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The clinical signicance of interleukin-6, C-reactive protein, and procalcitonin in the early recognition of nosocomial infections in preterm infants

Background: This study assessed the clinical value of interleukin-6 (IL-6), C-reactive protein (CRP), and procalcitonin (PCT) for early recognition of nosocomial infections in preterm infants. Methods: 120 neonates were nally analyzed, 89 with sepsis were compared with 31 noninfected controls. IL-6, CRP and PCT were measured at certain stages. Receiver-operating characteristic (ROC) curve analysis was used to determine the best cutoff values of IL-6, CRP, and PCT for diagnosing sepsis. Results: In the sepsis group, IL-6 peaked 6 hours after the infection onset and dropped to normal 24–48 hours after the infection was controlled. The best cutoff values of IL-6, CRP, and PCT were >99.6 pg/mL, >9.27 mg/L, and >2.33 ng/mL, respectively. The areas under the ROC curves were 0.888, 0.823, and 0.953, respectively. When PCT was combined with IL-6 or IL-6 and CRP, the sensitivity and specicity were >85%. Conclusion: IL-6, CRP, and PCT are reliable indicators for early diagnosis of nosocomial infections in preterm infants. Combining PCT and IL-6, CRP with IL-6 and PCT levels could improve the accuracy of clinical diagnosis.

The risk of developing sepsis is higher in neonates due to their immature immune systems, low leukocyte phagocytic activity, poor humoral immunity, weak skin barrier, and the use of invasive procedures such as mechanical ventilation and central venous catheterization [4,5]. In 2013, the World Health Organization announced the results of an assessment of the causes of death of 2.8 million neonates worldwide. There were an estimated 420,000 neonatal deaths caused by sepsis per year, accounting for 15.0% of neonatal deaths. The incidence of neonatal sepsis has been gradually increasing worldwide, and the mortality rate was relatively high [6]. The onset of neonatal sepsis is relatively insidious. However, it progresses rapidly and is often accompanied by severe complications, such as shock and multiple organ dysfunction. Therefore, early diagnosis is essential to reducing the mortality of neonatal sepsis and improving the prognosis. At present, the laboratory tests that are used for the diagnosis of sepsis include bacterial culture, white blood cell counts, platelet counts, neutrophil percentages, cell surface antigen and bacterial gene detection. However, because of unclear diagnostic thresholds, complex detection methods, falsenegative results, and other reasons, there is no ideal indicator for the early diagnosis of neonatal sepsis.
The gold standard for the diagnosis of sepsis is blood culture, but the reporting of results may take 24-48 hours. The accuracy of clinical diagnosis is often affected by an insu cient blood sample, administration of antibiotics before blood collection, and irregular operations. In addition, out of a fear of missing a diagnosis of sepsis, which might result in shock and other serious complications, physicians often administer broad-spectrum antibiotics empirically and adjust them after the results of drug susceptibility tests are reported, which can lead to antibiotic abuse, the presence of multidrug-resistant bacteria, fungal infections, and impaired intestinal immune functioning [7]. Therefore, testing methods with good accuracy, high sensitivity and speci city, and quick results are urgently needed to guide the clinical diagnosis and treatment of neonatal sepsis. This study assessed the best cutoff value of IL-6, the diagnostic accuracy of CRP, IL-6, PCT, singly and in combination, for the early diagnosis of nosocomial infections in preterm infants.

Participants
This study was conducted among preterm infants admitted to the Department of Neonatology, Shengjing Hospital of China Medical University, from July 2019 to July 2020. The inclusion criteria included an age > 3 days in preterm infants whose gestational age was ≥ 30 and ≤ 34 weeks at birth, with stable vital signs and hemodynamic parameters. The exclusion criteria included the mother's use of glucocorticoids before delivery; diagnosis with a community-acquired infection; incomplete clinical records; antibiotic use before the rst blood collection; and a suspicion that the infant had inherited metabolic diseases, congenital malformations, prenatal or intrapartum asphyxia, or immunode ciency.
This study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983 and was approved by the Ethics Committee of Shengjing Hospital of China Medical University.
Informed consent was obtained from the guardians of the study participants, and sample collection was performed in accordance with the standards of diagnosis and treatment.

