C-Reactive Protein, Procalcitonin, and a Novel Pathogenesis and Therapeutic Target of Thrombocytopenia in Sepsis.

Objective: The aim of the study is to analyze the clinical characteristics, pathogen distribution, and drug sensitivity information of adult sepsis, and to provide reference for empirical clinical use; to explore the relationship between C-reactive protein (CRP) and calcitonin (PCT) The clinical value in the diagnosis of adult sepsis.
Methods: We collected 455 cases of hospitalized patients with positive blood culture, including 352 cases with sepsis and 103 cases without sepsis; 1609 cases of hospitalized patients with suspected infection and negative blood culture, including 287 cases of sepsis, and 518 cases of non-infectious systemic inflammatory response syndrome (SIRS) and 804 cases of local infection. Age, gender, route of admission, admission status, CRP, PCT, and white blood cell (WBC) levels were collected from the patients. The differences between the factors were statistically analyzed, and the receiver operating characteristic curve (ROC curve) was plotted to obtain the optimal cut-off values of CRP and PCT and their area under the curve (AUC), and to compare the CRP, PCT and PCT, and the CRP + PCT tandem to diagnose sepsis sensitivity and specificity.
Results: (1) 387 pathogenic strains were isolated from blood cultures of patients with sepsis, 71.06% Gram-negative, 26.87% Gram-positive and 2.07% fungi. (2) Among Gram-positive bacteria, Staphylococcus aureus was 87.5% resistant to penicillin and sensitive to vancomycin, milantropine, and teicoplanin; among Gram-negative bacteria, Escherichia coli was resistant to piracillin The resistance rate was 73.1%, fully susceptible to viraemia (100%) and resistant to imipenem, amikacin, and lacillin/tazole. (3) Among patients with positive blood cultures, CRP and PCT levels were higher in patients with sepsis than in those without sepsis. Pairwise comparison of ROC curves showed that the diagnostic value of PCT was greater than that of CRP (P=0.016).
Conclusion: CRP and PCT have a good reference value for diagnosis of sepsis patients and determination of the degree of infection in septic patients, especially PCT is more valuable for diagnosis of sepsis.

1. Introduction

Bacterial sepsis is an infectious disease that, if not controlled and treated in a timely manner, can develop into severe sepsis, posing a serious threat to the life and health of patients [1]. In recent years, the incidence of this disease has not only increased gradually but also the morbidity and mortality rate has become high, with 500,000 bacterial sepsis patients losing their lives worldwide every year [2]. A study [3] showed that the morbidity rate of sepsis patients in ICU was 8.68%, mortality rate was 44.7%, and the per capita medical cost was about $502/day. A study [4] found that mortality in patients with sepsis in the ICU has surpassed myocardial infarction as the leading cause. Although much research and progress has been made in recent years on its mechanisms and treatment, the mortality rate of sepsis remains high, reaching 20%–50% [5, 6]. Therefore, early and correct diagnosis and assessment of the severity of the disease, how to prevent the misuse of antibiotics and improve the prognosis of patients through individualized treatment are the focus of clinical research.

PCT, first proposed in 1984, is a pro-peptide substance of calcitonin, which is mainly produced by thyroid C cells under physiological conditions; it contains a protein of 116 amino acids with a molecular mass of 14.5 kD, and PCT has a half-life of 25–30 h in the blood [7, 8]. The study [9] found that the level of PCT correlates well with the severity of the infection, and the more severe the infection, the higher its level. It is generally considered that PCT levels less than 0.5 ug/L are within the normal range; PCT levels are greater than 2 ug/L. Serum PCT levels are slightly higher when the body has a fungal infection or a parasitic infection, but are significantly higher in the case of non-bacterial infections. When the body is invaded by a virus, serum PCT levels generally do not increase. Therefore, PCT can be used for the differential diagnosis of bacterial or viral infections and provide some help in clinical treatment, but it cannot effectively identify bacterial infections or sepsis caused by nonbacterial infections [10].

