SuPAR in Adult Patients With Covid-19

BACKGROUND

Soluble urokinase plasminogen activator receptor

The biomarker soluble urokinase plasminogen activator receptor (suPAR) is the soluble form of
the cell membrane-bound protein urokinase plasminogen activator receptor (uPAR), which is
expressed mainly on immune cells, endothelial cells, and smooth muscle cells. uPAR is
released during inflammation or immune activation, and therefore the suPAR level reflects the
extent of immune activation in the individual. All human beings have a baseline level of
suPAR that is individually determined and increases with age. Studies have shown that the
suPAR level is associated with morbidity and mortality in a number of acute and chronic
diseases and in the general population. The suPAR level is elevated across diseases, and not
solely associated with one specific disease. Therefore, suPAR is applicable as a prognostic
marker and not as a diagnostic marker. This characteristic may be utilized for risk
stratification in unselected patients.

Originally, uPAR was proven a receptor for urokinase (uPA) which splits plasminogen into
active plasmin. Moreover, uPAR interacts with other proteins and plays a role in several
important cell processes like migration, adhesion, angiogenesis, proliferation, and
chemotaxis. The suPAR protein was discovered in 1991, when it was found to be a marker of
cancer progression. In recent years, several studies have shown that suPAR is associated with
a number of chronic diseases (including cardiovascular, hepatic, renal, and pulmonary
diseases), and that the level is a predictor of a negative outcome of various infectious
diseases (tuberculosis, HIV, malaria, sepsis, meningitis, pneumonia) and in critically ill
patients.

Across diseases, the suPAR level discriminates non-survivors from survivors. suPAR reflects
the level of chronic inflammation, and therefore it has been studied as a potential marker of
development of diseases, and studies have shown that an elevated level predicts development
of chronic diseases and cancer in the general population. The suPAR blood level is stable
with no diurnal variation and no changes following fasting, while suPAR can be measured in
blood, plasma, urine, cerebrospinal fluid, ascites fluid and pleural fluid. The level
increases and decreases with progression and improvement of a disease, respectively, but more
slowly compared to e.g. C-reactive protein (CRP). The normal suPAR plasma level is 2-3 ng/mL
in healthy individuals, about 3-4 ng/mL in unselected patients in emergency departments, and
about 9-10 ng/mL in critically ill patients.

suPAR in intensive care

In critically ill patients, the suPAR level is significantly increased. suPAR is an
independent prognostic marker, and the change over time correlates with organ dysfunction.
suPAR is elevated and has a prognostic value in patients with: SIRS (systemic inflammatory
response syndrome), sepsis/septic shock, burn injuries, and traumatic brain injuries. The
suPAR level reflects the body's immune response to infections, and the level increases with
the severity of the infection. In patients with organ dysfunction, the suPAR value is often a
two-digit value. In particular hepatic and renal dysfunction affects the suPAR level. suPAR
has been studied in patients with SIRS who were acutely admitted to the emergency department
(n=902). The studies showed that suPAR is a stronger marker of 2-day, 30-day, and 90-day
mortality than age, CRP, IL-6, creatinine, and procalcitonin. However, for diagnostic
purposes, IL-6 and CRP are superior to suPAR in predicting a positive blood culture. A Greek
multicenter study including 1914 patients with sepsis showed that suPAR is a strong predictor
of mortality, and that a suPAR level above 12 ng/mL is linked to a >80% sensitivity for
mortality and a negative predictive value of 94.5%. In addition, the prognostic value of
suPAR in patients with sepsis is independent of relevant covariates like APACHE score, CRP,
etc.

In patients with burn injuries and inhalation trauma requiring mechanical ventilation, the
plasma suPAR level and bronchoalveolar lavage fluid level correlate to IL-6 and coagulation
factors. An elevated plasma suPAR level is associated with prolonged Intensive Care Unit
(ICU) stay and the duration of mechanical ventilation. The suPAR level is elevated in
patients with traumatic brain injury. In trauma patients who suffered a brain injury within
12 hours prior to blood sampling, the mean suPAR level is 14.9 ng/mL ± 6.9 vs. 2.8 ng/mL ±
0.7 in control subjects. In these patients suPAR is associated with severity of the brain
injury and with mortality.

suPAR and acute respiratory distress syndrome

Mortality rates of patients with the acute respiratory distress syndrome (ARDS) are high.
Biological markers as well as disease severity scoring systems may be useful for risk
stratification and the creation of homogenous patient groups in clinical trials. However,
currently no single biological marker has been found to have sufficient discriminative value
for accurate prediction of mortality or bedside decision making in ARDS patients in the
clinical setting The first study that investigated the association between plasma suPAR and
mortality in a cohort of ARDS patients was published in 2015. In that study, plasma levels of
suPAR were higher with increasing severity of ARDS, and higher in patients who died in the
ICU. Furthermore, higher plasma suPAR levels were associated with less ventilator- and
ICU-free days.

