








key: cord-347280-jpwf55l6
authors: Skevaki, Chrysanthi; Fragkou, Paraskevi C.; Cheng, Chongsheng; Xie, Min; Renz, Harald
title: Laboratory characteristics of patients infected with the novel SARS-CoV-2 virus
date: 2020-06-21
journal: J Infect
DOI: 10.1016/j.jinf.2020.06.039
sha: 
doc_id: 347280
cord_uid: jpwf55l6

A subgroup of COVID-19 patients develop very severe disease with requirement for ICU treatment, ventilation, and ECMO therapy. Laboratory tests indicate that the immune and clotting system show marked alterations with hyper-activation, hyper-inflammation, cytokine storm development. Furthermore, organ-specific biomarkers demonstrate the involvement of cardiac muscle, kidney, and liver dysfunction in many patients. In this article the use of laboratory biomarkers is discussed with regard to their use for diagnosis, disease progression, and risk assessment.

Although only a minority of COVID-19 patients show critical disease progression from moderate to severe stages of the disease including requirement for ventilation and ECMO therapy, this subgroup of COVID-19 patients requires particular attention. Data collection from several regions of the world including China, Europe, and the United States clearly demonstrate that COVID-19 is not only a disease of the lung and the airways. Many other organ systems are involved and contribute to disease variety and progression. With regard to the immune system, hyper-inflammation together with the development of exorbitant increased cytokine production represents a hallmark of severe patients requiring ventilation. Some of these patients develop bacterial superinfections with increased levels of sepsis markers. Another important systems which recently caused increased attention is the clotting system. This is particularly highlighted by the detection of increased levels of D-dimers. Organ dysfunction has been reported in many patients including the heart (myocardial muscle damage), the kidney, and the liver. Laboratory diagnostics play not only an important role in disease diagnosis, but also in assessing progression and severity in these patients. Furthermore, laboratory diagnostics allow early detection of organ dysfunction in many cases. Moreover, biotests are used to assess an increased mortality risk in severe lethal patients. In this article we summarize the most prominent findings in COVID-19 patients and discuss the use of these markers for diagnosis, disease progression, and risk assessment.

Retrospective analyses from China demonstrated that leukocyte counts were higher among non-survivors compared to recovered patients 1, 2 ; in particular, Zhou et al. reported that COVID-19 patients who did not survive had a median of 9.8x10 9 /L WBC count compared to 5.2x10 9 /L among those who survived (p<0.0001), although the exact time point of measurement was not defined in their methods. Furthermore, another study of 140 hospitalized patients in Wuhan, demonstrated significantly higher leukocyte counts among those with severe COVID-19 disease, compared to patients with milder infection (p=0.003) 3 . Finally, a series from the same center, and possibly overlapped populations with the previous study, reported significantly higher WBC counts upon hospital admission among patients requiring critical care, although median values were within normal range (WBC count median 6.6x10 9 /L for ICU vs 4.3x10 9 /L for non-ICU admission, p=0.003) 4 .

The observed leukocytosis is attributed to an elevation of neutrophils, as the other WBC populations seem to drop in severely ill and eventually fatal COVID-19 cases 5 . Absolute lymphopenia is commonly observed in patients with COVID-19, but pronounced lymphocyte depletion is a cardinal marker of enhanced disease severity and an indicator of imminent death, that has been consistently depicted by almost all currently published reports, coming mainly from China 1-7 . Importantly, not only the degree of lymphocyte drop, but also the persistence of low lymphocyte counts throughout the disease course have been associated with critical illness and death 1, 2, 4 . In contrast to previous reports for SARS-CoV, peripheral blood smears reveal the presence of reactive lymphocytes, including some lymphoplasmacytoids, in the majority of COVID-19 patients [7] [8] [9] . Severe SARS-CoV-2 infection depletes all lymphocyte subsets, including CD4+ T cells, CD8+ T cells, B cells and natural killer (NK) cells, but CD4+/ CD8+ ratio is not inverted as seen in other viral infections [10] [11] [12] [13] . Not only the absolute numbers of T-cells are reduced, but also receptors suppressing their cytotoxic effects, like the CD94/NKG2A receptor, are up-regulated leading to diminished defense mechanisms against the virus 10 .

Monocyte, eosinophil and basophil counts are also decreased in COVID-19, but the magnitude of this reduction has not been associated with disease severity, in currently published data from Chinese centers 3,10,14 .

Moreover, pro-inflammatory cytokines are known to blunt erythropoiesis 15 .

However, aside from one study that found significantly higher frequencies of decreased hemoglobin concentrations among severe (43.6%) and critical cases (37.2%) compared to mild/moderate ones (23.1%) (p<0.001), solid evidence of significant hemoglobin reduction in severe COVID-19 has not been consistently

reported as yet 5, 11, 16 . In one particular study, lower hemoglobin concentration was associated with increased odds for lack of disease improvement but not death (odds ratio 1.731, p=0.008) 16 .