Study design
Participants were assigned to groups according to a 2019 expert consensus [8] on the diagnosis and treatment of neonatal sepsis. In the sepsis group, 1 mL of venous blood was drawn to measure the IL-6 level when the infection was rst clinically suspected (before the administration of antibiotics); 6 hours and 12 hours after infection onset; and 24-48 hours, 3 days, and 7 days after infection control. Another 1 mL of venous blood was drawn to measure the CRP and PCT levels at infection onset (before the administration of antibiotics) and 7 days after infection control. Additionally, 1 mL of blood was drawn for bacterial culture before the administration of antibiotics.
In the control group, according to the admission routine, 2 mL of venous blood was drawn after admission for measurement of IL-6, CRP, and PCT.
This study adheres to CONSORT guidelines.
Index and methods IL-6 was measured by enzyme-linked immunosorbent assay with a reagent provided by Xiamen Huijia Biotechnology Co., Ltd. (Xiamen, China), and ≥ 96.52 pg/mL was considered positive [9]. CRP was measured by immune scattering turbidimetry with a reagent provided by Shanghai Jiemen Biotechnology Co., Ltd. (Shanghai, China), and ≥ 8 mg/L was considered positive. PCT was quantitatively measured by enzyme-linked uorescence assay with a reagent produced by BRAHMS Co. (Hennigsdorf, Germany), and ≥ 0.5 ng/mL was considered positive.
De nitions and diagnostic criteria A con rmed sepsis diagnosis was de ned as clinical manifestations of sepsis ( respiratory distress, tachycardia or bradycardia, poor perfusion, abnormal body temperature, et al) and a positive blood or cerebrospinal uid (or other sterile body cavity uid) culture [8].A clinical sepsis diagnosis was de ned as clinical manifestations of sepsis with any of the following: more than two positive nonspeci c blood detection indicators (total leukocyte count, platelet count, CRP, PCT, et al), cerebrospinal uid showing signs of purulent meningitis; or the detection of pathogenic bacterial DNA in the blood [8].
A nosocomial infection was de ned as an infection occurring ≥ 48 hours after hospital admission, including infections occurring during hospitalization and those occurring after discharge, as a result of hospital exposure to pathogens [10].
Statistical analysis SPSS 22.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis of the data. Continuous data were expressed as means ± standard deviations, and t-tests were used for comparisons between groups. Discrete data were expressed as frequencies, and chi-square tests were used for comparisons between groups. The best cutoff values of IL-6, CRP, and PCT were determined using receiver-operating characteristic (ROC) curve analysis. The sensitivity, speci city, positive predictive values (PPVs), negative predictive values (NPVs), and areas under the ROC curves (AUCs) of the three in ammatory marker levels were calculated singly and in combination. The correlation analysis used single-factor regression analysis, with the levels of the indicators as independent variables. A P value of < 0.05 indicated that the difference was statistically signi cant. This case control study was retrospectively registered at 2 September 2020 in the China Clinical Trials Registry (http://www.chictr.org.cn/listbycreater.aspx, reference number: ChiCTR2000037817).

Participant characteristics
A total of 262 cases were enrolled in the study. Among these infants, 136 were excluded because of congenital malformations (n = 18), neonates showing sepsis-like symptoms but ultimately undiagnosed (n = 7) and mother's use of glucocorticoids (n = 111). In all, 126 infants enrolled in the study; 5 infants in sepsis group and 1 infant in control group dropped out of the programme because of parent's wish to withdraw the infants from the trial. Finally, 89 cases of sepsis (47 cases of con rmed sepsis and 42 cases of clinical sepsis) and 31 controls (noninfected neonates hospitalized during the same period) could be analyzed (Fig. 1). There were twenty cases of low birth weight, four cases of neonatal hyperbilirubinemia, two cases of neonatal anemia, two cases of gastrointestinal bleeding, and three cases of arrhythmia in control group.
Participant characteristics according to group are shown in Table 1. There were no statistically signi cant differences in gestational age, age, sex, Apgar score, or birth weight between the neonates in the three groups. Blood culture A total of 47 neonates in the sepsis group had positive blood cultures, and the distribution of pathogens was as follows: 23 cases of gram-negative bacilli (48.9%), 20 cases of gram-positive cocci (42.5%), and four cases of fungi (8.6%) ( Table 2).

