key: cord-0932837-rybjc58j
authors: Carmo, Anália; Pereira‐Vaz, João; Mota, Vanda; Mendes, Alexandra; Morais, Célia; da Silva, Andreia Coelho; Camilo, Elisabete; Pinto, Catarina Silva; Cunha, Elizabete; Pereira, Janet; Coucelo, Margarida; Martinho, Patrícia; Correia, Lurdes; Marques, Gilberto; Araújo, Lucília; Rodrigues, Fernando
title: Clearance and Persistence of SARS‐CoV‐2 RNA in COVID‐19 patients
date: 2020-06-02
journal: J Med Virol
DOI: 10.1002/jmv.26103
sha: 3c6419fba5fe2c4c422469939d12a63c531322f9
doc_id: 932837
cord_uid: rybjc58j

COVID‐19 patients may be discharged based on clinical resolution of symptoms, and evidence for viral RNA clearance from the upper respiratory tract. Understanding the SARS‐CoV‐2 viral clearance profile is crucial to establish a re‐testing plan on discharge and ending isolation of patients. We aimed to evaluate the number of days that a patient needed to achieve undetectable levels of SARS‐CoV‐2 in upper respiratory tract specimens (nasopharyngeal swab and/or an oropharyngeal swab). The clearance and persistence of viral RNA was evaluated in two groups of positive patients: those who achieved two negative RT‐PCR tests and those who kept testing positive. Patients were organized thereafter in two subgroups, mild illness patients discharged home and inpatients who had moderate to severe illness. Results from RT‐PCR tests were then correlated with results from the evaluation of the immune response. The study evidenced that most patients tested positive for more than two weeks and that persistence of viral RNA is not necessarily associated with severe disease but may result from a weaker immune response instead. This article is protected by copyright. All rights reserved.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded RNA virus that crossed species barriers and caused the outbreak of coronavirus disease 2019 (COVID- 19) in humans [1] [2] [3] [4] .

The incubation period of SARS-CoV-2 ranges from 1 to 14 days, with a mean of 5 to 6 days. The first studies indicated that the viral load persisted up to 8 days after the onset of symptoms in mild cases and peaked in day 11 in more severe cases 4, 5 . However, upon the resolution of clinical symptoms the clearance of SARS-CoV-2 from respiratory samples remains unclear, making the establishment of patient discharge and the ending isolation criteria difficult 2,4,6 .

According to recommendations, clinically recovered COVID-19 patients may be discharged after two negative RT-PCR tests from respiratory specimens This article is protected by copyright. All rights reserved.

within a 24 hours interval 2, 5 . However, there are several reports of prolonged viral RNA detection requiring the consumption of numerous tests 1,2,6 . COVID-19 outbreak had a major impact on the management of clinical virology laboratories and caused a reduced availability of laboratory consumables and reagents limiting the testing capacity. The need to repeat tests to detect viral RNA poses an additional challenge. Therefore, to achieve a rational use of human and laboratory resources it is important to establish the adequate time to determine viral clearance.

We aimed to evaluate the number of days that a patient needed to achieve undetectable levels of SARS-CoV-2 in upper respiratory tract specimens (nasopharyngeal swab and/or an oropharyngeal swab).

Retrospective and cross-sectional analysis of laboratory data obtained from nasopharyngeal/oropharyngeal swabs and serum samples received at the Clinical Pathology Unit of Centro Hospitalar e Universitário de Coimbra, Portugal.

The samples were collected from symptomatic patients (fever, cough, chills and dyspnea) or from asymptomatic patients who had contact with infected patients.

Swabs samples were used to detect SARS-CoV-2 RNA and serum samples were used to evaluate IgM and IgG antibodies against SARS-CoV-2. Laboratory data and patient's characteristics such as age, sex and provenance (ward, emergency department, home) were collected anonymously from our laboratory database from March 1 to April 30, 2020.

Detection of SARS-CoV-2 RNA Nasopharyngeal and oropharyngeal samples were collected using swabs immediately placed in standard viral transport medium. Viral RNA was extracted from 400 µL of respiratory samples and eluted in 50 µL of elution buffer using the EMAG automated nucleic acid extraction platform (Biomérieux). Detection of SARS-CoV-2 RNA was performed by an adapted previously described real-time This article is protected by copyright. All rights reserved.

RT-PCR assay targeting regions of the virus nucleocapsid (N) gene, and also targeting the human RNase P gene for sample quality control 7 Quantitative variables were assessed for normality with the Shapiro Wilk test.

