Transmission Potential of Asymptomatic and Paucisymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Infections: A 3-Family Cluster Study in China

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), emerged in December 2019 in Wuhan, China [1]. It has since been declared a global pandemic with > 2.4 million cases reported as of 22 April 2020 [2]. Person-to-person transmission has been established [3–8], and asymptomatic transmission of SARS-CoV-2 has been reported [9]. However, studies on the potential transmission of SARS-CoV-2 by asymptomatic persons and those with mild illness have been limited [10]. Herein, we report a 3-family cluster study of 8 patients associated with asymptomatic and paucisymptomatic (1 with mild symptoms only) SARS-CoV-2 transmission in Shandong Province, China.

METHODS

Epidemiological Investigation

The first SARS-CoV-2–positive patients in this cluster were identified on 21 January 2020, triggering an epidemiological investigation by the local Center for Disease Control and Prevention. To identify the possible infective source, the epidemiological investigation focused on exposure history before the onset of illness, such as travel history to Wuhan or Hubei provinces, visiting live animal markets, and contact history with febrile persons. Medical records were also closely reviewed to verify the timelines of events and clarify clinical progressions.

To examine possible environmental contamination of SARS-CoV-2 in households, select surfaces that may be frequently touched by family members were sampled in the bedroom (door handle, bedside light switch, and sliding of wardrobe door), kitchen (door handle, faucet handle, light switch, rice cooker plug), and bathroom (door handle, handrail, surface of the toilet bowl, sink). One swab per site (room) with multiple surfaces was collected.

All close contacts of SARS-CoV-2–positive patients were traced, including family members who lived with the patients and individuals who had contact with the patients within 1 m without wearing proper personal protection. Close contacts were quarantined at home and monitored for fever (≥ 38°C) and symptoms. In addition, nasopharyngeal (NP) swabs of close contacts were collected every 24 hours from day 1 to day 14 to detect SARS-CoV-2 by molecular assay. If any close contact had positive detection of SARS-CoV-2, they were sent to a hospital for isolation and treatment.

SARS-CoV-2 Molecular Detection, Sequencing, and Phylogenetic Analysis

All collected environmental and patient samples were stored at –80°C before being transported using cold chain to a Biosafety Level 2 enhanced laboratory to perform molecular detection of SARS-CoV-2. A real-time reverse-transcription polymerase chain reaction (rRT-PCR) test kit (GZ-D2RM, Shanghai GeneoDx Biotech Co, Ltd) targeting the ORF1ab and N genes of SARS-CoV-2 was used. A cycle threshold (Ct) value < 37 was interpreted as positive for SARS-CoV-2 RNA, and a Ct value of ≥ 40 was defined as a negative test. A medium load (weakly positive), defined as a Ct value of 37 to < 40, required confirmation by retesting. Positive samples were sequenced directly from the original specimens as previously described [11]. The maximum likelihood phylogenetic tree of the complete genomes was conducted by using RAxML software (version 8.2.9) [12] with 1000 bootstrap replicates, employing the general time-reversible nucleotide substitution model.

RESULTS

Description of SARS-CoV-2–Positive Patients

Patient 1 (62-year-old woman) and patient 2 (65-year-old man) were a couple who lived with their son (patient 3), daughter-in-law (patient 4), and 2 grandchildren. Patient 1 presented with cough, rhinorrhea, and sputum on 12 January 2020 (Figure 1). On 15 January, she visited a health clinic and was diagnosed with a common cold. She was prescribed intravenous infusions of ampicillin and sulbactam, ribavirin, and traditional medicine for 5 days. On 16 January, she developed a fever (38°C). On 17 January, patient 2 reported symptoms of fever (37.8°C), cough, sputum, earache, and upset stomach. He was also diagnosed with a common cold at the health clinic and received the same prescriptions as patient 1 for 3 days. However, their symptoms did not resolve at the conclusion of the treatment regimen, leading both to seek care at a local hospital on 21 January. NP swabs were collected from both patients at the hospital and confirmed positive for SARS-CoV-2 by rRT-PCR. Thereafter, they were admitted to the isolation ward of the hospital for treatment. Major symptoms during hospitalization for both patients included fever, cough, and fatigue (Figure 1).

Figure 1.

