SARS-CoV-2 RNA in plasma samples of COVID-19 affected individuals: a cross-sectional proof-of-concept study

Background Recent studies showed that plasma SARS-CoV-2 RNA seems to be associated with worse COVID-19 outcome. However, whether specific population can be at higher risk of viremia are to date unexplored. Methods This cross-sectional proof-of-concept study included 41 SARS-CoV-2-positive adult individuals (six affected by haematological malignancies) hospitalized at two major hospital in Milan, for those demographic, clinical and laboratory data were available. SARS-CoV-2 load was quantified by ddPCR in paired plasma and respiratory samples. To assess significant differences between patients with and patients without viremia, Fisher exact test and Wilcoxon test were used for categorical and continuous variables, respectively. Results Plasma SARS-CoV-2 RNA was found in 8 patients (19.5%), with a median (IQR) value of 694 (209–1023) copies/mL. Viremic patients were characterized by an higher mortality rate (50.0% vs 9.1%; p = 0.018) respect to patients without viremia. Viremic patients were more frequently affected by haematological malignancies (62.5% vs. 3.0%; p < 0.001), and had higher viral load in respiratory samples (9,404,000 [586,060-10,000,000] vs 1560 [312–25,160] copies/mL; p = 0.002). Conclusions Even if based on a small sample population, this proof-of-concept study poses the basis for an early identification of patients at higher risk of SARS-CoV-2 viremia, and therefore likely to develop severe COVID-19, and supports the need of a quantitative viral load determination in blood and respiratory samples of haematologic patients with COVID-19 in order to predict prognosis and consequently to help their further management. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-021-05886-2.

In this study, we systematically quantified SARS-CoV-2 load in paired plasma and respiratory samples of 41 well characterized patients, with the aim of defining whether the presence of viremia could be correlated with clinical and/or laboratory characteristics, and COVID-19 outcome.

Data collection
This proof-of-concept cross-sectional study included 41 SARS-CoV-2-positive adult patients hospitalized at Grande Ospedale Metropolitano Niguarda and at Fondazione IRCCS Policlinico Ca′ Granda (Milan, Italy) since March 01, 2020, and who have either died (N = 7) or been discharged (N = 34) by July 01, 2020, for those paired respiratory (nasopharingeal swabs or bronchoalveolar lavages) and plasma samples were available. Demographic and clinical data were retrospectively collected thanks to pseudonymized electronic forms. The severity of the disease was classified in mild, moderate, severe and critical in line with WHO scale [12].
SOFA score and Charlson Comorbidity index were computed from the patients' electronic medical records.
The study protocol was approved by the Ethical Committees of ASST Grande Ospedale Metropolitano Niguarda and IRCCS Ca′ Granda Ospedale Maggiore Policlinico Foundation (Prot. Numbers 92-15,032,020 and 0008489) and conducted in accordance with the principles of the 1964 Declaration of Helsinki. Study participation was by written informed consent. The requirement for informed consent was waived for ICU patients for those communication of information was impossible or characterized by disproportionate efforts [13].

SARS-CoV-2 quantification
Total RNA was extracted from 280 ul of respiratory samples or plasma samples using QIAamp viral RNA mini kit (Qiagen) following manufacturer's instruction. SARS-CoV-2 genomic RNA was quantified by means of the QX200™ Droplet Digital™ PCR System (ddPCR, Biorad) using an home-made protocol targeting the RNA dependent RNA polymerase (RdRp) of SARS-CoV-2 and the housekeeping gene RNAse P [14,15]. All results were further confirmed by using a second assay adapted for ddPCR and targeting two different portions of RdRp (https://www.who.int/docs/default-source/ coronaviruse/real-time-rt-pcr-assays-for-the-detection-ofsars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2). SARS-CoV-2 quantification was finally expressed in number copies/mL.

Statistical analysis
Descriptive statistics are expressed as median values and interquartile range (IQR) for continuous data and number (percentage) for categorical data. To assess significant differences between patients with and patients without viremia, Fisher exact test and Wilcoxon test were used for categorical and continuous variables, respectively.
SPSS statistical software (version 23.0, IBM, Armonk, NY, USA) was used for statistical analysis.

Patients' characteristics
From March 01 through July 01, 2020, a total of 1801 patients received a diagnosis of SARS-CoV-2 infection at two of major hospital in Milan, Lombardy. Forty-one patients with available paired respiratory and plasma samples for SARS-CoV-2 detection were included in the analysis. Six of them (14.6%) were affected by haematological malignancies. Demographic, clinical, and laboratory, findings at admission, stratified by presence of SARS-CoV-2 in plasma, are reported in Table 1.
Thirteen patients (30.2%) were male, and median age was 59 (IQR: 50-67) years. Chest radiographs or CT scan confirmed a classical bilateral interstitial pneumonia in 95.1% of cases (39/41). Looking at the outcome available, 7 patients (17.1%) died in a median time of 28 (27-36) days from symptoms onset, and 34 (82.9%) were discharged in a median time of 54 (30-76) days from symptoms onset at the study endpoint.
Stratifying the population by plasma SARS-CoV-2 RNA, viremic patients were characterized by an higher mortality rate (50.0% vs 9.1%; p = 0.018) respect to no viremic patients. Respiratory failure was the leading cause of death in 3 out of 4 patients with SARS-CoV-2 viremia and in the 2 out 3 patients without evidence of SARS-CoV-2 in plasma. The underlying disease found in the remaining death with viremia was a T-cell lymphoma at advanced stage.

