Table 2.

Characteristics of included studies

Study (the calendar time period of collection)	Study population	Brief description of study	Screening/testing methods for COVID-19 infection	Case definition(s) including cut-off values for PCR or other laboratory tests	Symptom ascertainment, including follow-up ascertainment methods	Evidence of the chain of transmission (probability)	Notes
Arons (Feb-March 2020)	Skilled nursing home facility; residents with mean age 78 years, Washington, USA.	Repeated point prevalence survey in a care home. First case was identified then a survey done 10 days later, followed by a second survey 7 days subsequently.	NP and OP swabs taken and subjected to testing for SARS-CoV-2, using rRT-PCR, viral culture and gene sequencing.	Positive testing residents were categorized as symptomatic with typical symptoms (fever, cough, or shortness of breath), symptomatic with only atypical symptoms, presymptomatic or asymptomatic.	Standardized symptom: assessment form completed by nurses for each resident tested, on the survey day. Interview and medical records were used to ascertain symptoms for the previous 14 days. Asymptomatic positive-testing residents were reassessed for symptoms 7 days later.	Probable/Likely Viral growth was observed for specimens obtained from 17/24 presymptomatic residents, 24 presymptomatic residents had a median rRT-PCR Ct value of 23.1. More than half of residents with positive test results were asymptomatic at the time of testing and most likely contributed to the transmission. Staff and residents were being actively screened for signs and symptoms and either promptly isolated (residents) or excluded from work (staff) if any were present.	Direct evidence about transmission not presented, indirect evidence reported about spread within the care home. Cognitive impairment was present in 28/48 (58%) patients that were positive for SARS-CoV-2, reducing the reliability of their self-reporting.
Borges (Summer 2020)	Non-COVID-19 hospital ward patients and staff, Lisbon and Tagus valley, Portugal.	Investigation of a nosocomial outbreak, with in-depth contact tracing and testing. After cases were identified within the hospital, 348 HCWs and 92 patients were screened. Laboratory tests were performed in 245 individuals	NP and OP swabs were collected from patients and HCWs. Positive SARS-CoV-2 RNA samples were subjected to virus genome sequencing.	Not reported.	Symptoms of fever, cough or shortness of breath, were recorded at the time of testing.	N/A	The transmission was not tracked according to symptomatology. Study participants were not subjected to serial PCR testing and culture was not attempted. Asymptomatic is not clearly defined, and only referred to as at the time of testing, so may not be persistently asymptomatic.
Cordery (Oct-Dec 2020)	School staff, pupils and their households, London, UK	Sequential longitudinal sampling of infected children, their contacts, and surfaces at school and home. Nose and throat swabs were taken, faecal samples were collected where possible. Samples with Ct value < 30 were inoculated into Vero cells for culture.	Case follow-up: separate nose, throat, and hand swabs, saliva samples and gingival crevicular fluid swabs were obtained from each case up to 5 times within 14 days, then weekly over a second period of up to 14 days; also, faecal samples collected where available. Close contact follow-up: combined nose–throat swabs and gingival crevicular fluid swabs were obtained from each participating contact on the same day or as soon as possible (<48 hr) after case sampling, then weekly for up to 28 days. Samples with high viral load (Ct value < 30) were inoculated into Vero cells for culture.	Combined nose–throat swab testing positive by RT-PCR.	Symptom description and contact history of cases were collected by questionnaire, completed by parent or guardian.	Probable/Likely Three asymptomatic cases were detected in week 2 of screening. No evidence of wider transmission among children remaining in school, except the 1 unexpected cluster of 3 asymptomatic cases in 1 school in the same class. In 1 of the asymptomatic cases, the viral load rose on repeat testing to >4 million copies per swab and another asymptomatic household member was identified as infected (case A Cts: 26.3, 22.3,28.2). The case remained asymptomatic however despite viral shedding continuing for at least a week after an initial positive test.	Environmental sampling was also done in homes and schools.
De Laval (Feb–March 2020)	Military support facility staff, France	Outbreak investigation using a testing strategy according to pre-test probability, after identification of a severely ill index case. Case finding and contact tracing with testing of at-risk contact persons who had any relevant symptoms. Only symptomatics were tested.	NP and OP swabs were taken. Samples positive by PCR were gene sequenced. One month subsequently, serology was done on all staff, which was potentially able to indicate past infections that ppts did not report symptoms for (i.e., asymptomatic).	Confirmed cases were participants with positive RT-PCR test results and/or positive serology	The interviewer-administered a standardized questionnaire using an in-depth interview to ascertain symptoms and date of onset, also information about contacts in the 14 days prior to symptom onset.	Possible Three cases were asymptomatic. Contact tracing results did not identify any transmission from asymptomatic to symptomatic cases in this cluster.	Only symptomatic individuals had swabs collected. Serology done 1 month later was able to identify participants who remained asymptomatic.
