Correlation of serum interleukin-6 levels and neutrophil-lymphocyte ratio in the severity of COVID-19

Introduction

Coronavirus disease 2019 (COVID-19) is atypical pneumonia caused by the novel coronavirus of the coronaviridae class, i.e. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The World Health Organisation (WHO) declared this disease a pandemic.^1,2 SARS-CoV-2 can fatally lead to acute respiratory distress syndrome (ARDS) or multiple organ distress syndrome (MODS).^1,3

Based on WHO and the Indonesian Ministry of Health data for COVID-19 as of 19 August 2021, the number of confirmed cases globally reached 209,201,939, with a total of 4,390,467 deaths (case fatality rate [CFR] 2.1%).^4,5 In Indonesia, as of 19 August 2021, 3,930,300 confirmed cases, 3,472,915 recovered cases and 122,633 deaths from COVID-19 were reported (CFR 3.1%). South Sulawesi recorded 99,791 confirmed COVID-19 cases, 86,349 recovered cases and 1,807 deaths from COVID-19.⁴

The main characteristic of people with COVID-19 is an increase in the production of pro-inflammatory cytokines.⁶ Excessive increase in cytokines and inflammatory mediators such as interleukin (IL)-1β, IL-2, IL-6, IL-7, IL-8 and IL-10 and chemokines (CXCL 10 and CCL2) can exacerbate COVID-19 symptoms, which is characterised by a picture known as a ‘cytokine storm’.^6,7 In patients with COVID-19, high IL-6 levels were found as an immunological response to SARS-CoV-2.⁸ Similarly, Coperchini et al. (2020) and Liu et al. (2020) demonstrated high serum IL-6 levels in patients with COVID-19 and circulating levels were positively related to disease severity.^6,8 Several studies have found high IL-6 levels in severe conditions or ARDS compared with mild cases; thus, IL-6 could be a biomarker of COVID-19 severity.^9,10

Luo et al. (2020) and Liu et al. (2020) stated that the combination of IL-6 levels >20 pg/mL and CD8+ T lymphocytes <165 cells/μL is a prognostic indicator and can predict COVID-19 death.^11,12 An increase in IL-6 levels of more than 10 times correlated with severe COVID-19, and decreased levels were related to successful therapy.^11,12

Studies have also found that the neutrophil–lymphocyte ratio (NLR) increased in COVID-19. This ratio is a new marker for assessing inflammation, including COVID-19. The NLR is obtained from a comparison of neutrophil and lymphocyte counts from a complete blood count.¹³ A high NLR is caused by an imbalance in the expressions of inflammatory cytokines and the upregulation of genes involved in the apoptotic pathway of lymphocyte cells during SARS-CoV-2 infection.^14,15 A high NLR in patients with severe COVID-19 is characterised by lymphopenia and increased absolute neutrophil count; thus, the NLR may reflect an imbalance in inflammatory and immune responses in patients with COVID-19.¹⁶

Ongoing research on IL-6 and NLR in COVID-19 reveals a correlation between IL-6 and NLR with COVID-19 severity. Several studies have also reported different cut-offs. However, currently, no study in Indonesia has reported cut-offs for IL-6 and NLR. The results of this study are expected to provide information regarding the body’s immune response and inflammation in COVID-19, which is characterised by differences in serum levels of the pro-inflammatory cytokine IL-6 and an imbalance of inflammatory cells with the NLR. The results can be included in COVID-19 management.

The hypothesis is:

Methods

Results

During the study period, 96 patients with COVID-19 met the inclusion criteria. Five patients were excluded because they did not meet the sample criteria, namely, one patient was lipemic, two involved lysate and two had small serum volumes; thus, they could not be examined. In total, the sample included 91 patients, consisting of 57 (62.6%) with mild COVID-19 and 34 (37.4%) with severe COVID-19. As shown in Table 1, 46 (50.5%) patients were male and 45 (49.5%) were female, and the age range 21–40 years had the highest number of patients. Patients were divided into mild and severe COVID-19, and no significant difference in sex was found among these patients (Table 2).