Comparisons of quantitative variables between two groups were performed with t-Student or Mann-Whitney tests. Correlations between quantitative variables were assessed by computing Pearson or Spearman correlation coefficients, depending on whether normality requirements were met or not. The level of significance adopted was 0.05. This cohort was divided in two groups: those who achieved two consecutive negative tests and those who maintained RT-PCR tests positive. The former comprised a total of 116 patients (55.2%) and the later comprised a total of 94 patients (44.8%).

In the group of patients with two consecutive negative tests, the first negative RT-PCR test was achieved 24.8 ± 9 days (range: 7 -46 days) after the first positive test. In men, the first negative test took 24 ± 9 days (range: 7 -46) and in women it took 25 ± 9 days (range: 9 -44), P>0.05, In an attempt to understand why some patients maintained positive tests for longer, we correlated the detection of SARS-CoV-2 RNA with the host immune response to virus infection. Therefore, we analyzed the production of specific IgM and IgG antibodies. 

Therefore, understanding the SARS-CoV-2 viral clearance profile is crucial to establish a re-testing plan on discharge and ending isolation of patients.

We analyzed the presence of viral RNA in two groups of patients: those who achieved two negative RT-PCR tests and those who kept testing positive.

Patients were organized thereafter in two subgroups, mild illness patients discharged home and inpatients who had moderate to severe illness.

The results evidenced that most patients from either group tested positive for more than two weeks. In fact, 69.8% of the patients that tested negative twice needed more than 20 days to achieve the first negative test. Moreover, 34% of patients that kept RT-PCR tests positive at the end of our study, tested positive for 40 days. These results may indicate that in a situation of lack of testing resources, patients might be re-tested only after the 20th day from the first positive test and beyond.

Surprisingly, mild illness patients discharged home took longer to become negative than inpatients. Our results are in accordance with the results from Lan et al. 10 but in disagreement with several studies pointing that delayed viral clearance is associated with severe illness 1, 5, 11 .

In previous studies the correlation between virus persistence and severe disease was based on the detection of viral RNA in non-survivors until death 8, [11] [12] [13] . It is important to emphasize that RT-PCR does not evaluate the infectious capacity of the detected RNA. In fact, no live virus was isolated from sample cultures obtained 8 days after the onset of symptoms 5, 14 . Therefore, the persistence of viral RNA may not be associated with disease severity but may indicate that the immune response is unable to promote the virus RNA clearance.

To evaluate the host immune response, we determined SARS-CoV-2 specific IgM and IgG in the same day of viral RNA detection. In accordance with previous studies, patients maintain RT-PCR tests positive even after seroconversion 5, 14 .

Our results also showed that the patients discharged home from both groups presented a significantly lower IgG titer than the inpatients, which is This article is protected by copyright. All rights reserved.

probably associated with the disease severity. In accordance with other studies, moderate/severe illness patients appear to have higher antibody titers than those with milder disease. Although increased titers of specific antibody may induce the expression of pro-inflammatory factors they also contribute to the inactivation and clearance of virus 12,15-17 . Our study evidenced that patients discharged home were younger, had mild disease, presented low IgG titer and maintained viral RNA for a longer period of time. On the other hand, patients in the inpatient group were older, moderately to severely ill, presented a higher titer of IgG and had a better capability to achieve viral RNA clearance.

Since mild illness is associated with a low viral load 4, 18, 19 , we hypothesize that the exposition of patients discharged home to SARS-CoV-2 did not elicit an effective immune response, explaining the milder disease and the need of more time to viral RNA clearance.

The lack of information regarding persistence of virus RNA and infectivity, disease severity and immune response, supports the current guidance of viral clearance confirmation prior to patient transference out of dedicated COVID-19 wards and of ending isolation in mild illness patients.

In conclusion, our study highlights that viral RNA may persist for a long period of time in respiratory samples; mild illness patients present a weak immune response; viral RNA may remain even after the rise of IgG titer; and persistence of viral RNA is not necessarily associated with severe disease but may result from a weaker immune response instead. Data are reported as mean ± standard deviation, (minimum -maximum). There was a significant difference between the mean number of days to achieve the first negative test in the inpatients and in the patients discharged home, P=0.027. The inpatients were older than the patients discharged home, P<0.001. 

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Conflict of interest in the manuscript: Authors disclose any financial or other conflict of interest Author contribution statement in the manuscript: All authors contribute to the article