Asymptomatic SARS-CoV-2 Carriers: A Systematic Review and Meta-Analysis

Asymptomatic cases of SARS-CoV-2 can be unknown carriers magnifying the transmission of COVID-19. This study appraised the frequency of asymptomatic individuals and estimated occurrence by age group and gender by reviewing the existing published data on asymptomatic people with COVID-19. Three electronic databases, PubMed, Embase, and Web of Science (WoS), were used to search the literature following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The study population for this review included asymptomatic individuals infected with SARS-CoV-2 reported in original articles published up to 30 April 2020. A random effects model was applied to analyze pooled data on the prevalence of asymptomatic cases among all COVID-19 patients and also by age and gender. From the meta-analysis of 16 studies, comprising 2,788 SARS-CoV-2 infected patients, the pooled prevalence according to the random effect size of asymptomatic cases was 48.2% (95% CI, 30–67%). Of the asymptomatic cases, 55.5% (95% CI, 43.6–66.8%) were female and 49.6% (95% CI, 20.5–79.1%) were children. Children and females were more likely to present as asymptomatic COVID-19 cases and could act as unknown carriers of SARS-CoV-2. Symptom-based screening might fail to identify all SARS-CoV-2 infections escalating the threat of global spread and impeding containment. Therefore, a mass surveillance system to track asymptomatic cases is critical, with special attention to females and children.


INTRODUCTION
Coronaviruses are common respiratory pathogens causing illness in humans around the world (1). By the end of 2019, six different coronaviruses contagious to humans had been reported (2,3). SARS-CoV-2 was identified as the seventh virus of the coronaviridae family causing infection to humans and the World Health Organization (WHO) announced "COVID-19" as a new disease on 11 February 2020 (4). In response to the rapid global spread of SARS-CoV-2, WHO declared COVID-19 as a Public Health Emergency of International Concern (PHEIC) on 30 January 2020 and a pandemic on 11 March 2020 (5). As of 26 May 2020, there were 5,508,904 confirmed cases of COVID-19 with 346,612 deaths globally (6).
(Supplementary Table 1) was followed in conducting this study. Medical Subject Headings (MeSH) and key words, using "OR" and "AND, " were used to search published articles in the electronic databases. The following search terms were used: "Novel coronavirus 2019, " or "2019 nCoV, " or "COVID-19, " or "Asymptomatic cases/carriers of COVID-19, " or "Asymptomatic infections with COVID-19, " and "SARS-CoV-2." Articles published in English between 1 January 2020 and 30 April 2020 were included.

Selection Criteria
Authors GS, PD, SB and DRJ evaluated the search results and independently determined the eligibility of studies. Any dissonance regarding inclusion and exclusion of the studies was resolved by discussion between the authors. Any discrepancy during the review of full articles was resolved with a majority vote.

Eligibility Criteria
The inclusion criteria set for the selection of study articles were: • study population: patients with SARS-CoV-2 infections confirmed by RT-PCR but without symptoms at time of screening • study design: prospective/retrospective cohort study, case-control studies, and case reports published in peer-reviewed journals.
The exclusion criteria were: • studies without a proportion of asymptomatic SARS-CoV-2 infected patients • review and opinion articles, published protocols, metaanalyses of primary data, editorials, and cases published in languages other than English.

Study Selection
The title and abstract of the articles selected from the initial search strategy were scrutinized to screen for relevant articles. Then, the full text of relevant articles was inspected using the inclusion and exclusion criteria. Studies that reported the proportion of asymptomatic SARS-CoV-2 infected patients were included for quantitative analysis.

Data Extraction
Elements recorded from the screened articles included first author, type of study, publishing institution, date of publication, site of study, sample size ( Table 1), age and gender of patients, and ratio of asymptomatic and symptomatic cases (Supplementary Table 3).

Outcome Measurements
The primary finding of this study was the prevalence of asymptomatic cases among total SARS-CoV-2 infected individuals. The secondary outcome measures included prevalence of asymptomatic cases by gender and age group and prevalence of true asymptomatic cases within total asymptomatic cases.

