key: cord-1002980-hos10td9
authors: Cogliati Dezza, F; Oliva, A; Cancelli, F; Savelloni, G; Valeri, S; Mauro, V; Calabretto, M; Russo, G; Venditti, M; Turriziani, O; Mastroianni, CM
title: Determinants of prolonged viral RNA shedding in hospitalized patients with SARS-CoV-2 infection
date: 2021-02-12
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2021.115347
sha: d602b1c0f5e0f8857e106b93f9a73808671e2fac
doc_id: 1002980
cord_uid: hos10td9

OBJECTIVE: To evaluate determinants of prolonged viral RNA shedding in hospitalized patients with SARS-CoV-2 infection. MATERIALS AND METHODS: Hospitalized patients with SARS-CoV-2 positive nasopharyngeal RT-PCR were included in a single-center, retrospective study. Patients were divided in two groups according to the timing of viral clearance [≤14 days, “early clearance (EC)” and >14 days, “late clearance (LC)”]. RESULTS: 179 patients were included in the study (101 EC, 78 LC), with median age 62 years. Median time of viral shedding was 14 days (EC/LC 10 and 19 days, respectively, p<0.0001). Univariate analyses showed that age, male gender, receiving corticosteroids, receiving tocilizumab, ICU admission, low albumin and NLR ratio were associated with late viral clearance. In the multivariable analysis, older age (p=0.016), albumin level (p=0.048), corticosteroids (p=0.021) and tocilizumab (p=0.015) were significantly associated with late viral clearance. CONCLUSIONS: Age, albumin, tocilizumab and corticosteroid treatment were independently associated with a prolonged SARS-CoV-2 RNA shedding.

The novel β-coronavirus emerged in Wuhan, China Understanding the kinetics of infectious viral shedding in relation to possible transmission risk is crucial to guide infection prevention and control strategies [3] , even if the correlation between infectivity and viral persistence is still unclear [4] . Long-term shedding of viral RNA has been reported in COVID-19 patients putting serious problems on timely discharge from hospital or home isolation [5, 6] in order to mitigate the risk of secondary community transmission. Although this topic is currently under consideration in SARS-CoV-2 infection [7] , only few studies investigating factors possibly influencing the viral shedding duration have been performed so far, with conflicting results. Disease severity, age, sex and treatment have been correlated with the timing of viral persistence, but no general key determinants have been clearly demonstrated to prolong the viral shedding [7] [8] [9] [10] [11] .

Hence, it is of urgent need to elucidate the determinants of viral shedding duration among patients with COVID-19 in order to optimize the public health policy about the discharge strategies and to achieve a rational use of laboratory resource establishing an adequate time of the viral clearance.

Based on these premises, a retrospective, single-center study was conducted with the aim of identifying clinical and/or laboratory determinants associated with prolonged viral persistence in the nasopharyngeal samples of patients with SARS-CoV-2 infection.

Over a 2-month period (March-May 2020), a retrospective single-center study including all adult patients with diagnosis of SARS-CoV-2 infection and hospitalized at Azienda Policlinico Umberto I, Sapienza University of Rome was performed. Nasopharyngeal swab samples were collected and SARS-CoV-2 RNA was detected by using real time RT-PCR assay (RealStar SARS-CoV2 RT-PCR, Altona Diagnostics). Following logistical and clinical needs, other molecular methods were used (GeneFinder COVID-19 Plus RealAmp Kit, Elitech; DiaSorin Molecular Simplexa COVID-19

Direct EUA assay, DiaSorin Molecular; Xpert Xpress SARS-CoV-2 assay, Cepheid). All tests and procedures were performed following the manufacturers' protocols. Definition of pneumonia or severe pneumonia was based on the WHO interim guidance and included clinical signs of pneumonia (fever, cough, dyspnea, fast breathing) with or without signs of severe pneumonia such as respiratory rate > 30 breaths/min, severe respiratory distress, or SpO2 < 90% on room air [12] .

For each subject, laboratory, clinical and radiological data at hospital admission, as well as treatment data and SARS-CoV-2 RNA time of detection were collected and recorded anonymously in an electronic database. As for treatment data, antiviral therapy as well as the choice of using tocilizumab or steroids was based on the national, regional and local guidelines available at the time [13, 14] and on clinical judgement. In particular, corticosteroids were used in patients with moderate or severe Acute Respiratory Distress Syndrome (respiratory failure with PaO2/FiO2 <200 mmHg or worsening of respiratory conditions during hospitalization or at ICU admission or during ICU stay).

Tocilizumab was used in patients with COVID-19 pneumonia and PaO2/FiO2 <300 mmHg.

Specific contraindications for the use of tocilizumab were hypersensitivity to tocilizumab, concomitant treatment with anti-rejection drugs, concomitant active infections, neutrophils level <500 cell/mL, platelets <50.000 cell/mL, alanine/aspartate aminotransferases >5-fold the normal value, intestinal perforation. The study was approved by the local Ethics Committee (ID Prot. 109/2020).

