key: cord-0729770-r8mn4lzv
authors: Kermali, Muhammed; Khalsa, Raveena Kaur; Pillai, Kiran; Ismail, Zahra; Harky, Amer
title: The role of biomarkers in diagnosis of COVID-19 – A systematic review
date: 2020-05-13
journal: Life Sci
DOI: 10.1016/j.lfs.2020.117788
sha: eee1f875fb06dd47be76cd3421724fbf3932434e
doc_id: 729770
cord_uid: r8mn4lzv

AIMS: As of the 28th April 2020, the COVID-19 pandemic has infiltrated over 200 countries and affected over three million confirmed people. We review different biomarkers to evaluate if they are able to predict clinical outcomes and correlate with the severity of COVID-19 disease. METHODS: A systematic review of the literature was carried out to identify relevant articles using six different databases. Keywords to refine the search included ‘COVID-19’, ‘SARS-CoV2’, ‘Biomarkers’, among others. Only studies which reported data on pre-defined outcomes were included. KEY FINDINGS: Thirty-four relevant articles were identified which reviewed the following biomarkers: C-reactive protein, serum amyloid A, interleukin-6, lactate dehydrogenase, neutrophil-to-lymphocyte ratio, D-dimer, cardiac troponin, renal biomarkers, lymphocytes and platelet count. Of these, all but two, showed significantly higher levels in patients with severe complications of COVID-19 infection compared to their non-severe counterparts. Lymphocytes and platelet count showed significantly lower levels in severe patients compared to non-severe patients. SIGNIFICANCE: Although research is still in its early stages, the discovery of how different biomarkers behave during the course of the disease could help clinicians in identifying severe disease earlier and subsequently improve prognosis. Nevertheless, we urge for more research across the globe to corroborate these findings.

On December 2019, Wuhan City in China, became the epicentre of unexplained cases of pneumonia. On January 2020, Chinese scientists identified this as a novel coronavirus, temporarily labelled as, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)(1). Its name was then changed to coronavirus disease 2019 by the World Health Organization in February 2020 as the disease spread worldwide (2) .

It has been suggested the outbreak has a zoonotic origin, and like other respiratory pathogens, it spreads through human-to-human transmission such as coughing and sneezing (3) . Although limited, research suggests a possibility of transmission even amongst the asymptomatic (4) .

As of the 28 th April 2020, over 200 countries have been affected by the COVID-19 disease with over three million confirmed cases leading to over 200,000 deaths. Yet it is believed many remain unreported in certain areas of the world(1).

J o u r n a l P r e -p r o o f 6 D-dimer, platelet count, high-sensitivity troponin I (hs-TnI) and renal markers. Table   1 summarises the study's characteristics.

CRP is a plasma protein produced by the liver and induced by various inflammatory mediators such as IL-6. Despite being non-specific, this acute phase reactant is used clinically as a biomarker for various inflammatory conditions; a rise in CRP levels are associated with an increase in disease severity (7) .

The application of CRP in COVID-19 has been highlighted by a retrospective singlecentre study in Wuhan, China, where the majority of patients in the severe cohort showed significantly higher levels compared to the non-severe cohort (57.9 mg/L vs 33.2 mg/L, P < 0.001) (8) . A second retrospective cohort study found the likelihood of progressing to severe COVID-19 disease increased in patients with CRP levels greater than 41.8mg/L (9) . Both studies suggest CRP levels are a strong indicator to reflect the presence and severity of COVID-19 infection.

Furthermore, a study from unpublished observations suggests CRP is one of the first biomarkers within blood plasma that changes to reflect physiological complications; if accepted CRP will be the most effective biomarker to predict the progression of COVID-19 infection. Contrastingly, the same study illustrated some cases of infection which showed changes in serum amyloid A (SAA) instead of evoking significant CRP changes thus requiring further evaluation (10) .

J o u r n a l P r e -p r o o f 7 Pathologically, computed tomography (CT) scans can identify lung lesions relating to COVID-19. Nonetheless, a study conducted in China revealed CT scores could not differentiate mild cases from severe. However, compared to erythrocyte sedimentation rate (ESR), CRP levels were significantly greater during early periods of severe cases and proved to be a more sensitive biomarker in reflecting disease development(12).

