Association of Baseline Levels of C-Reactive Protein and Neutrophil to Lymphocyte Ratio in Assessing Severity and Mortality among COVID-19 Patients in Three Waves: A Cross-sectional Study

1. Associate Professor, Department of Biochemistry, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India. 2. Junior Resident, Department of Pulmonology, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India. 3. Professor, Department of Biochemistry, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India. 4. Associate Professor, Department of Biochemistry, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India. 5. Assistant Professor, Department of Biochemistry, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India. 6. Assistant Professor, Department of Biochemistry, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India.

Abstract

Introduction: The Coronavirus Disease 2019 (COVID-19) infection has experienced three peaks in India, with possibilities of reinfections and future peaks. A comprehensive understanding of the disease, particularly in terms of inflammatory markers, is crucial. Neutrophil-to-Lymphocyte Ratio (NLR) and C-Reactive Protein (CRP) have been established as markers of disease severity in COVID-19 during the first wave, but limited data exists regarding these markers in the second and third waves.

Aim: This study aims to investigate the association between baseline levels of CRP and NLR with disease severity and mortality among COVID-19 patients in three waves.

Materials and Methods: A cross-sectional study was conducted at a tertiary care hospital in Bangalore from March 2020 to March 2022. The study included clinical data from 1485 patients with COVID-19. CRP and NLR levels were measured on the day of hospital visit, and their association with severity and mortality was assessed. Continuous variables were compared using independent t-test, one-way analysis of variance (ANOVA), or Kruskal-Wallis test as appropriate. Categorical variables were compared using the Chi-square test.

Results: Wave 1 demonstrated a significant increase in CRP and NLR values among patients with critical illness (mean±SD: 11.96±11.9, 12.41±13.7) compared to other categories. The values of these two parameters in other categories of wave 1 were as follows: asymptomatic (2.28±6.0, 3.17±2.0), mild (1.67±3.4, 2.41±2.7), moderate (1.07±1.7, 3.71±2.4), and severe (6.47±7.2, 4.39±4.3). In wave 2, critical illness (10.52±8.9 and 10.73±7.8) showed elevated values compared to other categories (0.957±1.1, 2.61±1.7), (4.90±5.9, 4.27±4.1), (4.84±4.8, 4.03±2.4), (7.35±6.7, 7.10±6.7). In wave 3, the two parameters in critical cases (15.0±17.3 and 16.47±7.6) were elevated compared to other categories (3.67±2.9, 16.44±10.3), (3.3 and 13.9), (3.7±5.9, 19.10±1.6).

Conclusion: CRP and NLR were found to be useful early markers for assessing disease severity and mortality in COVID-19 patients across all three waves.

Acute-phase proteins, Complete blood counts, Inflammatory markers, Pandemic, Pneumonia

The COVID-19 infection is a pandemic disease characterised by diffuse lung inflammation, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus (1). Genetic recombination and mutations of SARS-CoV-2 have led to different variants that have varying pathogenesis and transmissibility (2). In an effort to reduce morbidity and mortality, age, gender, CRP levels, as well as NLR can be used as economical and practical clinical indicators to predict COVID-19 severity (2).

Feng X et al., found that inflammatory parameters such as white blood cells, lymphocytes, procalcitonin, CRP, and NLR can indicate the progression of COVID-19 (3). NLR takes into account the levels of both neutrophils and lymphocytes for a comprehensive, accurate, and reliable examination. Monitoring CRP and NLR may help physicians recognise high-risk patients. It is beneficial for forecasting disease intensity to reduce clinical outcomes in pneumonia cases (3).

CRP is commonly used as a prognostic marker that indicates ongoing inflammation; it appears in the blood 6-10 hours after tissue inflammation begins and starts decreasing 18-20 hours after the inflammation reduces [4-6]. Furthermore, a complete blood count test, which includes the neutrophil count and lymphocyte count ratio, is an economical predictor of systemic inflammation.

Studies have been conducted to investigate the association between CRP and NLR ratio during the first wave period [7-10], but there are only a few studies (11),(12) available regarding this association during the second or third wave periods. Ziuzia-Januszewska L et al., found that high NLR and CRP levels were predictors of poor outcomes of SARS-CoV-2 infection among the Polish population during the second and third waves of the pandemic (12).

In an effort to identify severe patients irrespective of the variant of SARS-CoV-2 in an early stage to reduce morbidity and mortality, parameters such as age, gender, CRP levels, as well as NLR can be used as economical and practical clinical indicators to predict COVID-19 severity in three different waves of the disease.

Hence, the present study was conducted to evaluate the association of baseline levels of CRP and NLR with disease severity and mortality among COVID-19 patients in three waves.