Time of viral shedding was defined as the number of days from the first viral detection by RT-PCR on nasopharyngeal specimen until the first of two consecutive negative results within 24 hours.

Patients with a first negative sample followed by a positive nasopharyngeal specimen within 24 hours were excluded from the study. According to the duration of viral shedding, the study population was further divided in two groups: "early clearance (EC)" group (median ≤14 days) and "late clearance (LC)" group (median >14 days).

The data, unless otherwise stated, were given as medians with interquartile ranges (IQR, 25th-75th percentile) for continuous variables and as simple frequencies, proportions, and percentages for categorical variables. Mann-Whitney test was used for unpaired samples. Dichotomous variables were compared using Fisher's exact tests or chi-square test statistics, as appropriate. Log-rank test and univariate Cox regression were used for categorical or continuous variables, respectively. Multivariable Cox regression model was performed to tease out the independent predictors of prolonged viral persistence. P-value analyses were two-sided and a p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed with SATA/IC software (StataCorp) version 15.

Demographic and clinical characteristics of the patients are shown in Table 1 respectively]. The median time from symptoms onset to first viral detection was 7 days (4-9.9), whereas the median time of viral shedding was 14 days (9-19, range 2-45 days), with a median time of 10 days (7.75-12) and 19 days (17) (18) (19) (20) (21) (22) (23) (24) in the EC and LC groups, respectively (p<0.0001).

Overall, 124 (69.3%) patients presented with at least one underlying disease, with no significant differences between the two groups. Among laboratory findings, albumin values were significantly lower in the LC group [3.7 g/L (3.4-4) vs 3.9 (3.5-4.2), p=0.023]. All patients received antiviral therapy with hydroxychloroquine and/or protease inhibitor (162/179, 90.5%, and 67/179, 37.4%, respectively) whereas azithromycin and teicoplanin were given in 81/179 (45.2%) and 23/179 (12.8%) subjects, respectively. Again, no significant differences as for the antiviral treatment between the 2 groups were observed, with the exception of teicoplanin use which was more frequent in the LC group ( 

Severity of disease (expressed by ICU transfer), receiving tocilizumab or steroids as a part of infection treatment, male sex, age, albumin level and NLR ratio were associated with persistence of viral positivity at nasopharyngeal specimen (Figure1). In the multivariable analysis, age (p=0.016), albumin level (p=0.048), receiving corticosteroids (p=0.021) and tocilizumab (p=0.015) were significantly associated with late viral clearance (Table 2 ).

Despite the multitude of studies published during the ongoing COVID-19 pandemic, knowledge about factors possibly influencing prolonged viral shedding is still limited. In this study, it was found that age, albumin levels and receiving corticosteroids or tocilizumab were independently associated with SARS-CoV-2 RNA shedding.

Consistent with previous findings, we observed that median SARS-CoV-2 RNA shedding in upper respiratory specimens was 14 days (IQR 9-19) [16] , although a wide variability among different studies was observed, reflecting the heterogeneity of populations [17] [18] [19] .

The main finding of the present research is the association between tocilizumab therapy and the duration of viral shedding. To our knowledge, no studies focusing on this aspect have been published so far. Tocilizumab, an interleukin-6 (IL-6) inhibitor with anti-inflammatory properties, has been used for the treatment of SARS-CoV-2 infection with the aim of reducing and controlling the cytokine storm present during the infection. In fact, during severe SARS-CoV-2 infection pathogenic T-cells and inflammatory monocytes incite inflammatory response with large amount of IL-6 production [20] . Furthermore, in rheumatoid arthritis patients tocilizumab was shown to induce a significant reduction in the peripheral memory B cell, with a decline of serum immunoglobulin levels [21] , which might have a role in slowering viral clearance during the course of COVID-19.

Therefore, the interference of the drug with the immune system might possibly explain this phenomenon. Nevertheless, further studies are needed to understand the immunological mechanisms involved in the SARS-CoV-2 viral clearance and to reveal how blocking IL-6 receptor is associated to a late viral shedding.

Similarly, receiving corticosteroids resulted to be an independent factor associated with viral persistence. This finding is consistent with other recent observational studies of patients with COVID-19 [10, 22] , confirming past reports that showed a link between corticosteroids therapy and a prolonged viral shedding in different viral infections, such as seasonal influenza, SARS and MERS [23] [24] [25] . The corticosteroid immune modulatory action and the impairment of T-cell immunity might be the reasons explaining this finding. However, the effect of corticosteroids on duration of viral shedding is still controversial, with few studies no confirming this association [9, 26, 27] . These conflicting data could be explained by the heterogeneity of study populations and, possibly, by the different types and dosages of corticosteroids, suggesting the need of additional investigations. This is especially true considering that recent clinical trials clearly demonstrated the advantage of using corticosteroids during SARS-CoV-2 infection [28] . Therefore, with the expectation of an increasing use of steroids for COVID-19, understanding the relation between steroids and viral shedding appears to be crucial.

