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Blood myeloperoxidase‐DNA, a biomarker of early response to SARS‐CoV‐2 infection?

Convergent arguments suggest that innate immunity related to neutrophils, and in particular neutrophil extracellular traps (NETs), could play a key role in response to SARS-CoV-2 infection.


Blood myeloperoxidase-DNA, a biomarker of early response to SARS-CoV-2 infection?
To the Editor, Convergent arguments suggest that innate immunity related to neutrophils, and in particular neutrophil extracellular traps (NETs), could play a key role in response to SARS-CoV-2 infection. 1 The neutrophil-to-lymphocyte ratio is a predictor of severity. 2 Neutrophils are involved in the innate response to pulmonary viral infections. 3 The production of NETs (NETosis) is increased by inflammatory mediators and activates macrophages in pulmonary alveoli. 3,4 Despite these arguments, no direct evidence of the spread of NETs into circulation has been produced in the early symptomatic phase of COVID-19 and other viral infections.
We assayed myeloperoxidase-DNA (MPO-DNA) and histone-DNA complexes, two serum markers of circulating NETs, and blood cell counts in 60 consecutive ambulatory subjects attending a screening center for COVID-19 RT-PCR examination of nasopharyngeal swab samples. 5 The study was approved by the Institutional Ethical Committee. All subjects were symptomatic with at least two very recent symptoms among fever, dry cough, and dyspnea since <1 week and most had recent contact with infected cases. They were compared to matched asymptomatic controls recruited several months before the epidemy. The sera were used after completion of biochemical testing ordered by the clinician. The remaining samples were stored in the same conditions among groups, at 20°C in the 24 hours following blood withdrawal. Histone-DNA and MPO-DNA complexes were measured in serum by the Cell Death Detection ELISA Kit (Roche Diagnostics, Sigma Aldrich), using a biotinylated antibody against histones H1, H2A, H2B, H3, and H4 or MPO and a soluble peroxidase-labeled anti-DNA monoclonal antibody, respectively, as described previously. 6 Values were reported in 450 nm absorbance units (AU). Blood cell counts were determined in all subjects. Routine biochemical markers, including hemoglobin, bicarbonate, potassium, sodium, chloride, serum total proteins, C-reactive protein, urea nitrogen, creatinine, bilirubin, conjugated bilirubin, aspartate aminotransferase, alanine aminotransferase, lactico-dehydrogenase, gamma-glutamyl transpeptidase, alkaline phosphatase, and creatine kinase, were assayed in a Cobas 8000 Analyzer (Roche Diagnostics).
We observed a higher level of MPO-DNA complexes in symptomatic subjects with positive vs negative RT-PCR (1.31 ± 0.18 vs 0.89 ± 0.21 AU, P < .001, Figure 1). The levels of MPO-DNA complexes were 3.1-fold and 2.1-fold higher in the two groups, compared to asymptomatic control subjects recruited before the epidemy (0.42 ± 0.08 AU), respectively (P < .001). Eleven symptomatic PCR-negative subjects had MPO-DNA levels higher than the 0.95 AU upper limit reported in negative symptomatic subjects. We also observed a clear increase of histone-DNA complex level in symptomatic subjects compared to controls (0.93 ± 0.25 vs 0.05 ± 0.01, P < .001) but no difference between positive and negative cases (0.94 ± 0.29 vs 0.92 ± 0.20, P = .9). We reported significant associations of MPO-DNA and histone-DNA complexes with markers of blood cell counts predicting severity, including neutrophil, lymphocyte, and platelet counts, and neutrophil-to-lymphocyte ratio ( Figure 2) and no correlation with RT-PCR. 2 MPO-DNA was negatively correlated with lymphocytes and platelets, while histone-DNA was positively correlated with neutrophils and neutrophil-to-lymphocyte ratio. The subjects with positive vs negative RT-PCR had decreased blood counts of leucocytes, platelets, neutrophils, and lymphocytes and increased sodium and bicarbonate. We reported no difference in other routine biochemical markers between the two symptomatic groups (Table S1) and no correlation of these biochemical parameters with blood levels of MPO-DNA and histone-DNA complexes.

Cytokine storm
The increase of MPO-DNA and histone-DNA complexes in blood shows that the release of NETs is involved in the early host answer to COVID-19. The release could result from neutrophil exposure to virus particles, cytokine production, and/or response to host damage-associated molecular patterns (DAMPs) produced by infected cells, as reported in other pulmonary viral infections. 3,4 Our data also show that MPO-DNA blood level is a sensitive marker of the early phase of COVID-19. All positive patients had a level dramatically higher than that observed in controls. Our data suggest to further evaluate MPO-DNA as a early marker, which could help to discriminate symptomatic patients with false-negative RT-PCR from those with negative RT-PCR related to another viral lung infection, in addition to computed tomography. 7 We estimated the number of

CO N FLI C T S O F I NTE R E S T
All authors have no conflict of interest to declare. host immune response will we be able to rationally manage future outbreaks.

FU N D I N G I N FO R M ATI O N
The immune response to SARS-CoV-2 is currently under study and needs to be better characterized. However, it has been previously reported that viral infection involves activation of CD8 + cytotoxic cells, antibody-producing B cells, and innate immune response that in some patients triggers a so-called "cytokine storm". 3 Moreover, whether immune responses to SARS-CoV-2 generate long-term memory or whether immunized patients have long-term sterilizing immunity is still unknown.
Spain has been devastated by the COVID-19 pandemic with more than 280 000 confirmed cases, from which more than 67 000 were in Madrid, causing a huge personal, health system, and economic burden. 4 In fact, more than 20% of infected subjects were healthcare workers. 4 We aimed to generate an immune response map to SARS- Healthcare workers were recruited and classified in two subgroups depending on whether they were diagnosed or not for COVID-19 by RT-PCR (Appendix S1).