SARS-CoV-2 detection in the lower respiratory tract of invasively ventilated ARDS patients

Background Data on SARS-CoV-2 load in lower respiratory tract (LRT) are scarce. Our objectives were to describe the viral shedding and the viral load in LRT and to determine their association with mortality in critically ill COVID-19 patients. Methods We conducted a binational study merging prospectively collected data from two COVID-19 reference centers in France and Switzerland. First, we described the viral shedding duration (i.e., time to negativity) in LRT samples. Second, we analyzed viral load in LRT samples. Third, we assessed the association between viral presence in LRT and mortality using mixed-effect logistic models for clustered data adjusting for the time between symptoms’ onset and date of sampling. Results From March to May 2020, 267 LRT samples were performed in 90 patients from both centers. The median time to negativity was 29 (IQR 23; 34) days. Prolonged viral shedding was not associated with age, gender, cardiac comorbidities, diabetes, immunosuppression, corticosteroids use, or antiviral therapy. The LRT viral load tended to be higher in non-survivors. This difference was statistically significant after adjusting for the time interval between onset of symptoms and date of sampling (OR 3.78, 95% CI 1.13–12.64, p = 0.03). Conclusions The viral shedding in LRT lasted almost 30 days in median in critically ill patients, and the viral load in the LRT was associated with the 6-week mortality.


Background
The SARS-CoV-2 disseminated in Europe in late February 2020, causing the largest pandemic due to any respiratory viruses any respiratory viruses in recent history [1]. Several authors suggested that viral shedding and severity of disease might be correlated [2], but they mostly focused on viral presence in upper respiratory secretions [3,4]. Viral shedding from upper respiratory tract appeared to be higher soon after symptoms' onset, but during the course of disease, the shedding originates predominantly from the lower respiratory tract (LRT) [5]. To date, data on viral replication in distal airways are scarce. Only one small study partly investigated the role of viral presence into LRT [6]. Moreover, the association between SARS-CoV-2 viral load in LRT and mortality remains unevaluated [7]. Our objectives were (1) to describe the viral shedding and the viral load in LRT and (2) to determine THE ASSOCIATION BETWEEN VIRAL PRESENCE AND MORTALITY in critically ill COVID-19 patients.

Study design and population
We conducted a binational study merging prospectively collected data from two COVID-19 reference centers in France and Switzerland. We included all COVID-19 patients under mechanical ventilation who underwent regularly to LRT sampling for SARS-CoV-2. The Bichat University Hospital served as a reference center for the Northern Paris Region, whereas the Locarno Regional Hospital was an entirely COVID-19-dedicated hospital for all (n = 7) public hospitals of southern Switzerland (Canton of Ticino).

Data collection
All data of COVID-19 patients were prospectively collected in two specific standardized institutional databases. We collected demographic characteristics, medical history, characteristics at hospital and intensive care unit (ICU) admissions, organ support procedures, administration of antivirals and other agents, and biological and virological results. Of note, prescription of antivirals and anti-inflammatory drugs varied among countries, availability, and choice of physician.

Study procedures
All patients had a SARS-CoV-2 positive nasopharyngeal swab before ICU admission. During the ICU stay, all patients underwent a regularly monitoring of LRT samples (LRTS) until the detection of two consecutive negative real-time polymerase chain reaction (RT-PCR) results [8]. All patients underwent regularly to LRT RT-PCR reevaluation until removal of the endotracheal tube or death. Endotracheal aspirates, bronchoalveolar leakage fluid, and plugged telescoping catheter were all considered as LRTS. The sampling was performed according to a predefined protocol. All specimens were sent to two accredited reference virology laboratories and used for RNA extraction and amplification by RT-PCR techniques utilizing validated commercial kits that were previously described [9]. A cycle threshold (Ct) value of < 40 was defined as positive for SARS-CoV-2 RNA and ≥ 40 was defined as negative (see supplementary material). Then, RT-PCR values were transformed in viral load as previously published [10]. The French and the regional Swiss Ethics Committees approved this study.

Definitions and objectives
The primary objective was the description of viral shedding duration (i.e., time to negativity) in LRTS and was defined as the time between onset of symptoms and viral clearance process (i.e., first negative detection of viral RNA in LRT). Of note, we previously showed that the association of the first negative RT-PCR with a second negative result was very high (i.e., 97%) in LRTS [11]. Secondary objectives were (1) the analysis of RT-PCR viral load in LRTS and (2) the assessment of 6-week mortality. Patients discharged before 6-weeks were considered alive.