In a prospective, multicenter, observational study including mechanically ventilated ICU
patients, low concentrations of suPAR were predictive for survival and high concentrations
for renal replacement therapy and mortality. The authors concluded that SuPAR may be used for
screening for patients with potentially good survival, while its association with renal
replacement therapy may supply an early warning sign for acute renal failure. Several other
authors have evaluated the predictive value of suPAR regarding the risk of ARDS in sepsis
patients, and reported a correlation/association with disease severity, inflammation and
mortality.

AIM

SPARCOL is a multi-center observational study aiming to investigate if suPAR measured at
admission can predict the risk of future complications and mortality in adults patients with
Covid-19.

METHODS

Design

This is a prospective observational study designed in accordance with the declaration of
Helsinki. The study will be registered at Clinical Trials.gov and has been approved by the
Institutional Review Board of the University Hospital of Larisa, under the reference number
17543.The study will be approved by the IRB of each of the participating centers.

Patient eligibility

Inclusion criteria will be: (1) adult (≥18 years old) patients hospitalized primarily for
COVID-19; (2) a confirmed SARS-CoV-2 infection diagnosed through reverse transcriptase
polymerase chain reaction test of nasopharyngeal or oropharyngeal samples; and (3) at least
one blood sample collected at admission and stored for biomarker testing. Patients with
confirmed SARS-CoV-2 infection who will not be primarily admitted for COVID-19 and patients
with incomplete data will be excluded.

Sampling and laboratory measurements

Participants will undergo sampling of peripheral venous blood, immediately after admission.
Blood samples drawn from all patients and EDTA plasma will be stored at -80° C until later
measurement. Plasma suPAR levels will be determined using the suPARnostic® ELISA assay
(ViroGates, Denmark), which is based on a simplified double monoclonal antibody sandwich
ELISA assay whereby samples and peroxidase-conjugated anti-suPAR are first mixed together and
then incubated in anti-suPAR pre-coated micro wells. The recombinant suPAR standards of the
kit are calibrated against healthy human blood donor samples. suPAR concentrations are
determined as ng/mL plasma.

Outcomes

The primary endpoint will be the presence of respiratory complications [need of non-invasive
ventilation (CPAP-BiPAP), hi-flow nasal oxygen, or mechanical ventilation], admission to ICU,
and survival at 30 days.

Secondary endpoints will be hypotension (defined as systolic blood pressure <90 mmHg or mean
arterial pressure <65 mmHg for 3 minutes or more), need for vasoactive drugs (need for
vasoactive drugs not planned before and/or continuous infusion), and/or acute new
cardiovascular disorder (atrial fibrillation, sustained ventricular tachycardia,
supraventricular tachycardia, myocardial infarction, pericarditis, myocarditis, cardiogenic
shock, and/or cardiac arrest), renal failure, duration of mechanical ventilation,
reintubation, hospital length of stay, length of stay in ICU and readmission.

Data Collection and Monitoring

Data analysis will be based on predefined data points on a prospective data collection form.
The staff will be blinded to measurements until the end of the study and all data are
analyzed. Clinical monitoring throughout the study will be performed to maximize protocol
adherence, while an independent Data and Safety Monitoring research staff will monitor
safety, ethical, and scientific aspects of the study. Data collection will also include
demographics, general blood count, C-reactive protein, APACHE II, and SOFA.

Data management

The goal of the clinical data management plan is to provide high-quality data by adopting
standardized procedures to minimize the number of errors and missing data, and consequently,
to generate an accurate database for analysis. Remote monitoring will be performed to signal
early aberrant patterns, issues with consistency, credibility and other anomalies. Any
missing and outlier data values will be individually revised and completed or corrected
whenever possible.

Sample size

This study will include approximately 500 patients. Based on international and national
epidemiological data, we anticipate that approximately 30% of the included patients will
reach the primary endpoints.

Ethics and dissemination

The study will be performed according to national and international guidelines. The study
will begin after approval has been obtained from the Hospital's Institutional Review Board,
according to local regulation. Prospective written informed consent will be requested before
inclusion of all eligible patients based on the Review Board's decision.

Competing interests None declared.