INTRODUCTION:

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has various degrees of severity ranging from asymptomatic to severe. COVID-19 causes an innate and adaptive immune response. This massive increase in cytokine storm attacks the lymphoid organs. In inflammatory conditions, neutrophils release large amounts of Reactive Oxygen Species (ROS), which induce damage to the cell's DNA and cause free viruses to leave the cell.

Neutrophils are stimulated by inflammatory factors associated with viruses, such as IL-6, IL-8, TNFα, granulocyte colony-stimulating factor, and interferon-gamma factors, which are produced by lymphocytes and endothelial cells, causing a state of neutrophilia in day^1,2,3,4. In addition, the human immune response caused by viruses mainly depends on lymphocytes. Under conditions of systemic inflammation, it significantly suppresses cellular immunity, thereby reducing CD4+ T lymphocyte levels and increasing CD8+ suppressor T lymphocyte levels. Inflammation triggered by a virus will increase NLR. An increase in NLR will trigger the progress of COVID-19.⁵ Neutrophil-to-lymphocyte ratio is one of the inflammatory markers obtained from the calculation of the absolute neutrophil value divided by the absolute lymphocytes. A study by Liu et al. argues that COVID-19 has a good prognosis if the NLR is < 3.13, and the prognosis will be worse if the NLR is ≥ 3.13.⁶Patients with severe COVID-19 have higher NLR than those with mild clinical features and can be utilized to predict the severity of COVID-19.⁷

Cytokine storms reduce platelet synthesis by destroying bone marrow progenitor cells. Inflammation triggers the formation of autoantibodies and immune complexes, which can lead to platelet destruction. Platelet inflammation activated during lung injury activates thrombus formation, so this mechanism will decrease the number of platelets.⁸ COVID-19 patients have thrombocytopenia 36.2%. Virus-induced inflammation will increase PLR, and an increase in PLR triggers the progress of COVID-19. Platelet-to-Lymphocyte Ratio is a new marker of inflammation, which is inexpensive and available in the clinical setting. PLR is obtained from the calculation of the absolute platelet values divided by the absolute lymphocytes, PLR has been utilized in cardiovascular diseases and autoimmune diseases, as a prognostic for inflammation and death.^9,10 Due to the rapid involvement of the inflammatory process in COVID-19, patients with severe COVID-19 have shown elevated PLR on admission^6,7,11,12. Studies show statistically significant higher PLR upon admission in severe COVID-19 patients compared to non-severe COVID-19 patients.¹³ An increase in the PLR indicates an overactive inflammatory response and a worse prognosis.¹⁴ This study aims to assess the two inflammatory markers, NLR and PLR, as predictors in determining the severity of COVID-19. These studies suggest that the parameters of inflammation can be reflected in NLR and PLR. This study aims to determine whether NLR and PLR can help determine the severity of COVID-19. This study aims to determine the correlation between the NLR and the severity of COVID-19 and the relationship between the PLR and the severity of COVID-19.

METHODS:

The subjects of this study included COVID-19 patients who were treated at Dr. Hasan Sadikin Central Public Hospital, Bandung, from May 2020 to December 2020. This cross-sectional study applied a correlation and comparative analytic design. Secondary data were collected from medical record data and Laboratory Information System using a retrospective data collection method. Data collected for this study were those of patients with confirmed COVID-19 through nasopharyngeal and oropharyngeal swab tests using a Polymerase Chain Reaction tool and treated in the COVID-19 isolation ward at Dr. Hasan Sadikin Central Public Hospital, Bandung. Researchers obtained a total of 274 medical record data of COVID-19 patients that met the inclusion and exclusion criteria. The inclusion criteria for the subjects of this study included (1) COVID-19 patients aged > 18 years with positive SARS-CoV-2 RT-PCR swab results and (2) those with hematology laboratory results data during the first treatment. The exclusion criteria for the subjects of this study included (1) COVID-19 patients with hematological disorders such as leukemia. Patient characteristics taken included gender and age.

The data accessed in this study included data on patient characteristics based on sex, age, degree of disease severity, and presence or absence of comorbidities. Sex and age categories followed the hospital database. The severity of COVID-19, based on the criteria of WHO and the Ministry of Health of the Republic of Indonesia, is divided into two categories, namely: (1) Severe COVID-19 characterized by a SpO2 of <90%, a respiratory rate of > 30x, and signs of respiratory distress. Severe COVID-19 includes Critical ill COVID-19 characterized by ARDS, sepsis, and septic shock; (2) Moderate/Non-Severe COVID-19, those who do not meet the criteria for Severe COVID-19 and Critical ill COVID-19.¹⁵ In this study, the cut-off value for PLR was 180, based on a study by Ai-ping et al.⁹ The severity of the disease was expressed in two categories, non-severe and severe, referring to hospital medical record data. The severity of COVID-19 was diagnosed early in the patients' treatment period.