CRP is a plasma protein produced by the liver and is considered to be a marker of inflammatory response. Studies [11, 12] first identified CRP as a substance that precipitates with C polysaccharide (CPS), a nonspecific bacterial polysaccharide component of Streptococcus pneumoniae, in the serum of patients with Streptococcus pneumoniae. Studies [13, 14] showed that its biological function may be to clear pathological substances or pathogens from the body by binding to phospholipid components of apoptotic and necrotic cells or invading bacteria, fungi, parasites and other pathogens and activating the complement and mononuclear phagocyte systems. Studies [15, 16] have shown that serum concentrations are below 10 mg/L in normal subjects, but in patients with severe infections or sepsis, CRP concentrations rise rapidly 6 hours after the onset of stimulation and reach a maximum within approximately 48 hours, with concentrations decreasing rapidly and returning to normal within 1 week, whereas concentrations do not increase in virally infected individuals, but this also provides an extremely important basis for determining the type of early infection The basis for this study was the following.

In this study, we included patients with negative and positive blood cultures, analyzed clinical data from patients with sepsis, compared the differences in CRP and PCT levels between patients, and investigated the role of these two indicators in the diagnosis of sepsis. Optimal threshold values were obtained, and the sensitivity and specificity of the indicators for the diagnosis of sepsis at the threshold values and the relationship between CRP and PCT levels and the severity of sepsis were analyzed.

2. Materials and Methods

2.1. Study Subjects and Groupings

Cases of blood culture-positive inpatients and suspected infectious, blood culture-negative inpatients admitted to our hospital from January 2015 to July 2021 were collected for retrospective analysis. The study population was divided into blood culture-positive sepsis, blood culture-positive non-septic, blood culture-negative sepsis, blood culture-negative non-infectious SIRS, and blood culture-negative localized infection (Table 1), according to the diagnostic criteria for sepsis established by the American Thoracic Society/American Academy of Critical Care Medicine consensus conference, and the diagnostic criteria for SIRS (Table 2).

Table 1

Grouping of study subjects and diagnostic criteria.

Grouping	Diagnostic criteria
Blood culture positive sepsis	Determination of SIRS caused by pathogen infection
Blood culture positive non sepsis	It was determined that pathogen infection did not cause SIRS
Blood culture negative sepsis	Sirs caused by suspected infection or local infection
Blood culture negative non infected SIRS	No evidence of infection and SIRS
Blood culture negative local infection	Etiology showed that local infection did not cause SIRS

Table 2

Diagnostic criteria for SIRS.

SIRS	At least 2 symptoms are met
Symptom 1	Body temperature >38°C or <36°C for two consecutive times
Symptom 2	Heart rate >90 beats per minute
Symptom 3	Respiratory rate >20 times/min or PaCO2 <32 mmhg
Symptom 4	WBC >12 × 109/L or <4 × 109/L or immature cells >10%

2.2. Inclusion Criteria

The inclusion criteria were as follows:

Hospitalized patients over 18 years old.

The etiological data are complete.

The disease course information recorded in medical records is complete.

2.3. Exclusion Criteria

The exclusion criteria were as follows:

Have a hospitalization record and have used antibiotics 2 weeks before admission.

Patients who have been included in study cohort with repeated admissions.

Died or discharged within 24 hours after admission.

Emergency biochemical and procalcitonin examinations were not performed.

2.4. Pathogen Detection and Drug Resistance Detection

5 mL of fasting elbow venous blood was collected from all patients within 24 h after admission, placed in EDTA-Na anticoagulated EP tubes, centrifuged at 3 500 r/min for 10 min, and the serum was separated and placed at −70°C for measurement. The PCT level was measured by electrochemiluminescence with a Roche e411; the CRP level was measured by immunoturbidimetric method, and platelet-related parameters including platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), and large platelet ratio (P-LC) were measured by hematocrit analyzer in all patients after the onset of disease. R). All operations were performed in strict accordance with the standard instructions. After identification of the samples, drug sensitivity analysis was performed using the Beidou Phoenix IM-100 fully automated microbiological analysis system; some drug sensitivity tests were performed using the K-B method or MIC broth dilution method, and the results were judged according to the American Clinical Laboratory Standardization Institute (CLSI) standards, and the specific instruments and manufacturers are shown in Table 3.