The excellent performance of CRP as a biomarker is reflected in the 'area under curve' in the receiver operating analysis of 0.87 (95% CI, 0.10-1.00) where values 83% and 91% represent sensitivity and specificity, respectively. Hence compared to CT scans alone, CRP values are more reliable for earlier identification of case severity (12). Table 2 summarises the studies used in analysing CRP and COVID-19.

Cytokine release syndrome (CRS) is an over-exaggerated immune response involving an overwhelming release of pro-inflammatory mediators. This mechanism underlies several pathological processes including acute respiratory distress syndrome (ARDS) (13). Studies investigating the role of cytokines in SARS and MERS have had also found a link between CRS and disease severity (14) .

Understanding their role in COVID-19 disease may help facilitate the design of novel immunotherapies.

Studies have revealed that levels of IL-6, the most common type of cytokine released by activated macrophages, rise sharply in severe manifestations of COVID-19 (15) . However, since most studies to date have been observational, it is difficult to J o u r n a l P r e -p r o o f 8 extrapolate if the rise is significant enough to cause the manifestations seen in severe forms.

One meta-analysis reviewing six studies show mean IL-6 concentrations were 2.9fold higher in patients with complicated COVID-19 compared to those with noncomplicated disease (n=1302; 95% CI 1. 17-7.19) (16) . In its analysis, the outcomes of the studies include ICU admission, onset of ARDS and mortality. Since the proportionate rise of IL-6 is correlated with disease severity, this study can prove ground-breaking. Although clinicians can use this to identify severity earlier and commence oxygen therapy sooner, the varying outcomes makes it somewhat difficult to ascertain what level of IL-6 corresponds to what negative outcome.

Furthermore, many studies recruited participants from the same centre, giving rise to the potential of selection bias. Table 3 summarises the studies used in analysing IL-6 and COVID-19.

White blood cells (WBCs), known as leucocytes are a component of blood generated from bone marrow and lymphoid tissue. They are divided into two major groups, granulocytes and agranulocytes. Within the granulocyte group are eosinophils, basophils and neutrophils (NC), whereas lymphocytes (LC) and monocytes are present in agranulocytes. A disproportionate number of these cells may reveal an underlying infection and hence can be measured using blood tests, producing a WCC. However, the reliability of WCC as a biomarker for COVID-19 remains unproven.

J o u r n a l P r e -p r o o f 9 0.001). NCs were predominantly driving this increase as the severe set (4.3 vs 3.2 × 10 9 /L ; P < 0.001). Interestingly, the levels of lymphocytes, monocytes, basophils and eosinophils were less, resulting in a greater neutrophil-to-lymphocyte ratio (NLR; 5.5 vs 3.2; P < 0.001). NLR is an infamous biomarker, high in wide-spread inflammatory conditions and can be used to reflect disease severity. However, a larger study is needed to clarify NLR's effectiveness as a biomarker.

Another conducted in China concludes similar findings of high NC and low LC count in severely affected patients, suggesting NLR could be a potential biomarker for early detection of severe COVID-19 (17) .

However, other factors may disrupt the accuracy of the WCC results observed.

These include glucocorticoid therapy and other underlying viral/bacterial infections (11) .

LC is separately addressed in the literature, secondary to NLR. A descriptive study in China reported depleted LC levels in the majority of COVID-19 patients (15) .

Another study has found low blood lymphocyte percentage (LYM%) in critically ill patients, suggesting low LC count indicates poor prognosis. However, since the virus can target lymphoid tissue and mechanisms of IL-6, other causes of low LC count must be investigated (18) . Similar to NLR, the clinical benefits of LC count as a biomarker for COVID-19 remains uncertain. Table 4 summarises the studies used in analysing WCC and COVID-19.

In glucose metabolism, the enzyme LDH converts pyruvate to lactate. LDH secretion is triggered by necrosis of the cell membrane, hinting to viral infection or lung Journal Pre-proof J o u r n a l P r e -p r o o f 10 damage, such as the pneumonia induced by SARS-CoV-2 (19) . There is convincing evidence linking LDH levels to the development of COVID-19 disease (20) .

