key: cord-1017759-rlonudx9
authors: Choreño-Parra, José Alberto; Jiménez-Álvarez, Luis Armando; Ramírez-Martínez, Gustavo; Cruz-Lagunas, Alfredo; Thapa, Mahima; Fernández-López, Luis Alejandro; Carnalla-Cortés, Martha; Choreño-Parra, Eduardo M; Mena-Hernández, Lourdes; Sandoval-Vega, Montserrat; Hernández-Montiel, Erika Mariana; Hernández-García, Diana Lizzeth; Ramírez-Noyola, Jazmín Ariadna; Reyes-López, Cynthia Estefania; Domínguez-Faure, Andrea; Zamudio-López, Guillermo Yamil; Márquez-García, Eduardo; Moncada-Morales, Angélica; Mendoza-Milla, Criselda; Cervántes-Rosete, Diana; Muñoz-Torrico, Marcela; Luna-Rivero, Cesar; García-Latorre, Ethel A; Guadarrama-Ortíz, Parménides; Ávila-Moreno, Federico; Domínguez-Cherit, Guillermo; Rodríguez-Reyna, Tatiana Sofía; Mudd, Philip A; Hernández-Cárdenas, Carmen Margarita; Khader, Shabaana A; Zúñiga, Joaquín
title: Expression of Surfactant protein D (SP-D) distinguishes severe pandemic influenza A(H1N1) from COVID-19
date: 2021-03-01
journal: J Infect Dis
DOI: 10.1093/infdis/jiab113
sha: 4cdaae153935c562a531519d616a8e2ca4448d96
doc_id: 1017759
cord_uid: rlonudx9

The differentiation of influenza and COVID-19 could constitute a diagnostic challenge during the ongoing winter due to their clinical similitude. Thus, novel biomarkers that enable distinguishing both diseases are required. Here, we evaluated whether the surfactant protein D (SP-D), a collectin produced at the alveolar epithelium with known immune properties, was useful to differentiate pandemic influenza A(H1N1) from COVID-19 in critically ill patients. Our results revealed high serum SP-D levels in severe pandemic influenza but not COVID-19 patients. This finding was validated in a separate cohort of mechanically ventilated COVID-19 patients who also showed low plasma SP-D levels. However, plasma SP-D levels did not distinguish seasonal influenza from COVID-19 in mild-to-moderate disease. Finally, we found that high serum SP-D levels were associated with mortality and renal failure among severe pandemic influenza cases. Thus, our studies have identified SP-D as a unique biomarker expressed during severe pandemic influenza but not COVID-19.

M a n u s c r i p t 6 

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues spreading despite the social distancing measures adopted worldwide. As of January 17 th of 2021, about 93.2 million new cases of coronavirus disease 2019 (COVID-19) and 2 million deaths have been reported globally [1] . In the absence of sufficient vaccination coverage to control the current pandemic, this could converge with the flu season in many Northern Hemisphere regions. Besides the hospitals' burden, this overlap will represent a diagnostic dilemma in emergency rooms. The impact could be further heightened by the emergence of the pandemic influenza A(H1N1)pdm09 virus, since this infection, like SARS-CoV-2, also causes severe disease with higher frequency than seasonal influenza viruses [2] . Moreover, the respiratory manifestations of pandemic influenza A(H1N1) and COVID-19 can be similar [3] [4] [5] [6] . Hence, novel biomarkers enabling to distinguish these two infections, especially in severely ill patients, are urgently needed.

A variety of cytokines measured in the peripheral circulation have shown some predictive value to differentiate between influenza and COVID-19 [5, 7, 8] . However, their levels could be modified by other inflammatory conditions. Thus, candidate biomarkers for clinical use must have a lung tissue-specific expression pattern and be differentially regulated during influenza and COVID- 19 . Previously, we demonstrated that the surfactant protein D (SP-D), an essential component of the pulmonary surfactant with immune properties, is translocated from the alveoli to the blood of patients with pandemic influenza A(H1N1) and its serum levels predict mortality [9] . Here, we report high SP-D levels in the blood of pandemic influenza A(H1N1) but not seasonal influenza and COVID-19 patients.

Furthermore, we identified an association between high serum SP-D concentrations and poor clinical outcomes in pandemic influenza subjects. Our results suggest a possible diagnostic A c c e p t e d M a n u s c r i p t 7 usage of SP-D to distinguish pandemic influenza A(H1N1) from COVID-19 in severe disease.

