Evaluation of time to positivity for blood culture combined with immature granulocytes, neutrophil‐to‐lymphocyte ratio, and CRP in identifying bloodstream coagulase‐negative Staphylococci infection in pediatric patients

Abstract Objective To evaluate the application value of time to positivity (TTP) for blood culture combined with inflammatory parameters that included immature granulocyte percentage (IG%), immature granulocyte count (IG#), C‐reactive protein (CRP), white blood cells (WBC) neutrophil percentage (NE%), and neutrophil‐to‐lymphocyte ratio (NLR), and to identify bloodstream infections from contamination with coagulase‐negative staphylococci (CoNS) in pediatric patients. Methods Data of 12 897 inpatients with blood culture CoNS were retrospectively collected and analyzed from January‐December 2019 at our hospital. According to pre‐defined criteria, they were divided into a CoNS infection group (132 cases) and a CoNS contamination group (124 cases). Infection with Staphylococcus aureus (SA, 27 cases) at the same period was considered a positive control group. ROC curve analysis assisted in determining the value of applying TTP combined with the above‐mentioned inflammatory parameters to distinguish CoNS infection from contamination. Results Among the 256 strains of CoNS, Staphylococcus hominis (55.1%), Staphylococcus epidermidis (32.0%), and Staphylococcus capitis (7.0%) were common. There was no significant difference in the subspecies distribution between the infection and contamination groups. The TTP of the CoNS infection group was significantly lower than the contamination group (P < .05). IG%, IG#, CRP, NE%, and NLR were all higher in the infected group as compared to the contaminated group (P < .05), while WBC was similar among groups. There was also no statistical difference in those parameters when comparing the CoNS infection and SA groups. ROC analysis showed that TTP value in identifying CoNS infection from contamination was the highest with area under the curve (AUC) of 0.913, and the sensitivity and specificity were 0.827 and 0.852, respectively, at the optimal cutoff value of 23.9 hours. This was followed by IG% (AUC = 0.712), with an optimal critical value of 0.55%, and a sensitivity of 0.519 and specificity of 0.797. All the AUC values of IG#, CRP, NE%, and NLR were <0.7. A combination of TTP with IG%, CRP, and NLR improved the AUC, sensitivity, specificity, accuracy, PPV, and NPV values to 0.977, 0.922, 0.957, 91.8%, 92.2%, and 91.3%, respectively. Conclusions TTP within 24 hours indicates likelihood of CoNS as the pathogenic agent in pediatric patient blood culture. The combination of TTP with IG% CRP and NLR might improve the diagnostic accuracy.


| INTRODUC TI ON
Coagulase-negative Staphylococci (CoNS) including Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus capitis among others, which cannot produce substances like plasma coagulase and hemolysin, are considered normal flora or opportunistic pathogens of the skin mucosa. In recent years, with the extensive use of biomedical devices and the widespread application of various broad-spectrum antibiotics, CoNS has emerged as an important pathogen of hospital infection and the most common bacteria that causes blood culture contamination. 1 Blood culture is the gold standard for bloodstream infections.
As a common parameter in blood culture, the clinical predictive value of time to positivity (TTP) on bloodstream infection has also attracted increased attention. 2,3 TTP refers to a period of time from the moment the blood is isolated from the body to the positive alarm of the blood culture instrument. This is related to the initial concentration of bacteria, and can be used to differentiate the diagnosis of whether a blood culture of CoNS is an infection or due to contamination.
Immature granulocyte IG is a new detection index for the diagnosis of infectious diseases, including promyelocytic, and middle and late granulocytes, which belong to the precursor stage of developing mature white blood cells. Immature granulocytes generally do not exist in the peripheral blood of healthy people; however, severe clinical conditions (such as sepsis and septicemia) can consume a large number of peripheral blood granulocytes, and the body compensates by releasing immature granulocytes from the bone marrow to the peripheral blood circulation. Therefore, peripheral immature granulocytes can be regarded as a new indicator to reflect the severity of inflammation.
Peripheral blood neutrophil-to-lymphocyte ratio as an indicator reflecting the body's systemic inflammatory response and immune status has attracted widespread attention in recent years. According to reports, it has proven its accuracy in predicting the outcome of patients with major cardiac events, ischemic stroke, cancers, sepsis, and infectious pathologies. 4 The mark is simple, easy to obtain and calculate, and easy to integrate into daily practice without paying extra.
In this current study, clinical data and laboratory inflammatory indicators of the included children were retrospectively analyzed to explore the value of blood culture positive alarm time TTP when combined with common inflammatory indicators (eg, WBC and CRP), including IG, NLR in the differential diagnosis of bloodstream infections, and contamination. 23.9 hours. This was followed by IG% (AUC = 0.712), with an optimal critical value of 0.55%, and a sensitivity of 0.519 and specificity of 0.