The data were statistically processed and then analyzed and calculated using SPSS version 25.0. The data are presented as means±standard deviation for continuous variables, namely NLR and PLR, and as proportions for categorical variables, namely moderate severity and critical severity.

The correlation between NLR and PLR values with the severity of COVID-19 was analyzed using Spearman's rank correlation coefficient. The criterion is significant if the p-value is <0.05 and the coefficient r ranges from 0-1, the closer to 1, the stronger the correlation (0-0.2: very weak correlation, 0.2-0.4: weak correlation, 0.4-0.6: moderate correlation, 0.6-0.8: strong correlation, 0.8-1.00: very strong correlation).

RESULT AND DISCUSSION:

Based on the characteristics of age, sex, comorbidities, and severity, the data of the 274 patients in this study were not normally distributed according to the normality test, and the data were analyzed using Spearman's rank correlation coefficient.

Table 1: Subject characteristics data

Characteristics	COVID-19 Severity Rates		P-value
Characteristics	Moderate n = 177	Severe n = 97	P-value
Age (years)
Median	48 (33–59)	57 (46–63)	< 0.001^a*
Sex n (%)
Male	94 (53.1)	56 (57.7)	0.462^b
Female	83 (46.9)	41 (42.3)	0.462^b
Having comorbid, n (%)	15 (8.45)	27 (27.3)	< 0.001^b*

Description: n = amount, % = percentage

The p-values were generated from the ^aMann-Whitney and ^bChi Square tests

Based on age, most of the subjects with confirmed COVID-19 belonged to the fifth-decade age group with severe COVID-19 compared to those belonging to the fourth-decade age group with moderate COVID-19. Based on sex, the number of male subjects was higher than that of female subjects, namely 54.7% and 45.3%, respectively.

Based on the characteristics of those who had comorbidities, 27 patients (27.3%) had severe COVID-19, more than those who had moderate COVID-19, amounting to 15 patients (8.45%).

Table 2 shows the correlation between the two inflammatory markers, NLR and PLR, with the severity of COVID-19. NLR correlates with the disease severity (p < 0.001*) as PLR does (p < 0.001*).

Table 2: Differences in the PLR and NLR of COVID-19 patients treated at Bandung Dr. Hasan Sadikin Central Public Hospital

Inflammatory Markers	Total n = 274	COVID-19 Severity Rates		p-values
Inflammatory Markers	Total n = 274	Moderate n = 177	Severe n = 97	p-values
NLR
Median	4.17 (2.18–8.44)	3.44 (1.82–6.58)	7.17 (3.34–12.81)	< 0.001*
PLR
Median	207.4 (137.6–313.2)	182.4 (131.6–281.2)	254.4 (167.5–377.8)	< 0.001*

Note: the p-values were generated from Kruskal-Wallis’s test, and the r coefficient was generated from Spearman's rank correlation coefficient, *significant

Table 3.2 Correlation between the PLR and NLR of COVID-19 patients treated at Bandung Dr. Hasan Sadikin Central Public Hospital

Inflammatory Markers	Total n = 274	COVID-19 Severity Rates		p-values	r coefficient
Inflammatory Markers	Total n = 274	Moderate n = 177	Severe n = 97
PLR
≤180	112 (40.9)	86 (48.6)	26 (26.8)	< 0.001*	0.198
> 180	162 (59.1)	91 (51.4)	71 (73.2)
NLR
≤ 3.13	100 (36.5)	78 (44.1)	22 (22.7)	< 0.001*	0.308
> 3.13	174 (63.5)	99 (55.9)	75 (77.3)

Note: p-values were generated from the Chi-Square test, *significant

From the results of the above analysis, PLR>180 and NLR >3.13 are more common in severe COVID patients than in moderate COVID-19 patients and are statistically significant (p<0.001). The r coefficient of NLR reached 0.308, which means it has a weak correlation, while the r coefficient of PLR reached 0.198, indicating a very weak correlation.