We conducted a cohort study in mechanically ventilated patients with laboratory- swab samples, bronchial aspirates (BA), or bronchoalveolar lavage (BAL) specimens as described before [10] . Study participants were not co-infected with the human immunodeficiency virus (HIV). Solid-organ transplant recipients and patients with cancer or autoimmune diseases were not eligible.

Clinical and demographic data of all study participants were retrieved by reviewing their medical records. These data included age, gender, anthropometrics, comorbidities, symptoms, triage vital signs, admission Sequential Organ Failure Assessment (SOFA), and Acute Physiology And Chronic Health Evaluation II (APACHE II) scores, as well as initial laboratory tests (the first test results available, typically within 24 hours of admission). Initial laboratory tests included white blood cell (WBC) counts, liver and kidney function, procalcitonin (PCT), blood gases, and other tissue injury markers. Patients were followed until their hospital discharge or death. The incidence of specific complications, requirement A c c e p t e d M a n u s c r i p t 8 of antibiotics, corticosteroids, antivirals, choroloquin/hydroxychloroquine, azithromycin, and specific intensive care interventions during the follow-up period were registered.

Peripheral blood samples were obtained from all participants at their hospital admission by puncturing a superficial vein using yellow top collecting serum tubes without anticoagulant. A second blood sample was taken after seven days of hospitalization only from pandemic influenza patients. The serum was collected and aliquoted from whole blood after centrifugation at 400 x g for 10 minutes and stored at -80 ºC until use. Serum samples from ten volunteer donors were used as healthy controls (HC). Also, we obtained serum from 20 patients with pulmonary tuberculosis (PTB) before initiation of anti-tuberculosis drugs and 17 individuals with stable non-exacerbated chronic obstructive pulmonary disease (COPD) that attended INER and were considered as disease controls.

A separate group of patients with mild-to-severe COVID-19 (n = 47) and individuals with mild-to-moderate type A(H1N1/H3N2) and B seasonal influenza (n = 41; hereinafter referred to as seasonal influenza) from the EDFLU study were recruited at the emergency room of the Barnes Jewish Hospital in St. Louis, MO, USA [11] , and served as a validation cohort. The levels of SP-D in these patients were analyzed in plasma. Briefly, peripheral blood samples were obtained in the emergency department or within 48 hours of patient admission to the hospital. Blood was obtained by standard phlebotomy techniques into EDTA-anticoagulated collection tubes. Blood samples were layered over Ficoll in the laboratory and centrifuged at 400 x g for 30 minutes at room temperature. The plasma layer was removed, centrifuged for 5 minutes at 350 x g to remove residual peripheral blood mononuclear cells (PBMCs), and then stored at -80 ºC until analysis.

A c c e p t e d M a n u s c r i p t 9 Both in the validation cohort and the original Mexican cohort, the respiratory disease severity was defined as follows: (a) mild, patients with an acute respiratory illness that did not require hospitalization; (b) moderate, patients that were admitted to the hospital but did not require mechanical ventilation (MV); (c) severe, patients requiring MV and ICU admission.

The SP-D levels in serum and plasma were determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Human Surfactant Protein D ELISA, BioVendor, USA). Briefly, standards, quality controls, and serum/plasma specimens were brought to room temperature and incubated in microplate wells precoated with polyclonal anti-human SP-D antibodies for 2 hours, followed by a 1-hour incubation with biotin-labeled anti-human SP-D antibodies and 1-hour incubation with a streptavidin-HRP conjugate. The plate was thoroughly washed five times with the manufacturer's washing buffer between each step of the assay. Finally, the plate was incubated with the substrate solution for an additional 15 minutes. The reaction was interrupted by adding a stop acid solution, and the absorbance of each well was determined using a microplate reader set at 450nm, with the reference wavelength set at 630 nm. Results were calculated by subtracting readings at 630nm from readings at 450nm, then interpolating each well's substracted absorbances to the standard curve's absorbances using a four-parameter logistic regression model.

The current study was reviewed and approved by the Institutional 

Descriptive statistics were used to characterize the study population clinically. 

We enrolled 93 patients with pandemic influenza and 54 with COVID-19. Their median age was 47 years. Both diseases preferentially affected males (73% and 64%, respectively). Overall, study participants attended the hospital after a median of 7 days since onset of symptoms. All patients required MV and ICU care. Fever, dyspnea, cough, fatigue, myalgia, and arthralgia were the most frequent symptoms presented by both patient groups. The clinical manifestations (Table 1 ) and laboratory profiles ( Table 2) were similar in patients with both diseases. These data indicate that the discrimination of these diseases by clinical characteristics is complex. However, some significant differences deserve to be mentioned. For instance, pandemic influenza differed from COVID-19 with regards to a higher prevalence of obesity, increased frequency of fever, myalgia, arthralgia, rhinorrhea, sputum production, and higher levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), PCT, SOFA, and APACHE II scores.