Table 3

Main instruments and manufacturers used in test process.

Main instruments	Manufacturer
BD Phoenix-100 Automatic Microbial Identification Instrument	Becton Dickinson
BD Bactec™ FX Blood Culture System	Becton Dickinson
BD Bruker Mass Spectrometer Microflex Lt/Sh System Electrothermal Incubator	Brooke Dalton company
Electric constant temperature incubator	Shanghai Xinmiao
Ultralow temperature freezer	Fuzhou Keze
Biological safety cabinet	Shanghai Lixin
Clean workbench	Suzhou Antai Air Technology Co., Ltd
Micropipette	Eppendorf, Germany
High-speed cryogenic centrifuge	Eppendorf, Germany
Automatic biochemical analyzer cobas C501-2	Roche
Automatic electrochemiluminescence immunoassay analyzer cobas e601	Roche
Sysmex XN-3000 automatic blood cell analyzer	Sisen Meikang medical electronics (Shanghai) Co., Ltd
SLAN-96S real-time PCR system	Zhishan biotechnology Co., Ltd

2.5. Detection of CRP, PCT, and WBC

Samples for CRP, PCT, and WBC testing were obtained from fresh or during hospitalization of patients. CRP was detected by automatic biochemical analyzer Cobas C501-2; PCT was detected by automatic electrochemiluminescence immunoassay analyzer Cobas e601; WBC was detected by Sysmex XN-3000 automatic blood analyzer. The specific instruments and manufacturers are shown in Table 3.

3. Results

A total of 387 strains of pathogenic bacteria were isolated from blood culture of 352 patients, including 275 strains (71.06%) of gram-negative bacteria, 104 strains (26.87%) of gram-positive bacteria, and 8 strains of fungi (2.07%). The top three pathogenic bacteria with composition ratio are as follows: 134 strains (34.63%) of Escherichia coli, 79 strains (20.41%) of Klebsiella pneumoniae and 40 strains (10.34%) of Staphylococcus aureus. The distribution of pathogenic bacteria detected in blood cultures of 387 adult septic patients is shown in Table 4 and Figure 1.

Table 4

Distribution of pathogenic bacteria detected in 387 adult blood cultures.

Pathogenic bacteria	Number of cases (n = 387)	Composition ratio (%)
Total G− bacteria	275	71.06
Escherichia colibacillus	134	34.63
Klebsiella pneumoniae	79	20.41
Pseudomonas aeruginosa	23	5.94
Acinetobacter baumannii	15	3.88
Burkholderia	5	1.29
Citrobacter	4	1.03
Enterobacter spp.	2	0.52
Myxobacteria	2	0.52
Klebsiella oxytoca	2	0.52
Proteus mirabilis	2	0.52
Aeromonas hydrophila	2	0.52
Salmonella typhi	2	0.52
Corynebacterium striatum	1	0.26
Salmonella enteritis	1	0.26
Pseudomonas putida	1	0.26
Total G+ bacteria	104	26.87
Staphylococcus spp.	54	13.95
Staphylococcus aureus	40	10.34
Staphylococcus epidermidis	5	1.29
Staphylococcus haemolyticus	4	1.03
Staphylococcus capitis	2	0.52
Staphylococcus hominis	2	0.52
Staphylococcus saprophyticus	1	0.26
Streptococcus spp.	31	8.01
Streptococcus pneumoniae	11	2.84
Streptococcus sanguis	4	1.03
Streptococcus viridans	3	0.78
Group G Streptococcus	3	0.78
Streptococcus constellatus	3	0.78
Streptococcus oralis	2	0.52
Streptococcus anginosus	2	0.52
Streptococcus bovis	1	0.26
Streptococcus salivarius	1	0.26
Streptococcus anoxy	1	0.26
Enterococcus spp.	18	4.65
Enterococcus faecium	10	2.58
Enterococcus faecalis	6	1.55
Enterococcus gallinarum	1	0.26
Enterococcus caseliflavus	1	0.26
Arcanobacterium pyogenes	1	0.26
Fungus	8	2.07
Candida spp.	7	1.81
Cryptococcus neoformans	1	0.26

Figure 1

Distribution of pathogenic bacteria detected in blood culture of 387 adult sepsis patients.