Ferreria (April–May 2020)	HCWs, a large hospital, Toronto, Canada	Over a 6-week period, HCWs were prospectively enrolled and underwent 1 to 6 serial NP swabs for SARS-CoV-2 PCR testing; study participants were required to be asymptomatic and not have a previous diagnosis of COVID-19.	Serial NP swabs were taken and subjected to PCR. Serological testing for IgG was performed on a subset of asymptomatic HCWs with no prior known exposure to SARS-CoV-2. Genome sequencing was performed on positive swab specimens.	PCR Ct count cut off not reported. For serology, ratio of ppt sample: calibration interpreted as: <0.8 negative; 0.8 to <1.0 borderline; and 1.1 IgG positive.	Symptoms compatible with COVID-19 included fever, headache, new or worsening cough, shortness of breath, sore throat, rhinorrhoea, diarrhoea, anosmia, myalgias and conjunctivitis.	N/A	The main aim of the study was to assess the prevalence of asymptomatic, positive-testing HCWs.
Francis (until November 2020)	Patients attending hospital, and associated staff contacts, Nottingham, UK	Hospital patients were screened on admission irrespective of symptomatology; hospital staff were tested if symptomatic or a local outbreak occurred.	NP swabs were subjected to RT-PCR; positive tests from samples with some epidemiological evidence of linkage were subject to genome sequencing; also surveillance of sequences was done using samples with PCR Ct < 30.	Not reported.	No methods for symptom assessment were reported.	N/A	The study was set up to examine how whole genome sequencing can help identify and control outbreaks. Clusters of infections are reported, with evidence on epidemiology, PCR, and genome sequencing. Transmission from specific individuals not reported; transmission from individuals established to be asymptomatic therefore not reported.
Gettings (Dec 2020–Jan 2021)	Students and staff in schools, Georgia, USA	Index cases and their close contacts in schools were identified by the school and public health staff. Epidemiology and WGS were used to identify transmission patterns.	In-school contacts: symptoms assessed, RT-PCR test on anterior nasal swab offered. WGS was done on PCR- positive samples with a Ct of <32 cycles.	Case is defined as a student or staff member who attended school in person within ≤2 days before testing PCR or antigen test positive.	Symptoms at the time of testing were recorded, and for 14 days, daily through daily text-message based symptom monitoring.	N/A	The study aimed to assess the extent and settings of transmission in and related to schools.
Hershow (Dec 2020–Jan 2021)	Students and staff in elementary schools reopening after pandemic related closure, Utah, USA.	The screening was offered to close contacts of identified index cases. Samples were collected 5–10 days after exposure.	Saliva samples (or nasal samples, if saliva is not available) are subjected to RT-PCR. WGS was performed on positive samples.	Index case defined as a student or staff member with laboratory-confirmed SARS-CoV-2 infection who had attended in-person school while infectious for at least 1 day.	Symptoms and exposures information were collected by questionnaire.	Possible Low transmission in schools despite substantial community transmission. Among the five persons with school-associated cases, 3 persons were asymptomatic and 3 were exposed to asymptomatic index patients; 4 cases were attributed to student-to-student transmission, and 1 was to student-to-teacher transmission.	Community transmission was relatively high at the time. In-school mask use and 3ft/2 m distancing were in place. Reports low transmission in schools despite substantial community transmission.
Jeffery Smith (April 2020)	Care homes, London, UK	Study designed to look at asymptomatic transmission, using serology and comparing findings from 7 care homes without outbreaks (single case or no cases) to 6 care homes with recognised outbreaks (2 cases or more).	7 non-outbreak homes investigated with nasal swabbing for SARS-CoV-2 RT-PCR and serology for SARS-CoV-2 antibodies five weeks later. WGS was performed on RT-PCR positive samples.	Case definition not reported.	Staff self-reported symptom status during preceding 14 days and at the time of swabbing; residents' symptoms were recorded by staff. Daily monitoring of each care home by study staff to identify any newly symptomatic individuals. Typical COVID-19 symptoms classed as fever 37.8°C, shortness of breath/cough; atypical symptoms included (but not restricted to) new confusion, reduced alertness, fatigue, lethargy, reduced mobility and diarrhoea.	Possible The finding of asymptomatic SARS-CoV-2 infection in care homes that did not report a single case of COVID-19 and genomic evidence of a small cluster of staff and residents infected with the same SARS-CoV-2 lineage in care home F. It was not possible to extract direct information on transmission from identified asymptomatic or pre-symptomatic index cases identified to contacts.	This study aimed to investigate asymptomatic transmission; it is phase 3 of a series of investigations in care homes.
