key: cord-0430181-02kky1fe
authors: Pereson, Matias J.; Badano, Maria Noel; Aloisi, Natalia; Chuit, Roberto; de Bracco, MME; Bare, Patricia
title: TNF-α levels in respiratory samples are associated with SARS-CoV-2 infection
date: 2021-07-13
journal: bioRxiv
DOI: 10.1101/2021.07.12.452071
sha: 827376355334189383e01561e7f4539f4d718b0d
doc_id: 430181
cord_uid: 02kky1fe

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.

The coronavirus disease 2019 caused by the severe acute respiratory 58 syndrome coronavirus 2 (SARS-CoV-2) is one of the current major health concerns. After 15 59 months since the emergence of the pandemic SARS-CoV-2, up to 178 million cases were 60 5 Concentrations of IL-6 (standard curve range: 0-300 pg/mL) and TNF-α (standard curve 109 range: 0 -500 pg/mL) in swabs were determined using commercial reagents based on enzyme 110 linked immunosorbent assay (ELISA) (BD-Biosciences, San Diego, California, United States). 111

Recommendations of the supplier were followed for procedure. Duplicates were performed in 112 selected samples to verify the accuracy of the results. 113 114

Mann-Whitney U test was used for testing differences between two groups. Categorical 116 variables were compared with Chi-square test or Fisher's exact test. Confidence intervals were 117 set at 95% (CI95). In all cases, a p value <0.05 was considered significant. Data and graphs 118 were performed using the GraphPad Prism 9. 

Combined oro-and nasopharyngeal swabs from 127 patients submitted for diagnosis of 136 COVID-19 were examined for SARS-CoV-2 genome presence and IL-6 and TNF-α 137 concentration as described above. Since samples were drawn for SARS-CoV-2 infection 138 diagnostic purposes, an important aspect of this study consisted in the timing of cytokine 139 concentration assessment, performed during the first 10 days of symptoms in most of these 140 patients. The most prevalent symptoms included malaise, myalgia, headache and fever. 141

Characteristics of the study population are shown in Table 1 . 142 143

Normal ranges of IL-6 and TNF-α for swab samples were set up using a group of 145 asymptomatic COVID-19 (-) individuals. In this group, IL-6 median value was 3.18 pg/ml (0.78-146 8.39) and TNF-α median value was 4.72 pg/ml (3.19-9.69). 147 IL-6 was increased in both groups of symptomatic patients compared to asymptomatic 148 patients (Fig. 1a) . However, the difference between COVID-19 (+) and (-) symptomatic 149 patients, with median values of 5.40 (1.7-467) pg/ml and 6.07 (1.57-466.6) pg/ml respectively, 150 did not reach statistical significance (Fig. 1a) . Despite both groups of symptomatic patients 151 showed higher TNF-α levels compared to the asymptomatic group, no significant differences 152 were observed (Fig. 1b) . However, TNF-α levels were greater in COVID (+) symptomatic Among the group of COVID-19 (+) individuals, correlation between IL-6 or TNF-α levels and 164 viral load (Ct) was not found. Furthermore, older age and gender did not seem to be related 165

with higher values of IL-6 or TNF-α among the COVID-19 (+) group. In this study, we found a statistically significant association between the production of local 212 TNF-α and the presence of the virus in early stages of infection. Therefore, measurement of 213 local TNF-α, and probably some others mediators, could contribute to identify those individuals 214 that may benefit from immunomodulatory and cytokine inhibiting therapies. 215

An increase of IL-6 and TNF-α and other cytokines in respiratory samples has been already 216 described in numerous respiratory viral infections and has been associated with a worse 217 prognosis of the disease [22, 25] . Our findings highlight the wide and pleiotropic role of IL-6 in 218 local inflammation process and show that TNF-α estimation and its association to viral 219 presence might be a valuable biomarker that could contribute to disease evolution prognosis. 220

Through our results, we could hypothesize that a proportion of individuals might take 

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