Statistical analysis
The statistical plan had three steps. First, we explored the duration of viral shedding in LRT using Kaplan-Meier curves for different clinical relevant patient populations (i.e., age, diabetes, cardiac comorbidities, immunosuppression, and corticosteroids during the ICU stay) which are thought to influence viral shedding in upper respiratory tract samples [2,[12][13][14][15]. A log rank test was performed; a specific subgroup had prolonged viral shedding if the log rank test between the two groups (e.g., diabetic versus non-diabetic) was statistically significant. Second, we evaluated viral load in LRT during the ICU stay (i.e., quantitative RT-PCT) using graphical and descriptive statistics (i.e., Wilcoxon test) between survivors and non-survivors. Third, we determined the association between viral presence in LRT and mortality using mixed-effect logistic models for clustered data (PROC GLIMMIX of SAS) and adjusting for the time between symptoms' onset and date of sampling. All collected samples were considered for this analysis. These models take into account the clustering effect of multiple sampling per patient and the center effect (i.e., random effect). We performed two sensitivity analyses: the first using only endotracheal aspirates and the second adjusting for sex and age. Tests were twotailed, with p < 0.05 being considered significant. All analyses were performed using SAS (version 9.4) and R (version 3.5.3). The current analysis complied with the STROBE guidelines for observational studies [16]. Prolonged viral shedding was not associated with age (p = 0.32), cardiac comorbidities (p = 0.93), diabetes (p = 0.16), immunosuppression (p = 0.14), nor corticosteroid use (p = 0.99, Fig. 1).

Results
However, using a graphical description, the LRT viral load tended to be higher in non-survivors (Fig. 2).
This difference was statistically significant using mixed-effect models and after adjusting for the time interval between onset of symptoms and date of sampling, patient, and center random effect (OR 3.78, 95% CI 1.13-12.64, p = 0.03). A sensitivity analysis using only endotracheal aspirates (i.e., without bronchoalveolar leakage fluid and plugged telescoping catheter) showed similar results (OR 2.96, 95% CI 0.78-11.25, p = 0.10). After adjustment for age and gender, we observed again similar results (OR 3.25, 95% CI 0.95-11.17, p = 0.06).

Discussion
Our findings have several clinical, pathophysiological, and infection prevention implications. First, we showed that viral load in the LRT was associated with the 6week mortality. Therefore, positive LRTS may be used as a prognostic marker in COVID-19 patients. Second, COVID-19 is characterized by an initial phase of viral replication in the upper respiratory tract, followed by a second pulmonary phase driven especially by the host inflammatory response [17], and this latter phase probably determines the prognosis of disease. For this reason, some investigators administered immunomodulatory drugs in severe COVID-19 patients [18]. However, our data may suggest a possible direct cytopathic role of virus in the LRT, supported by the significant association between viral load and mortality. Third, we showed that the viral shedding in LRT lasted almost 30 days in median, and we did not identify specific risk factors for prolonged viral shedding in the LRT. Several studies illustrated that viral shedding in the upper respiratory tract in non-critically ill patients varied from 8 to 24 days [2,5,[12][13][14][15]19]. We observed that the viral shedding in critically ill patients may be longer, probably due to a prolonged replication in the LRT. Interestingly, any of the well-known risk factors for upper respiratory tract appeared to be able to influence the viral shedding in LRT. Critically ill patients may, therefore, require prolonged infection prevention measures during their hospitalization. Our study has several limitations. First, we performed an observational study using surveillance data and we showed only an association between viral presence and mortality. Second, viral cell cultures (i.e., the gold standard) were not routinely performed during the study: the results on viral shedding might have been different if this method would have been used and firm assumptions on infectivity cannot be made. Third, all aspirates (endotracheal samples, BAL, and telescope catheters) were categorized as LRTS and a comparison between them may be debatable. However, a sensitivity analysis using only endotracheal samples showed similar results. Moreover, the LRTS dilution was not standardized between the two participating centers and a dilution marker was not used. Finally, we did not routinely collect nasopharyngeal samples and the true viral excretion (e.g., from the upper respiratory tract) concomitantly with LRT replication remains unknown; therefore, assumptions on infectivity based on LRT analyses should be interpreted with caution.

Conclusions
The viral shedding in LRT lasted almost 30 days in median in critically ill patients, and the SARS-CoV-2 viral presence in the LRT was associated with the 6week mortality.