IL-6, CRP, and PCT levels
Before antibiotic treatment, the levels of IL-6, CRP, and PCT in the sepsis group were signi cantly higher than those in the control group. After effective infection control (7 days), the levels of IL-6, CRP, and PCT in the sepsis group were signi cantly reduced (Table 3).

IL-6 at each stage of infection
Previous studies have clari ed the dynamic trends of CRP and PCT [11]. The IL-6 levels peaked at 6 hours after the onset of infection and decreased to normal levels 24-48 hours after the infection was effectively controlled (Table 4, Fig. 2). Comparison of the diagnostic values of IL-6, CRP, PCT, and combined testing The ROC curves (Figs. 3 and 4) revealed that the best cutoff values of IL-6, CRP, and PCT were > 99.6 pg/mL, > 9.27 mg/L, and > 2.33 ng/mL, respectively. PCT had a high sensitivity, speci city, PPV, NPV, and AUC value, making it an ideal single index for the early identi cation of nosocomial infection in preterm infants. When combining PCT and IL-6 or using a combination of CRP, IL-6, and PCT for testing, the sensitivity and speci city values were over 85%, and the predictive values were high (Table 5).

Discussion
Cytokines are proteins or small molecular polypeptides secreted by cells. They are powerful mediators of in ammation and are essential for body defense, damage repair, and signal transduction. The detection of cytokines provides new possibilities for the diagnosis and treatment of neonatal sepsis. Among them, the proin ammatory cytokine IL-6 is currently a focus of research [12]. The IL-6 gene is located on human chromosome 7 and encodes a glycoprotein with multiple biological activities. IL-6 is a multi-effect proin ammatory factor. Under normal circumstances, the body's IL-6 content is extremely low (< 10 pg/mL). After pathogens invade the body, lymphocytes, monocytes-macrophages, and broblasts are stimulated and a large amount of IL-6 is produced. The IL-6 then stimulates the production of other in ammatory factors and acute phase response proteins [13]. Newborns with a gestational age less than 30 weeks or whose mothers used glucocorticoids before delivery have signi cantly reduced IL-6 levels [14]. In addition, IL-6 can also be markedly increased due to certain tumors, autoimmune diseases, and transplant rejection. However, the above conditions are rare in the neonatal period. Therefore, when IL-6 is markedly increased in a newborn, it indicates an infection largely. Under the action of bacterial cell wall substances, such as endotoxin, IL-6 is quickly released, reaching a peak within a few hours: the more serious the infection, the greater the increase. The half-life of IL-6 is about 100 minutes, and it decreases signi cantly 24-48 hours after the appearance of clinical signs, which means that it can be used for the effective diagnosis of bacterial infections and can be used to accurately judge the severity and prognosis of the infection [15][16][17]. Verboon et al. [18] found that 90% of children with enterovirus infections had normal levels of IL-6, indicating that IL-6 could be used as an important indicator to distinguish between bacterial and viral infections. Moreover, a prospective, randomized control study showed that IL-6 levels increase signi cantly 2 days before the onset of signs of bacterial infection, which is before other laboratory indicators indicated positive results [19]. The IL-6 level was negatively correlated with the time of blood collection, and the timing of its peak after infection was earlier than that of PCT. Combined with the nding that it had no physiological peak, the IL-6 level could be regarded as a sensitive indicator for the early diagnosis of neonatal sepsis. However, there are certain limitations to using IL-6 for guiding the empiric administration of antibiotics. IL-6 has a short half-life and peaks early. If the blood sample is not collected in time, the test may provide false-negative results. Moreover, the IL-6 level can return to normal while infection still persists [20]. Hatzidaki et al. [21] tested the IL-6 levels in cord blood, maternal blood during delivery, and serum on the fourth day after birth in 109 newborns who were born prematurely due to the premature rupture of membranes. The results showed that when the cutoff value of IL-6 was 108.5 pg/mL, the sensitivity for diagnosis of sepsis was 95% and the speci city was 100%, suggesting that IL-6 levels in the cord blood of neonates with a high risk of neonatal sepsis could help predict EOS. The cutoff value range of IL-6 was 3.6 to 300 pg/mL in a meta-analysis by Hu et al. [22]. In this study, the best cutoff value of IL-6 was 99.6 pg/mL. The IL-6 level peaked at 6 hours after infection, which was much earlier than the levels of CRP and PCT, and returned to normal 24-48 hours after the onset of infection and showed the highest sensitivity. Therefore, if preterm neonates have an elevated IL-6 level, physicians should be alerted to the possibility of a nosocomial infection, even if signs of infection are not present and levels of other in ammatory indicators are not abnormal, Natural immune cells can express Toll-like receptors or nucleotide-binding oligomerization domain-like receptors to identify various invading pathogens and release tumor necrosis factor alpha (TNF-α) and proin ammatory factors, such as IL-1β and IL-6. Then, these proin ammatory factors promote the release of acute phase proteins (such as CRP and PCT). CRP is mainly synthesized in the liver. It is an acute response protein produced by body tissues after various injuries or in ammatory stimuli and is mainly regulated by TNF-α and IL-6 secreted by fat cells. CRP binds to the teichoic acid of Gram-positive bacteria and binds to the lipopolysaccharides of gram-negative bacteria. It is released 4-6 hours after the onset of infection, reaches its peak in 24-48 hours, and has