Although the aim of the study was to find factors associated with a prolonged viral shedding and not to assess whether tocilizumab or corticosteroids were beneficial for patients, we also analysed mortality rates in patients receiving or not tocilizumab or corticosteroids and we found no statistical differences.

Another interesting result of the present research is that albumin might possess a role in determining the duration of viral shedding, further highlighting the increasing attention towards this molecule in the setting of SARS-CoV-2 infection. In fact, not only hypoalbuminemia was associated with worse outcome and coagulopathy [29, 30] , but also a recent report showed that patients with decreased albumin levels experienced prolonged viral RNA shedding, confirming what observed by our group [31] . The anti-oxidant and anti-inflammatory properties of albumin as well as its ability to downregulate the expression in the cells of the human angiotensin-converting enzyme 2 receptors [32, 33] might explain this finding.

Age was another independent factor associated with RNA persistence in nasopharyngeal samples.

Apart from being a well-known determinant of poor prognosis in COVID-19 [34] , previous studies have also demonstrated that elderly is associated with late SARS and SARS-CoV-2 negativity [16, 18, 22, 35] . One possible explanation could be the age-dependent dysfunction of innate and adaptive immune response that could lead older patients to a more difficult pathogen eradication, thus predisposing to a prolonged viral shedding [36] . Although age is commonly associated with comorbidities, in our study the presence of comorbidity did not have an influence on RNA persistence, suggesting that age per se might influence the viral shedding [11, 22, 37] .

In contrast to recent studies, in the present report neither sex nor disease severity, expressed as ICU transfer, were factors independently associated with viral persistence. Male gender has been linked with more severe symptoms, higher mortality rate and a more prolonged shedding than female in SARS-CoV-2 infection [9, 11, 34] . The specific mechanism of sex-related difference in SARS-CoV-2 infection is unclear; however, the underlying mechanisms may be related to sex-hormones, which affect different components of the immune system [38] , and to the effect of gender on the expression of ACE-2 [39, 40] . Therefore, being aware that sex might have an important role in influencing SARS-CoV-2 RNA shedding, we strongly believe that additional studies with higher number of patients are warranted.

Similar conflicting data could be found in the literature regarding the impact of infection severity on duration of viral positivity. In fact, a significant difference of viral shedding time in ICU and non-ICU patients has been described [7, 9, 41] , whereas a recent paper [8] surprisingly showed that mild non-hospitalized cases presented a longer duration of viral shedding than inpatients.

Although the use of antiviral drugs might hypothetically lead to an early viral clearance, as it was shown for lopinavir/ritonavir [27, 31, 42] , we did not find a relation between antivirals and duration of viral shedding, in agreement with Shi et al (2020) and Hraiech et al (2020) [9, 43] .

This study has some limitations. In fact, RT-PCR for SARS-CoV-2 RNA on nasopharyngeal swab in the absence of viral cultures does not discriminate between viable and non-viable viruses.

Therefore, detection by RT-PCR represents only a surrogate marker for infectivity, making the great dilemma, "shedding vs infectivity" more and more actual [4] . Unfortunately, we were not able to perform viral load quantification during the first phase of pandemic. We acknowledge that this might represent an additional limit of the present investigation and that further studies are needed to test whether viral load at admission and thereafter could be responsible for the prolonged viral shedding. Then, having data on RT-PCR only at the time of hospitalization rather than immediately from symptoms onset might have underestimated the time needed for viral clearance.

Age, albumin levels and receiving corticosteroids or tocilizumab represent key determinants of prolonged SARS-CoV-2 RNA shedding. A better understanding of viral kinetics might be useful in the hospital clinical management in order to early identify patients at risk of prolonged viral shedding and to provide crucial insights into a rational use of laboratory resource.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 

All authors revised and approved the revised version of the manuscript.

The data used to support the findings of this study are available from the corresponding author upon request.

Figure1. Cumulative proportion of patients with detectable SARS-CoV-2 RNA by day after first positive RT-PCR between patients who received tocilizumab (blu line) and patients who did not received tocilizumab (red line) (A), between patients who received steroids (blu line) and patients who did not received steroids (red line) (B), between patients who were transferred to ICU (blu line) and patients who were not transferred to ICU (red line) (C) and between male (blu line) and female patients (red line) (D).

Cumulative proportion of patients with detectable SARS-CoV-2 RNA by day after first positive RT-PCR between patients who received tocilizumab (blu line) and patients who did not received tocilizumab (red line) (A), between patients who received steroids (blu line) and patients who did not received steroids (red line) (B), between patients who were transferred to ICU (blu line) and patients who were not transferred to ICU (red line) (C) and between male (blu line) and female patients (red line) (D).

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