Версия для печати

SARS-CoV-2. INFLAMMATION MARKERS Ustyantseva I.M., Zinchenko M.A., Guselnikova Yu.A., Kulagina E.A., Aliev A.R., Agadzhanyan V.V.

Kuzbass Clinical Center of Miners’ Health Protection, Leninsk-Kuznetsky, Russia,

Kemerovo State Medical University, Kemerovo, Russia

 

The pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiated the significant increase in amount of scientific studies of various aspects of the problem, beginning from research of SARS-CoV-2 genome, epidemiological and clinical patterns of COVID-19, to development of actual therapeutic strategies and vaccines [1-4]. The new scientific information accelerates the procedures for prevention, diagnosis and treatment of the new coronavirus infection [5, 6], as well as realization of studies of COVID-19 complications [7, 8, 9].

SARS-CoV-2 presents the enveloped single-chain RNA-virus of Coronaviridae family of Betacoronaviruses genus. All coronaviruses are similar in organization and expression of their genome, which codes 16 non-structural proteins and 4 structural proteins: spike (S), enveloped (E), membrane (M) and nucleocapsid (N). The viruses of this family are of zoonotoc origin. They cause a disease, symptoms of which may vary from mild respiratory symptoms to more severe conditions such as severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS) and COVID-19 [7, 10].

SARS-CoV-2 is passed from human to human mainly by means of airborne way, but indirect contact transmission is possible [11, 12]. SARS-CoV-2 can be separated from the samples of materials of smears from mucosa of nasopharynx or oropharynx or from sputum. The virus enters the cell-targets through receptors of angiotensin converting enzyme of type 2 (ACE-2) which are mainly in the lungs [13].

It is considered that the incubation period of COVID-19 is 2-14 days after contact with the agent. In most cases, the symptoms appear approximately after 4-5 days. The interval when a person with COVID-19 is contagious is not clear yet. There were some cases of transmission from persons with symptoms, from persons shortly before occurrence of symptoms and from persons without symptoms. However, these findings are not final and require for future confirmation [14].

The infected persons can demonstrate such symptoms as increasing temperature, cough, fatigue, sputum discharge, anosmia, apnoea [15, 16, 17]. The range of symptoms of the infection varies from mild manifestations to critical ones, but most cases are not severe. The severe form of the disease is characterized by such symptoms as apnoae, hypoxia, > 50 % of lung infiltration within 24-48 hours. The severe form is mainly observed in adult patients of older age or with concurrent diseases such as arterial hypertension, diabetes mellitus and cardiovascular diseases [16, 17]. The main serious complication in patients with severe form of the disease is acute respiratory distress syndrome (ARDS). Critical conditions are characterized by respiratory insufficiency, shock and/or multiple organ dysfunction or failure. The proportion of severe and fatal cases varies highly in dependence on territorial distribution [18, 19, 20].

COVID-19 can be considered as systemic inflammatory response, which is characterized by life-threatening hyperinflammation, which finally causes multiple organ failure. More detailed understanding of COVID-19 pathogenesis can result in improvement.

Objective – to estimate the clinical and metabolic manifestations of systemic inflammatory response in patients with new coronaviral infection COVID-19 complicated by community-acquired pneumonia in critical conditions.

 

MATERIALS AND METHODS

The study was conducted after informed consent of patients (or their relatives if a patient was unable to communicate). It corresponds to Helsinki Declare (2013), and to the Rules for Clinical Practice in the Russian Federation (the Order by Health Ministry of RF, 19 June, 2003, No. 266) and has been approved by the ethical committee of Kuzbass Clinical Center of Miners' Health Protection, Leninsk-Kuznetsky.

A retrospective analysis included clinical cases of treatment of COVID-19 complicated by community-acquired pneumonia: 49 patients (28 men and 21 women in the intensive care unit at Kuzbass Clinical Center of Miners' Health Protection from July, 2020, to November 30, 2020.

The cases were considered according to the registration date of positive test of COVID-19.

All required variables used in this study, individually for each critically ill patient were received from the database of Medical Information System (MIS) on the data of positive result of RNA of SARS-Cov-2.

A degree of severity of the new coronavirus infection was estimated according to Temporary Methodical Recommendations. Prevention, Diagnosis and Treatment of New Coronavirus Infection (COVID-19) of Health Ministry of RF. The version 9, 1.10.2020.

The demographic data (sex, age), comorbid status (0, 1-2, 3+ concurrent disease), all cases of complications (acute respiratory distress syndrome (ARDS), disseminated intravascular clotting (DIC), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF), sepsis, septic shock), vital functions (temperature, respiratory rate (RR), heart rate (HR), systolic, diastolic arterial pressure (SAP, DAP), SpO₂, PaO₂ /FiO_2, changes in lungs on CT images) were registered.

qSOFA [21] was used for clinical description of patients and for estimation of organ dysfunction. Glasgow Coma Scale (GCS) was used for estimation of consciousness. Presence of sepsis signs was identified in compliance with Sepsis-3 criteria [21].