The results showed that moderate and severe COVID-19 patients were dominated by male patients. It is in line with the results of a study by Khumar et al.showing that the number of male patients was higher than that of female patients.³

The age distribution data depicts the domination of the fifth-decade age group. It is in accordance with a study by Liu et al.showing that the average age distribution of the patients was >50 years. It is because, in old age, a person's immunity decreases, and comorbid diseases such as hypertension and diabetes mellitus are most often found in individuals aged > 50 years and above.³

The number of COVID-19 patients with comorbidities is higher than those who have moderate COVID-19. This is in accordance with a study by Liu et al.in which most of the comorbidities are diabetes mellitus and hypertension, which can further exacerbate the severity of COVID-19 disease.³ Higher susceptibility to patients who have comorbidities. Diabetes mellitus is one of the strongest risk factors for the severity of SARS-CoV-2 infection. In a study by Zheng et al. intensive care patients with COVID-19 in China, 22% of 32 patients who died had diabetes as a comorbidity. The risk of admission to intensive care, as a result of disease severity, doubles in patients with diabetes. A study by Lim et al. states that ROS and viral activation of the Renin-Angiotensin-Aldosterone System through increased expression of angiotensin II cause damage to pancreatic beta cells, resulting in insulin resistance. This mechanism causes disruption of glucose regulation, may increase in blood glucose levels. Hyperglycemia results in a weakened innate immune response, vascular dysfunction, and a prothrombotic state in COVID-19 patients and worsens the clinical situation.¹²

A study by Ai-Ping Yang et al. indicated that the higher the NLR and PLR, the more severe the COVID-19.¹³ It is consistent with this study where the median NLR in severe COVID-19 reached 7.17, higher than that in moderate COVID-19, reaching 3.44. There is a significant correlation between NLR and the severity of COVID-19, even though it is a weak correlation (r = 0.308). Simple hematology laboratory tests such as NLR measurements are used to assess the degree of inflammation. An increase in NLR can reflect an increased inflammatory process and can be associated with a poor prognosis.⁸ In inflammatory conditions, neutrophils are the main component of leukocytes that actively migrate toward the immune system or organs. Neutrophils secrete large amounts of ROS, which induce damage to the cell's DNA and allow viruses to freely leave the cell. Antibody-Dependent Cell-Mediated Cells can kill viruses directly and trigger humoral immunity. Neutrophils are stimulated by virus-associated inflammatory factors, such as IL-6, IL-8, TNFα, granulocyte colony-stimulating factors, and interferon-gamma factors. The human immune response caused by viruses mainly depends on lymphocytes. Under conditions of systemic inflammation, it significantly suppresses cellular immunity, thereby reducing CD4+ T lymphocyte levels and increasing CD8+ suppressor T lymphocyte levels. Inflammation triggered by a virus increases NLR, and an increase in NLR triggers the progression of COVID-19.12 Liu et al. states that COVID-19 has a good prognosis if the NLR is < 3.13 and the prognosis will be worse if the NLR in ≥ 3.13.¹²

This study also showed that the median PLR for severe COVID-19 reached 254.4, higher than that for moderate COVID-19, reaching 182.4. There is a statistically significant correlation between PLR and the severity of COVID-19, although it is a very weak correlation (r = 0.198). Cytokine storms reduce platelet synthesis by destroying bone marrow progenitor cells. Inflammation triggers the formation of autoantibodies and immune complexes, which can lead to platelet destruction. Platelet inflammation that is activated during lung injury activates thrombus formation decreases the number of platelets.^11,13,15,16 Inflammation triggered by a virus increases PLR, an increase in PLR triggers the progress of COVID-19.^9,10 Due to the rapid involvement of the inflammatory process in COVID-19, patients with severe COVID-19 have shown elevated PLR on admission.⁴

The higher the PLR and NLR, the more severe the COVID-19 (r = 0.198 vs. r = 0.308, p < 0.001). It can be seen that the r coefficient for NLR is greater than that of PLR, meaning that NLR has better accuracy for determining the severity of COVID-19. This study found a weak correlation because neutrophilia occurred on the third-seventh day of treatment, while the assessment was performed at first day of admission with clinical symptoms on day 1-3.There are limitations in this study, namely the non-uniform admission days of COVID-19 patients admitted to Dr. Hasan Sadikin Central Public Hospital and data on the history of drug consumption (corticosteroids).

CONCLUSION:

It can be concluded from this study that there is a weak and significant positive correlation between NLR and PLR with the severity of COVID-19. Even though both NLR and PLR have a statistically significant correlation with the severity of the disease, these inflammatory markers cannot be utilized independently to determine the severity of COVID-19. They can be combined with other inflammatory markers that have a greater correlation.

ACKNOWLEDGEMENTS:

We thank Jalan Tengah (http://jalantengah.site), Indonesia for editing the manuscript.

CONFLICT OF INTEREST:

The authors declare that there are no conflict of interest.

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Received on 30.01.2023 Modified on 23.03.2023

Research J. Pharm. and Tech 2024; 17(1):323-326.

DOI: 10.52711/0974-360X.2024.00050