DO LABORATORY BIOMARKERS PREDICT SURVIVAL IN SEVERE COVID-19? A CROSS-SECTIONAL STUDY

Background: This study aims to compare lymphocyte count, C-reactive protein (CRP), ferritin, Lactate Dehydrogenase (LDH) and D-dimer among survivors and non-survivors of severe COVID-19. Methods: This retrospective cross-sectional analytical study included 69 patients for whom a record of the biomarkers and survival status was available. Baseline and peak values were selected for serum CRP, ferritin, LDH and D-Dimer. Baseline and trough lymphocyte counts were selected. Data were analyzed using SPSS version 21. Mean and standard deviation were used to compare the biomarkers with paired t-test. p- value <0.05 was taken as significant. Results: The mean age of the study population was 55.5±9.1 years and 50 (72.5%) were male. Among survivors, the increase in CRP level was not significant (from 15.80±9.8 mg/dl to 17.87±8.4 mg/dl, p=0.45) while it was significant in non-survivors (from 16.68±10.90 mg/dl to 20.77±12.69 mg/dl, p=0.04). There was no significant rise in LDH levels in survivors (from 829.59±499 U/L to 1018.6±468 U/L, p =0.20) while it increased significantly in non-survivors (from 816.2±443.08 U/L to 1056.61±480.54 U/L, p =0.003). The decrease in lymphocyte count and increase in D-Dimers in both the groups was significant ( p =0.001). There was no significant elevation in ferritin in both the groups ( p >0.05). Conclusion: In severe COVID-19 patients, serum CRP and LDH can be used for risk stratification and predicting survival. Lymphopenia, increase in serum ferritin and D-dimers may not predict survival. suggests that is biomarkers


INTRODUCTION
Corona Virus Disease 2019 (COVID-19) originated as unusual pneumonia in the visitors of a seafood market which was also selling live wild animals in Wuhan, China. Soon it was realized that human to human transmission does occur as similar symptoms appeared in those who got in contact with the cases & never visited the market. 1 Shortly after China, it spread across the globe. It was declared as a pandemic on March 11, 2020. 2 As of December 22, 2020 there are 76,023,488 confirmed cases globally and 1,694,128 deaths worldwide. 3 In Pakistan, there are 460,672 confirmed cases of COVID-19, and death tally of 9,474. 4 Corona Virus Disease 2019 has posed medical, social and scientific challenges all over the world due to its complex and unpredictable clinical course. Multiple factors have been proposed for the complex and ill-defined course of the illness. 5 In the most of the cases, the disease is fairly mild, while in the rest it results in severe disease leading to multiorgan failure and death. 6 Apart from gender, age, blood groups, and co-morbidities, a plethora of laboratory markers have been investigated to predict the course and outcome of the disease. [7][8][9][10] Infection with SARS CoV-2 leads to the release of cytokines and chemokines from monocytes, lymphocytes and macrophages resulting in an inflammatory response. 11 The common laboratory parameters which are supposed to be linked with worse outcome in COVID-19 include CRP, interleukin-6 (IL-6), D-dimer, fibrinogen, LDH, Cardiac Troponin, lymphocyte count, serum ferritin, serum amyloid A (SAA) and erythrocyte sedimentation rate (ESR). 12 However, there is no clarity as the results of most of the studies are inconsistent. 1,5,10,12,13 Laboratory predictors for outcome in terms of survival need a comprehensive investigation.
The course of SARS-CoV-2 in Pakistan is different from the developed world. The cases have declined (from 6,825 new cases per day on June 13 th , 2020 to 450 new cases on August 24 th , 2020) 4 in the last few weeks. The seropositivity is reported as 11% 14 in the general population which indicates that herd immunity is still to be achieved. On the other hand, adherence to standards operating procedures (SOPs) for SARS-CoV-2 were also not visible in most of the areas. This discrepancy from the rest of the world could be linked to the genetics of the population as well as that of the virus. Given the different course that COVID-19 has taken in Pakistan, it is likely that the pattern of immune and inflammatory response to SARS-CoV-2 infection in our population is distinct. Keeping this in view, this study aims to analyze the pattern of different laboratory parameters between survivors and nonsurvivors of severe COVID-19 and compare the results with published literature.

MATERIAL AND METHODS
This study was approved by Institutional Review Board of Khyber Girls Medical College / Hayatabad Medical Complex, Peshawar, Pakistan. Electronic record of the patients was used for this crosssectional study. All confirmed cases of severe COVID-19 as per WHO criteria 15 in COVIDintensive care unit (COVID-ICU) of Hayatabad Medical Complex (HMC), Peshawar were eligible for inclusion in the study. The data were retrieved from Hospital Information System (HIS). The laboratory markers included in the study were lymphocyte count, CRP level, serum ferritin level, serum LDH level and serum D-dimer level. The outcome of patients was classified as survivor and non-survivor. Survivor was defined as a patient who was discharged from the hospital following improvement in symptoms. Non-survivor was defined as a patient who died during hospital stay with COVID-19 as a predominant contributory factor in death. Sixty-nine patients admitted in COVID-ICU, for whom data of proposed laboratory markers and outcome was available, were included in the study. All patients received standard treatment for severe COVID-19. For every patient, baseline and peak values were selected for CRP level, serum ferritin level, serum LDH level and serum D-Dimer level. Similarly, baseline and trough levels were selected for lymphocytes. Analysis was carried out in SPSS version 21.0. Qualitative data like gender, comorbidities, and age groups were expressed as frequency and percentages while quantitative variables like age and laboratory biomarkers' levels were calculated as mean±S.D. Paired t-test was applied for comparison of means of study variables (lymphocyte count, serum CRP level, ferritin, LDH and d-dimer levels) in each group (survivors and nonsurvivors). p value below 0.05 was considered significant.

