The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (Covid-19) has rapidly spread from China to the rest of the world. The resulting pandemic has challenged societies and their health care systems and requested unprecedented political, medical and scientific actions. The long-term societal and economic consequences of the pandemic per country start to emerge.

SARS-CoV-2 is an RNA virus of the Coronaviridae family, which is closely related with SARS (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV). These viruses are airborne and they all infects epithelium (primarily lung epithelium) through a high affinity interaction between the viral spike protein and cell surface expressed angiotensin converting enzyme 2 (ACE2). Viral entry is finally facilitated through proteolytic cleavage of ACE2 via the host type II transmembrane serine metalloprotease TMPRSS2 (Heurich et al., 2014) which equally cleaves the SARS-CoV-2 spike protein (Hoffmann et al., 2020).

SARS-CoV-2 infection leads to disease courses varying from none or mild symptoms for the majority of infected individuals to severe or critical disease. Mild cases most often recover without medical intervention. Among the minority with severe symptoms a non-negligible fraction, particularly among older and comorbid individuals, has fatal outcome (Verity et al., 2020).

SARS-CoV-2 infected individuals will undergo a non-symptomatic incubation period lasting on average 6.4 days (Lescure et al., 2020). During this incubation period the virus will replicate and the exposed individual will transition from non-infectious to infectious. Infected individuals generally suffer a mild disease course associated with viremia in the respiratory tracts for 8-10 days. These patients will either recover or transition towards a more severe disease phase characterized by prolonged viremia (Lescure et al., 2020) and a pronounced pro-inflammatory signature (Chen et al., 2020). The disability to control the cytokine storm ravaging during the severe phase of the disease is believed to be the primary cause of fatality.

Despite efforts to develop therapeutic approaches the world has largely faced the first 9-12 months of pandemic with no efficient prophylactic or therapeutic medical intervention available. Indeed, the primary purpose of the health care system has been to provide life support to ensure the time for the patient to naturally clear the virus. Life support consists primarily of providing oxygen and sometimes mechanical ventilation.

During a pandemic, it is of outmost importance not to surpass the capacity of the health care system for a given country. This is accommodated at two levels; the hospital level and the political level. At the hospital level medical expertise should ensure that hospital resources are distributed effectively and according to patient needs. At the political level one can install temporary reinforced socioeconomic regulations to dampen the propagation of disease through isolation of infected individuals or even confinement of entire populations. The latter strategy has been adopted by most developed countries and serves to extend the period of the pandemic to reduce the number of infected individuals during the peak of the pandemic (Sheikh et al., 2020; Zanin et al., 2020). Meanwhile, overwhelmed hospital wards require tools for patients’ severity stratification.

Stratification of Covid-19 patients on the basis of cellular biomarkers is an unmet need in patient care. The primary objective of this study is to characterize the relationship between neopterin blood levels at hospital admission and clinically assessed severe subsequent evolution. Indeed, coronavirus infections in the past have been characterized by the onset of a cytokine storm. The hallmark of a cytokine storm is an abnormal regulation and uncoordinated release of several pro-inflammatory cytokines at inappropriate time intervals during infection, such as interleukin (IL)-6, IL-1β, Tumor necrosis factor (TNF-α) and type-I interferons (IFNs) (Huang et al., 2005; Hadjadj et al., 2020; Dorgham et al., 2021; Mahat et al., 2021) resulting in an uncontrolled inflammatory response, acute respiratory distress syndrome, respiratory failure, shock, organ failure, and death (Huang et al., 2005; Batisse et al., 2020; Chen et al., 2020; Mortaz et al., 2020; Remy et al., 2020; Wang et al., 2020). It is therefore reasonable to postulate that the inflammatory response measured both at cellular and molecular levels would represent a main prognostic signature for the disease.