Table 1 shows that in the mild COVID-19 group, the highest number of patients were in the age range of 21–40 years, whereas in the severe COVID-19 group, most of the sufferers were in the age range of ≥41 years. A significant difference was found between the two groups, and this indicates that older age can be a comorbid factor in the development of COVID-19, which becomes more severe.

Based on the NLR cut-off category of 3.13, most of the patients (n = 57 [78.9%]) in the mild COVID-19 group had NLR below the cut-off; in the severe COVID-19 group (n = 34 [94.1%]), the NLR values were above the cut-off (P <0.005). In a 2020 study in China, Yang found that old age and NLR were significantly related to COVID-19 severity.¹⁸

In this study, the IL-6 level and NLR were not normally distributed (Kolmogorov–Smirnov); thus, the differences in IL-6 levels and NLR in the two COVID-19 groups were analysed using the Mann–Whitney test. The median serum IL-6 in the mild COVID-19 group was significantly lower (3.59 pg/mL [min–max of 1.50–638.30]) than that of the severe COVID-19 (28.82 pg/mL [min–max of 5.52–926.30], P <0.005). The median NLR in the mild COVID-19 group was significantly lower (2.18 [min–max of 0.69–15.58]) than that of the severe COVID-19 group (8.13 [min–max of 2.24–30.90, P <0.005]).

In Figure 1 in the mild COVID-19 group, two patients had IL-6 levels higher than the average IL-6: a female patient (aged 68 years) who had an IL-6 level of 638.3 pg/mL and NLR of 2.81, who was diagnosed with mild COVID-19 accompanied by diabetes mellitus and hypertension; a male patient (aged 29 years) who had an IL-6 level of 485.4 pg/mL and NLR of 1.26, who was diagnosed with mild COVID-19 accompanied by fever, cough and nausea.

Figure 1. (A) Differences in Interleukin-6 (IL-6) levels in mild and severe COVID-19; (B) Differences in neutrophil–lymphocyte ratio (NLR) values in mild and severe COVID-19.

In the severe COVID-19 group, three patients had IL-6 levels higher than the average IL-6: a male patient (aged 73 years) who had an IL-6 level of 926.3 pg/mL and NLR of 30.9 and C-reactive protein (CRP) of 340.2 mg/dL, who was diagnosed with severe COVID-19 accompanied by bilateral pneumonia, cardiomegaly and aortic dilatation; a female patient (aged 60 years) who had an IL-6 level of 515.8 pg/mL and NLR of 2.82, who was diagnosed with severe COVID-19 accompanied by diabetes mellitus and hypertension; and a female patient (aged 54 years) who had an IL-6 level of 105.7 pg/mL, NLR of 17.68 and CRP level of 399.88 mg/dL, who was diagnosed with severe COVID-19 accompanied by hematochezia with suspected malignancy. In the severe COVID-19 group, two patients had an NLR higher than the existing average: a male patient (aged 73 years), with NLR of 30.9, who also had very high IL-6 levels, and was diagnosed with severe COVID-19 accompanied by bilateral pneumonia, cardiomegaly and aortic dilatation and a male patient (aged 58 years) who had an NLR of 23.68 and was diagnosed with severe COVID-19 accompanied by pneumonia and aortic atherosclerosis.

Figure 2 shows the IL-6 and NLR cut-off; the ROC curve was used. In the IL-6 ROC curve, the area under the ROC curve (AUC) was 0.89 (95% CI 0.818–0.957; p <0.0001, and the IL-6 cut-off was >6.99 pg/mL). In the NLR ROC curve, the AUC was 0.93 (95% CI 0.881–0.984; p <0.0001), and the cut-off NLR was >4.18.

Figure 2. (A) Interleukin-6 receiving operator characteristic (ROC) curve; (B) Neutrophil-lymphocyte ratio ROC curve.

The relationship between IL-6 and NLR and COVID-19 severity was assessed using the chi-square test. The results are shown in Tables 3 and 4. As shown in Table 3, more patients with mild COVID-19 (n = 43, 75.44%) had IL-6 levels below the cut-off compared with those (n = 14, 24.56%) who had levels above the cut-off. In the severe COVID-19 group, more patients (n = 33, 97.06%) had IL-6 levels above the cut-off. In the chi-square test, a significant relationship was found between IL-6 levels and COVID-19 severity (P = 0.004; P <0.05). Patients with high IL-6 levels have 29 times the risk of suffering from severe COVID-19.