Risk of Bias (Quality) Assessment in Individual Studies
The potential risk of bias in each included study was assessed using the Newcastle-Ottawa Scale (NOS) for observational studies (case-control, cohort, and cross-sectional studies) (39). Studies were graded out of 10 points (stars) as shown in Supplementary Table 2. The mean score of two reviewers (GS, SB) was considered to make the decision. Any variation in individual scores was checked and resolved by a third author (DRJ). As there is no standard cut-off score, we included all the studies with an arbitrary score value ≥ 6. This quality assessment was performed to assess the systematic error and external validity of studies and ultimately reduce the risk of biases.

Publication Bias Assessment
The potential publication bias was assessed by plotting a standard error and precision with Logit event rate as funnel plots. The absence of publication bias among the included studies was also confirmed by Begg's (Begg and Mazumdar Rank Correlation) and Egger's test (regression intercept) with p > 0.05 indicating no publication bias (40,41).

Data Analysis
Comprehensive Meta-Analysis version 3 statistical software (https://www.meta-analysis.com/) was used for the statistical analysis. Percentages were calculated to describe the distributions of categorical variables. The prevalence of asymptomatic cases of SARS-CoV-2 infection was expressed as a proportion with a 95% confidence interval using a random effects model and was presented as a Forest plot. The Cochran Q test and inconsistency index (I 2 ) were used to detect heterogeneity among studies, with a p < 0.05 indicating significant heterogeneity. I 2 values of <25, 25-75, and >75% indicate low, moderate, and high heterogeneity respectively (42).

Subgroup Analysis
Subgroup analysis was performed in targeted patient age groups (children, adults, and elderly), gender groups (male and female) and clinical outcome of asymptomatic cases (presymptomatic and true asymptomatic). Individuals 18 years and younger were considered children as recognized by the United Nations Convention on the Rights of the Child (43). Asymptomatic cases without typical symptoms associated with COVID-19 during the time of hospitalization were categorized as "true asymptomatic cases" and cases not showing symptoms during a 14-day incubation period but later exhibiting symptom were categorized as "pre-symptomatic cases" (44)(45)(46). Only studies that reported age, gender and true cases of asymptomatic COVID-19 patients were included in the subgroup analysis of age, gender, and clinical outcome of asymptomatic cases.

Study Characteristics
This systematic review included 16 studies published between 1 January and 30 April 2020. Most studies were from China (n = 11), two were from Japan and three were from the USA, with a total of 2,788 SARS-CoV-2 infected patients ( Table 1). This review integrated results from both cross-sectional and observational cohort studies.

Demographic Characteristics and Clinical Manifestation of COVID-19 Patients
Of the SARS-CoV-2 infected patients, 54

Publication Bias
The funnel plot (Supplementary Figure 4)

