An immunocompetent 169-day prolonged SARS-CoV-2 shedding patient with high neutralizing antibody


 Information with prolonged SARS-CoV-2 shedding among immunocompetent patients is limited. We describe a twice repositive 169-day prolonged SARS-CoV-2 shedding in an immunocompetent patient and explore potential factors from clinical, immunological and genomic perspectives. We found that continuous viral replication and infectivity could exist in an immunocompetent COVID-19 patient with high neutralizing antibody.


Introduction
Previous identi cation of immunocompromised cases with 105-and 153-day persistent RNA shedding suggest immunocompromised patients are more likely to have prolonged virus shedding 1, 2 . Here, we described a twice repositive immunocompetent COVID-19 case in Beijing with 169-day viral shedding.

Results
Using data available in the patient's medical record, the 64-year-old male patient had underlying diseases of coronary atherosclerotic heart disease, hypertension and type 2 diabetes with a history of daily smoking and alcohol consumption for 40 years. He was identi ed from a family cluster of SARS-CoV-2 infection on Feb 12 (Fig. 1A), in which 4 out of 7 were con rmed as COVID-19 cases whose viral sequences were highly homologous. While his 3 family members recovered and were discharged by March, the patient who had non-severe pneumonia (i.e., moderate severity 3 ) was quarantined at the hospital due to persistent positivity until August with the exception of two 2-week periods when he tested negative.
After identi cation as a COVID-19 case, he was immediately admitted to the hospital for quarantine. On February 25, he was discharged following two consecutive negative PCR tests (> 24hr apart) 4 but readmitted due to a repositive result from routine follow-up testing on March 13th. Because he was still positive on day-105, he was transferred May 16 to Ditan Hospital which has more experience treating COVID-19 cases. He was discharged July 14 after two consecutive negative tests but was readmitted when found repositive again July 28. He was discharged permanently the next week (August 5; Fig. 1A).
He was treated with antivirals, interferon, and traditional Chinese medicine during the whole course of illness.
The patient reported chills, fever (38.6°C), sore throat and loss of appetite from February 1 to rst admission on Feb 12. Clinical examination revealed decreased WBC and lymphocytes (Table S1). CT images showed patchy ground-glass opacity in the upper lobe and the lower lobe under the pleura of both lungs and the middle lobe of the right lung ( Figure S1). Throughout his hospitalization, no obvious abnormality or changes were observed in his liver or kidney functions or in routine blood examinations (Table S2). All CD4 absolute counts were above 350/µl, CD4/CD8 was above 1, and he was HIV negative, indicating he was immunocompetent, although CD8 cell count and Natural Killer (NK) cells were relatively low at some timepoints (Table S2).
The patient's neutralizing antibody was 1:2048 13 days from disease onset, peaked at 1:8192 on day 24 and declined in the follow-up period but remained at 1:384 for over 9 months (day 299) (Fig. 1B). The highest viral loads as assessed by cycle threshold (Ct) in both sputum (Ct 17.4) and nasopharyngeal specimens (Ct 23.4) occurred 4 to 5 months after disease onset (Tables S5-S6

Discussion
To our knowledge, we reported the longest period of viral shedding (169 days) and intrahost variants (151 days). The intrahost mutation rate was comparable to that of other interhost variants 6, 7 . However, unlike previous studies which were of shorter infection periods, the mutations identi ed in this case appeared across the whole genome rather than select hotspots, such as S and ORF8 1, 3 .
Although previous studies suggest repositive COVID-19 patients have lower or no infectiousness 8, 9 , our evidence suggests infectiousness may last until 151 days after symptom onset. Although lack of lab facilities prevented virus isolation and culture was not performed, the observed accumulated mutations as well as positive sgRNA 3-4 months after infection suggests on-going viral replication in the course of infection and therefore potential for transmission 1 . More attention to the infectiousness of repositive patients is therefore warranted.
The patient did not have severe clinical symptoms which documents that prolonged viral shedding can occur in moderately ill cases 10,11 . Whether the administration of traditional Chinese medicine or interferon for COVID-19 treatment in uences the occurrence of repositivity is unknown. Because such frequent nucleic acid testing is only done in people with COVID-like symptoms, we may be underdetecting the frequency of these long-term virus shedding cases 4 .
To our knowledge, this is the rst case of long-term shedding in an immunocompetent case, which previously has only been reported in immunocompromised cases 1, 2 . We are aware of only this one case so the evidence might have limited generalizability, but occurred despite his neutralizing antibody titer being much higher than that of other patients evaluated at our facility 3

Methods
Before enrollment, a written informed consent was obtained from this patient.

Clinical presentations
All available data on white blood cell (WBC), lymphocyte, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL) from liver function tests; albumin (ALB), blood urea nitrogen (BUN) and creatinine level (Cr) from kidney function tests, and computerized tomography (CT) images were collected from the past clinical charts and tests results.

Laboratory examinations
Neutralizing Antibody A total of 10 blood samples were collected. Antibodies were determined with a modi ed cytopathogenic neutralization assay (NA) based on live SARS-CoV-2. The dynamics of neutralizing antibody levels at different time points were analyzed. We conducted neutralization assay (NA) to evaluate antibody level according to the Reed-Muench method on day 5. The presence of neutralizing antibody was determined by a modi ed cytopathogenic assay. Serum samples were inactivated at 56°C for 30 minutes and serially diluted with cell culture medium in 2-fold steps. The diluted serums were mixed with a virus suspension of 100 median tissue culture infective dose in 96-well plates at a ratio of 1:1, followed by 2 hours' incubation at 36.5°C in a 5% carbon dioxide (CO2) incubator; 1-2 × 104 Vero cells were then added to the serum-virus mixture, and the plates were incubated for 5 days at 36.5°C in a 5% CO2 incubator. Cytopathic effect of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution number of 50% protective condition. A titer of ≥ 1:4 indicated seropositivity.
Viral Load Viral loads were obtained from 33 nasopharyngeal swabs and 20 sputum samples collected at different time points during the patient's hospitalization. All available Cycle threshold (Ct) values for determining viral load were abstracted from 7500 software v2.3 from two hospitals.
In addition, subgenomic RNA (sgRNA) was monitored to evaluate the transcription of SARS-CoV-2. 15 nasopharyngeal swabs and four sputum samples collected from day 100 to day 157 were analyzed.

Phylogenetic analysis
Longitudinal nasopharyngeal swabs and sputum samples were collected after diagnosis, and then sequenced by Next Generation Sequencing (NGS). A total of 14 viral genome sequences were obtained from serial samples, including nine genomes from sputum samples and three genomes from nasopharyngeal swabs collected from the study case, and two from nasopharyngeal swabs collected from his two family members. Single nucleotide polymorphic variants were ltered for quality (QUAL) > 200 and quality by depth (QD) > analysis was conducted using MAFFT v7 16, 17 . A maximum likelihood tree was inferred by NJ model with bootstrap of 1000, including the patient SARS-CoV-2 genomes, the reference genome sequence (GenBank: MN908947.3) and 494 representative genomes randomly selected from NCBI virus dataset by regions of interest. The nal gure was made using iTOl (https://itol.embl.de/). The viral genomes reported in this study have been deposited in the GISAID (https://www.gisaid.org).

Data Availability
The data that support this study are available on request from the corresponding author [LW and QW] upon reasonable request. Figure 1 The