During the study period, 40, 5, 4, 2, 2, 2, and 1 cases were infected with Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus cephalosporus, Staphylococcus humanus, and Staphylococcus saprophyticus, respectively. Among the Gram-positive bacteria, S. aureus was 87.5% resistant to penicillin and sensitive to vancomycin, mirantolimus, and teicoplanin; other types of staphylococci were more resistant to penicillin, benzocillin, and erythromycin and sensitive to rifampicin, linezolid, vancomycin, mirantolimus, and teicoplanin, as shown in Table 5; streptococci were up to 80% resistant to erythromycin and clindamycin. The resistance rate to penicillin, cephalosporin, linezolid, and vancomycin was low, and the sensitivity rate was over 90%, as shown in Table 6. Among Gram-negative bacteria, E. coli had a resistance rate of 73.1% to piracillin, complete susceptibility (100%) to viraemia, and resistance to imipenem, amikacin, and laracillin/tazole. The susceptibility rate of bactam was above 95%; Klebsiella pneumoniae was 24.1% resistant to piroxicillin and relatively sensitive to amikacin with a sensitivity of 96.2%; Pseudomonas aeruginosa was resistant to amikacin, fully sensitive to gentamicin (100%) and had a relatively low antibiotic resistance rate; Acinetobacter baumannii had a relatively high antibiotic resistance rate and was 53.3% sensitive to amikacin, as Table 7 shows.

Table 5

Resistance rate of Gram-positive Staphylococcus aureus (%).

Antimicrobial agents	Staphylococcus aureus (n = 40)		Drug resistance
	Sensitive	Intermediary
Penicillin	10.0	2.5	87.5
Erythromycin	50.0	7.5	42.5
Ciprofloxacin	77.5	2.5	20.0
Clindamycin	80.0	2.5	17.5
Gentamicin	82.5	0	17.5
Oxacillin	85.0	0	15.0
Compound xinruoming	95.0	0	5.0
Li Fuping	92.5	2.5	5.0
Linezolid	97.5	0	2.5
Vancomycin	100	0	0
Furantoin	100	0	0
Teicoplanin	100	0	0

Table 6

Resistance rate of Gram-positive other staphylococci (%).

Antimicrobial agents	Other staphylococci (n = 14)		Drug resistance
	Sensitive	Intermediary
Penicillin	21.4	0	78.6
Erythromycin	28.6	0	71.4
Oxacillin	28.6	0	71.4
Ciprofloxacin	42.9	0	57.1
Gentamicin	64.3	0	35.7
Compound xinruoming	78.6	0	21.4
Clindamycin	85.7	0	14.3
Li Fuping	100	0	0
Linezolid	100	0	0
Vancomycin	100	0	0
Furantoin	100	0	0
Teicoplanin	100	0	0

Table 7

Gram-positive Streptococcus drug resistance rate (%).

Antimicrobial agents	Streptococcus (n = 31)		Drug resistance
	Sensitive	Intermediary
Erythromycin	19.4	0	80.6
Clindamycin	19.4	0	80.6
Levofloxacin	87.1	0	12.9
Chloramphenicol	87.1	0	12.9
Cefotaxime	90.3	0	9.7
Penicillin	93.5	0	6.5
Linezolid	96.8	0	3.2
Vancomycin	96.8	0	3.2

A total of 455 hospitalized patients were included, including 352 patients with sepsis and 103 patients without sepsis. There was no difference in gender and age between sepsis and nonseptic patients (P > 0.05), but there was a statistical difference in admission route. Patients with toxemia had a higher proportion of emergency admissions and were relatively critically ill on admission, while nonseptic patients had relatively average admissions. In addition, levels of CRP (146.4, 115.9) mg/L in sepsis were higher than nonseptic CRP (58.9, 65.1) mg/L (P < 0.001), and levels of PCT in sepsis (12.6,45.0) ng/mL level was higher than PCT in non-septic (0.4, 0.8) ng/mL (P < 0.001), WBC in κ toxemia (10.7, 9.2) × 109/L and non-septic WBC (10.7, 9.2) × 109/L There was a statistical difference between WBC (8.2, 6.2) × 10°/L in toxemia (P < 0.001), and WBC was converted into a categorical variable for analysis, see Table 8 and Figure 2 for details.