Material and Methods

A cross-sectional, retrospective study was conducted at a tertiary care hospital, which is a state government-approved centre for the care of COVID-19 patients and an Indian Council of Medical Research (ICMR) approved COVID-19 testing centre in Bangalore. Comparative data analysis was performed between COVID-19 patients during March 2020 to February 2021, considered as the first wave of the COVID-19 disease, with the second wave patients from April 2021 to September 2021 and the third wave from November 2021 to March 2022. Waves were determined based on the number of daily new cases recorded in India from internet data sources. The study was approved by the Institutional Ethics Committee (IEC:VIEC/2021/APP/016), and a waiver for permission was granted.

Inclusive criteria: Patients aged ≥18 years, who tested positive for SARS-CoV-2 by Reverse Transcription Polymerase Chain Reaction (RT-PCR) method using nasal and oropharyngeal swabs, within the study duration, were included.

Exclusion criteria: Patients with comorbid conditions such as heart attacks, trauma, infections, burns, chronic inflammatory diseases like lupus, vasculitis, rheumatoid arthritis, and inflammatory bowel diseases were excluded.

Sample size estimation: In this study, the data were collected from the medical record section. The total data of 1485 patients were retrieved and investigated for CRP and NLR levels. Out of that, data of 1027 patients during the first wave period, 419 patients during the second wave, and 39 patients during the third wave were collected.

Study Procedure

Data regarding demographic variables (age, gender), serum CRP, NLR levels within 24 hours after hospitalisation, clinical status, and the need for oxygen and ventilator support were retrieved. The data on epidemiological, clinical, laboratory, radiological findings, and outcomes were collected using a data collection checklist from electronic medical records.

After collecting the data, patients were categorised based on disease severity at the time of hospitalisation. Classification was done based on the new guidelines for COVID-19 disease severity by the National Institute of Health (NIH) issued by the Government of India, as described below (13):

• Asymptomatic or presymptomatic infection: Individuals who tested positive for SARS-CoV-2 using a virological test (i.e., nucleic acid amplification test or antigen test), but have no symptoms.
• Mild illness: Individuals with symptoms such as fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell, but without any respiratory symptoms such as shortness of breath, dyspnea, or abnormal chest imaging.
• Moderate illness: Individuals with a saturation level of oxygen (SpO2) ≥94% on room air at sea level.
• Severe illness: Individuals with SpO2 <94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) <300 mmHg, respiratory frequency >30 breaths per minute, or lung infiltrates >50% (13).
• Critical illness: Individuals with respiratory failure, septic shock, and/or multiple organ dysfunctions.

Serum CRP was estimated by the Nephelometry method, using the Immage-800 Beckman Coulter fully automatic instrument. The coefficient of variation for the assay was 6.5%. The normal serum CRP levels in adult males and females were <0.8 mg/dL [14,15]. Blood NLR was estimated by the calculation method from neutrophil and lymphocyte counts, which were collected from the complete blood count test. Neutrophil and lymphocyte counts were estimated by the volume conductivity light scatter method in the DXH 900 Beckman Coulter instrument. The normal blood NLR levels in adult males and females were 2-2.33 (15).

Statistical Analysis

The collected data was entered into Microsoft Excel. Continuous variables were presented as mean±standard deviation. Qualitative variables were presented as frequency and percentage. The continuous variables were compared using an independent t-test, one-way ANOVA, or Kruskal-Wallis test as appropriate, whereas the qualitative variables were compared using the Chi-square test. A two-tailed p-value <0.05 was considered statistically significant.

Results

In the present study, out of a total of 1485 COVID-19 positive cases, most of them (752, 73.2%) had mild illness during the first wave period, 221 (52.74%) during the second wave period, but in the third wave, most of them (23, 58.97%) were critically ill cases. In the third wave, there were no asymptomatic cases, and only 1 (2.56%) subject with moderate illness visited the hospital (Table/Fig 1).

The mean age of patients significantly increased as the disease progressed in all three waves of the COVID-19 disease (Table/Fig 2). In all categories, the number of male patients was significantly higher than females in the first and second waves of the COVID-19 disease. In the third wave, males outnumbered females in all categories, but the difference was not found to be significant (Table/Fig 3).

The results indicate that the mean CRP and NLR levels were elevated in asymptomatic cases, followed by lower mean values in mild cases and moderate cases. They were again markedly elevated in severe and critically ill categories during the first wave. Mean CRP and NLR values increased with disease severity and positively correlated with each other in asymptomatic cases, mild cases, moderate cases, and severe cases in the second wave. The mean CRP levels maintained the same levels in mild, moderate, and severe cases and were markedly elevated only in critically ill cases. Mean NLR values were elevated in mild cases, further elevation was found in severe cases, and they maintained the same levels as mild cases in critically ill cases. Mean CRP and NLR values were positively correlated with each other in mild cases, severe cases, and critically ill cases (Table/Fig 4).

