key: cord-1010937-rekm4qgu
authors: Radulescu, Amanda; Istrate, Alexandru; Flonta, Mirela; Lupse, Mihaela
title: Antibody and viral RNA kinetics in SARS-CoV2 infected patients admitted in a Romanian University Hospital of Infectious Diseases
date: 2021-04-24
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.04.067
sha: 019ec101ead8421d7c36ed60c7537e75cb111434
doc_id: 1010937
cord_uid: rekm4qgu

OBJECTIVES: To assess the antibody and viral kinetics in asymptomatic/mild confirmed SARS-CoV-2 infections compared to more severe patients. MATERIAL AND METHODS: Retrospective analysis of data obtained from adult patients with a confirmed SARS-CoV2 infection having at least one SARS-CoV-2 pair of specific IgM/IgG test, admitted in The University Hospital of Infectious Diseases Cluj-Napoca, Romania (28 February to 31 August 2020). Data base also included: demographic, clinical, chest X-ray and/or CT scan results, RT-PCR SARS-CoV-2 and dexamethasone treatment. A total of 469 patients were evaluated as “asymptomatic/mild” and “moderate/severe/critical” cases. RESULTS: The median time since confirmation to SARS-CoV-2 PCR negativity was 15 days [95% CI: 13-18] in asymptomatic/mild cases and 17 days [95% CI: 16-21] in moderate/severe ones. The median time to seroconversion for both IgM and IgG was 13 days [95% CI: 13-14] in asymptomatic/mild cases and 11 days [95% CI: 10-13] in moderate/severe ones. For both antibody types, the highest reactivity was significantly associated with more severe presentation (IgM: OR = 10.30, IgG: OR = 7.97). CONCLUSION: The asymptomatic/mild COVID-19 cases had a faster RT-PCR negativity rate compared to moderate/severe/critical patients. IgG and IgM dynamics was almost simultaneous, more robust for IgG in more severe cases and at one month after confirmation, almost all patients had detectable antibody titres.

In Romania, from the beginning of the pandemic until 23 rd June, hospitalization was mandatory even in asymptomatic and mild infections and two negative consecutive RT-PCR SARS-CoV-2 tests were required before discharge. Later, hospitalization was mandatory for at least 10 days. Therefore, many of the study subjects had a prolonged hospitalization allowing us to perform repeated molecular and antibody testing (Ministry of Health, 2020).

The aim of our study was to compare the viral kinetics and antibody response in asymptomatic/mild and moderate/severe/critical SARS-CoV-2 patients.

We retrospectively evaluated the antibody profiles and RT-PCR SARS-CoV-2 kinetics in asymptomatic/mild confirmed SARS-CoV-2 infections compared to more severe patients. Out of 1349 admitted cases, 469 adults fulfilled the inclusion criteria. Mild cases were defined by the presence of some symptoms and normal chest imaging, while moderate/severe/critical cases by the evidence of pneumonia, pneumonia and respirator y failure, pneumonia and mechanical ventilation or septic shock or multiple organ dysfunction syndrome, respectively (WHO 2020b, WHO 2020c). 

Besides the descriptive analyses of clinical and laboratory tests, log2-transformed data and geometric mean ± standard deviation were used for antibody index assessment. Hypothesis testing was performed using odds ratio with 95% confidence intervals and p-value from Fisher tests or Mann-Whitney test, as appropriate, followed by logistic regression for higher IgM/IgG reactivity by age, severity and lung area abnormalities. Cumulative distribution plot, hazard ratio and a log-rank test were used for the RT-PCR test result by time and severity. A probit regression model was used to estimate the average proportion of patients with detectable antibodies or negative RT-PCR result at individual time-points after confirmation. Statistical analysis was performed by R 4.0 (R Core Team, 2020). Statistical significance was considered for a p-value of <0.05.

The dataset included 469 patients, 208 with asymptomatic/mild infections and 261 with moderate/severe/critical disease. Patient characteristics are presented in Table 1 . Higher male proportion, older age and longer hospitalization were observed in more severe cases (p<0.01). Figure 1A ). At 28 days after confirmation, the cumulative proportion of cases with at least one negative RT-PCR test was 93.1% in asymptomatic/mild cases and 75.8% in more severe ones (p<0.01) (Supplementary Table 1 ).