Table 8

Comparison of clinical data between sepsis and nonsepsis patients.

	Total	Sepsis	Non sepsis	P
Number of cases	455	352	103	—
Gender
Male	249	192 (54.3%)	57 (55.5%)	—
Female	206	160 (45.3%)	46 (44.5%)	0.888
Age	63.4 ± 17.6	64.4 ± 17.3	59.9 ± 18.4	0.732
Admission route
Emergency treatment	195	173 (49.3%)	22 (21.6%)	—
Outpatient department	260	179 (50.7%)	81 (78.8%)	<0.001
Admission
Critical	76	70 (19.7%)	6 (5.9%)	—
Urgent	131	119 (33.6%)	12 (11.5%)	—
Commonly	248	163 (46.5%)	85 (82.3%)	<0.001
CRP (IQR) mg/L	124.3 (119.5)	146.6 (115.7)	58.7 (65.3)	<0.001
PCT (IQR) ng/mL	6.4 (37.7)	12.5 (45.2)	0.6 (0.7)	<0.001
WBCa (IQR) × 109/L	10.7 (9.4)	11.5 (10.4)	8.4 (8.4)	<0.001
WBCb	—	—	—	—
>12 × 109/L or <4 × 109/L	253	218 (61.7%)	35 (34.2%)	—
4 × 109/L∼12 × 109/L	202	134 (38.2%)	68 (66.2%)	<0.001

Figure 2

Comparison of CRP, PCT, and WBC levels between blood culture-positive sepsis and non-sepsis. Note: CRP: C-reactive protein; PCT: procalcitonin; WBC: white blood cell count; : P < 0.001. CRP, PCT and WBC levels were compared using Wilcoxon rank sum test for two independent samples.

In patients with positive blood cultures, CRP and PCT levels were higher in sepsis than in non-sepsis. ROC curve analysis showed that the ACC for CRP, PCT and CRP + PCT tandem for the diagnosis of sepsis were 0.85 (95% CI, 0.80–0.89), 0.90 (95% CI, 0.88–0.94) and 0.92 (95% CI 0.89∼0.94). When the critical value of CRP was 97.05 mg/L, the sensitivity and specificity were 77.8% and 80.7%, respectively; when the critical value of PCT was 1.34 ng/mL, the sensitivity and specificity were 86.5% and 79.5%, respectively; the sensitivity and specificity of CRP + PCT tandem diagnosis were 84.3% and 87.5%, respectively. Pairwise comparison of ROC curves indicated that the diagnostic value of PCT was greater than that of CRP (P=0.016). CRP and PCT levels were higher in patients with severe sepsis/septic shock. When CRP and PCT were 197.01 mg/L and 28.55 ng/mL, respectively, the AUC was 0.67 (95% CI, 0.62–0.73) and 0.98 (95% CI, 0.96–1.00), and the diagnostic sensitivity and specificity of CRP was 50.5% and 79.7%, respectively, and that of PCT was 92.8% and 96.1%, respectively as shown in Figure 3 and Table 9.

Figure 3

ROC curves of CRP, PCT, and CRP + PCT in diagnosis of sepsis. Note: CRP: C-reactive protein; Pct: procalcitonin. Statistical method: ROC curve analysis.

Table 9

Diagnostic value of CRP, PCT and CRP + PCT tandem for sepsis.