Lewis (April 2020)	Households, Utah, USA	Within-household transmission study following identification of an index case within each household.	NP swabs were taken daily, blood samples at day 0 and day 14. Swabs tested by RT-PCR. All positive or inconclusive according to PCR were subjected to viral culture.	Symptoms classified as classic (cough, shortness of breath, or discomfort while breathing), non-classic (>2 of measured or subjective fever, chills, headache, myalgia, sore throat, loss of taste, or loss of smell), and asyndromic (symptoms other than classic or non-classic). PCR Ct values were categorized as low (<20), medium (20–30), and high (>30).	Index patients and household members completed a daily symptom diary;	Unlikely Five households enrolled. Eligibility entailed an identified positive index case resulting from testing due to symptom onset within each household; secondary transmission was observed in 2 households. WGS for the second household (HH 05-00 symptomatic) indicated the likely chain of transmission was from 05-00 and/or 05-03 (symptomatic) who had genetically identical infections and were exposed to the same community contact. WGS indicates that the infections across all 4 household members in HH-2 were essentially genetically identical, suggesting that the index case, 02–00 (symptomatic), transmitted to all remaining household members.
Murata (Feb 2020)	Passengers disembarking from the Diamond Princess cruise ship, Japan	Observational study of a cohort of asymptomatic passengers and crew members who tested positive for SARS-CoV-2 and their cabin-mates who tested negative and were transferred from the cruise ship to on-shore hospitals in Japan for isolation.	Screening RT-PCR of nasopharyngeal or throat swabs. Samples with 2 or more positive PCR test results were subjected to viral culture.	Ct value of 40 is used as a cut-off for positivity.	Asymptomatic status was determined at the time of testing based on the absence of fever (temperature of ≥37.5°C) and clinical symptoms (cough, dyspnoea, chest pain, sore throat, and nasal discharge) by physicians and nurses	N/A	This study was designed to examine the shedding of viable viruses from asymptomatic carriers.
Pray (Sept–Oct 2020)	University campuses, Wisconsin, USA	Evaluated performance of an antigen (immunoassay) test compared with RT-PCR for SARS-CoV-2 detection among asymptomatic and symptomatic persons.	Nasal swabs were collected from all consenting participants and tested using rapid antigen and RT-PCR. All specimens testing positive were subjected to viral culture.	Case definition cited; laboratory test cut-off values not reported.	Cross-sectional study, no follow-up.	N/A	Study was designed to investigate performance of a rapid antigen test, using RT-PCR as the standard. Onward transmission was not investigated.
Soto (April–May 2020)	Emergency childcare centre contacts, Quebec, Canada	Outbreak study in an emergency childcare centre, including 120 children, employees and household contacts of confirmed COVID-19 cases.	NP swabs were subjected to RT-PCR. Nucleic acids were extracted from NP samples and subjected to reverse transcription for phylogenetic analyses.	Algorithm for deciding cases is reported; PCR Ct cut-offs not reported.	Definition of symptoms collected not reported and asymptomatic not defined	N/A	Epidemiology (social network analysis) and phylogeny were used. Unclear but assume screening of all children and staff at the childcare centre; report states that within household contacts, only symptomatics were tested.
Speake (March–April 2020)	Passengers on a 5-hr domestic flight, Australia; some passengers had arrived from abroad, including from cruise ships.	To investigate the possible transmission of SARS-CoV-2 on a commercial airline flight, using whole genome sequencing to support evidence on chains of transmission.	PCR testing was applied to throat swabs and bilateral NP or deep nasal swabs from symptomatic individuals. Genome sequencing performed where possible (if multiple samples were available from a participant, samples with lowest Ct values used). Virus culture was attempted for all samples sent to 1 of the 2 laboratories used.	Case definition according to symptomatology and/or a closely matching virus genomic sequence.	Symptoms compatible with COVID-19 led to testing.	N/A	People were only tested for SARS-CoV-2 if they had significant symptoms (testing capacity not sufficient to include people without symptoms).