The results of the correlation analysis between NLR and COVID-19 are shown in Table 4. In the mild COVID-19 group, more patients (n = 50, 87.72%) had NLR values below the cut-off than those (n = 7, 12.28%) who had levels above the cut-off. In the severe COVID-19 group, 31 (91.18%) patients had NLR values above the cut-off. In the chi-square test, a significant relationship was found between the NLR and the degree of COVID-19 severity (P = 0.000; P <0.05). Patients with a high NLR had 26 times the risk of suffering from severe COVID-19.

The correlation between the serum IL-6 level and the NLR in COVID-19 was analysed using the Spearman correlation test, and the results are shown in Table 5 and Figure 3. After the Spearman correlation test, a P value of <0.001 was obtained, which indicated a significant correlation between the IL-6 level and the NLR in patients with COVID-19. A correlation coefficient of 0.612 (95% CI 0.459–0.729) was obtained indicating a moderate positive correlation. This suggested that an increase in the NLR is associated with an increase in IL-6 levels. However, this relationship may still be influenced by other internal factors. Our raw data has been published on figshare (https://www.doi.org/10.6084/m9.figshare.22736474).¹⁹

Figure 3. Correlation of Interleukin-6 (IL-6) and neutrophil–lymphocyte ratio (NLR) levels in COVID-19.

Discussion

The COVID-19 guidelines in Australia and New Zealand, released in March 2020, identified lymphopenia and neutrophilia as prognostic markers of severe COVID-19, and the Centers for Disease Control and Prevention in the United States released guidelines emphasising that leukopenia (9%–25%), leukocytosis (24%–30%) and lymphopenia (63%) are the results of routine blood tests in patients who are hospitalised for COVID-19 (https://www.who.int/).²⁰

Haematological parameters in patients with COVID-19 demonstrated significant changes including decreased lymphocyte, haemoglobin and platelet counts, whereas combined parameters such as NLR, monocyte–lymphocyte ratio (MLR) and platelet–lymphocyte ratio (PLR) in patients with COVID-19 significantly increased, indicating that changes in routine blood parameters have clinical significance.^21,22 In patients with severe COVID-19 and poor outcomes, the lymphocyte, eosinophil and platelet counts increased, whereas neutrophil, NLR, IL-6, procalcitonin, D-dimer, ferritin, amyloid A protein and CRP levels increased.^23,24

The NLR was identified as a marker of systemic inflammation and a prognostic factor for mortality in patients with COVID-19. An imbalance in neutrophil and lymphocyte counts can indicate severe inflammation leading to complications such as sepsis, MODS and ARDS.^14,25

A high NLR can be caused by the dysregulation of inflammatory cytokine expression and the upregulation of genes involved in the lymphocyte apoptosis pathway caused by SARS-CoV-2 infection.^26,27 A high NLR in patients with severe COVID-19 is characterised by an increase in neutrophil count and a decrease in lymphocyte count. The NLR may reflect an imbalance in the inflammatory and immune response in patients with COVID-19.^23,27 Zhu et al. (2020) reported that the AUC for IL-6 was 0.84 (95% CI 0.708–0.962) and NLR was 0.69 (95% CI 0.537–0.842), which were slightly lower than our values, but similarly showed better sensitivity in predicting severe COVID-19.

Haematological parameters in patients with COVID-19 change significantly, including a decrease in lymphocyte, haemoglobin and platelet counts. Meanwhile, combined parameters such as the NLR, MLR and PLR in patients with COVID-19 increased significantly.^6,15 This suggests the clinical significance of changes in routine blood parameters. This is reflected in the results of this study, revealing higher NLR in patients with severe COVID-19.