a half-life of about 24 hours [23,24]. CRP increases with the increase of in ammatory factors and decreases after infection control. It is positively correlated with the severity of infection. Therefore, a signi cant increase can be used as an early sign of bacterial sepsis. However, some non-infectious factors, such as neonatal asphyxia, ischemic or traumatic tissue damage, meconium aspiration syndrome, immunization, and hemolysis, could also cause an increase in CRP, resulting in low speci city of CRP as an indicator of sepsis [25]. Hengst et al. [26] reported that dynamic monitoring of the CRP trend could assist in the diagnosis of infectious diseases and in evaluating the clinical e cacy of antibiotics. Moreover, a retrospective analysis by Cui and Xue [11] showed that CRP peaked 24-48 hours after infection occurred and returned to normal 7 days after the infection was effectively controlled, indicating its dynamic trend is of great signi cance in guiding the rational administration of antibiotics and monitoring of clinical e cacy. In this study, the level of CRP before treatment in the sepsis group was signi cantly higher than that in the control group, indicating that CRP could be used for the diagnosis of neonatal sepsis. The ROC curve analysis found that the best cutoff value of CRP was 9.27 mg/L. Compared with PCT and IL-6, CRP had lower sensitivity and speci city; therefore, it should not be used as an independent marker for the early diagnosis of nosocomial infection in preterm infants.
Serum PCT is a glycoprotein without hormone activity that is synthesized by liver cells and monocytes and is a precursor of calcitonin. In bacterial infections, the PCT level increases under the stimulation of endotoxin and proin ammatory factors (IL-6, TNF-α, and IL-1β). However, in viral infections, interferon gamma inhibits the synthesis and release of PCT [27]. PCT is not affected by factors such as maternal PCT level and stress and is only related to the extent of bacterial infection. The level of PCT in healthy people is extremely low (< 0.1 ng/mL), and PCT is only secreted by thyroid neuroendocrine C cells. When the body is infected, it can be released from the cells of almost all tissues, and a level ≥ 0.5 ng/mL is de ned as abnormal. A study by Lee et al. [28] showed that PCT started to increase 4 hours after the onset of infection and remained at the peak level for 8-24 hours. After control of the infection, the PCT level decreased by 50% per day. However, Cui and Xue [11] found that PCT peaked 12 hours after the onset of infection and returned to normal within 3 days after infection control. This study found that in the sepsis group, the serum PCT level before the initiation of antibiotic treatment was signi cantly higher than that after treatment and was higher than the level in the control group, suggesting that the PCT level was positively correlated with the severity of infection. This is consistent with the results of a study by Canpolat et al. [29]. Auriti et al. [30] conducted a prospective, controlled study on 762 neonates from multiple centers. It was found that the serum PCT levels of neonates with sepsis were signi cantly higher than those of healthy neonates, with the best cutoff value of 2.4 ng/mL, which had a sensitivity of 62% and speci city of 84%. In this study, the best cutoff value of PCT was 2.33 ng/mL, with a sensitivity of 84.0%, speci city of 97.8%. Therefore, serum PCT is of higher value for the early diagnosis of nosocomial infection in preterm infants, and the administration of antibiotics can be determined in the early stage of infection. Especially when the PCT level continues to remain high, it is necessary to consider replacing antibiotics. However, there is a physiological increase of PCT after birth. It is about 0.7 ng/mL at birth, peaks after 24 hours, and drops to below 0.1 ng/mL within 72 hours. This physiological pattern limits the diagnostic accuracy of serum PCT for the diagnosis of EOS [31]. Cord blood PCT has been reported to have good diagnostic accuracy for EOS, with a sensitivity of 82% and a speci city of 86% [32]. Moreover, a retrospective analysis by Joram et al. [33] showed that the average cord blood PCT level of neonates with EOS was 3.03 ng/mL, which was signi cantly higher than the 0.16 ng/mL of noninfected neonates.
With the cutoff value of cord blood PCT as 0.6 ng/mL, the NPV of PCT was 99%, which made it a useful indicator for diagnosing EOS.
This study found that when the PCT result was combined with the IL-6 or with both IL-6 and CRP results, the sensitivity increased to 96.6% and 86.5%, respectively; and the speci city increased to 87.1% and 96.8%, respectively. In addition, the PPVs for PCT in combination with IL-6 or with both IL-6 and CRP were 98.3% and 98.2%, respectively; the NPVs were 78.4% and 77.6%, respectively; and the AUCs were 0.966 and 0.965, respectively. These two combinations could both improve the accuracy of PCT for the early diagnosis of nosocomial infection in preterm infants.
The incidence of neonatal sepsis has steadily increased, and the prevalence of drug resistance has also increased. The incidence of methicillin-resistant, coagulase-negative Staphylococcus aureus and Escherichia coli has increased, and Acinetobacter baumannii and multidrug-resistant bacterial infections have been appearing in neonates. The results of this study showed that gram-negative bacteria were isolated signi cantly more frequently than gram-positive bacteria in the blood cultures of the neonates with sepsis. The three most common pathogenic bacteria were Klebsiella pneumoniae (34.0%), Staphylococcus epidermidis (21.3%), and Escherichia coli (12.8%), showing that gram-negative bacteria are the main cause of neonatal sepsis and that Klebsiella pneumoniae was the dominant bacteria, which is consistent with the results of Wei [34].