Conversely, COVID-19 was characterized by dry cough, gastrointestinal symptoms, higher number of white blood cells (WBC), neutrophils, percentage of oxygen saturation (SO 2 %), and PaO 2 /FiO 2 ratio values.

Importantly, COVID-19 patients showed higher mortality, despite presenting similar rates of complications (Table 1) . All pandemic influenza patients were A c c e p t e d M a n u s c r i p t 12 treated with oseltamivir, whereas 31.5% of COVID-19 patients received oseltamivir, 24% lopinavir/ritonavir, 59% chloroquine/hydroxychloroquine, and 42% azithromycin. Both patient groups received antibiotics, although individuals with COVID-19 required more antibiotics (Table 1) . Notably, COVID-19 patients received more corticosteroids compared to pandemic influenza subjects (51% vs. 13%, p < 0.0001), especially those enrolled after the publication of the RECOVERY trial [12] . Approximately 50% of pandemic influenza and COVID-19 patients were ventilated in the prone position. Finally, 8% and 4% of pandemic influenza and COVID-19 patients were subjected to extracorporeal membrane oxygenation (ECMO), respectively.

The clinical characteristics of the comparative and validation cohorts are summarized in Table 1 and Supplementary Table 1 , respectively.

SP-D plays an essential role in innate lung defenses, and its production is dysregulated during inflammatory pulmonary disorders [13] . We previously observed that SP-D translocates from alveoli to the blood during pandemic influenza A(H1N1) [9] . Hence, this molecule may be a useful readout of lung injury secondary to infections. To address this hypothesis, we measured the serum SP-D levels in pandemic influenza and COVID-19. We observed that serum SP-D levels were elevated in pandemic influenza patients (Figure 1a) compared to HC, as previously described [9] . Age, gender, BMI, and laboratory parameters of pandemic influenza patients did not influence their serum SP-D levels (data not shown). Also, differences in serum SP-D levels between severe pandemic influenza patients and HC As aforementioned, SP-D can also be induced during chronic pulmonary diseases.

Hence, we also tested serum SP-D levels in patients with PTB and COPD. Interestingly, the serum SP-D levels of these disease controls were significantly lower than severe pandemic influenza patients (Figure 1a ). Together, these results suggest that SP-D can be used as a specific marker to differentiate pandemic influenza from COVID-19 in severe disease and other chronic infectious or inflammatory lung conditions as well.

Despite these findings, the analysis of SP-D in the plasma of non-mechanically ventilated seasonal influenza and COVID-19 patients showed no differences between groups ( Figure 1d ). In fact, using the cut-off value determined in the serum of the Mexican cohort, the sensitivity and specificity of plasma SP-D to differentiate seasonal influenza from COVID-19 was 4.9% and 78.7% in the whole USA cohort, respectively. Furthermore, when restricted to only hospitalized patients, this protein showed a 8.7% sensivity and 75.68%

A c c e p t e d M a n u s c r i p t 14 specificity. These data indicate that the diagnostic potential of SP-D observed in severe pandemic influenza does not extend to seasonal influenza in patients with mild-to-moderate conditions. However, we could not rule out possible differences in SP-D levels between seasonal influenza and COVID-19 in severe disease.

As reported before [9] , here we observed that serum SP-D levels tended to be higher among patients that died of pandemic influenza compared to survivors (Figure 2a) 

Due to the diagnostic and prognostic potential of SP-D in severe pandemic influenza, we evaluated longitudinal changes in serum SP-D levels during the course of the disease in 56 pandemic influenza patients from which we obtained a second blood sample after seven days of hospitalization. Interestingly, we found that the dynamics of serum SP-D levels A c c e p t e d M a n u s c r i p t 15 differed between patients that survived (n = 49) and died of pandemic influenza (Figure 3a ).

In fact, five out of seven patients who succumbed to the infection showed a longitudinal increase in SP-D levels, whereas most survivors showed stable or decreasing levels during the first week of hospitalization (Figure 3b ). This finding suggests that serum SP-D levels could be used for monitoring treatment response in patients with severe pandemic influenza, although this hypothesis should be validated in larger cohorts.