Preliminary reports imply that high neutrophil counts and persistently deep lymphocyte nadir counts during hospitalization as well as high neutrophil to lymphocyte ratios (NLR) are indicators of adverse outcomes such as ICU admission and death 10 . A retrospective Chinese study reported that NLR, along with the SARS-CoV-2 IgG levels, could be used as a simple discriminative tool for severity between COVID-19 patients, and further predict the clinical outcome of these patients 14 . However, whether these indices can actually risk stratify patients and predict poor outcomes, most importantly at an early stage of the disease, remains to be addressed and validated in large prospective trials.

The regulation of ferritin synthesis is cytokine-controlled 17 Only scarce data have contextualized the erythrocyte sedimentation rate (ESR) kinetic in patients with COVID-19. One study reported that fatal cases had a tendency for higher ESR compared to those who recovered (median ESR 38.5 vs 28 mm/h) without reporting the statistical significance of the observed difference among the two groups 1 . A similar trend was also depicted for C-reactive protein (CRP) concentration by the same study, with median levels being 4-fold higher among non-survivors (median concentration 113 vs 26.2 mg/L) 1 . Between severe and non-severe cases, reported CRP differences are not that striking (median (IQR): 47.6 mg/L (20.6-87.1) vs 28.7 mg/L (9.5-52.1), p<0.001), but significantly increased frequency of higher concentrations among severe and critical cases compared to mild/moderate ones are nevertheless evident (mild/moderate cases: 50.5%, severe cases: 79.2% and critical cases: 92%, p<0.001) 3, 16 . Finally, one Chinese study with 663 COVID-19 patients reported that higher CRP levels are inversely associated with disease improvement (odds ratio 4.697, p<0.0001) 16 .

Individual studies demonstrate that greater procalcitonin (PCT) concentrations (usually ≥0.05 ng/ml) can significantly distinguish between non-severely from severely ill and fatal cases, thus possibly acting as a prognostic marker [2] [3] [4] 6, 18, 19 . However, a meta-analysis found that severe from non-severe COVID-19 could be differentiated by a marginally higher PCT (by 0.2 ng/ml) 5 . Increments of both CRP and PCT may be associated, not only with the immense inflammatory response, but also with the higher frequency of bacterial superinfections among critically ill COVID-19 patients (up to 50% rate among non-survivors) 5 Albumin is a negative acute phase reactant whose synthesis is down-regulated by inflammatory cytokines 21 . Therefore, it is not surprising that hypoalbuminemia (usually <30 g/L) has been persistently noticed among patients with severe or fatal COVID-19 1, 2, 6, 11 . Moreover, one study demonstrated that low albumin concentration was associated with lack of disease improvement (odds ratio 2.377, p<0.0001), while hypoalbuminemia was also introduced as a risk factor, among other parameters, in a proposed risk prediction nomogram for severe COVID-19 16, 19 .

Serum amyloid A (SAA) is another acute phase reactant inhibiting monocyte mobilization, platelet activation and various chemotactic pathways 21 .

High concentrations of SAA among all COVID-19 patients have only been reported by Zang et al., without a significant difference between severe and non-severe cases 3 .

Exuberant release of pro-inflammatory cytokines is associated with multi-organ injury and acute respiratory distress syndrome (ARDS), which is inevitably fatal if left untreated 22 . Fulminant hypercytokinemia has been increasingly recognized among critically ill COVID-19 patients. Moreover, the connection of viral spike protein to ACE2 receptor, down-regulates ACE2 levels in lungs; this in turn, increases the angiotensin II (AngII) levels, reduces angiotensin 1-7 (Ang-(1-7) ), and imbalances the renin-angiotensin system in the lung, leading to vasoconstriction 28 . These data are in concordance with a notably distinct type of ARDS with highly compliant lungs, which is seen in a major subset of COVID-19 patients; this manifestation is quite possibly consistent with an underlying vasoconstriction and microvasculature injury leading to loss of lung perfusion regulation 29 . Though neither histopathology specimens nor lung ACE2 or AngII levels are easily obtainable in daily clinical practice, they would definitely be useful in research settings in order to elucidate the disease's pathophysiology and may assist diagnosis in the future.

Cardiac troponin I and T are highly sensitive and specific biomarkers of myocardial injury which can be caused by myocardial ischemia, inflammation, immune response, and toxin 30 

According to a cohort study 34 of 701 patients with COVID-19, the proportion of proteinuria, hematuria, abnormal serum creatinine and urea nitrogen at admission and were 43.9, 26.7, 14.4 and 13.1%, respectively. In addition, there was a high prevalence (5.1%) of acute kidney injury (AKI) during the study period. The result showed proteinuria, hematuria, and elevated serum creatinine/urea nitrogen at admission and acute kidney injury (AKI) during hospitalization over stage 2 were associated with in-hospital death. However, the other largest retrospective study to date found that the prevalence of serum creatinine abnormalities and AKI was only 1.6% and 0.5% 18 . This may be due to the different proportions of severe patients between the two studies and the different definitions of the normal reference range for serum creatinine. From the result of autopsy of 26 COVID-19 patients 35 , the histopathology of the kidney revealed significant acute tubular injury and found that the tubular epithelial cells were directly infected by SARS-CoV2. Therefore, SARS-CoV2 may cause kidney injury or exacerbate existing kidney disease. Attention should be paid to monitoring renal function and the occurrence of AKI.