Focus on hematological malignancies
As mentioned before, 5 out 6 patients affected by hematological malignancies were viremic for SARS-CoV-2 (Supplementary Table 1). Three of these 5 viremic patients were affected by large B-cell lymphoma (60.0%) and 4 received chemotherapy during COVID-19 (80.0%), with one patient receiving Bendamustine in combination with Gemcitabine and Vinorelbine (BEGEV). The median time to SARS-CoV-2 clearance in respiratory tract and plasma was of 6.5 (4.4-6.5) and 1.7 (1.5-3.0) weeks, respectively. The longer time for viral clearance in respiratory and peripheral tracts was observed in one patient

Discussion
In our study plasma SARS-CoV-2 RNA was found in 8 (19.5%) individuals, 6 (75.0%) of whom had a severe or critical COVID-19 manifestation and 4 (50.0%) died. This is in line with studies published so far, describing plasma SARS-CoV-2 RNA detectable in different percentage of COVID-19 patients, ranging from 0 to 73%, and frequently and significantly associated with worse clinical outcomes [2][3][4][5][6][7][8][9][10][11]. It should be noted that SARS-CoV-2 was not the first respiratory viral agent to be detected in blood of critical individuals. The viremic phase was frequently correlated with the severity of the disease and worse outcome in most viral infections, like those sustained by Rhinoviruses, Respiratory syncytial virus, Adenovirus, SARS-CoV, and Metapneumovirus [16][17][18]. Now that more evidence suggests a close relationship between viremia and severe COVID-19, the identification of specific factors or conditions that can define a population at risk of viremia is of outmost importance. In our study, plasma SARS-CoV-2 load was frequently detected in patients affected by hematological malignancies (62.5% vs. 3.0%, p < 0.001), and with higher viral load in respiratory samples (9,404,000 [586,060-10,000, 000] vs. 1560 [312-25,160] copies/mL, p = 0.002), as compared to patients without viremia (Table 1). Previous studies on series of hospitalised patients showed that the concurrent haematological malignancies and COVID-19 are strongly related to mortality [19], and to prolonged viral shedding in the respiratory tract and serum samples [1,20]. An episode of prolonged plasma SARS-CoV-2 RNA started prior to the onset of symptoms and diagnosis of COVID-19 was also recently described in a 51-year-old patient with acute myeloid leukemia, and thus with a heavily compromised immune status [21]. Consistent with these earlier reports, we found that viremia was detected since 12 (9)(10)(11)(12)(13)(14) days from symptom onset, and viral undetectability in the blood occurred while SARS-CoV-2 in respiratory samples was still present. All these evidences may pose this fragile population at higher risk of developing a systemic infection, suggesting a potential utility of plasma SARS-CoV-2 RNA testing as a prognostic indicator in severe immunocompromised patients.
Our proof-of-concept study has some limitations. a) The small sample size and the high proportion of individuals with chronic comorbidities (80.5% of patients) prevented to appreciate a clear association between viremia and severe COVID-19, that preliminary data have already shown [7][8][9][10], as well as correlations between viremia and IL-6 or other knows COVID-19 prognostic markers (i.e. lymphocytes count, lactate dehydrogenase or D-dimer level) [5,22,23]. b) The correlation between viremia and the outcome was based only on 4 definitive events, requiring a confirmation on larger sample size. c) The real infectivity of the virus found in plasma was not assessed. Thus, it cannot be determined at present whether detectable RNA represents intact infectious virus or inactive, non-replicating nucleic acid. d) The study design, the heterogeneity of hematological malignancies and chemotherapies prevented to draw certain conclusions about correlations between these parameters and time to SARS-CoV-2 clearance. Even if the prolonged respiratory shedding and persistent viremia observed in one patient treated with the bendamustine is in line with recent findings, [24,25] this observation requires further evaluation by ad hoc designed studies.

Conclusion
Our cross-sectional proof-of concept study provides evidence for plasma SARS-CoV-2 load detection in the 19.5% of patients (n = 8), 50% of whom died, confirming the association between viremia and worse outcome [7][8][9][10][11]. Patients with SARS-CoV-2 in plasma were frequently characterized by haematological malignancies, and elevated viral load in respiratory samples. Although the associations here observed are based on a small population and need to be confirmed on a larger sample size, they pose the basis for an early identification of patients at higher risk of SARS-CoV-2 viremia, and therefore likely to develop severe COVID-19. This proof-ofconcept study also supports the need of a quantitative viral load determination in blood and respiratory samples of haematologic patients with COVID-19 in order to predict prognosis and consequently to help their further management.