Neopterin (1’, 2’, 3’-D-erythro-trihydroxypropylpterin) belongs to the family of pteridines and is involved in a variety of oxidation-reduction reactions in the body. Neopterin is derived in vivo from guanosine triphosphate (GTP). The reaction is catalyzed by the enzyme GTP-cyclohydrolase-I (GCH I) in activated monocytes, macrophages, dendritic cells, and endothelial cells upon stimulation mainly by IFN-γ (Fuchs et al., 1993; Murr et al., 2002). Its release by macrophages in response to cytokines released by T lymphocytes and natural killer cells make neopterin an indicator of activation of cell-mediated immunity, which is tightly associated with hyper-inflammation. Therefore, the measurement of neopterin reflects an elevated and likely sustained immune activation taking place during viral assault and cytokine storm. Noteworthy, it has been described that neopterin was associated with the clinical progression of patients infected with SARS-CoV and that the plasmatic levels observed during SARS-CoV infection were predictive of the severity of respiratory syndromes independently of viremia (Zheng et al., 2005).

Here we demonstrate that increased serum neopterin levels at hospital admission are predictive of extended symptomatic disease duration and non-survival post SARS-CoV-2 infection. Neopterin therefore constitutes a relevant prognostic biomarker to optimize Covid-19 patient’s management.

Evidence to date is that approximately 80% of individuals with Covid-19 have a mild disease, i.e. a respiratory tract infection with or without pneumonia, and most of them recovered. In 14% of cases, Covid-19 develops into a more severe disease requiring hospitalization while the remaining 8% of cases experience critical illness requiring intensive care. The mortality of patients hospitalized due to Covid-19 in Europe is approximately 4% and more than 10% in the oldest population (source: ECDC). Monitoring and estimating resource-needs is crucial to ensure that healthcare systems have the capacity to respond to acute surges in cases. Today, clinical decision-making during Covid-19 disease is mainly based on medical parameters since no biomarkers can be used to evaluate the risk for respiratory distress or to decide on adapted care for vulnerable patients.

Here, we demonstrate that neopterin can serve as a predictive biomarker of mortality post SARS-CoV-2 infection, enabling discrimination between patients who will recover and those at risk of fatal clinical outcome. Of note, neopterin level was measured at arrival to hospital before any treatment could interfere. The fact that the study was conducted on two independent cohorts from Spain (n=309) and France (n=65), which could imply different initial health status and medical follow-up, makes us confident that neopterin is a reliable prognostic biomarker for stratifying SARS-CoV-2 infected patients according to disease severity. Indeed, it has been reported that serum neopterin levels measured in patients suffering from mild disease was lower than the concentration found in severe patients (Robertson et al., 2020; Ozger et al., 2021). Another recent study based on 115 patients from Austria showed that SARS-CoV-2-infected patients exhibiting high level of systemic neopterin (>45nM) were more prone to require mechanical ventilation and were at higher risk of intensive care unit admission (Bellmann-Weiler et al., 2021).

Neopterin measurement at hospital admission, identifying patients at risk of a severe disease course (high neopterin levels, >53nM), can serve as an urgently required tool to guide the triage of infected patients and ensure that critical care beds and specialized hospital wards are dedicated to their treatment (Hailemichael et al., 2021). In doing so, healthcare resources will be put to best use during pandemic peak periods and the pressure on specialized hospital wards will be reduced. Infected patients with low levels (<53nM) of neopterin would be considered at low risk and can return to home quarantine and remote follow-up. With a 53nM cut-off value neopterin is able to predict all fatal clinical outcomes (100% sensitivity), but at this threshold value the specificity is 64%. Therefore, a number of false positives are to be expected, which makes it relevant to consider a third stratification level representing patients with intermediate levels of neopterin who should be kept under perpetual observation in a classic hospital wards to assure their follow-up.

