key: cord-0779315-x3l29yj9 authors: Boef, Anna G.C.; van Wezel, Esther M.; Gard, Lilli; Netkova, Kala; Lokate, Mariëtte; van der Voort, Peter H.J.; Niesters, Hubert G.M.; Buter, Coretta Van Leer title: Viral load dynamics in intubated patients with COVID-19 admitted to the intensive care unit date: 2021-04-22 journal: J Crit Care DOI: 10.1016/j.jcrc.2021.04.010 sha: 2249f48a6e429bbabf710bbbd45e8ca3223b1064 doc_id: 779315 cord_uid: x3l29yj9 BACKGROUND: Prolonged viral RNA detection in respiratory samples from patients with COVID-19 has been described, but the clinical relevance remains unclear. We studied the dynamics of SARS-CoV-2 on a group and individual level in intubated ICU patients. METHODS: In a cohort of 86 patients, we analysed SARS-CoV-2 RT-PCR results on nasopharyngeal and sputum samples (obtained as part of clinical care twice a week) according to time after intubation. Subsequently, we performed survival analyses. RESULTS: 870 samples were tested by RT-PCR. Overall viral load was highest in the first week (median nasopharynx 3.1, IQR 1.4–4.0; median sputum 4.0, IQR 3.1–5.2) and decreased over time. In 19% of patients a relapsing pattern was observed. Nasopharyngeal and sputum PCR status on day 14 was not significantly associated with survival up to day 60 in this small cohort. CONCLUSION: In general SARS-CoV-2 RNA levels in respiratory samples in patients with severe COVID-19 decrease after the first week after intubation, but individual SARS-CoV-2 RNA levels can show a relapsing pattern. Larger studies are needed to address the association of clearance of SARS-CoV-2 RNA from respiratory samples with survival, because we observed a trend towards better survival in patients with early clearance from sputum. Coronavirus disease 2019 , caused by infection with SARS-CoV-2, has a broad clinical spectrum of disease, ranging from asymptomatic infection to severe respiratory disease requiring intensive care unit (ICU) admission and mechanical ventilation. For diagnosis of COVID-19, guidelines recommend SARS-CoV-2 nucleic acid amplification testing (NAAT) on respiratory samples (nasopharyngeal/oropharyngeal samples and sputum) (1, 2) . Although respiratory samples have the greatest yield, SARS-CoV-2 can also be detected in non-respiratory samples, like stool samples (3, 4) . A pooled analysis of 28 studies reporting on SARS-CoV-2 RNA shedding showed a median duration of shedding of 18.4 days in respiratory samples from mainly hospitalized patients (5) . Prolonged shedding has been described in patients with mild and severe disease, including RNA detection in respiratory samples for more than 3 months after onset of symptoms (5, 6) . Severe disease has been shown to be associated with prolonged shedding (7) (8) (9) . In addition multiple cases have been reported of patients testing positive after being discharged from isolation. To date the question remains whether this is caused by intermittent low load RNA shedding or reinfection with SARS-CoV-2 (10). Whether prolonged (17) ), date of hospital discharge, date of transfer from or to another hospital and date of death. Comorbidity was categorized in 10 categories: diabetes mellitus, hypertension, hypercholesterolemia, cardiovascular disease, chronic pulmonary disease, malignancy, renal insufficiency, thromboembolic disease, auto-immune disorders or solid organ transplantation. Respiratory sample SARS-CoV-2 RT-PCR results and CRP levels of these patients were extracted from the laboratory information system. ICU capacity during the inclusion period was sufficient for the relatively small number of patients from our own region and for transfer of patients from other regions in The Netherlands (the majority of patients included). Most patients were transferred due to ICU capacity problems and a few patients transferred because extra corporeal life support was considered. The first diagnostic samples of transferred patients were tested elsewhere and these initial Ct values were therefore not available for most patients. All samples were collected and tested as part of clinical care. For all ICU COVID-19 patients nasopharyngeal samples and sputum samples (in intubated patients) were routinely collected twice a week. The use of samples and data was approved by the review board of the UMCG (METc 2020/344). The objection register for use of data and biological materials was checked for all patients. Data were pseudonymised after collection from the electronic patient files and laboratory information system and the pseudonymisation key was stored separately. Nasopharyngeal swabs were preserved in 3 ml of universal viral transport medium prior to testing. Sputum samples were transported to the laboratory in native conditions. In some viscous sputum samples 1ml of viral transport medium was added. For RNA extraction 190μl of sample was used. All samples were tested within 24 hours and stored at 4°C prior to testing. Total nucleic acid was extracted on the NucliSENS® EasyMAG® (Biomerieux, France) according to the instructions of the manufacturer. RT-PCR was performed by using an inhouse assay targeting the E-gene of SARS-CoV-2 on the Applied Biosystems 7500 Real-time To investigate whether unexpected increases in viral load were attributable to variations in sample quality human housekeeping gene RNAse P (included as an endogenous internal control in the GeneFinder COVID-19 Plus RealAmp assay) was used as