Abstract
Introduction
Many viruses through aerosols, droplets, and droplet nuclei utilize the respiratory passages to establish not only localized respiratory tract infections but also systemic disease. The coronaviruses (CoV) are no exception. The two most common illnesses that occurred in the recent past were severe acute respiratory syndrome (SARS, 2003) and the Middle East respiratory syndrome (MERS, 2012).1 The current pandemic, which broke out in late December 2019, has been a major threat to global public health due to significant morbidity and mortality, akin to snapping of Thanos’ fingers. The novel coronavirus was initially named the 2019-novel CoV (2019-nCoV), but because of nearly 80% genetic homology to SARS-CoV, the Coronavirus Study Group of International Committee rechristened this virus as SARS-CoV-2.1 The disease was named coronavirus disease 2019 (COVID-19) on January 12, 2020, by the World Health Organization (WHO).2 According to the Advisory Committee on dangerous pathogens UK, COVID-19 is assigned as a hazardous group-3 organism, meaning that it can cause severe human disease.3 The novel coronavirus was named the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, 2019-nCoV) due to its high homology (~80%) to SARS-CoV, which caused acute respiratory distress syndrome (ARDS) and high mortality during 2002–2003.4 The outbreak of SARS-CoV-2 was considered to have originally started via a zoonotic transmission associated with the seafood market in Wuhan, China. Later it was recognized that human-to-human transmission played a major role in the subsequent outbreak.5 The most common clinical manifestations of COVID-19 include fever, cough, dyspnea, fatigue, and myalgia. A few patients have developed severe pneumonia and they may present with acute respiratory distress syndrome (ARDS), extrapulmonary organ dysfunction, or even death. SARS-CoV-2 virus primarily affects the respiratory system, although other organ systems are also involved. Lower respiratory tract infection-related symptoms including fever, dry cough, and dyspnea were reported in the initial case series.6 In addition, headache, dizziness, generalized weakness, vomiting, and diarrhea were observed.7 It is now widely recognized that respiratory symptoms of COVID-19 are extremely heterogeneous, ranging from minimal symptoms to significant hypoxia with ARDS. The heterogeneous disease course of COVID-19 is unpredictable with most patients experiencing mild self-limiting symptoms. However, up to 30% require hospitalisation, and up to 17% of these require intensive care support for acute respiratory distress syndrome (ARDS), hyperinflammation, and multiorgan failure.8-10 A cytokine storm in patients with severe disease was identified in the early reports of Wuhan patients and is intrinsic to disease pathology. In this cohort, elevated plasma interleukin (IL)-2, IL-7, IL-10, granulocyte colony-stimulating factor (GCSF), interferon γ-induced protein 10 (IP10, monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein 1-alpha (MIP1A), and tumor necrosis factor-alpha (TNF-α) levels in ICU patients were identified.6 Studies have shown that severe or fatal cases of COVID-19 disease are associated with an elevated white cell count, blood urea nitrogen, creatinine, markers of liver and kidney function, C-reactive protein (CRP), interleukin-6 (IL-6), lower lymphocyte (<1000/µL) and platelet counts (<100 × 109/L) as well as albumin levels compared with milder cases in which survival is the outcome. Subsequent studies have implicated IL-6 as a valuable predictor of adverse clinical outcome and a potential therapeutic target.11,12 One or more clinical and wet biomarkers may enable early identification of high-risk cases, assisting disease stratification and effective use of limited specialist resources. Age is a strong risk factor for severe illness, complications, and death.13,14 Patients with no underlying medical comorbid conditions have an overall case fatality rate of <1%. Case fatality is higher for patients with comorbidities. The severe cases are associated with elevated levels of inflammatory biomarkers such as serum lactate dehydrogenase, creatine kinase, C-reactive protein (CRP), d-dimer, procalcitonin, and ferritin.15 Since laboratory medicine has always supported clinical decision-making in various infectious diseases, it is important to assess the ability of laboratory-derived biomarkers to facilitate risk stratification of COVID-19 disease. This study will comprehensively explore clinical disease features and routine laboratory tests associated with COVID-19 disease and its complications, to address their association with disease severity and outcome. Hence, the present retrospective study will be done at our tertiary care centre to assess the association between different laboratory biomarkers and disease severity and outcomes in COVID-19 patients.
Aims and objectives
Clinical correlation of biomarkers and disease severity in COVID-19 patients – a retrospective study.