DISCUSSION
The results of the present study were based on laboratory parameters of 69 confirmed cases of COVID-19 admitted to ICU. Lymphocyte count was significantly decreased in both survivors and nonsurvivors (p<0.001). Similar findings were reported by Huang and Pranata 16 who observed that lymphopenia is found to be the key predictor for the severity of COVID-19 and is associated with poor outcomes. They reported that cases with worse outcome have a lower lymphocyte count with a mean difference = -361.06 per μL (95% CI -439.18, -282.95; p<0.001;) when compared to cases with favourable outcome. Research conducted by Zhou et al. 1 revealed that cases with COVID-19 who did not survive had lower lymphocyte count in comparison to survivors (0.6 vs 1.1x10 9 /L, p<0.0001). In contrast to Zhou et al. where patients with all stages of COVID-19 were studied, the patients included in this study had only severe disease. Moreover, we determined the maximum drop in lymphocyte count from baseline in both groups. The potential mechanisms of lymphopenia in COVID-19 are; direct destruction of lymphocytes by the virus, injury to the lymphatic organs, 16 abnormalities of cytokines leading to lymphocyte depletion, 17 and lactic acidosis secondary to hypoxemia. 18 In the present study significant rise in CRP was found to be a marker of non-survival. There was significant increase (from 16.68±10.90 mg/dl to 20.77±12.69 mg/dl, p=0.04) in non-survivor as compared to survivors (p=0.45). The association of CRP with poor outcomes in COVID-19 has already been reported by Li et al., Feng et al.,Chen et al and Wang. 7,8,19,20 Research suggests that CRP is one of the most important biomarkers to predict the prognosis of COVID-19. 10 The serum ferritin increased from the baseline in survivors and non-survivors, but the rise in both groups was not significant. (p=0.31 and p=0.12, respectively). This contrasts with Zhou et al. 1 who have reported significantly elevated serum ferritin in non-survivors as compared to survivors (1435 ng/ml and 503 ng/ml, respectively; p<0.0001). Similarly, Chen et al. 12 have observed higher serum ferritin in patients with severe illness as compared to those with moderate disease (serum ferritin >800 ng/ml in 100% of patients with severe disease versus 30% in those with moderate disease, p=0.003). The difference could be due to a difference in the case selection. We included only patients with severe disease while 93% of survivors in the study conducted by Zhou et al. 1 had mild disease (CURB score 0-1) and 48% of the participants included by Chen et al. 12 were having non-severe disease.
There was a statistically significant increase in serum LDH level in non-survivors (from 816.2±443.08 U/L to 1056.61±480.54 U/L, p=0.003) as compared to survivors (from 829.59±499 U/L to 1018.6±468 U/L, p=0.20). According to a study from China 1 , serum LDH level was significantly higher among non-survivors (p<0.001). Similarly, it has been reported that high LDH level is associated with Acute respiratory distress syndrome, admission to intensive care unit, progression of the disease and higher mortality. 1,9,[21][22][23] The D-Dimer level increased significantly in both survivors and non-survivors (from 7.2±9.8 µg/ml to 28.8±35.4 µg/ml, p=0.01 and from 8.75±14.8 µg/ml to 29.52±37.96 µg/ml, p=0.001, respectively). Published literature indicates that D-Dimer levels were significantly higher among those with severe disease. 1,5,7,10,21 This discrepancy may be due to the homogenous nature of our study population (severe COVID-19 patients admitted to ICU) while the majority of other studies had patients with mild, moderate and severe disease. It has been reported that patients with non-severe disease have a lower level of D-Dimer as compared to those with severe disease. 24 Our sample was a homogenous one including only cases with severe disease admitted to intensive care unit. To the best of our knowledge, this is the first study on the role of biomarkers in COVID-19 patients admitted to intensive care unit. It had a relatively small sample size and it was a single-center experience.

CONCLUSION
Serum CRP and LDH levels predict mortality in patients with severe COVID-19 and can be used for risk stratification. Lymphopenia, increase in serum ferritin and D-dimers are frequently observed in patients with severe COVID-19 but these may not predict survival. Clinicians caring for patients with COVID-19 should use serum CRP and LDH levels for risk stratification and prognosis.

Acknowledgments
We acknowledge the efforts of Prof. Akhtar Sherin (Chief Editor, KMUJ) for reviewing the manuscript. We also acknowledge the help of Muhammad Irfan (Medical Student) in data collection.

Disclosure
The manuscript was submitted to BMC Infectious Disease and was under consideration for publication with a preprint available on Research Square on their request with DOI -10.21203/rs.3.rs-67563/v1. We anticipated the publication charges but were not aware of the hidden charges to deposit dataset in their repository. We gave them the choice if we could deposit the dataset on free repository https://www.openicpsr.org/openicpsr/covid19 as we could not afford the heavy charges of repository in addition to the publication charges. Therefore, our article was not considered for further processing.
Funding / grant disclosure This research did not receive any specific grant or funding. Medical Teaching Institute / Hayatabad Medical Complex, Peshawar is a public sector, nonprofit organization. Charges of all laboratory investigations were borne by the hospital.

Conflicts of interest
All authors declare no conflicts of interest.