Biochemical predictors for Sars-Cov-2 severity

It is of interest to assess the inflammatory marker profile in SARS-CoV-2 patients and to correlate the levels of systemic inflammatory biomarkers such as neutrophil-to-lymphocyte ratio (NLR), C-Reactive Protein CRP, Ferritin, Creatine kinase (CK), Lactate dehydrogenase (LDH) and liver function analytes total serum proteins, albumin, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) with the severity of SARS-CoV-2 infections. A total of 1000 COVID-19 positive patient's data were collected. Laboratory assessments consisted of NLR (neutrophil-lymphocyte ratio) by cell counter, C Reactive Protein (CRP) by immunoturbidimetry, Ferritin by electrochemiluminescence (ECLIA) and Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Total Protein and Albumin by spectrophotometry. The mean plasma CRP levels, NLR, ferritin, CK and LDH levels were higher in severe cases than in non-severe cases, and the difference was statistically significant (p<0.05). All liver function tests such as the total and direct bilirubin, AST, ALT, ALP, total protein and albumin were higher in severe patients than non-severe patients and the difference was statistically significant (p<0.05). Data indicate that NLR, CRP, Ferritin, CK, LDH and liver function analytes have a crucial role as prognostic markers for SARS-CoV-2 infections and hence should be routinely recommended for risk assessment and stratification of the patients to reduce the associated morbidity and mortality.


Background:
The COVID-19 pandemic has led to dramatic loss of lives worldwide with unprecedented burden on the medical fraternity. It has displayed a broad spectrum of clinical symptoms and varied susceptibility across ages and populations. As soon as patients progress to the severity or critical stage, the risk for poor outcomes increases significantly [1,2]. It is estimated that around 10-15% of mild COVID-19 patients advance to severe disease and 15-20% of severe cases progress to become critical, with many of the individuals in the critical category needing treatment in intensive care units (ICU) [1,3]. Hence, it becomes imperative that severity predictive biomarkers be identified for early stratification, triage and efficient utilisation of resources in the treatment of individuals with SARS-CoV-2 infections.
The portal of entry of the virus into the host cell is through the angiotensin-converting enzyme-2 (ACE-2) receptors expressed by various cells [4]. On binding of the viral spike protein to the ACE-2 receptors on the host cell surface, conformational changes occur in the spike protein, which leads to fusion of the viral envelope ©Biomedical Informatics (2021) with the host cell membrane [5,6]. Also, the virus is identified by pathogen recognition receptors like Toll like receptors (TLR), the nucleotide-binding oligomerization domain (NOD)-Leucin Rich Repeats (LRR)-containing receptors (NLR), the retinoic acidinducible gene 1 (RIG-1) -like receptors (RLR) and the C-type lectin receptors (CLR) which detects the microbe-associated molecular patterns and activates adapter proteins such as Myeloid differentiation primary response 88 (MyD88), Tollinterleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP), and TRIF-related adaptor molecule (TRAM) [7,8]. These proteins further activate Nuclear Factor kappa-light-chainenhancer of activated B cells (NF-kB) and Mitogen-activated protein kinases (MAPK) signalling pathways which stimulate cellular responses and release of inflammatory mediators like interleukins, interferons, Tumour necrosis factor-A, C-reactive proteins [9]. The cytokines and chemokines thus released cause a cytokine storm with associated systemic inflammation, increased risk of Acute Respiratory Distress Syndrome, Disseminated Intravascular Coagulation leading to Multiple Organ Dysfunction Syndrome Analysing the available evidence, it may be postulated that SARS-CoV-2 infections may also express a similar phasic pattern of disease progression. Various studies have suggested that higher levels of inflammatory markers such as WBC, CRP, PCT, ESR, IL-6, and IL-10 are associated with the severity of COVID-19 [1,[12][13][14][15][16]. Therefore, It is of interest to assess the inflammatory marker profile in SARS-CoV-2 patients and to correlate the levels of systemic inflammatory biomarkers such as neutrophil-tolymphocyte ratio (NLR), CRP, Ferritin, Creatine kinase (CK), Lactate dehydrogenase (LDH) and liver function analytes total serum proteins, albumin, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) with the severity of SARS-CoV-2 infections.