Review of Literature
Xia et al.16 in 2020 defined disease stages and identified stages’ determining factors are instructive for the definition of standards for home quarantine. The authors demonstrated pulmonary involvement on a chest CT scan in 97.9% of cases. It took 16.81 ± 8.54 (3–49) days from the appearance of the first symptom until 274 patients tested virus-negative in naso- and oropharyngeal (NP) swabs, blood, urine, and stool, and 234 (83%) patients were asymptomatic for 9.09 ± 7.82 (1–44) days. Subsequently, 131 patients were discharged. One hundred and sixty-nine remained in the hospital; these patients tested virus-free and were clinically asymptomatic because of widespread persisting or increasing pulmonary infiltrates. Hospitalization took 16.24 ± 7.57 (2–47) days; the time interval from the first symptom to discharge was 21.37 ± 7.85 (3–52) days. The authors concluded that with an asymptomatic phase, disease courses are unexpectedly long until the stage of virus negativity. NP swabs are not reliable in the later stages of COVID-19. Pneumonia outlasts virus-positive tests if sputum is not acquired. Imminent pulmonary fibrosis in high-risk groups demands follow-up examinations. Investigation of promising antiviral agents should heed the specific needs of mild and moderate COVID-19 patients.
Keddie et al.17 in 2020 investigated the routine laboratory tests and cytokines implicated in COVID-19 for their potential application as biomarkers of disease severity, respiratory failure, and need for higher-level care. The authors found CRP, IL-6, IL-10, and LDH were most strongly correlated with the WHO ordinal scale of illness severity, the fraction of inspired oxygen delivery, radiological evidence of ARDS, and level of respiratory support. IL-6 levels of ≥3.27 pg/mL provide a sensitivity of 0.87 and specificity of 0.64 for a requirement of ventilation, and a CRP of ≥37 mg/L of 0.91 and 0.66. The authors concluded that reliable stratification of high-risk cases has significant implications on patient triage, resource management, and potentially the initiation of novel therapies in severe patients.
Malik et al.18 in 2020 in a systematic review and meta-analysis assessed the role of biomarkers in evaluating the severity of disease and appropriate allocation of resources. Studies having biomarkers, including lymphocyte, platelets, d-dimer, lactate dehydrogenase (LDH), C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, procalcitonin (PCT), and creatine kinase (CK), and describing outcomes were selected with the consensus of three independent reviewers. The authors found lymphopenia, thrombocytopenia, elevated d-dimer, elevated CRP, elevated PCT, elevated CK, elevated AST, elevated ALT, elevated creatinine, and LDH were independently associated with a higher risk of poor outcomes. The authors concluded a significant association between lymphopenia, thrombocytopenia, and elevated levels of CRP, PCT, LDH, d-dimer, and COVID-19 severity. The results have the potential to be used as an early biomarker to improve the management of COVID-19 patients, by identification of high-risk patients and appropriate allocation of healthcare resources in the pandemic.
Tjendra et al.19 in 2020 assessed specific laboratory parameters and summarized the currently available literature on the predictive role of various biomarkers in COVID-19 patients. The authors summarised that although the markers are considered non-specific, acute-phase reactants, including C-reactive protein (CRP), ferritin, serum amyloid A (SAA), and procalcitonin, were reported as sensitive markers of acute COVID-19 disease. Significantly elevated white blood cell count; marked lymphopenia; decreased CD3, CD4, or CD8 T-lymphocyte counts; high neutrophil count; thrombocytopenia; and markedly elevated inflammatory biomarkers were associated with severe disease and the risk of developing sepsis with rapid progression. Trends observed by serial laboratory measurements during hospitalization, including a progressive decrease of lymphocyte count, thrombocytopenia, elevated CRP, procalcitonin, increased liver enzymes, decreased renal function, and coagulation derangements, were more common in critically ill patient groups and associated with a high incidence of clinical complications. Elevated interleukin 6 level and markedly increased SAA were most often reported in severely and critically ill patients. Indicators of systemic inflammation, such as neutrophil to lymphocyte ratio, systemic immune-inflammation index, or COVID-19 Severity Score, may be used to predict disease severity, outcome, and mortality.