a comparison gene if the amount of sample was sufficient for an additional PCR. Patient characteristics were described as numbers with percentages (categorical variables) or median with interquartile range (continuous variables). To calculate the duration of admission, the date of discharge home or to a physical rehabilitation hospital was used. Differences in SARS-CoV-2 load between duplicate samples on a single day were calculated as a measure of replicability (see Supplementary Table 1 ). For all further analyses, the mean of the duplicate samples on one day was used. Categories of 1 week were made to plot the viral load in nasopharynx and sputum over time. If more than one sample of a patient was tested within this time period of 1 week, a value of a random sample within this week was taken for the analysis. an increase of 50mg/l in the interval from 2 days before to 3 days after a SARS-CoV-2 peak was used. We compared survival of patients with at least 1 negative SARS-CoV-2 PCR on a nasopharyngeal swab by day 14 after intubation to survival of patients without a negative another hospital were censored on the day of transfer. Patients who were discharged home or to a physical rehabilitation hospital were presumed to be alive on day 60. Survival curves were estimated using Kaplan-Meier's methodology. To assess the differences between the estimated survival curves the log-rank test was used. All analyses were performed in StataSE 14 and/or GraphPad Prism 7.0. Eighty-six patients with proven COVID-19 admitted to the ICU were included in the study. Patient characteristics are listed in Table 1 . Most patients were transferred from a different region in The Netherlands (n=84, 98%) and intubated before or on the day of transport Next we examined the dynamics of viral load through time in all individual patients. In most patients viral loads were high on admission to the intensive care unit and decreased over time (typical example shown in Figure 2A) . However, in a considerable proportion of J o u r n a l P r e -p r o o f patients a relapsing pattern was observed (example shown in Figure 2B ). Sixteen out of 86 patients (19%) had a relevant increase in nasopharyngeal SARS-CoV-2 RNA load (defined as an increase of >2 log copies/mL in 1 or 2 increments, with a minimal "peak" load of 2 log copies/mL). Four out of 86 patients (5%) had a relevant increase in sputum SARS-CoV-2 viral load. For 6 patients with an increase in nasopharyngeal SARS-CoV-2 RNA load, the CT-value of housekeeping gene RNAse P was available for both the peak sample and the pre-peak sample. The difference in RNAse P CT-value between the peak sample and the pre-peak sample ranged from -2.6 (i.e. more human DNA in the peak sample) to +1.7 (i.e. less human DNA in the peak sample). Therefore the increases in viral load are not clearly caused by differences in sample quality. In 2/16 patients the increase in SARS-CoV-2 RNA nasopharyngeal load was accompanied by a simultaneous CRP increase (more than 50 mg/l in any interval between 2 days before and 3 days after the peak in viral load). Furthermore, in 1 patient a positive nasopharyngeal PCR (>2 log copies/ml) was observed following ≥2 consecutive negative nasopharyngeal PCRs. Overall 60 day survival as estimated by Kaplan-Meier in this cohort of patients with severe COVID-19 was 76% (Figure 3) . Most deaths occurred within the first 3 weeks after intubation Figure 4B shows the survival curve according to nasopharyngeal PCR status. Survival from 14 days to 60 days was 80% in the group of patients with positive nasopharyngeal swabs only up to day 14, while survival was 78% in the patients with at least 1 negative swab at 14 days after intubation (p=0.81) . Fourteen days after intubation 39 patients had positive sputum samples and 9 patients had 1 or more negative test results. Figure 4C Most patients had high viral loads in the first week after intubation and these loads gradually decreased over time. A peak in viral load in the first week and gradual decrease of viral load over time has been described by several authors in mixed populations of patients with severe and mild disease (7, 12, (14) (15) (16) . Furthermore, we observed a higher load in sputum samples than in nasopharyngeal samples in the first 2 weeks after intubation, which also has been described previously (12, 14, 16, 18) . In 19% of patients a relapsing pattern was observed. After an initial low and/or decreasing viral load, a sudden increase of ≥ 2 log copies/ml was observed. Although previous studies have described viral dynamics primarily on a group level and have not commented on the patterns of individual viral dynamics, upon inspection of published individual viral load J o u r n a l P r e -p r o o f graphs similar viral load increases can be observed (19, 20) . The phenomenon of more than one peak in the viral load versus time curve is recognized for Influenza A, where it is thought to be the result of target cell depletion, temporal dynamics in interferon production by infected cells or developing adaptive immunity (21) . More recently it was shown that subgenomic RNA from double walled vesicles may be detected for prolonged periods of time following SARS-CoV-2 infections (22) . The significance of the increases in viral load after periods of decrease or low load which we describe in this study remains uncertain. More research is necessary to investigate if this