Materials and Methods:
This was a retrospective, single-centered cohort study conducted at the Rajiv Gandhi Government General Hospital, Chennai which is the designated hospital for the treatment of COVID-19 patients. Data of patients were collected from June 2020 to August 2020 from the Institute of Biochemistry and Pathology, Rajiv Gandhi Government General Hospital. Ethical clearance was obtained from the institutional ethical committee before the start of the study. A total of 1000 COVID-19 positive patient's data were collected from June 2020 to August 2020, of which (35%) 350 were female patients and (65%) 650 were male patients.
Only laboratory-confirmed COVID-19 patients with mild symptoms at the time of admission were enrolled in this study. A confirmed case of COVID-19 was defined as a positive result on real-time Reverse-Transcriptase-Polymerase-Chain-Reaction (RT-PCR) assay using nasal or pharyngeal swab specimens. Laboratory assessments consisted of NLR (Neutrophil-Lymphocyte Ratio) by cell counter, C Reactive Protein (CRP) by immunoturbidimetry, Ferritin by electrochemiluminescence (ECLIA) and Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Total Protein and Albumin by spectrophotometry. Demographic information, clinical signs and symptoms, clinical outcomes and laboratory findings on admission were extracted from the electronic medical records. Only the baseline laboratory data at the time of admission before undergoing treatment were collected regardless of cases receiving multiple laboratory testing.
In the present study only, the inclusion criteria were patients confirmed with SARS-CoV-2 infection by real-time reverse transcription polymerase chain reaction (RT-PCR), COVID-19 positive patients with presence of lung involvement confirmed by CT-CHEST findings and COVID-19 positive patients of all age groups. COVID-19 positive patients for whom the treatment had already been started and COVID-19 positive patients without lung involvement were excluded from the study. Non-probability convenience sampling technique was used. Chi square association test was used to analyse the associations.

Results:
The mean age of the study participants was 52.6 ± 15.8 years. The most common age group affected was 50-75 years, which can be attributed to the associated comorbidities in this age group. The female participants were significantly younger (mean age: 48.4 ± 16.4 years) than the male participants (mean age: 54.6 ± 15.2 years).
The mean plasma CRP levels, NLR, ferritin, CK and LDH levels were higher in severe cases than in non-severe cases, and the difference was statistically significant (p<0.05). All liver function test analytes -Total and direct bilirubin, AST, ALT, ALP, total protein and albumin were higher in severe patients than nonsevere patients and the difference was statistically significant (p<0.05).   On comparison of all liver function analytes with the inflammatory markers, it was observed that direct bilirubin, AST, ALT and albumin had significant positive correlation with the inflammatory markers. Total bilirubin had positive correlation with all inflammatory markers except NLR. ALP had positive correlation only with CRP and CK while it had a negative correlation with NLR, ferritin and LDH. Total protein had a positive correlation with NLR, CK and Ferritin while it had a negative correlation with CRP and LDH levels.

Discussion:
Managing COVID-19 infections worldwide has become a huge task for the medical fraternity. The rapid progression of the disease from being mild symptomatic to severe breathlessness with reduced oxygen saturation and with lack of adequate medical infrastructure it becomes paramount that early symptom profiling and stratification based on prognostic biomarkers be done for all patients to reduce the associated morbidity and mortality. The present study was conducted to analyse and correlate the biochemical inflammatory markers like NLR, CRP, Ferritin with the clinical symptoms of the patients. CK, LDH and liver function analytes were assessed and correlated with the inflammatory markers, which was predictive of multi-organ involvement.
Neutrophil-to-lymphocyte ratio (NLR) is the most well established inflammatory marker that reflects systemic inflammatory response and is easily obtainable through routine blood count analysis C-reactive protein is an acute-phase inflammatory protein produced by the liver and regulated at the transcriptional level by the cytokine IL-6 and IL-1 [20]. It is an important index for diagnosing and evaluating severe pulmonary infectious diseases [1,21]. In this study, CRP levels correlated with severity of COVID-19 infections. These findings are supported by recent meta-analysis by Roshan Kumar Mahat et al. and Pan Ji et al. [1,12]. It is also noteworthy that our results are also in keeping with those of previous studies [12,22,23,24]. The National Health Commission of the People's Republic of China included elevated inflammatory factors such as IL-6 and CRP as potential early warning indicators of severe disease in its widely used "COVID-19 diagnosis and treatment plan" [12,25].  [32][33][34][35]. This evidence highlights the importance of monitoring the liver function analytes level to determine the progression of the infection and prevent multiorgan involvement. It also suggests that ALP and total protein levels might not have greater significance as a prognostic biomarker.

Conclusion:
Data shows that NLR, CRP, Ferritin, CK, LDH and liver function analytes have a crucial role as prognostic markers for SARS-CoV-2 infections. This should be routinely recommended for risk assessment and stratification of the patients to reduce the associated morbidity and mortality. Similar data is needed among various other populations at a larger scale to provide more insight regarding the role of biochemical markers in predicting the course of the disease.