Surie (June–Aug 2020)	Convenience sample of elderly care home residents with underlying health conditions, Arkansas, USA	To estimate the infectious period of SARS-CoV-2 in elderly care home residents with underlying conditions, using symptom recording. 17/39 nursing home residents (all PCR positive, all eventually symptomatic) were followed prospectively to examine viral shedding duration, and viral culture was done to assess infectivity.	OP and anterior nares swabs and saliva samples collected and tested using RT-PCR. All positive samples were subjected to viral culture. Where CPE in viral culture was observed RT-PCR was used to confirm the presence of SARS-CoV-2. Collection of blood (for serology) attempted at enrolment and at visit days 6, 12, 21, and 42.	Not reported.	Symptoms of shortness of breath, cough, malaise, muscle pain, dizziness, diarrhoea, vomiting, sore throat and headache assessed by HCW (before enrolment). At enrolment and each subsequent visit, participants were interviewed using the CDC standard list of symptoms, to which chest and abdominal pain were added. Participants followed for 42 days after enrolment in study. Symptom assessment, medical record review, sample collection done at each study visit.	Possible Whole-genome sequencing on eligible specimens (Ct < 30) showed there were only 2–3 single nucleotide variant differences among the entire set of sequenced genomes, which implied they were likely from the same source and a single introduction to the nursing home. There were 6 symptomatic participants, only 1 was among the 9 (53%) participants who had replicable, infectious virus isolated. The authors consider that the findings underscore the potential role of pre-symptomatic carriers in transmission.	Infectivity was defined as isolation of replication-competent virus from a specimen in cell culture
Taylor (March–May 2020)	Outbreak investigation at army barracks, London, UK	Study to monitor SARS-CoV-2 infection and antibodies in soldiers, their family and civilians; also to correlate SARS-CoV-2 infection and antibody positivity with clinical symptoms and signs.	Screening of army personnel, their families and civilian contacts was done twice 36 days apart. Nasal swabs, throat swabs and blood samples taken. Respiratory samples subjected to rtRT-PCR. Positive samples with Ct < 35 subjected to WGS and to virus isolation on Vero E6 cells; virus detection confirmed by CPE up to 14 days after inoculation. Serum samples analysed for SARS-CoV-2 antibodies.	Not reported	Participant recalled symptom onset and timing, assessed by questionnaire (including fever, cough, sore throat, runny nose, sneezing, breathless, drowsy, lethargic, seizures, coma, muscle aches, rash, vomiting, diarrhoea, loss of appetite, conjunctivitis, headache, loss of smell, loss of taste, blurred vision, other (state).	Probable/Likely There were 4 cases that all remained asymptomatic throughout with 0 base difference (genetically indistinguishable), these 4 cases had a link through 1 common workplace location. There were another 4 (different) cases although 2 later developed symptoms also with 0 base difference, other than visiting the same shop and using a common entrance to the barracks no common links in the workplace/barracks setting could be found.	No onward transmission from asymptomatics documented; samples from asymptomatic individuals were assessed for infectivity via viral culture, but timeline of symptoms to exclude previous or subsequent symptoms is not reported.
Van Hensbergen (March 2020)	99 residents of a long-term care facility, aged 64 to 97 years, The Netherlands.	Cross-sectional outbreak investigation in a care home.	Throat and NP swabs taken for PCR testing. Cycle count numbers determined. WGS performed on samples. Residents with relevant symptoms or recent contact or epidemiological history were tested; additionally, 12 random samples from residents without symptoms were taken.	Clinical case definition reported.	On the day of survey, HCWs performed semi-structured oral interviews of all residents, to collect information on age, sex, new or unusual signs and symptoms of disease, complemented with comorbidity information from their patient records and taking their temperature (rectally) in the morning and the evening (subfebrile: 37.5–38°C; fever: 38.0°C and above). Symptoms: fever, subfebrile temperature, cough, fatigue, malaise, vomiting, loss of appetite, nausea and dizziness	N/A	Some residents had some impaired cognition, which may have prevented full recording of signs and symptoms.
Wallace (May–June 2020)	Detainees in a detention centre, Louisiana, USA.	Prospective cohort study using serial testing of detainees initiated 2 to 4 weeks after identification of SARS-CoV-2 infection in staff and detainees. Dormitories had shared toilets and bathroom facilities.	NP swabs were tested by rRT-PCR. Ct < 40 considered positive and those specimens were subjected to culture; also nucleic acid extracted and sequenced.	Ct < 40 considered positive	Symptom questionnaire, self-administered on each survey day.	Probable/Likely 12/52 asymptomatic had positive viral culture results. A large number of asymptomatic infections, and shedding of replication-competent virus in asymptomatic participants. The phylogeny indicates within-dormitory transmission. Individuals are described as asymptomatic, but they could have been presymptomatic.	Authors state: “… detained persons might have limited recall of mild symptoms and symptom timing, particularly symptoms occurring >2 weeks before testing, potentially resulting in an overestimation of the prevalence of asymptomatic infection.”

HCW, healthcare worker; N/A, not applicable/not assessed; NP, nasopharyngeal, OP, oropharyngeal; WGS, whole-genome sequencing.