A study found significantly higher levels of LDH in ICU patients than non-ICU patients (248 U/L vs 151 U/L, p=0.002). Since high levels of LDH continued in the ICU patients number of days post-admission (160 U/L vs 218 U/L, p=0.002), LDH may be a predictive biomarker of severe disease. However, the one centre study may be prone to selection bias which could potentially reduce its validity (21).

A multi-centre study involving 1099 patients reported supporting evidence correlating extent of tissue damage and inflammation with increasing levels of LDH (22) . Furthermore, when LDH levels were correlated with CT scans, significantly higher levels reflected the severity of pneumonia (23) .

There is increasing confidence in using LDH as a biomarker to measure severity of COVID-19 infection. Another study found that there was a significant rise in LDH levels among refractory COVID-19 patients (24). Table 5 summarises the studies used in analysing LDH and COVID-19.

D-dimer originate from the lysis of cross-linked fibrin with rising levels indicating the activation of coagulation and fibrinolysis (25) . Early studies have associated COVID-J o u r n a l P r e -p r o o f 11 Studies have reported that nearly 90% of inpatients with pneumonia had increased coagulation activity marked rising D-dimer levels (28) .

Furthermore, Huang et al. found that levels of D-dimer on admission could be used to triage patients into critical care (5) . The researchers found that median D-dimer levels were higher in ICU patients compared to non-ICU patients (2.4 mg/L vs. 0.5mg/L; p=0.0042). This, along with the previous study, suggest that D-dimer levels can be used as a prognostic marker and help clinicians monitor those who are likely to deteriorate earlier. However, this study confirmed the diagnosis of COVID-19 using lower respiratory tract specimens and did not use paired nasopharyngeal swabs to investigate the viral RNA detection rate between the upper and lower respiratory tract specimens. Secondly, with a cohort size of 41 patients, it is difficult to assess predictors of disease severity and mortality with multivariable-adjusted methods. Table 6 summarises the studies used to analyse D-dimer and COVID-19.

As seen with previous coronavirus outbreaks, COVID-19 infection leads to severe haematological changes leading to thrombocytopenia. A retrospective study which used Cox proportional hazard regression analysis found that platelet count is an independent risk factor for mortality among COVID-19 patients, where a 50x10 9 /L increase is associated with 40% deceased mortality (HR 0.60, 95%CI: 0.43, 0.84) (30) . Here, thrombocytopenia at admission was more likely to occur in non-survivors than in survivors. Although many risk factors were accounted for in this study, the possibility for unmeasured confounder cannot be excluded.

Another study corroborates the previously documented work. The nadir platelet count was significantly associated with mortalityand the lower the nadir, the stronger the association(31). Again, thrombocytopenia was more likely to occur in non-survivors than survivors. This study is from adequate sample sizes providing statistical power, however, similar to the previous studies, they are all retrospective making the correlation seen difficult to extrapolate from.

Testing the platelet count is a routine part of laboratory tests and the literature suggests it has inherent value in providing more detail on the patient's condition. Table 7 summarises the studies used in analysing platelet count and COVID-19.

There is growing evidence of higher mortality rates among those with underlying cardiovascular disease due to COVID-19 infection (22) . Some have investigated the use of high-sensitivity cardiac troponin I (hs-TnI) as a marker of disease progression and mortality. based on SARS-CoV-2 RNA detection, revealed a univariable odds ratio for death at 80.1 (95% CI 10.3-620.4, p<0.0001) for hs-TnI(32). This risk was higher compared to other biomarkers such as D-dimer and lymphocyte count. Another study of 416 hospitalised patients with COVID-19 reported that hs-TnI was elevated in 1 in 5 patients on presentation (33) . These patients were more likely to require invasive (22% vs 4%, p<0.001) or non-invasive (46% vs 4% , p<0.001) ventilation, develop ARDS (59% vs 15%, p<0.001) or acute kidney injury (9% vs 0%, p<0.001).

Early recognition of myocardial injury indicated by elevated hs-TnI aid in appropriate triage to a critical care area and inform the use of inotropes and vasopressors.