DISCUSSION
The pooled prevalence of asymptomatic cases of COVID-19 from this meta-analysis of 2,788 infected patients was 48.2% as estimated by random effect size. In a case series of 78 patients from 26 transmission clusters in Wuhan, China, a similar estimate (42.3%) of asymptomatic carriers in SARS-CoV-2 infected individuals was reported (22). However, considering the basic reproduction number (R 0 ) of 2.5, a slightly lower asymptomatic SARS-CoV-2 infection rate of 35% was estimated by the US Centers for Disease Control and Prevention (CDC) (47). This indicates a large number of asymptomatic cases of COVID-19 are in the community seeding potential outbreaks which requires vigilant control strategies to prevent future outbreaks. Thus, mask wearing, hand washing, physical distancing, and extensive testing, followed by quarantine of infected asymptomatic individuals, are essential to contain the rapid spread of SARS-CoV-2 locally and globally (48). Due to the high prevalence of asymptomatic cases, it is important to seek reasons for SARS-CoV-2 infection in people without visible symptoms (49). Some studies have highlighted that the cross-reactive T-cell response with exposure to other coronaviruses might have contributed to the asymptomatic phenotype in SARS-CoV-2 infected individuals (50)(51)(52)(53). This is supported by the 67% homology in the sequences of epitope between common cold coronaviruses and SARS-CoV-2, and an increased number of CD4+ T cells in asymptomatic patients compared to symptomatic patients (54,55).
SARS-CoV, SARS-CoV-2, and MERS, despite all belonging to the genus Betacoronavirus, differ in their disease transmission and clinical features (56)(57)(58). SARS and MERS were mainly associated with nosocomial spread whereas SARS-CoV-2 is being widely disseminated both in the community and hospital environments (59). The prevalence of asymptomatic infection was comparatively lower in MERS and SARS, at about 9.8% (60) and 13% (61), respectively. The higher asymptomatic infection of SARS-CoV-2 may be related to the high replication efficiency of this virus, which is three times greater than SARS-CoV (62,63). Thus, SARS-CoV-2 rapidly disseminates to the pharynx and sheds prior to the activation of the innate immune response and presence of symptoms (64). Another contributing factor could be a less significant induction of host interferon and pro-inflammatory response, which also distinguishes SARS-CoV-2 from other SARS-CoV strains (64). The proportion of asymptomatic cases of COVID-19 has gradually increased since the first reported outbreak Frontiers in Public Health | www.frontiersin.org in Wuhan, China (65). This may be because of decreased pathogenicity of SARS-CoV-2 in the process of the long chain of transmission (36).
The communicable period of asymptomatic cases of COVID-19, defined as the interval between the 1st day of positive nucleic acid tests to the 1st day of continuous negative tests, can last for a month (27). Thus, asymptomatic cases of COVID-19 may carry the virus for a long period of time and spread it unknowingly. Potential SARS-CoV-2 transmission from asymptomatic individuals has been documented in many studies (19)(20)(21)27). A high viral load was detected when SARS-CoV-2 was assessed in the upper respiratory specimens of asymptomatic, pre-symptomatic and symptomatic patients, suggesting potential for transmission regardless of symptoms (66). The biological evidence for this was supported by a study in a skilled nursing facility where infectious SARS-CoV-2 culture was grown from the upper respiratory tract specimens of pre-symptomatic and asymptomatic patients 6 days before the development of COVID-19 related symptoms (67).
Of the total SARS-CoV-2 infected patients included in this meta-analysis, 54.4% were male and 45.6% were female. This implies that men were more susceptible to SARS-CoV-2 infection than women. The COVID-19 pandemic has shown a striking gender bias with more cases and a higher mortality rate in men than in women (68). Data from the WHO and Chinese studies indicate that ∼1.7% of females who contracted SARS-CoV-2 have died compared to 2.8% of males (69). Increased male susceptibility might be explained by biological and behavioral factors (70). Biological factors include men's high level of testosterone that inhibits antibody production, and the presence of angiotensin-converting enzyme 2 (ACE2) receptors (cell receptors which play an essential role in SARS-CoV-2 entry) that facilitate viral replication (71). Similarly, social, behavioral, and lifestyle factors include men's higher rates of smoking and alcohol consumption, and low level of hand-washing practices (72), although there is no clear evidence these behavioral factors have any impact on COVID-19 transmission. In general, women have a stronger immune response against viral infections than men (73), which possibly gives women some immunological protection (74). Gender-based hormones also regulate and influence aspects of viral entry including expression and activity of ACE2 and trans-membrane serine protease 2 (TMPRSS2) (69). The gene expression of ACE2, a, together with TMPRSS2 may also influence intensity of SARS-CoV-2 infection. Subgroup analysis of asymptomatic COVID-19 patients revealed that children are the largest age group of asymptomatic carriers of SARS-CoV-2 (49.6%), followed by adults (30.3%), and the elderly (16.9%). A higher incidence of asymptomatic cases of COVID-19 in children can be related to both exposure and host factors. It is known that children's immune systems are not well-developed, and the maturity and binding ability of ACE2 in children may be lower than in young adults (75). However, children often experience numerous viral infections, and their repeated viral exposure is anticipated to aid their immune response to SARS-CoV-2 (76). Meanwhile, elderly people with a weakened immune system (77,78) are less likely to be asymptomatic carriers. In contrast, adults who most likely have a stronger immune system can be infected and remain asymptomatic carriers (79). However, a detailed mechanism for the differences in asymptomatic manifestation of SARS-CoV-2 among these three age groups (children, adults and the elderly) is yet to be explored.