The ICU stay was estimated with consideration of amount of days of artificial lung ventilation (ALV).

Also the discharge status (survival or death) was considered. The survival group was 39 patients, deceased − 10 (the table 1). For etiological laboratory diagnosis of COVID-19, we used the samples of nasal and/or oropharyngeal smears, for identification of RNA SARS-CoV-2 − loop isothermal amplification with reagents Isoterm SARS-CoV-2 RNA-screen according to technical conditions 21.20.23-069-26329720-2020 (AO GENERIUM, Vladimir region, Volginskiy town).

Doubtful biological samples were tested again within 48 hours in the virological laboratory at Center for Hygiene and Epidemiology in Kemerovo Region (Kemerovo) with polymerase chain reaction with hybridization-fluorescent detection with reagents Vector- PCR- 2019 − nCoV − RG according to technical conditions 21.20.23 -088-05664012-2020 (Novosibirsk, Koltsovo township).

The study program was realized with use of laboratory methods.

The samples of peripheral venous blood in test tubes with K₃EDTA anticoagulant (Becton Dickinson) were studied with hematological analyzer Sysmex XN-1000 (Sysmex Co., Japan) within 2 hours after collection of samples. The main parameters, including the calculation of leukocytes, absolute and relative number of neutrophils and immature granulocytes (IG), as well as extended inflammation parameters (NEUT-GI − neutrophil granulation intensity; NEUT-RI − neutrophil reactivity intensity; RE-LYMP − calculation of reactive lymphocytes; AS-LYMP − calculation of antibody synthesizing lymphocytes), were measured.

At the same time, serum samples were estimated for urea, creatinine, electrolytes, glucose, AST, ALT, bilirubin, total protein, albumin, high sensitive troponin (hT_s), ferritin, C-reactive protein (CRP) on the analytic module platform Cobas 6000 SWA (Switzerland). The values of acid-alkaline state (pH, pO₂, pCO₂) and lactate in whole venous blood were estimated with analyzer of critical states Cobas b221 (Germany).

The coagulogram parameters (APTT, PT, fibrinogen, D-dimer) were measured with automatic hemostasis system STA Compact Max (France).

The statistical analysis of data was conducted with IBM SPSS Statistics 21 (Statistical Product and Service Solutions – SPSS).

The qualitative signs are presented as absolute and relative (%) values. The quantitative variables are presented as mean arithmetic (M) and standard deviation (SD) in view of Me (LQ-UQ), with Me − median, (LQ-UQ) − interquartile range (IQR) (LQ − 25 %, UQ − 75 % quartiles). Kolmogorov-Smirnov test was used for testing of character of distribution of quantitative values between groups according to quantitative signs with use of non-parametrical Mann-Whitney U-test. Qualitative signs were compared with use of Fisher's exact test and χ²-test. The critical level of significance (α) for testing statistical hypotheses was 0.05. The differences were statistically significant at p < 0.05.

 

RESULTS AND DISCUSSION

The mean age of patients with COVID-19 complicated by community-acquired pneumonia was Me (IQR) 65 (55-72) years. Most of them were men (57 %). The groups had some differences in age. The deceased patients were 1.15-fold older as compared to survived ones (p < 0.05) (the table 1).

Table 1

     Characteristics of patients with new COVID-19 complicated by community-acquired pneumonia

Patients (n)	Survivors (39)	Deceased(10)	p
Age, y., median (IQR)	60 (55-62)	69 (65-72)	0.05
Sex, n (%)
Male	22 (56)	6 (60)	1.00
Female	17 (44)	4 (40)
Comorbidity (concurrent diseases), % 1
No concurrent diseases	12	0	< 0.001
1-2 concurrent diseases	72	59.8	< 0.001
3+ concurrent diseases	16	40.2	< 0.001
Lung lesion volume in CT, n (%):
Minimal or average, CT 1-2	25 (64 %)	2 (20 %)	< 0.001
High or subtotal, CT 3-4	14 (36 %)	8 (80 %)	< 0.001

Note: Me – median, (IQR) – interquartile range; * Fisher’s exact test and χ²-test. 

¹ Percentage for each comorbidity is calculated with consideration of all patients with at least one concurrent disease. 

Estimation of comorbidity categories showed no concurrent diseases in 12 %, 72 % of patients had comorbidity category 1-2, 16 % − 3 and more concurrent diseases. The group of deceased patients had 3 concurrent diseases 2.5 times more often than survived patients (p < 0.001) (the table 1). The most common diseases were hypertonia (70 %), ischemic heart disease (59 %), diabetes mellitus (28 %), obesity of degree 3-4 (28 %).