Conclusion
In summary, IL-6, CRP, and PCT could be used as reliable indicators for the early identi cation of nosocomial infections in preterm infants. IL-6 levels peaked 6 hours after the onset of infection, much earlier than those of CRP and PCT. Therefore, when IL-6 is elevated, physicians should be highly alert to the possibility of nosocomial infection in preterm infants. The ROC curve analysis revealed that the best cutoff values of IL-6, CRP, and PCT were > 99.60 pg/mL, > 9.27 mg/L, and > 2.33 ng/mL, respectively. In single-marker measurements, the sensitivity, speci city, PPVs, NPVs, and AUC values of PCT were high, making PCT an ideal index for the early identi cation of nosocomial infections in preterm infants. Combined testing using PCT combined with IL-6 or with both CRP and IL-6 could improve the accuracy of clinical identi cation of nosocomial infections in preterm infants. Testing these markers is fast and convenient and is conducive to early diagnosis and treatment. Large sample sizes and multicenter studies are needed to con rm this result.

Declarations
Authors' contributions CX and FJ: designed the research; CX, ZX, and SY: conducted the research and collected data; CX: wrote the manuscript; XX and SY: directed the writing of the manuscript; FJ: had primary responsibility for nal content. All authors designed, supervised, and analyzed the study and assisted with the preparation of the manuscript. All authors read and approved the nal manuscript.

Funding
None.