The pooled prevalence of asymptomatic SARS-CoV-2 infection was observed to be higher in females (55.5%) than in males (44.5%) from subgroup analysis. Aligning with our results, more women (66.7%) were observed to be asymptomatic among 78 close contacts of COVID-19 patients in Wuhan, China (22). Although there was no significant subgroup difference by gender, higher prevalence of asymptomatic cases in females could arise from host factors in women (80). Compared to men, women exhibit stronger innate, cellular, and humoral immune responses due to increased activation effects of female sex hormones and the presence of immune response genes on sex chromosomes (81). These enhanced innate and adaptive immune responses could have contributed to the increased asymptomatic SARS-CoV-2 infection in women.
Of the total asymptomatic SARS-CoV-2 cases included in the subgroup analysis, 39% cases did not have any symptoms while 15.3% were pre-symptomatic cases with mild COVID-19 related symptoms including fever, malaise and cough 3 days after a RT-PCR nucleic acid test positive (28,30). This data inferred that a small fraction of asymptomatic cases may eventually become symptomatic. Thus, in order to evaluate the actual prevalence of true COVID-19 asymptomatic cases, longitudinal observations of cases for at least 14 days is recommended.
A recent review reported that as the surveillance and contact tracing of MERS progressed over time, the rate of asymptomatic MERS infected patients increased to 28.6% (60). The increase in asymptomatic infection of MERS was inversely proportional to the case fatality rate (82). This clearly demonstrates the importance of mass surveillance and contact tracing in the detection of asymptomatic SARS-CoV-2 infected individuals in the community and hospitals to reduce the disease fatality rate and dissemination of COVID-19. Transmission of COVID-19 by asymptomatic people is the weakness of COVID-19 control and prevention strategies (17). Hence, mass testing for SARS-CoV-2 in asymptomatic people should be increased especially in mass living conditions such as prisons, hospitals, camps, nursing, and elder care facilities. To contain the rapid spread of COVID-19, screening and testing of SARS-CoV-2 asymptomatic carriers along with other control strategies must be prioritized at the community level.
The strengths of this study include comprehensive analysis of 16 studies adding precision to the estimation of the prevalence of asymptomatic SARS-CoV-2 infection. Subgroup analysis revealed that children and females were the most likely asymptomatic carriers of SARS-CoV-2, highlighting the necessity of focusing on these target populations to control the pandemic. An additional strength of this study was the evaluation of asymptomatic and pre-symptomatic cases which helped understand the dynamics of this pandemic.
Despite strong findings, this study has some limitations. As asymptomatic patients are very likely to be unnoticed and not detected, the pooled prevalence rate in this study might be under-reported. Moreover, the majority of the studies in this meta-analysis were from China, with few ethnic groups other than Chinese, because the meta-analysis was conducted only 3 months after the pandemic started. More comprehensive studies including a wider range of ethnicities from global communities are needed to provide a more precise estimate of the prevalence of asymptomatic SARS-CoV-2 cases in a broader context.

CONCLUSION
About half of the SARS-CoV-2 infected patients were asymptomatic at the time of screening. Despite the limitation, the findings of this study highlight that females and children were the predominant groups without symptoms of COVID-19. However, in general, asymptomatic infections can occur in any age range and either gender. As asymptomatic carriers play a critical role in the spread of the COVID-19 pandemic, understanding the actual prevalence of asymptomatic cases is important for setting control measures in both the community and health care centers. The high prevalence of asymptomatic COVID-19 cases suggests that screening based only on symptoms might fail to identify a large proportion of SARS-CoV-2 infections, escalating the threat of rapid spread. Thus, mask wearing, extensive testing for identification, and the quarantine of infected asymptomatic individuals are essential to curb this pandemic.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
BR conceived the idea and BR, GS, and DJ designed the study. GS, PD, and SB reviewed the literature, extracted data, performed data analysis, and drafted the manuscript. DJ, BR, LS, and RT revised and reviewed the manuscript. BR and GS substantially revised the manuscript. DJ supervised the overall data analysis and writing. All the authors read and approved the final manuscript.