TNF-α Levels in Respiratory Samples Are Associated with SARS-CoV-2 Infection

Purpose Increased serum levels of IL-6 and TNF-α have been proposed as biomarkers for COVID-19 progression. However, the role and the implication of these cytokines in SARS-CoV-2 infection remain controversial. The aim of this study was to measure levels of IL-6 and TNF-α in swab samples from individuals with symptoms compatible with COVID-19 and analyze their association with SARS-CoV-2 presence. Methods SARS-CoV-2 detection was performed using the CDC (USA) real-time RT-PCR primers, probes and protocols. Cytokine concentrations were measured using commercial reagents based on enzyme linked immunosorbent assay (ELISA). Results TNF-α median levels were greater in COVID19 (+) symptomatic group (5.88 (1.36 - 172.1) pg/ml) compared to COVID19 (−) symptomatic individuals (2.87 (1.45 – 69.9) pg/ml) (p=0.0003). No significant differences were shown in IL-6 median values between COVID-19 (+) and (−) symptomatic patients (5.40 (1.7 - 467) pg/ml and 6.07 (1.57 – 466.6) pg/ml respectively). In addition, increased TNF-α levels (greater than 10 pg/ml), but not IL-6, were associated with SARS-CoV-2 presence (OR= 5.7; p=0.006; 95% CI= 1,551 to 19,11). Conclusions IL-6 concentration showed high levels in swabs from some symptomatic patients, suggesting the presence of immune response at viral entry site. However, IL-6 levels were independent from SARS-CoV-2 presence and viral load, individual’s age and gender. On the contrary, TNF-α evaluation confirmed the presence of inflammatory response but mostly related to COVID-19. More studies are required in order to characterize the cytokine profile expressed at the site of infection of SARS-CoV-2 and its implications in disease outcomes.

T he coronavirus disease 2019 (COVID-19) is one of the current major health concerns. High systemic cytokine levels were correlated with increased morbidity and mortality in COVID-19 patients (1). However, lower plasma cytokine levels were reported in severe COVID-19 compared with other inflammatory conditions (2). Consequently, cytokine contribution in disease progression remains contradictory.
The aim of this study was to evaluate concentrations of IL-6 and TNF-a in swab samples from individuals with symptoms compatible with COVID-19 and analyze their association with SARS-CoV-2 genome presence.
The study was conducted on 127 combined nasopharyngeal and oropharyngeal swabs, referred to the laboratory for detection of SARS-CoV-2. Cytokine levels of symptomatic individuals with detectable (n = 52) or undetectable (n = 33) SARS-CoV-2 were measured. A group consisting of asymptomatic individuals and negative for SARS-CoV-2 genome (n = 42) were included to obtain cytokines' normal range.
Genomic extraction and real time-based methodology were performed for viral genome detection using the CDC RT-qPCR protocol, 2019-nCov CDC USA. Viral load was calculated considering Ct values.
Concentrations of TNF-a (standard curve range: 0 to 500 pg/mL) and IL-6 (standard curve range: 0 to 300 pg/mL) in swabs were determined using commercial reagents based on enzyme linked immunosorbent assay (BD-Biosciences). Considering the minimum detectable dose of each cytokine and the cross-reactivity with other cytokines in our system, the limit of detection for TNF-a and IL-6 was determined to be 5 pg/mL. All values between 0 and 5, were considered as 5 pg/mL for statistical analysis purposes.
Mann-Whitney U test and chi-square test were used for data analysis. In all cases, a P value ,0.05 was considered significant.
Characteristics of the study population are shown in Fig. 1A. Normal ranges of TNFa and IL-6 in swab samples are shown in Fig. 1B.
Cytokine levels have been studied in patients with COVID-19 to analyze their value as biomarkers but also, because cytokine inhibitors have been suggested for the treatment of acute respiratory distress syndrome patients based on the concept of "cytokine storm" in COVID-19 (3). Reports from patients on anti-TNF therapy showed a reduced rate of COVID-19 morbidity and death (4,5). Our findings, evaluating the concentration of TNF and IL-6 at the entry site of SARS-CoV-2, highlight the pleiotropic role of IL-6 in local inflammation process and show that TNF-a could contribute to disease evolution prognosis as a biomarker. Measurement of TNF-a in nasopharyngeal swabs at diagnosis could identify those individuals that may benefit from immunomodulatory and cytokine inhibiting therapies.  Mean 6 SEM for TNF-a (i) and IL-6 (ii) concentrations are expressed in log (pg/mL). Cytokines were measured in asymptomatic individuals negative for SARS-CoV-2 genome to set up normal ranges. All values below the limit of detection were considered as 5 pg/mL for statistical analysis purposes. Some aspects of this investigation could not have been fulfilled without the generous contribution of the Fundación René Baron, Buenos Aires, who provided financial support. P.B. conceived the study; M.N.B. and M.J.P. designed the study protocol and carried out the clinical assessment; P.B. made the analysis and interpretation of the database. P.B., M.N.B., and M.J.P. drafted the manuscript; M.M.E.B. and R.C. critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.
Experimental protocols and procedures have been approved by the Biosafety Review board and Ethical Committee of the Academia Nacional de Medicina.