However, elevated levels are common in hospitalized patients and are likely to be due to non-ischaemic causes of myocardial injury. This may lead to inappropriate use of cardiology consultation and downstream testing and increased risk to cardiac physiology staff. Table 8 summarises the studies used in analysing hs-TnI and COVID-19.

There is also evidence that chronic kidney disease is associated with severe forms of COVID-19 infection (34) .

Studies have demonstrated significantly higher levels of renal biomarkers such as serum urea, creatinine and markers of glomerular filtration rate in severe cases (35) .

Since these results stem from the analysis of 28 patients, extrapolation across larger cohorts is more difficult.

A larger study of 701 patients revealed that elevated serum creatinine levels on admission correlated with severity due to significant abnormalities in the coagulation J o u r n a l P r e -p r o o f 14 pathway (36) . They also found that these patients were more likely to require mechanical ventilation or be placed in intensive care. Univariate Cox regression analysis found elevated creatinine levels was also associated with in-hospital mortality (HR 2.99, 95% CI: 2.00, 4.47). Proteinuria, haematuria and elevated urea levels had similar, if not larger, hazard ratios.

Interestingly, another study showed a potential role for urinalysis over serum markers of kidney function (37) . Here, abnormalities in the routine urine test on admission correlated strongly with disease severity. They go on to suggest that urinalysis may reveal kidney impairment more readily than evaluation of serum renal biomarkers. However, these tests were only carried out on admission and so patients in earlier stages of the infection had changes in serum levels obscured by compensatory kidney function. Hence renal abnormalities on admission may indicate higher risks of deterioration, ensuring appropriate triaging. Table 9 summarises the studies used in analysing renal markers and COVID-19.

COVID-19 is a rapidly spreading pandemic increasing the burden on medical facilities. Symptoms vary from mild fever to ARDS complicating diagnosis, prognosis, and monitoring. Hence it is vital to ascertain a patient's condition in a timely manner.

Biomarkers are quantitative measurements used clinically for many conditions reflecting pathological development. A summary of the biomarkers discussed in this review can be found in Table 10 .

When assessing a patient with COVID-19 infection, biomarkers can be useful to clinicians in starting treatment and close monitoring. Though biomarkers may help J o u r n a l P r e -p r o o f improve prognosis and outcomes, their significant variability between patients could affect the findings of the studies.

Compared to other biomarkers, there is hesitancy in using WCC alone as it is influenced by many factors such as glucocorticoid treatment which increases it.

Although WCC encompasses many cell types, NCs and LCs are most clinically relevant biomarkers. Multiple studies on COVID-19 have concurred high NLR in severe cases compared to non-severe cases due to high NC and low LC. The use of LC independently has been suggested as a potential biomarker of COVID-19 as patients have consistently low LC, with significant lymphopenia reported in critically ill patients (18) . Therefore, further research on WCC accuracy for assessing disease progression is necessary.

Although, most of the studies referenced in this review are single centred studies originating in Wuhan, China, the virus is now a global pandemic thus requiring international studies. As a copious amount of data was collected from the same location, it is possible patients may have been used in more than one study. Findings of these studies has discovered changes in biomarker levels and may potentially be useful in creating a therapeutic intervention. For instance, one study has reviewed the use of anticoagulation therapy in patients with coagulopathy or marked rise in D-dimers in the setting of COVID-19 (38) . Low molecular weight heparin was found to be associated with better prognosis in severe cases. To further asses the role of anti-coagulants as a treatment, we encourage large interventional trails to study this.

Many studies in this review were limited due to small sample size and selection bias if conducted at one centre. To improve reliability and reproducibility, more research into the prognostic value of biomarkers is necessary.

In conclusion, the work to date suggests that there is clear evidence of how the levels of biomarkers may change according to severity of COVID-19 infection. This can be used as an adjunct in clinical practice to guide treatment and admission to ICU. By doing so, it may improve prognosis and minimise the mortality rates. Journal Pre-proof J o u r n a l P r e -p r o o f 33 Hs-TnI = high sensitivity troponin I 

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Records identified through database searching (n=397)

Additional records identified through other sources (n=17)