Pandemic influenza A(H1N1) and COVID-19 are the two most recent global epidemics associated with the emergence of previously unknown zoonotic viruses. The first one appeared in 2009, and ever since, it has acquired a seasonal pattern of transmission in North America. Meanwhile, the outbreak of COVID-19 began in December of 2019 and has continued spreading throughout 2020 and 2021, confining the world population into extended quarantine periods. Due to the lack of control of the current epidemic, it is almost inevitable that COVID-19, seasonal influenza, and pandemic influenza will converge during the current winter in the Northern Hemisphere. This implies that most clinicians in emergency departments will have to distinguish between these infections to provide specific therapeutics for each case. In settings of limited access to RT-PCR tests, solving this diagnostic dilemma will be challenging due to the clinical similitude between influenza and COVID-19. Indeed, only a few characteristics might differentiate both diseases, as demonstrated here and in previous studies [3] [4] [5] [6] .

The clinical manifestations of severe pandemic influenza and COVID-19 are related to dysregulated immune responses that cause secondary lung injury. Furthermore, COVID-19 is also characterized by an antiviral immune dysfunction and a skewed reaction with Th1/Th2 A c c e p t e d M a n u s c r i p t 16 components simultaneously elicited by the virus [5, 14] . These different immune responses triggered by both pathogens are translated into distinct lung morphological changes and captured as unique serum cytokine signatures in influenza and COVID-19 patients [5, 7, 8] .

However, it is not entirely understood whether the influenza A(H1N1) pdm09 virus, seasonal influenza viruses, and SARS-CoV-2 differentially regulate local innate lung defenses. In this context, here we evaluated whether the levels of SP-D, a protein component of the lung surfactant with immune properties [13] , differed between critically ill pandemic influenza and COVID-19 patients.

The molecule SP-D is a type-II pneumocyte-derived collectin that participates in the clearance of pathogens at alveolar spaces due to its opsonizing properties [13] . It also mediates anti-inflammatory activities [13] . Although SP-D production is mainly restricted to the alveolar epithelium, this molecule can be detected in the circulation of patients with different chronic inflammatory disorders of the lung [15, 16] , as well as during pulmonary infections [9, 17] . Accordingly, high serum levels of SP-D were observed in our cohort of pandemic influenza patients, as we also reported before in a prior group of individuals infected with the influenza A(H1N1) pdm09 virus [9] . Changes in the circulating levels of SP-D associated with lung disorders may reflect protein translocation and may serve as a readout of disruption of the alveolar-capillary membrane [15, 16] . As such, we observed slightly higher initial serum SP-D levels and a longitudinal increase of this marker during the first week of hospitalization in pandemic influenza patients that died but not in survivors, suggesting that this marker could predict poor outcome [9] . Perhaps, this observation reflects more severe lung damage in those who succumb to the infection than patients who recover from pandemic influenza. [19] . Third, SP-D induction in the lungs could be a very specific defense mechanism against influenza viruses. Indeed, it is known that this collectin inactivates seasonal influenza A viruses by binding to the viral hemagglutinin [20] . Our data only partially support this hypothesis, as we found induction of SP-D during severe pandemic influenza but not mild-to-moderate seasonal influenza.

Independently of the mechanism, our study demonstrates that serum levels of SP-D could be a biomarker with diagnostic applications to distinguish severe pandemic influenza and COVID-19 in the clinical setting. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 31 Figure 3 

World Health Organization. COVID-19 Weekly Epidemiological Update -19

Seasonal and pandemic influenza: 100 years of progress, still much to learn

Comparison of Hospitalized Patients With ARDS Caused by COVID-19 and H1N1

Systematically comparing COVID-19 with the 2009 influenza pandemic for hospitalized patients

Clinical and immunological factors that distinguish COVID-19 from pandemic influenza A(H1N1)

Comparative review of respiratory diseases caused by coronaviruses and influenza A viruses during epidemic season

Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections

Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm

Serum surfactant protein D (SP-D) is a prognostic marker of poor outcome in patients with A/H1N1 virus infection

High performance of rapid influenza diagnostic test and variable effectiveness of influenza vaccines in Mexico

Impaired Cellular Immune Responses During the First Week of Severe Acute Influenza Infection

Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report

Surfactant protein-D and pulmonary host defense

Longitudinal analyses reveal immunological misfiring in severe COVID-19

Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD

Pulmonary surfactant protein D in sera and bronchoalveolar lavage fluids

Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS

Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies

A systematic review of pathological findings in COVID-19: a pathophysiological timeline and possible mechanisms of disease progression

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t