There were some statistically significant differences in disease severity. In patients with lethal outcomes, severity of condition was severe and extremely severe almost 2.2 times more often as compared to survived patients (p < 0.0001). The differences were determined mainly by pattern and volume of lung lesion. So, in 80 % of lethal cases, the volume of lung lesion was significant and subtotal (CT 3-4) that characterized the course of severe bilateral multisegmental community-acquired pneumonia (the table 1).

The characteristics of clinical, physiological and laboratory parameters are presented in the table 2.

Table 2

Characteristics of clinical, physiological and laboratory parameters in patients with new COVID-19 complicated by community-acquired pneumonia  

Clinical Characteristics          

			Выжившие Survivors (39)			Умершие Deceased (10)
Physiology Measures		Mean (SD)		Median	IQR	Mean (SD)	Median	IQR	р
Systolic blood pressure, mm Hg		129 (18)				100 (35)			< 0.01**
Diastolic blood pressure, mm Hg		86 (19)				67 (24)			0.01**
Mean arterial pressure, mm Hg		100 (16)				78 (27)			0.01**
Heart rate, bpm		89.8 (18.9)				85.6 (25.9)			< 0.01**
Temperature, °C		37.3 (0.6)				38.4 (0.7)			< 0.05**
Respiratory rate,  breaths per min				22	18-25		32	25-36	< 0.05**
Oxygen saturation, %				93	90-96		85	83-90	0,04**
Clinical scores		Mean (SD)		Median	IQR	Mean (SD)	Median	IQR	р
Glasgow Coma Scale		13.5 (3.5)				7.9 (5.3)			0.06*
q SOFA		1.9 (0.43)				3.6 (0.54)			0.01*
Laboratory measures	Reference range	Mean (SD)		Median	IQR	Mean (SD)	Median	IQR	р
Glucose, mmol/l	3.9-6.1			8.7	12.2-16.5		9.34	13.1-18.8	0.41**
White blood cell count (×109/l)	4.0-10.6			8.8	9.8-21		14	12-25	0.60**
AS-LYMP, %				0	0.2 (0.1-0.3)		1.2	0.3-1.4	< 0.001**
RE-LYMP, %				0-5	1.3 (0.4-1.4)		1.8	0.3-2.4	1.0*
Creatinine, µmol/l	80-130			140	100-150		320	100-550	0.91**
Arterial blood gas, pH	7.39-7.42	7.33 (0.07)				7.64 (0.08)			0.02**
PaO2/FiO2		313 (137)				218 (100)			0.03**
Lactate, mmol/l	< 2	2.5 (1.8)				3.8 (3.0)			< 0.001**
CRP, mg/l	0-5	87.1 (51.2)				255.1 (138.2)			< 0.001**
hTs, pg/ml	1-15	23.9 (19.1)				29.5 (27.1)			< 0.05**
Ferritin, ng/ml	30-400	249.8 (167.3)				753.2 (520.8)			< 0.001**
Fibrinogen, g/l	2-4	5.73 (5.2)				11.37 (9.1)			< 0.001**
(%)  Prothrombin time (PTT), sec. (%)	70-120	65 (63)				60 (59)			< 0.05**
APTT, sec.	20-40	39.7 (31.5)				43.9 (41.5)			< 0.05**
D-dimer, µg/ml	≤ 0,5	0.78 (0.56)				9.54 (8.7)			< 0.001**
Outcomes				Median	IQR		Median	IQR	р
Length of stay, days				11	5-15		30	22-54	< 0.01*
ICU length of stay, days				3	1-4		17	12-25	< 0.01*
Ventilator-dependent days				0	0-1		16	9-21	< 0.01*

Note: M (SD) – mean value (quadratic deviation): Me – median, (IQR) – interquartile range; 
* Fisher’s exact test and χ²-test; ** Mann-Whitney’s U-test. 

The deceased patients demonstrated much lower mean arterial pressure, high HR, temperature and RR, as well as decreased indices of blood oxygen saturation by 9.4 % as compared to survived patients (p = 0.04) (the table 2).

The level of GCS was almost 1.7 times lower in deceased patients as compared to survived ones (p = 0.06). They also had higher level of qSOFA (1.9 times higher, p = 0.01) (the table 2).

The deceased patients had higher blood level of leukocytes and glucose as compared to survived ones (the table 2). pH values (p = 0.02) and РаО₂/FiO₂ (р = 0.03) showed some statistically significant intergroup differences. Lactate level was 1.5 times higher in deceased patients (p < 0.001).