Abnormal liver function tests, such as increased levels of ALT, AST, TBIL, GGT and decreased level of albumin were relatively common in patients with COVID-19, and 10-33% of these patients had abnormal ALT or AST 2,4,18,31,36 .

Although patients with severe COVID-19 seem to have higher rates of liver dysfunction, it is reassuring that the levels of ALT, AST, TBIL, GGT in COVID-19 patients were not significantly different in compared with hospitalized community-acquired pneumonia patients and even the median or average transaminase level in severe COVID-19 patients was lower than twice upper reference limit 4, 36, 37 . Therefore, the clinical effect of these elevated indicators may not be evident in COVID-19 patients. Liver dysfunction may be related to severe infection, inflammation induced liver injury, medication associated hepatotoxicity and hypoxia 38 .

D-dimer is a degradation product of fibrin. Elevated D-dimer levels were consistently reported in COVID-19 patients with prevalence ranging from 43-68% 2, 3, 31 . D-dimer>1ng/ml at admission were associated with increased severity and odds of death with COVID-19, and the gradual increasing of D-dimer during disease course was particularly associated with disease worsening and mortality 2, 4 . Serum D-dimer can reflect fibrinolytic activities and is also an inflammatory biomarker. Furtherly, recent studies found that severe cases of COVID-19 were commonly complicated with thrombosis 39, 40 , markedly elevated D-dimer was related to thrombosis and poor prognosis of severe COVID-19 patients. of fatal cases 45 . In fact, the fibrinogen would decrease when excessive consumption happened due to hypercoagulability or the worst disseminated intravascular coagulation occurred. Hence, the abnormality of the coagulation profile should be interpreted individually.

Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that is present in every tissue, and high serum concentrations indicate underlying organ damage.

Thus, LDH is expected to rise in severe COVID-19 cases, where multi-organ damage occurs 23 . Current data support that critically ill patients as well as fatal cases of COVID-19 have significantly higher LDH levels (usually >320 U/L) compared to moderate infections 1, 2, 5, 6, 11, 16 . Moreover, higher LDH quadruples the odds for lack of disease improvement (odds ratio: 4.381. p<0.0001) 16 . Lastly, greater LDH concentrations upon admission correlate with a higher risk for serious COVID-19, and therefore it has been added in a proposed early predictive tool for severe infection 19 . These data favor the utilization of LDH as a candidate prognostic marker for disease severity.

Hypertriglyceridemia is commonly encountered in hyperinflammatory states, like the CSS and the secondary HLH, due to the reduced lipoprotein lipase activity driven by the high TNF-α levels 46 bicarbonate concentration in patients who died, without reporting the statistical significance of this finding 1,2 . Importantly, but not surprisingly, in the latter study more than 50% of the deceased patients had arterial partial pressure of oxygen (PaO2) of <60mmHg (compared to 0% in the survivor group), while none in the same group had a partial pressure of oxygen to fraction of inspired oxygen ratio (PaO2:FiO2) of >300 1 . Hence, arterial blood gases constitute important prognostic tools for disease severity and poor outcomes, as they are directly associated with the degree of functional lung damage.

SARS-CoV-2 infection causes systemic disease, involving multiple organs and systems, including hyperactivation of the immune system, the nervous system and the clotting system. These in turn leading to pathologies in several organs, including the heart, liver and kidneys. In order to stratify patients at risk and to monitor high risk patients at intensive care units, tight laboratory diagnostics provide instrumental information. Laboratory tests can be used as prognostic markers for increased risk and mortality. The spectrum of currently available biomarkers is sufficient to fullfill this purpose. A major limitation of available studies is that the time point of sampling/biomarker assessment since onset of symptoms and/or presentation at health care facilities is not clearly mentioned.

Furthermore, currently, there are no internationally acceptable criteria regarding disease severity, which renders evaluation of data quite subjective, depending on individual study investigations. Over the next months and years, with the use of further knowledge on the pathogenesis of SARS-CoV-2 infections, an even more comprehensive list of suitable biomarkers will be developed.

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GM-CSF: Granulocyte-macrophage colony-stimulating factor, IL: Interleukin, IP10:Interferon gamma-induced protein 10, K: Potassium, MASP 2: mannose binding lectin associated serine protease 2. MCP1 (CCL2): Monocyte chemoattractant protein 1, MIP-1α (CCL3): Macrophage inflammatory protein 1-alpha), Na: Sodium,PaCO2:Arterial carbon dioxide partial pressure, PaO2: Arterial oxygen partial pressure, SIADH: Syndrome of inappropriate antidiuretic hormone secretion

INR, international normalized ratio

Hs-troponin I, high sensitivity troponin I