Timeline of relevant exposures and clinical symptoms of 8 patients with severe acute respiratory syndrome coronavirus 2 infection. Abbreviations: rRT-PCR, real-time reverse-transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

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Patient 3 (37-year-old man) and patient 4 (35-year-old woman), a young couple, were close contacts of patients 1 and 2. They were self-quarantined at home for 14 days starting on the day of the hospital admission of patients 1 and 2. Their NP swabs were collected on 23 January 2020 for SARS-CoV-2 testing. Patient 3 was confirmed positive that same day for SARS-CoV-2 by rRT-PCR, but had no symptoms. Patient 4 tested negative for SARS-CoV-2 but was later confirmed to be positive for SARS-CoV-2 on 25 January when a repeat NP swab was collected and tested. Upon hospital admission, patient 3 developed a slightly dry and itchy throat. The cough was a major symptom during hospitalization, and 2 days’ fever and 4 days’ fatigue of the total hospital stay were also recorded. Patient 4 had no identified clinical symptoms (Figure 1).

Patient 5, a 53-year-old woman, lived with her son (patient 6) and parents, and was a close contact of patients 3 and 4, being the mother-in-law of patient 3 and the mother of patient 4. She was self-quarantined at home beginning the day of patient 3’s confirmation (23 January 2020). On 24 January, her NP swab was collected to test for SARS-CoV-2 infection, which was later confirmed positive by rRT-PCR, despite her lack of symptoms. She also did not show any clinical symptoms of infection during hospitalization (Figure 1). Patient 6, a 28-year-old man, was identified as the close contact of his mother (patient 5). He was self-quarantined at home beginning the day of his mother’s (patient 5) confirmed infection (24 January). His NP swab was collected on 25 January and confirmed SARS-CoV-2 positive by rRT-PCR. Upon admission in the afternoon on 25 January, he developed a fever (37.5°C). Major symptoms during hospitalization included fever and cough, and the symptoms persisted > 2 weeks.

Patient 7, a 35-year-old man, lived with his wife and 3 children and was identified as the close contact of patient 3. He was self-quarantined at home beginning the day of patient 3’s confirmed infection (23 January 2020). On 25 January, his NP swab was collected and tested positive for SARS-CoV-2 by rRT-PCR. During hospitalization, he was paucisymptomatic, with only an occasional cough. Patient 8, a 3-month-old female infant, was the close contact of patient 7, her father. On 27 January, her NP swab was collected and was a weak positive for SARS-CoV-2. A repeat NP swab was collected on 29 January and was positive for SARS-CoV-2. The infant had no clinical symptoms before, during, or after hospitalization.

The chest computed tomographic images on admission or hospitalization showed that patients 1–6 had ground-glass opacities. However, no significant abnormalities were observed for patients 7 and 8 (Supplementary Figure 1). As of 17 February 2020, all patients recovered and were discharged to home isolation for 14 days.

Exposure Histories

Patients 1 and 2 traveled to their hometown in Xiaogan from 29 December 2019 to 15 January 2020 (Figure 1). Xiaogan is a city adjacent to the epidemic center of Wuhan and identified the first case on 1 January 2020. Moreover, they had changed trains at the Hankou railway station in Wuhan. Patients 3 and 4 had not traveled to Wuhan. They and their parents live together, eat together, and have frequent face-to-face interactions. Face masks or other personal protective equipment (PPE) were not used at home. Patient 5 had contact with patients 3 and 4 several times at a factory that they jointly operated. Patient 5 also visited the home of patients 1–4 on the evening of 21 January and stayed 1 night. On the morning of 22 January, patient 5 was driven home by patient 3. During these contacts, no face masks or other PPE were used. Patient 6 did not report close contact with known COVID-19 cases except for his mother. Patient 7 reported that he had a frequency of contact of 2–3 times daily with patient 3 at the factory from 15 to 18 January and dined with patient 3 and other colleagues on 18 January. He did not report close contact with any known COVID-19 cases except for patient 3. Except for contact with her father, patient 8 had no known contact with COVID-19 patients.

SARS-CoV-2 in Samples From Patients and Environments and Phylogenetic Analysis

SARS-CoV-2 was detected in NP swabs of all patients during hospitalization, including asymptomatic and paucisymptomatic patients (Figure 1). A total of 15 (5 per household) surface swab samples were collected from the bedrooms (n = 9; 3 per household), kitchens (n = 3; 1 per household), and bathrooms (n = 3; 1 per household) of patient homes. Two of 15 (13.3%) swabs (1 from the bedroom of patient 3 and 1 from his family kitchen) were positive for SARS-CoV-2 by rRT-PCR.

The full-genome sequences for 8 patients were obtained and have been deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database (accession numbers EPI_ISL_414934 through 414941). The full genomes for the 2 environmental swabs positive for SARS-CoV-2 were not obtained due to low-coverage genomes. The full genomes of 8 patients were > 99.9% identical across the whole genome. Phylogenetic analysis revealed that the viruses from patients were clustered in the same clade and were genetically similar to other SARS-CoV-2 sequences reported from China and other countries (Figure 2). No significant mutation site was identified in the 8 SARS-CoV-2 viral sequences compared with previous strains in China and other countries.