is observed by others and if it is associated with adverse outcome and longer total virus shedding. In many aspects, our study population was similar to previously described cohorts of ICU COVID-19 patients. Patients were admitted to the ICU between 4-22 days (mean 10 days) after onset of symptoms, which is similar to what has been reported before (23) . In 73 % of the patients at least one of the recognized risk factors for severe COVID-19 infections was present, which is also similar to what has been reported before (24) . A distinctive aspect of our cohort of patients is the large proportion of patients transferred from another hospital. This may affect generalizability of our results, because patients who were intubated, but had a clinical condition suitable for transport were transferred to our hospital. The slightly better survival than previously described (25) SARS-CoV-2 clearance from sputum before 14 days after intubation showed a trend toward better outcome, however not significantly due to small numbers of patients included in this analysis. Therefore, the association between survival and early clearance of SARS-CoV-2 needs to be investigated in larger prospective studies. Because we did not have follow-up data of patients discharged home or to a rehabilitation hospital, patients discharged before day 60 after intubation were assumed to be alive at day 60. Although there is a small chance that we may have underestimated mortality, this will have resulted in less bias than censoring all discharged patients (i.e. presuming that their risk of dying is the same as that of patients still in hospital). Sputum samples were infrequently obtained once patients were detubated, and nasopharyngeal swabs no longer obtained once patients were discharged. This could potentially lead to misclassification in survival analysis according to PCR status, due to patients with a last positive sample before detubation or J o u r n a l P r e -p r o o f discharge remaining in the positive category. However, because we performed the analysis on patients still intubated at day 14, the risk of misclassification in our analysis is low. In conclusion, we describe a relapsing viral load in a relatively large cohort of patients with severe COVID-19. We observed a higher viral load in sputum samples than in nasopharyngeal samples in the first two weeks after intubation and viral RNA was detected for more than 35 days in 5 patients. Whether prolonged or relapsing viral detection also has implications for infection control remains to be elucidated. Routinely monitoring of sputum viral load in severe COVID-19 patients may be beneficial for development of infection control guidelines and prediction of prognosis. J o u r n a l P r e -p r o o f Assessment tool for laboratories implementing SARS-CoV-2 testing: interim guidance Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Prolonged presence of SARS-CoV-2 viral RNA in faecal samples Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort: Systematic Review and Meta-analysis Understanding viral shedding of severe acute respiratory coronavirus virus 2 (SARS-CoV-2): Review of current literature Prolonged SARS-CoV-2 detection and reversed RT-PCR results in mild or asymptomatic patients Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province Factors Associated With Prolonged Viral RNA Shedding in Patients with Coronavirus Disease 2019 (COVID-19) Comparisons of viral shedding time of SARS-CoV-2 of different samples in ICU and non-ICU patients European Centre for Disease Prevention and Control. Reinfection with SARS-CoV: considerations for public health response: ECDC SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients Viral load of SARS-CoV-2 in clinical samples Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients Virological assessment of hospitalized patients with COVID-2019 SARS-CoV-2 Viral Load in Clinical Samples from Critically Ill Patients Medicamenteuze behandeling voor patiënten met COVID-19 (infectie met SARS-CoV-2) Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore Temporal dynamics in viral shedding and transmissibility of COVID-19 Viral Load Kinetics of SARS-CoV-2 Infection in First Two Patients in Korea Kinetics of Influenza A Virus Infection in Humans SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan Comorbidities and the risk of severe or fatal outcomes associated with coronavirus disease 2019: A systematic review and meta-analysis Mortality rates of patients with COVID-19 in the intensive care unit: a systematic review of the emerging literature Effect of Hydroxychloroquine in Hospitalized Patients with Covid-19 Corine Geurtsvankessel, Annemiek A. van der Eijk. 2020. Shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (COVID-19): duration and key determinants Boef: conceptualization, investigation, data curation, formal analysis, visualization, writing -original draft Wezel: conceptualization, investigation, data curation, formal analysis, visualization, writing -original draft Lilli Gard: investigation, writing -review & editing Kala Netkova: formal analysis, visualization, writing -review & editing Mariëtte Lokate: investigation, writing -review & editing van der Voort: resources, writing -review & editing Niesters: resources, supervision, writing -review & editing Buter: conceptualization, resources, investigation, supervision, writing-original J o u r n a l P r e -p r o o f