Intense generalized manifestation of systemic inflammatory response in deceased patients was testified by significant increase in blood level of CRP (by 2.9 times, p < 0.001), hT_s (by 1.2 times, p < 0.05), ferritin (by 3 times, p < 0.001), fibrinogen (by 2 times, p < 0.001), D-dimer (by 12.2 times, p < 0.001), as well as prolongation of APTT by 1.1 times as compared to survived patients (p < 0.05) (the table 2).

Estimation of functional activity of neutrophils with use of hematological analysis of extended inflammation parameters showed that development of inflammatory response in patients with lethal outcomes was characterized by increasing level of AS-LYMP (by 35 % on average, p < 0.001) as compared to survived patients (Fig. 1). At the same time, NEUT-RI, NEUT-GI and RE-LYMP were within the normal ranges in patients of the studied groups (Fig. 1). Only one case with a patient who died after sepsis showed a significant increase in such values: NEUT-RI = 86.5 FI, NEUT-GI = 169.2 SI, IG = 19 %.

Figure 1

The scattergram of distribution of populations of leukocytes in the patient P. with new coronaviral infection COVID-19 complicated by community-acquired pneumonia.

The scattergram of the clinical case shows AS-LYMP and RE-LYMP characterizing activated B-lymphocytes in quantitative form.   

 

Patients with lethal outcomes showed an increase in hospital stay (p < 0.01), including ICU stay, and increasing amount of ALV days (the table 2).

The causes of lethal outcomes were identified. The most common causes of death were cardiovascular complications (39.1 %), multiple organ failure (47.1 %) and sepsis (13.8 %).

 

DISCUSSION

The presented study, the main objective of which was determination of intensity of generalized inflammation in patients with COVID-19 complicated by community-acquired pneumonia, which was caused by SARS-CoV-2, showed that the course of this disease was accompanied by the cascade of abnormal processes relating to activation of systemic inflammatory response. As result, one can observe the fast development of multiple organ failure with predominant lesion of lungs, kidneys and liver [16, 17]. Moreover, concurrent diseases can partially explain the increase in mortality from COVID-19 [18-20]. Condition severity in deceased patients was determined by combination of age and presence of 3 and more concurrent diseases, mainly cardiovascular ones. So, an abnormal increase in cardiospecific troponin in the blood of deceased patients was identified 1.25 times more often (p < 0.05) as compared to survived patients. Along with progression of severity hypotension severity, we could observe an increase in lactatemia which was more intense in deceased patients.

Critical conditions are associated with fast worsening of oxygen status (oxygen delivery and consumption, pO₂, Hb (Hct); acid-base metabolism (pH, pCO₂, BE); levels of electrolytes (Na+, К+, Са++, Сl-); the main laboratory values of metabolism and hemostasis changes. Correspondingly, the basis of treatment of patients with critical conditions is correction of respiratory disorders, acid-base metabolism, water-electrolyte exchange, hemostasis and others.

COVID-10 pandemic enforced the medical society to revise a lot of common opinions, including ones, which relate to sepsis diagnostics. Ferritin, D-dimer, troponin, some interleukins, immunoglobulins, lymphocytes and other values have been added to traditional parameters: procalcitonin, CRP, white cell count.

One of the causes of hyperferritinaemia, which has the highest intensity in deceased patients with COVID, can be iron overload (both heme and free). Since it is known that systemic inflammation causes the effect of iron sequestration in macrophages with subsequent hyperactivation of and development of cytokine storm [22].

Moreover, we identified a significant increase in AS-LYMP − by 6 times (p < 0.001) in deceased patients as compared to survived. Probably, a combination of AS-LYMP and RE-LYMP parameters allows receiving additional information on cellular activation of inborn and adaptive immune response. The high values of fluorescence of these cellular populations testify a high cellular activity and changes in membrane composition. Therefore, they present a sign of cell-mediated or humoral immune response to pathogens [23, 24]. It allows differentiating viral and bacterial infections.

The presented study has some important methodological limitations. Particularly, this is a retrospective study with small number of cases that already presents some disadvantages. The heterogeneity of patients' populations with consideration of disease severity has not been considered. Moreover, the dynamic changes in inflammation markers were not estimated, and prehospital interventions and therapy were not considered.

 

CONCLUSION

The received data demonstrate that new coronaviral infection COVID-19 caused by SARS-Cov-2 can be considered as systemic inflammatory response, which is characterized by life-threatening hyperinflammation, hypercoagulation and disbalance in oxygen delivery/consumption, resulting in multiple organ failure. These abnormal processes are especially important in patients with concurrent diseases, which increase the risk of severe course of COVID-19 and its association with systemic inflammatory response.

 

Information on financing and conflict of interests

The study was conducted without sponsorship. The authors declare the absence of any clear or potential conflicts of interests relating to publication of this article.