Neopterin is induced through IFN-γ signaling. Its concentration might be considered an indicator of systemic immune activation of both lymphoid (T and NK cells) and myeloid cells (monocytes and macrophages). Moreover, neopterin can also indicate oxidative stress (Murr et al., 1999) since its synthesis has been shown to correlate with reactive oxygen species (ROS) production (Nathan, 1986). IFN-γ is an initiator of pro-inflammatory immunity. It is a primary inducer of TNF-α expression (Vila-del Sol et al., 2008). TNF−α is initially produced as surface bound precursor of TNF-α. Mature TNF-α is released through the proteolytic activity of TNF-α Converting Enzyme (TACE), also called ADAM-17. ADAM17 is a sheddase involved in a range of biological phenomena, which is tightly regulated through G-protein coupled receptor (GPCR) activity (Lorenzen et al., 2016). A number of ligands may induce this activity, including complement, nucleotides and hyperoxia (Oarhe et al., 2015). Once cleaved by ADAM17, mature TNF-α may induce signaling leading to activation of the NF-κβ pathway and inflammation. Interestingly, both IFN-γ and TNF-α induce the enzyme guanosine triphosphate cyclohydrolase I (GTPCH) which catalyze the transformation of guanosine triphosphate (GTP) to neopterin (Eisenhut, 2013). Neopterin may therefore serve both as a biomarker of an early inflammatory state based on IFN-γ signaling, which should be associated with a large quantity of surface bound TNF-α ready for secretion upon an acute event, such as viral infection and ADAM17 induction. This therefore suggests that high neopterin levels may be associated with a high risk of developing an intense inflammatory reaction, such as the cytokine storm observed in the late phase of severe SARS-CoV-2 infection (Hailemichael et al., 2021). Distinct cytokine profiles have been described to be associated with COVID-19 severity and differentially predictive of mortality according to oxygen support modalities (Dorgham et al., 2021). Based on cytokine profiling performed on our French patients, we observed multiple positive correlations between cytokines and neopterin which revealed 4 clusters. Neopterin was member of a cluster also including IFN-γ, IL-22, IL-18 and GM-CSF ( Supplementary Figure S3 ). This may suggest important innate pathways which could pinpoint physio-pathology mechanisms involved in Covid-19.

The expectation is that adjusting the level of care to the resilience status of infected patients facing Covid-19 will accelerate functional recovery, reducing the time spent at hospitals and the frequency of re-hospitalization, therefore having socio-economic benefits. Measurement of neopterin concentration in various body fluids is easily applicable to the standards of routine laboratories, especially since this fast procedure does not require high amount of biological material, nor specific high-cost equipment. This warrants the accessibility of this biomarker assessment to a large number of potential patients. Here, we measured neopterin in patients’ serum which results in an accurate estimation of disease and hence prognosis; however, this molecule is biologically and chemically stable in all body fluids; future studies should therefore assess if non-invasive salivatory or urinary measurements could also constitute a reliable assay to predict clinical outcome in this context for rapid testing. Preliminary results obtained from 10 patients did not show high level of neopterin in broncho-alveolar lavages ( Supplementary Figure S4 ). A recent study showed that the increased fecal neopterin parallels gastro-intestinal symptoms in Covid-19 (Grabherr et al., 2021). In Covid-19 patients with neurologic symptoms, an unusual pattern of marked central nervous system inflammation was observed in association with increased cerebrospinal fluid neopterin levels (Edén et al., 2021).

In conclusion, we propose that neopterin should be implemented as a rapid and easy clinical test to guide the allocation of health care resources during pandemics and aid in adapting the treatment for patients with an increased risk of adverse medical outcomes. It is presently unknown if neopterin and associated hyper-inflammation is a surrogate immune correlate or if it has causal impact on disease progression. Future studies should examine high-risk patients, who may gain most from more specialized medical care such as anti-inflammatory therapy.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by local ethical committee of Sorbonne-Université/Assistance Publique des Hôpitaux de Paris for standard hospitalized patients (n° CER2020-21) and ICU patients (n° CER2020-31). Similarly, ethical approval was obtained in Spain by local ethical committee (n°PI20-20). The patients/participants provided their written informed consent to participate in this study.

MC and KD performed experiments. ML designed research, analyzed the data and wrote the paper. PQ, AM, LR, HV, AG, BQ and JBa collected and analyzed clinical data. EM-C, AM, ZA, FA, JM, HV, JBo, and C-EL recruited patients GG, AM, AG, FA, and EM-C designed clinical protocol and ethical assessment. DS, AG, BC, CC, and GG provided financial support. DS designed research, performed experiments, analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.

This work was supported by the SARS-CoV-2 Program of the Faculty of Medicine from Sorbonne University (grant i-Covid), by Fondation de France “Tous unis contre le virus”, framework Alliance (Fondation de France, AP-HP, Institut Pasteur) in collaboration with Agence Nationale de la Recherche (ANR-Flash-Covid19).

ML and DS are inventors of patent EP20305794.8.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.