To determine IL-6 and NLR cut-offs, ROC curves are used (Figure 2). Very few studies have investigated IL-6 and NLR cut-offs as predictors of COVID-19 severity. Several existing studies have found different IL-6 cut-offs. For example, Harold et al. (2020) found that at an IL-6 cut-off of >80 pg/mL, the patient may experience respiratory failure and require a ventilator. Liu et al. (2020) reported that an IL-6 cut-off of >34.1 pg/mL indicated severe complications.^7,9 Meanwhile, Liu et al. (2020) obtained an NLR cut-off of >4.87 (95% CI 0.659–0.850) with a sensitivity of 56.52% and a specificity of 86.89%, which indicated an 8.5 times probability of experiencing critical/severe COVID-19 compared with a lower NLR.²⁸ The combination of NLR and CRP was recommended as a potential predictor of severe COVID-19. AP et al. (2020) obtained an NLR cut-off of >3.3, with a sensitivity of 88% and a specificity of 64%, and Liu et al (2020) reported that an NLR of >3.13 and age >50 years indicated severe COVID-19 that required intensive care unit (ICU) admission.^13,29

As shown in Table 3, the chi-square test revealed a significant relationship between IL-6 levels and COVID-19 severity (P = 0.004; P <0.05). Patients with high IL-6 levels have 29 times the risk of suffering from severe COVID-19.³⁰ Zhu et al. (2020) analysed several inflammatory markers in mild and severe COVID-19 and assessed the association of these markers with COVID-19 severity. They found that high IL-6 levels, CRP and hypertension were independent risk factors of COVID-19 severity. IL-6 levels are significantly higher in severe than in mild COVID-19 at the first onset and 5–10 days afterward.¹⁰ Liu et al. (2020) found that IL-6 was greatly increased in severe COVID-19 and was significantly associated with fever, increased CRP, LDH and ferritin. IL-6 was positively related to COVID-19 severity. Thus, IL-6 could be a marker for severe COVID-19.¹²

Fu et al. (2020) compared haematological and inflammatory markers in mild and severe COVID-19 and found that leukocytes (white blood cells), NLR, D-dimer and fibrinogen values were significantly higher in severe COVID-19 than in mild COVID-19. The highest NLR AUC value was obtained among the four parameters, with 0.88, and the value was slightly lower than our results; however, their value similarly showed a strong association between NLR and COVID-19 severity.³¹

Upon the entry of SARS-CoV-2 into the body, the immune system is activated, which causes an increase in several immune cells and the release of pro-inflammatory cytokines, resulting in an inflammatory response in the area of infection. Subsequently, the inflammation spreads throughout the body. Pro-inflammatory cytokines such as IL-6 are produced, and their levels are indirectly affected by the imbalance of neutrophils and lymphocytes. In severe COVID-19, IL-6 production and NLR values are also higher. Zhu et al. (2020) found a positive relationship between IL-6 and NLR (r = 0.428, P <0.001).³⁰

In patients with severe COVID-19 and poor outcomes, the lymphocyte, eosinophil and platelet counts decreased, whereas neutrophil, NLR, IL-6, procalcitonin, D-dimer, ferritin, amyloid A protein and CRP levels increased. The NLR was identified as a marker of systemic inflammation and a prognostic factor for mortality in patients with COVID-19. AP et al. (2020) and Liu et al. (2020) analysed patients with mild and severe COVID-19 in China and found that high NLR and age were closely related to disease severity and were markers of poor prognosis. Thus, the NLR can be an independent prognostic biomarker in the management of COVID-19. Moreover, Liu et al. (2020) set an NLR cut-off of 3.13 and an age of 50 years as risk factors of severe COVID-19 requiring ICU admission.

Conclusions

Serum IL-6 levels and NLR are higher in severe COVID-19 than in mild COVID-19 cases. Serum IL-6 levels and NLR are positively correlated with COVID-19 severity. If an IL-6 level is more than 6.99 pg/mL, then the patient has a 29 times risk of suffering from COVID-19 and if the NLR level is more than 4.18 pg/mL, then the patient has a 26 times risk. Serum IL-6 levels correlate with NLR in COVID-19.

This study was conducted using a cross-sectional design by collecting data when COVID-19 sufferers were hospitalised, no further observations were made on the course of the patient’s illness and the results of IL-6 and NLR examinations were not monitored. Thus, further research is needed to measure serum IL-6 levels and NLR in patients with COVID-19 using an IL-6 cut-off of >6.99 pg/mL and NLR of >4.18, which can be included in the laboratory parameters for the management of patients with COVID-19.

Consent

Written informed consent for publication of their clinical details was obtained from the patients.