In all three waves, the number of deaths in asymptomatic cases was nil. In mild cases during the first wave period, 6 deaths occurred, and during the second wave period, 13 deaths occurred. Among moderate cases during the second wave period, 2 deaths occurred. In severe cases during the first wave period, 6 deaths occurred, and during the second wave period, 19 deaths occurred. Markedly more deaths were found in critically ill cases during the first wave period (11 deaths) and the second wave period (23 deaths). In the third wave, there were no deaths in any category of the disease (Table/Fig 5). In the first and second waves, mean CRP and NLR values were significantly higher in deceased patients compared to survivors (Table/Fig 6).

Discussion

In the present retrospective study, more than 1000 subjects were involved (16),(17),(18),(19),(20),(21),(22),(23),(24),(25),(26),(27),(28),(29),(30),(31),(32),(33),(34).

Naess A et al., found that viral infections lead to lymphocytosis, contrary to the present study where SARS-CoV-2 led to lymphopenia (16). Wang D et al., Huang C et al., Guan W-J et al., Yang X et al., and Chan PKS et al., observed that COVID-19 infection presents with lymphopenia (17),(18),(19),(20),(21). The cause of this could be attributed to either viral particles attacking and damaging the cytoplasmic components of lymphocytes or intense cytokine storms inducing apoptosis of the lymphocytes. Similarly, the present study also observed lymphopenia across all categories of COVID-19 infection in all three waves.

Similarly, studies by Ali N, Sharifpour M et al., Clyne B and Olshaker JS, and Gabay C and Kushner I found elevated CRP levels in severe and critical illness across all three waves (22),(23),(24),(25). Alkhatip AAAMM et al., in their study found high NLR levels in all cases of COVID-19 across all three waves (26). Marked elevation of NLR values was seen as the severity progressed, as in studies by Liu J et al., Yang A-P et al., and Erdogan A et al., (27),(28),(29). Significant higher values were found among wave 3 cases compared to waves 1 and 2. These findings are consistent with the findings in the present study.

As found in studies by Shang W et al., Yufei Y et al., and Liu YP et al., an association was found between CRP and NLR values in the present study as well [30-32]. For wave three, due to the omicron variant of the COVID-19 disease, where most people had been vaccinated and the virus was not proving to be as fatal anymore, CRP and NLR still had positive correlations but not significant.

Similar to Peckham H et al., a higher number of males compared to females were infected across all three waves (33). Like in Bonanad C et al., and Yan X et al., this study also supports that for waves 1 and 2, age is an independent predictor of mortality, however not in wave 3 because people with no vaccine presented with more serious symptoms (34). It was also noted that CRP and NLR levels positively correlated with the mortality rate in waves 1 and 2, but no deaths were observed in wave 3.

A comparison of the findings in the present study with contrasting studies is shown in (Table/Fig 7) (16),(17),(18),(19),(20),(21),(22),(23),(24),(25),(26),(27),(28),(29),(30),(31),(32),(33),(34),(35).

Limitation(s)

This study has certain limitations. First, only one-time measured NLR and CRP levels within 24 hours of hospitalisation were used for analysis. Serial measurements throughout hospitalisation would have been more helpful. Secondly, there was only one moderate case in wave three. Thus, comparison between waves was limited due to a lack of data. Third, there was a huge difference in sample size among the three waves of different clinical categories of COVID infection. Fourth, genetic sequencing was not done among the three waves of COVID infection to confirm the different variants of the SARS-CoV-2 virus.

Conclusion

The study findings revealed that CRP levels did not differ much between the three different waves, but NLR levels greatly increased in wave 3 cases. An association was observed between CRP and NLR levels in each wave, regardless of the variant of the SARS-CoV-2 virus. This research confirms that a combination of older age and male sex, as well as elevated CRP and NLR levels, are independent predictors of disease severity and mortality among Asian Indian patients with COVID-19 in all three waves. As part of future studies, positive cases of COVID-19 will be sent for gene sequencing studies. Collecting specific variant data from gene sequencing and comparing it with the vaccine status of individuals can provide a clearer picture for understanding CRP and NLR variations.

Author contributions: SA supervised the acquisition of the data, analysed and interpreted the data. SD assisted in collecting clinical information about the patients and helped interpret the data. The manuscript was established by UA and JAN. The manuscript was critically reviewed by MNS. All authors edited and approved the final version of this manuscript.

Acknowledgement

We would like to thank our management for giving us the opportunity to conduct this research.

DOI and Others

DOI: 10.7860/JCDR/2023/62910.18271

Date of Submission: Jan 18, 2023
Date of Peer Review: Feb 23, 2023
Date of Acceptance: May 09, 2023
Date of Publishing: Jul 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. NA

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EMENDATIONS: 6