Variable	Cutoff value	AUC (95% CI)	Standard error	Poor sensitivity (%)	Specificity (%)	P
CRP	97.07 mg/	0.87 (0.82∼0.88)	0.023	77.8	80.7	<0.001
PCT	1.35 ng/mL	0.92 (0.87∼0.95)	0.014	86.6	79.7	<0.001
PCT + CRP	—	0.93 (0.88∼0.96)	0.016	84.5	87.7	<0.001

4. Discussion

Sepsis is a relatively common infectious disease that poses a serious threat to the quality of daily life and the health of patients. If timely diagnosis can be made at an early stage in patients with sepsis and early effective treatment can be taken for them, the treatment effect can be significantly improved and the morbidity and mortality rate of patients with sepsis can be reduced [17]. In the medical clinical tests for sepsis patients, the results of biological methods such as blood culture and urine culture are slow and therefore the degree of application is relatively low. Diagnostic assessment of diseases by detecting changes in the level of biomarkers is the result of recent discoveries in molecular biology, and this diagnostic method allows not only to explore the occurrence and development of diseases from a molecular point of view but also to make effective and accurate judgments in the early stages of disease development. Sepsis is a systemic inflammatory response caused by inflammatory infection, and the morbidity and mortality rate can be as high as 40%; early and effective diagnosis is the key to improving patient prognosis and reducing morbidity and mortality [18]. Currently, PCT and CRP are the most widely used diagnostic biomarkers for sepsis.

In this study, there were records in the hospital's electronic medical record system confirming that patients with pathogenic bacteria detected in clinical blood cultures were blood culture-positive patients. Studies [19, 20] have shown that SCT levels can be used to predict blood culture results and that a high SCT level increases the likelihood that a patient will have a positive blood culture. In systematic case studies, some patients had significant sepsis based on diagnostic criteria, but the diagnosis at discharge was usually recorded as a bloodstream infection or bacteremia, and the clinical diagnosis of sepsis was low. According to the study, a higher proportion of septic patients and a higher proportion of critically ill patients were admitted to the emergency department compared with non-septic patients. Because white blood cell counts are an indicator of SIRS, the results of this analysis also confirmed that a higher proportion of patients with sepsis had abnormal white blood cell counts compared with patients without sepsis. Studies [21, 22] reported that the best diagnosis was obtained when the white blood cell threshold was 1.49 ng/mL, which is similar to the threshold obtained in this study. When CRP and PCT were 97.05 ng/L and 1.34 ng/L, respectively, the diagnostic sensitivity and specificity of PCT levels were higher than CRP, and PCT had a better diagnostic value. The specificity of tandem CRP + PCT was 87.5%, which was higher than that of CRP (80.7%) and PCT (79.5%). A study [23] presented epidemiologic data and clinical outcomes of severe sepsis and septic shock, and studies [24, 25] showed that among patients with confirmed severe sepsis/septic shock, overall morbidity and mortality in intensive care units and hospitals reached 28.7% and 33.5% respectively, indicating severe clinical outcomes. In addition, severe sepsis/septic shock imposes a significant financial burden on patients, which is a serious problem for specialists.

In this study, sepsis was divided into a sepsis group, a severe sepsis group, and a septic shock group according to the severity of sepsis. When CRP and PCT levels were compared, sepsis was found to be associated with severe sepsis and septic shock. A statistical difference was observed in septic shock: CRP and PCT levels were higher in severe sepsis/septic shock than in sepsis. A level of 28.55 ng/mL had a better diagnostic value. PCT performed better than CRP in the diagnosis of sepsis compared to the area under the ROC curve.

In conclusion, the detection of serum PCT and CRP can effectively assess the development of sepsis patients, which has certain value for early diagnosis and prognosis, and the prognosis of patients is closely related to platelet count and related parameters, which has certain guiding significance for prognosis prediction. This study can provide some reference for the diagnosis of clinical sepsis, but there are some shortcomings in the study design. First, this part of the study was retrospective and failed to continuously monitor the dynamic changes of CRP and PCT and to follow up patients discharged from the hospital; therefore, the risk factors for sepsis-related mortality could not be analyzed. Second, because we did not have real access to the patients and the data came from information recorded in the electronic medical record system, data were missing for some patients, and a follow-up rigorous prospective study could be designed to compensate for the shortcomings of this study.