Study Design and Participants
To reduce the risk of introducing SARS-CoV-2 into basic training at Marine Corps Recruit Depot, Parris Island, in South Carolina, the Marine Corps established a 14-day supervised quarantine period at a college campus used exclusively for this purpose. Potential recruits were instructed to quarantine at home for 2 weeks immediately before they traveled to campus. At the end of the second, supervised quarantine on campus, all recruits were required 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 had completed the 14-day home quarantine, they presented to a local Military Entrance Processing Station, where a medical history was taken and a physical examination was performed. If potential recruits were deemed to be physically and mentally fit for enlistment, they were instructed to wear masks at all times and maintain social distancing of at least 6 feet during travel to the quarantine campus. Classes of 350 to 450 recruits arrived on campus nearly weekly. New classes were divided into platoons of 50 to 60 recruits, and roommates were assigned independently of participation in the CHARM study. Overlapping classes were housed in different dormitories and had different dining times and training schedules.
During the supervised quarantine, public health measures were enforced to suppress SARS-CoV-2 transmission (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). All recruits wore double-layered cloth masks at all times indoors and outdoors, except when sleeping or eating; practiced social distancing of at least 6 feet; were not allowed to leave campus; did not have access to personal electronics and other items that might contribute to surface transmission; and routinely washed their hands. They slept in double-occupancy rooms with sinks, ate in shared dining facilities, and used shared bathrooms. All recruits cleaned their rooms daily, sanitized bathrooms after each use with bleach wipes, and ate preplated meals in a dining hall that was cleaned with bleach after each platoon had eaten. Most instruction and exercises were conducted outdoors. All movement of recruits was supervised, and unidirectional flow was implemented, with designated building entry and exit points to minimize contact among persons. All recruits, regardless of participation in the study, underwent daily temperature and symptom screening. Six instructors who were assigned to each platoon worked in 8-hour shifts and enforced the quarantine measures. If recruits reported any signs or symptoms consistent with Covid-19, they reported to sick call, underwent rapid qPCR testing for SARS-CoV-2, and were placed in isolation pending the results of testing.
Instructors were also restricted to campus, were required to wear masks, were provided with preplated meals, and underwent daily temperature checks and symptom screening. Instructors who were assigned to a platoon in which a positive case was diagnosed underwent rapid qPCR testing for SARS-CoV-2, and, if the result was positive, the instructor was removed from duty. Recruits and instructors were prohibited from interacting with campus support staff, such as janitorial and food-service personnel. After each class completed quarantine, a deep bleach cleaning of surfaces was performed in the bathrooms, showers, bedrooms, and hallways in the dormitories, and the dormitory remained unoccupied for at least 72 hours before reoccupancy.
Within 2 days after arrival at the campus, after recruits had received assignments to platoons and roommates, they were offered the opportunity to participate in the longitudinal CHARM study. Recruits were eligible if they were 18 years of age or older and if they would be available for follow-up. The study was approved by the institutional review board of the Naval Medical Research Center and complied with all applicable federal regulations governing the protection of human subjects. All participants provided written informed consent.
At the time of enrollment, participants answered a questionnaire regarding demographic characteristics, risk factors for SARS-CoV-2 infection, symptoms within the previous 14 days, and a brief medical history; blood samples and mid-turbinate nares swab specimens were obtained for qPCR testing to detect SARS-CoV-2. Demographic information included sex, age, ethnic group, race, place of birth, and U.S. state or country of residence; information regarding risk factors included whether participants had used masks, whether they had adhered to self-quarantine before arrival, their recent travel history, their known exposure to someone with Covid-19, whether they had flulike symptoms or other respiratory illness, and whether they had any of 14 specific symptoms characteristic of Covid-19 or any other symptoms associated with an unspecified condition within the previous 14 days.
Study participants were followed up on days 7 and 14, at which time they reported any symptoms that had occurred within the past 7 days. Nares swab specimens for repeat qPCR assays were also obtained. 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 current study were tested for SARS-CoV-2 only at the end of the 2-week quarantine, unless clinically indicated (in accordance with the public health procedures of the Marine Corps). Serum specimens obtained at enrollment were tested for SARS-CoV-2–specific IgG antibodies with the use of the methods described below and in the Supplementary Appendix.
Participants who tested positive on the day of enrollment (day 0) or on day 7 or day 14 were separated from their roommates and were placed in isolation. Otherwise, participants and nonparticipants 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 instruction.
The qPCR testing of mid-turbinate nares swab specimens for SARS-CoV-2 was performed within 48 hours after collection by Lab24 (Boca Raton, FL) with the use of the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific), which is authorized by the Food and Drug Administration. Specimens obtained from nonparticipants were tested by the Naval Medical Research Center (Silver Spring, MD). Specimens were stored in viral transport medium at 4°C. The presence of IgG antibodies specific to the SARS-CoV-2 receptor-binding (spike) domain in serum specimens was evaluated with the use of an enzyme-linked immunosorbent assay, as previously described,10 with some modifications. At least two positive controls, eight negative controls (serum specimens obtained before July 2019), and four blanks (no serum) were included in every plate. Serum specimens were first screened at a 1:50 dilution, followed by full dilution series if the specimens were initially found to be 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 genome sequences. A custom reference-based analysis pipeline (https://github.com/mjsull/COVID_pipe) was used to assemble SARS-CoV-2 genomes with the use of data from Illumina, Ion Torrent, or both.11
SARS-CoV-2 genomes obtained from patients worldwide and associated metadata were downloaded from the Global Initiative on Sharing All Influenza Data EpiCoV database12 on August 11, 2020 (79,840 sequences), and a subset of sequences was selected from this database with the use of the default subsampling scheme of Nextstrain software13 with the aim of maximizing representation of genomes obtained from patients in the United States. Phylogenetic analyses of the specimens obtained from participants were performed with the v1.0-292-ga9de690 Nextstrain build for SARS-CoV-2 genomes with the use of default parameters. Transmission and outbreak events were identified on the basis of clustering of the SARS-CoV-2 genomes obtained from study participants within the Nextstrain phylogenetic tree, visualized with TreeTime.14 A comparative analysis of mutation profiles relative to the SARS-CoV-2 Wuhan reference genome was performed with the use of Nextclade software, version 0.3.6 (https://clades.nextstrain.org/).
The denominator for calculating the percentage of recruits who had a first positive result for SARS-CoV-2 by qPCR assay on each day of testing excluded recruits who had previously tested positive, had dropped out of the study, were administratively separated from the Marine Corps, or had missing data. The denominator for calculating the cumulative positivity rates included all recruits who had undergone testing at previous time points, including those who were no longer participating in the study. Only descriptive numerical results and percentages are reported, with no formal statistical analysis.