Xue et al.20 in 2020 in a retrospective study identified early diagnostic and prognostic biomarkers to determine the risk of developing a serious illness. The authors found patients with severe disease had reduced neutrophils and lymphocytes; severe coagulation dysfunction; altered content of biochemical factors (such as urea, lactate dehydrogenase); elevated high sensitivity C-reactive protein levels, neutrophil–lymphocyte, platelet-lymphocyte, and derived neutrophil–lymphocyte ratios, high sensitivity C-reactive protein-prealbumin ratio (HsCPAR), systemic immune-inflammation index, and high sensitivity C-reactive protein-albumin ratio (HsCAR); and low lymphocyte-monocyte ratio, prognostic nutritional index (PNI), and albumin-to-fibrinogen ratio. PNI, HsCAR, and HsCPAR correlated with the risk of severe disease. The nomogram combining the three parameters showed good discrimination with a C-index of 0.873 and reliable calibration. Moreover, HsCAR and HsCPAR correlated with the duration of hospital stay. The authors concluded that taken together, PNI, HsCAR, and HsCPAR may serve as accurate biomarkers for the prediction of disease severity in patients with COVID-19 upon admission/hospitalization.
Khan et al.21 in 2021 in a single-center, observational study evaluated the significance of inflammatory markers in different categories of COVID-19 in admitted patients. Infection biomarkers, including hs-CRP, serum ferritin, serum creatinine, ALT, ALP, cardiac troponin-I, and IL-6 were analyzed. The authors found median age was 61.3 years. 70% (35) were males while 30% (15) were females. We noted significantly increased hs-CRP (9.32 mg/dL ± 10.03) and ferritin levels (982.3 ng/mL ± 601.9). A noteworthy increase was observed in neutrophil count (11.05 × 109/L) and a decrease was observed in lymphocyte count (0.27 × 109/L), and the platelet count was borderline decreased (244.1 × 109/L). IL-6 levels were markedly increased in all admitted patients (100.2 ± 122.2 pg/mL). The authors concluded that serum levels of CRP, troponin-I, ALP, ALT, serum creatinine, and ferritin are markedly increased in COVID-19 patients. Increased CRP and ferritin levels were also associated with secondary bacterial infection and poor clinical outcomes.
Magdy et al.22 in 2021 in a retrospective study evaluated the role of semi-quantitative CT-severity scoring versus LDH as prognostic biomarkers for COVID-19 disease severity and short-term clinical outcome. The authors found CT severity score and LDH were significantly higher in severe and critical cases compared to mild cases (p value < 0.001). High predictive significance of CT severity score for COVID-19 disease course noted, with cut-off value ≥13 highly predictive of severe disease (96.96% accuracy); cut-off value ≥16 highly predictive of critical disease (94.21% accuracy); and cut-off value ≥19 highly predictive of short-term mortality (92.56% accuracy). CT severity score has higher sensitivity, specificity, positive, and negative predictive values as well as overall accuracy compared to LDH level in predicting severe, critical cases, and short-term mortality. The authors concluded that Semi-quantitative CT severity scoring has high predictive significance for COVID-19 disease severity and short-term mortality with higher sensitivity, specificity, and overall accuracy compared to LDH. Our study strongly supports the use of CT severity scoring as a powerful prognostic biomarker for COVID-19 disease severity and short-term clinical outcome to allow triage of need for hospital admission, earlier medical interference, and to effectively prioritize medical resources for cases with high mortality risk for better decision making and clinical outcome.
Materials and Methods
A hospital-based retrospective observational study will be done at our tertiary care centre on 200 patients to assess the association between different laboratory biomarkers and disease severity and outcomes in COVID-19 patients.
Study design
A hospital-based retrospective observational study.
Study duration
The study will be conducted from August 2020 onwards.
Study area
The study will be done at our tertiary care centre in the department of Pulmonology, Fortis Hospital, Vasant Kunj, New Delhi, a tertiary health care centre with all the required diagnostic modalities including research facility.
Study population
All COVID-19 patients admitted In COVID ICU from the month of August 2020 onwards at Fortis Hospital, Vasant Kunj, New Delhi who fulfilled the inclusion criteria.
Sample size
200 patients
Formula used for sample size calculation was:
N = (Z2 × P × (1 − P))/d2
Z2 = table value of alpha error from standard normal distribution table = 1.96 × 1.96 = 3.84
Power (P) = 0.05
(1 – P) = 0.95
Precision error of estimation (d) = 3%
d2 = 0.0009
N = (3.84 × 0.05 × 0.95)/0.0009 = 198.67
A similar study done by Keddie et al.17 investigated the routine laboratory tests and cytokines implicated in COVID-19 for their potential application as biomarkers of disease severity, respiratory failure the sample size was 203.