All patients were tested for IgM/IgG at least once after confirmation and half of them had the first detectable result for either IgM or IgG at 13 days [95% CI:13-14] in asymptomatic/mild cases and at 11 days [95% CI: 10-13] in more severe ones (p=0.001) ( Figure 1B, 1C ). At 28 days after confirmation, the cumulative proportion of cases who had at least one detectable IgM test was 85.0% in asymptomatic/mild cases and 94.0% in moderate to critical cases (p<0.01) and for at least one detectable IgG test was 96.7% in asymptomatic/mild cases and 94.2% in moderate to critical cases (p<0.01) (Supplementary Table 1 ).

RT-PCR positivity rate decreased faster among patients with milder presentation to 60% at 14 days and 25% at one month compared to more severe cases: 70% at 14 days and 50% at one month ( Figure 2A ).

Both antibody types had higher variability in patients with more severe clinical picture ( Figures 2B, 2C ).

The geometric mean (± geometric standard deviation), median and interquartile range [M (IQR)] for paired IgM and IgG tests and the RT-PCR tests are presented in Table 2 . Patients with higher IgM and IgG reactivity were men (albeit not significant), older, with a more severe presentation, >30% lung area abnormalities, higher fatality rate, longer hospitalization and ICU admission (p<0.01). In multivariate J o u r n a l P r e -p r o o f logistic regression, higher IgM and IgG reactivity were significantly associated with severity and >30% lung area abnormalities. Also, higher IgG reactivity was more often present in patients who received dexamethasone treatment while IgM reactivity showed a similar pattern but without statistical significance (Table 3) .

Both antibody types showed an upwards trend in positivity rate across time, IgG became positive after one month in almost all patients, regardless severity ( Figure 2B and 2C ).

In Romania, 87540 cases were confirmed and 3621 deaths (fatality rate 4.13%) from the end of February (Salameh et al., 2020) . Noteworthy, in our study, the relationship between disease severity ascertained by chest CT scan (lung area damage >30%) and a robust immune response appeared to be bidirectional. Since we found that higher antibody titres were correlated with an extended lung area damage, we hypothesize that unknown variables may cause a higher severity as well as higher immunological reactivity or the uncoordinated SARS-CoV-2 antigenspecific response fail to control the disease. These questions were raised by many authors but we do know that circulating memory B cell, CD4 + T cell, and CD8 + T cell memory to SARS-CoV-2 are of utmost importance for protection against reinfection as well as for vaccination strategy (Seow et al., do not negatively impact the humoral immune response (Masiá et al., 2021) . In univariate analysis, we found that dexamethasone treatment was correlated with higher antibody reactivity (IgM and IgG) and after adjustment for age and severity remained significant for IgG, suggesting that corticosteroids had a minimal effect on antibody response.

The main strength of our study is that viral and antibody kinetics were evaluated in patients with asymptomatic/mild disease for a prolonged hospitalization and, in comparison with more severe cases.

Since during the first four pandemic months, patients' discharge was possible only after two consecutive negative RT-PCR-based tests, we had a fairly good evaluation of the time when the SARS-CoV-2 was no longer detectable. Also, a parallel serological assessment was done in all cases allowing us to present a documented antibody dynamics.

The main limits of our study were that we evaluated only IgM and IgG antibodies without isotyping and virus-specific T-cell response was not assessed.

The asymptomatic/mild COVID-19 cases had a faster RT-PCR negativity rate compared to more severe patients and almost all cleared the virus in one month. RT-PCR tests remained positive in half of all patients for approximately two weeks after confirmation. IgG and IgM dynamics was almost simultaneous, more robust for IgG and more severe cases. At one month after confirmation, almost all patients had detectable antibodies. Both IgM/IgG high reactivity were correlated with disease severity mirrored by chest CT scan abnormalities.

Author contributions: AR drafted the initial manuscript. AR and ML contributed significantly to revision of the manuscript and its finalization for submission. AI, AR was responsible for conception and study design, analysis and interpretation of data. AI was responsible for data collection, data input and statistical analysis. AR, ML, and MF were responsible for critical analysis and editorial assistance. All authors have read and approved the final manuscript. confirmation and severity. Dates were calculated to the first event (negative PCR, detectable antibody). Inverse hazard -ratio (HR) comparing more severe presentations to asymptomatic/mild, 95% CI and log -rank test.

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Geometric mean ± geometric standard deviation

* total IgM & IgG paired determinations, relative to RT-PCR tests during the same period (both antibody types were measured simultaneously)

aOR = adjusted odds-ratio

We thank all clinical, laboratory and nursing staff who cared for the patients at Cluj-Napoca University Hospital of Infectious Diseases but are not responsible for the content of this article.J o u r n a l P r e -p r o o f