Figure 2.

Phylogenetic analysis of full-length genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 8 patients. Red text indicates SARS-CoV-2 detected in the patients in the present study.

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Close Contacts

Fifteen contacts of either patient 1 or patient 2 were identified, and 2 contacts (patients 3 and 4) were confirmed with COVID-19. A total of 88 contacts of patient 3 were identified, and 2 contacts (patients 5 and 7) tested positive for SARS-CoV-2. Seventy-three close contacts of patient 5 were identified, and 1 contact (patient 6) tested positive for SARS-CoV-2. Two contacts were identified for patient 6 and all tested negative. Twenty-one contacts of patient 7 were identified and 1 contact (patient 8) tested positive. Of 101 close contacts identified for patient 8, all tested negative. No other close contacts of the patients were identified during the 14-day follow-up. Thus, a crude estimation of the attack rate is 3.8% (4/105) for symptomatic and 1% (2/195) for asymptomatic and paucisymptomatic infection.

DISCUSSION

We report a unique 3-family cluster of infection with SARS-CoV-2, in which 8 of 15 members were confirmed with SARS-CoV-2 infection. Particularly interesting is that of 6 secondary patients, 2 were asymptomatic, 1 was paucisymptomatic, and 3 were symptomatic. Our findings show that the transmission of SARS-CoV-2 by individuals with asymptomatic or paucisymptomatic infections is possible. Patients 1 and 2 were likely first exposed to SARS-CoV-2 after visiting their hometown in Xiaogan, Hubei Province, China. Their son (patient 3, symptomatic) and daughter-in-law (patient 4, asymptomatic), whom they live with, were later found to be infected with SARS-CoV-2. Patient 5 (asymptomatic) was identified to be infected with SARS-CoV-2 after frequent contact with patients 3 and 4 during work and home visits. She transmitted the virus to her son (patient 6, symptomatic) whom she lives with. Patient 7 (paucisymptomatic) was found to be infected with SARS-CoV-2 after frequent contact with patient 3 during work. He likely transmitted the virus to his daughter (patient 8, asymptomatic). In addition, consistent with previous studies [5–8], the transmission of SARS-CoV-2 during the incubation period of patient 3 likely occurred. Patients 5 and 7 were infected after their exposures to a presymptomatic patient 3 during working or home visits. These findings may help explain the rapid spread of SARS-CoV-2 between persons.

The currently available evidence shows that SARS-CoV-2 is transmitted between people through droplets and close contact [13]. A recent study showed extensive environmental contamination by a SARS-CoV-2 patient [14], suggesting the contaminated environment as a potential medium of transmission. In this study, we detected SARS-CoV-2 in 2 environmental swabs from the household of patient 3. Such detection of SARS-CoV-2 in contaminated environments of the household may provide an additional contribution to virus transmission among family members as the virus can remain viable and infectious on the surface up to 7 days [15]. However, the direct research-based evidence describing exactly how SARS-CoV-2 is transmitted is limited, and further studies are required.

We cannot rule out the possibility of unknown COVID-19 patients (eg, asymptomatic carriers) transmitting the virus. However, according to screening protocols implemented by the provincial, municipal, and county-level Centers for Disease Control and Prevention, all close contacts were traced, and all patients with positive rRT-PCR results in this study were confirmed by whole-genome sequencing, including those who were asymptomatic or paucisymptomatic (patients 4, 5, 7, and 8).

CONCLUSIONS

The transmission potential by individuals with asymptomatic and paucisymptomatic infection and the detection of SARS-CoV-2 in contaminated environments create challenges in control and prevention for the disease. Further studies are needed to investigate the contribution of persons with asymptomatic or paucisymptomatic SARS-CoV-2 infection and the relationship with transmission of the virus in the household, occupational, and community settings.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. The authors thank all patients involved in the study, as well as Dr Hong-Guang Ren from the Beijing Institute of Biotechnology for generating the phylogenetic tree.

Disclaimer. The views expressed in this article are those of the authors and do not necessarily represent the official position of Shandong Provincial, Linyi Municipal, and Lanshan District Centers for Diseases Control and Prevention. All the authors have declared no relationships or activities that could appear to have influenced this work.

Financial support. This work was supported by the Special National Project on Investigation of Basic Resources of China (grant number 2019FY101502); the National Major Project for Control and Prevention of Infectious Disease of China (grant number 2017ZX10303401-006); the Key Research and Development Program of Shandong Province (grant number 2020SFXGFY02); and the National Natural Science Foundation of China (grant numbers 81773494 and 81621005).

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

References

Author notes