute to surface transmission; and they washed their hands regularly. They slept in double rooms with sinks, ate in shared dining rooms and used shared bathrooms. All recruits cleaned their rooms daily, disinfected bathrooms after each use with bleach wipes, and ate prepackaged meals in a dining room that was cleaned with bleach after each platoon had eaten. Most of the instructions and exercises were conducted outdoors. All movements of the recruits were supervised and one-way flow was implemented, with entry and exit points of the building designated to minimize contact between people. All recruits, regardless of study participation, were screened for temperature and symptoms daily. Six instructors assigned to each platoon worked in 8-hour shifts and applied quarantine measures. If recruits reported signs or symptoms consistent with Covid-19, they referred to sick call, underwent rapid qPCR tests for SARS-CoV-2, and were placed in solitary confinement pending test results.

Instructors were also confined to campus, had to wear masks, were provided prepackaged meals, and underwent daily temperature checks and symptom screening. Instructors assigned to a platoon where a positive case was diagnosed underwent a rapid qPCR test for SARS-CoV-2, and if the result was positive, the instructor was removed from service. Recruits and instructors were prohibited from interacting with campus support staff, such as housekeeping and catering staff. After each class completed quarantine, a deep bleach cleaning of surfaces was performed in bathrooms, showers, bedrooms and dorm hallways, and the dorm was unemployed for at least 72 hours prior to reoccupation.

Within 2 days of arriving on campus, after the recruits had received platoons and roommates assignments, they were offered the opportunity to participate in the longitudinal CHARM study. Recruits were eligible if they were at least 18 and available for follow-up. The study was approved by the Naval Medical Research Center Institutional Review Board and complied with all applicable federal regulations governing the protection of human subjects. All participants provided written informed consent.

Procedures

At enrollment, participants responded to a questionnaire regarding demographics, risk factors for SARS-CoV-2 infection, symptoms within the previous 14 days, and a brief medical history; Blood samples and swab samples of nostrils with medium turbinates were obtained for qPCR testing to detect SARS-CoV-2. Demographic information included gender, age, ethnic group, race, place of birth, and US state or country of residence; Risk factor information included whether participants had used masks, whether they had joined self-quarantine prior to arrival, their recent travel history, their known exposure to someone with Covid-19, whether they had symptoms similar to those of the flu or other respiratory illnesses and if they had had any of the 14 specific symptoms characteristic of Covid-19 or any other symptoms associated with an unspecified condition in the previous 14 days.

Study participants were followed up on days 7 and 14, at which time they reported any symptoms that had occurred in the past 7 days. Naresis swab samples were also obtained for repeated qPCR tests. Participants who had positive qPCR results were placed in isolation and were approached for participation in a related but separate study of infected recruits, which involved more frequent testing during isolation. All recruits who did not participate in the ongoing study were tested for SARS-CoV-2 only at the end of the 2-week quarantine, unless clinically indicated (in accordance with Marine Corps public health procedures) . Serum specimens obtained at enrollment were tested for SARS-CoV-2 specific IgG antibodies using the methods described below and in the Supplementary Appendix.

Participants who tested positive on enrollment day (day 0) or day 7 or day 14 were separated from their roommates and placed in solitary confinement. Otherwise, participants and non-participants were not treated differently: they followed the same safety protocols, were assigned to rooms and platoons regardless of participation in the study, and received the same formal instructions.

Laboratory methods

QPCR testing of medium turbinate nostril swab specimens for SARS-CoV-2 was performed within 48 hours of collection by Lab24 (Boca Raton, FL) using the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific), which is authorized by the Food and Drug Administration. Samples obtained from non-participants were tested by the Naval Medical Research Center (Silver Spring, MD). Samples were stored in viral transport medium at 4 ° C. The presence of specific IgG antibodies for the SARS-CoV-2 receptor binding domain (spike) in serum samples was assessed using a enzyme-linked immunosorbent assay, as described above,¹⁰ with some modifications. At least two positive controls, eight negative controls (serum samples obtained before July 2019) and four blanks (no serum) were included in each plate. Serum samples were first screened at a 1:50 dilution, followed by a full dilution series if the samples were initially positive.

Whole genome sequencing and assembly

SARS-CoV-2 sequencing was performed with the use of two sequencing protocols (an Illumina sequencing protocol and an Ion Torrent sequencing protocol) to increase the likelihood of obtaining complete genomic sequences. A custom reference-based analysis pipeline (https://github.com/mjsull/COVID_pipe) was used to assemble the SARS-CoV-2 genomes using data from Illumina, Ion Torrent, or both.¹¹

Phylogenetic analysis

SARS-CoV-2 genomes obtained from patients worldwide and associated metadata were downloaded from the EpiCoV Global Initiative on Sharing All Influenza Data database¹² on 11 August 2020 (79,840 sequences) and a subset of sequences was selected from this database using the Nextstrain software default subsampling scheme¹³ with the aim of maximizing the representation of genomes obtained from patients in the United States. Phylogenetic analyzes of the samples obtained from the participants were performed with the Nextstrain v1.0-292-ga9de690 build for SARS-CoV-2 genomes with the use of predefined parameters. Transmission and outbreak events were identified based on clustering of SARS-CoV-2 genomes obtained by study participants within the phylogenetic tree Nextstrain, visualized with TreeTime.¹⁴ A comparative analysis of mutation profiles related to the reference genome of Wuhan SARS-CoV-2 was performed using the Nextclade software, version 0.3.6 (https://clades.nextstrain.org/).

Data analysis

The denominator for calculating the percentage of recruits who achieved a first positive result for SARS-CoV-2 by the qPCR test each day of the test excluded recruits who tested positive previously, had dropped out of the study, were administratively separated from the Corps of the Marines or had missing data. The denominator for the cumulative positivity rate calculation included all recruits who had been tested at earlier times, including those who no longer participated in the study. Only descriptive and percentage numerical results are reported, without formal statistical analysis.