Hence, sample size of 200 patients was selected for the study of which 110 patients were mild/moderate cases and 90 patients were severe/critical cases.
Inclusion Criteria
All the patients of COVID-19 admitted in COVID ICU from August 2020 onwards.
Methodology
The study will be done at our tertiary care centre in the department of Pulmonology, Fortis Hospital, Vasant Kunj, New Delhi, on attending OPD/IPD after due permission from the Institutional Ethics Committee and Review Board and after taking Written Informed Consent from the patients.
After approval from the Institutional Ethics Committee, a valid informed consent will be taken. Once the patients will be enrolled for the study, a thorough history and physical examination will be done as per proforma. Informed consent will be taken in written form patients or patient’s attendants.
A total of 200 consecutive patients presenting with COVID-19 at our tertiary care centre in the department of Pulmonology, Fortis Hospital, Vasant Kunj, New Delhi irrespective of gender, race, religion, and socio-economic background will be enrolled in the study.
Detailed history, general physical examination, systemic examination, laboratory parameters, radiological findings will be incorporated as per proforma.
Demographic and epidemiological statistics, such as age, sex, and disease history, will be gathered upon admittance. For laboratory confirmation, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) will be used as gold standard, according to the recommended protocol of the hospital. All baseline serum samples will be collected immediately after admission for total blood count, ALT, ALP, CRP, aPTT, IL-6, and ferritin.
The severity of COVID-19 was assessed as per the Ministry of Health and Family Welfare (MOHFW) guidelines, Government of India.23
Mild cases were those COVID-19 patients who had uncomplicated upper respiratory tract infections with no evidence of hypoxia and breathlessness.
Moderate cases were those with radiological and clinical features of pneumonia with SpO2 in the range of 90–94%, with a respiratory rate of more or equal to 24 breaths per minute.
Severe cases were those who were meeting any of the following criteria: respiratory distress with respiratory rate ≥30/minute and oxygen saturation ≤90% at rest or respiratory rate ≥24 breaths per minute along with features of sepsis and septic shock.
The clinical classifications are as follows: (1) mild, minor symptoms and imaging shows no pneumonia. (2) moderate, with fever, respiratory tract symptoms, and imaging shows pneumonia. (3) Severe, meet any of the following: a) respiratory distress, respiratory rate ≥ 30 beats/minute; b) in the resting state, means oxygen saturation ≤ 93%; c) arterial blood oxygen partial pressure/oxygen concentration ≤300 mm Hg (1 mm Hg = 0.133 kPa); d) pulmonary imaging showed that the lesion progressed more than 50% within 24–48 h. (4) Critical, one of the following conditions: a) respiratory failure occurs and requires mechanical ventilation; b) shock occurs; c) ICU admission is required for combined organ failure.
Laboratory Investigation
Roughly 2∼4 mL of peripheral blood will be collected from subjects of the categorized groups, and serum will be separated by 2000 rpm/20 minutes centrifugation. Serum cytokines will be analyzed using the Elecsys IL-6 immunoassay and electrochemilumine scence immunoassay (ECLIA) performed on a COBAS e411 analyzer (Roche Ltd.). It is an in vitro diagnostic test for the quantitative determination of IL-6 in human blood. 25 μL of serum is incubated with biotinylated monoclonal IL-6 antibody, labeled with a ruthenium complex and streptavidin-coated microparticles, forming a sandwich complex with the antigens present in the sample, and the resulting chemiluminescence is then measured by using a photomultiplier.
Data collection
All suspected infection patients will be taken upper respiratory throat swab samples at admission and then to designated authoritative laboratories to detect the SARS-CoV-2. Bacterial and fungal detections of sputum or respiratory secretions and other laboratory tests will be completed in the clinical laboratory at the Department of Pulmonology. C-reactive protein (CRP) will be detected by the immunoturbidimetry method. Procalcitonin (PCT) was detected by the Roche electrochemiluminescence method. Erythrocyte sedimentation rate (ESR) was measured by Westergren’s international standard method.
Possible Risks Involved
Since the study is a retrospective study there will be no extra risk/complications.
Possible Benefits
Since laboratory medicine has always supported clinical decision-making in various infectious diseases, it is important to assess the ability of laboratory-derived biomarkers to facilitate risk stratification of COVID-19 disease. Therefore, in the present systematic review and meta-analysis, we assessed the association between different laboratory biomarkers and outcomes in COVID-19 hospitalised patients.