| Criteria for judging CoNS of bloodstream infections and contamination
According to the definition of nosocomial infection surveillance published by the US 5 Centers for Disease Control and Prevention (CDC) in 2016, at least one of the following one criteria are met: ① one or more sets of blood culture or identification of the CoNS pathogen through a non-culture microbiological detection assay, and blood microorganisms and other site infections being irrelevant; ② the patient had at least one of the following symptoms or signs: a body temperature of >38°C, chills, hypotension, and evidence that the same CoNS were isolated from blood culture on two or more occasions under different circumstances, and the above standard elements must occur in the window period of infection; that is, the day of positive blood sampling or 3 days before and after the window of infection; and ③ patients with an age of less than or equal to 1 years old, having at least one of the following symptoms or signs: evidence of fever (>38°C), or a low temperature (<37°C), apnea, bradycardia, and at least two positive blood cultures that detected the same CoNS. The detected microorganisms must also be confirmed not to be related to other site infections. According to this standard, the selected cases were divided into two groups, namely, the infection group and the contamination group. When the instrument indicated a positive alarm, blood plate and chocolate plate were transferred in a timely manner and cultured in a 5% CO 2 incubator for 18-24 hours to separate a single colony.

| Experimental method
A Vetak-2 automatic microbial identification instrument (Biomerere, France) was used for pathogen identification.
Specimen detection: the USES xn-1000 automatic hematological analyzer (Japan Sysmex Company) was used, and reagents were obtained from the original package of supporting reagents. IG was detected by sheath flow technology, electrical impedance technology, and nucleic acid fluorescence staining. In addition, relevant parameters such as IG percentage (IG%), IG count (IG#), total white blood cell (WBC) counts and classification, neutrophil-to-lymphocyte ratio (NLR) were recorded.

| Statistical analysis
The SPSS version 26.0 statistical software program was used to pro- were all calculated. An AUC > 0.9 has a highly prompt accuracy, a value of 0.7-0.9 has a medium prompt accuracy, and a value of 0.5-0.7 has low prompt accuracy. An alpha value of P < .05 for differences between data groups is considered statistically significant.

| Distribution of CoNS species in the infection and contamination groups
A total of 256 CoNS strains were isolated from 12,897 cases (see Table 1), among which the proportion of human Staphylococcus was the highest, with a total composition ratio of 55.1%. The others were Staphylococcus epidermis (32.0%), Staphylococcus cephalus (7.0%), Staphylococcus haemolyticus (2.3%), and Staphylococcus goat (1.2%).
There was no statistical difference in the distribution composition ratio of CoNS between the infection and the contamination groups (P > .05).

| Comparison of TTP and inflammatory parameters among different groups
In Table 2 Table 2).

| Efficacy of combined indicators to identify CoNS infection from contamination
According to the efficacy evaluation data of a single indicator (Table 3), although the area under the IG#, CRP, and NLR curves was lower than 0.7, considering that IG% and IG# were immature granulocyte parameters, CRP was a common inflammatory index in the laboratory, and NLR was an emerging and readily available inflammation parameter, and then, IG#, CRP, and NLR were included in the joint analysis. By contrast, NE% was not included because the AUC had not attained or exceeded a value of 0.6.
The ROC curve analysis shows ( Figure 1B) that TTP combined with IG%, IG#, or CRP can improve the discrimination efficiency of CoNS infection and peripheral toxemia as might be seen in sepsis/ septicemia; however, IG% was completely consistent with an improved efficacy of IG# (Table 4). Considering that IG# is susceptible to the WBC count, a combination of inflammatory indicators was not included. When the TTP was combined with IG%, CRP, and NLR, the AUC was 0.977(P < .001), and the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were increased to 0.922, 0.957, 92.2%, 91.3%, and 91.8%, respectively (234/256).

| D ISCUSS I ON
With the widespread use of immunosuppressants, broad-spectrum antibiotics, and the increasing number of interventional treatments, CoNS has gradually emerged as one of the most commonly isolated bacteria by blood culture. 6   In addition, in this study, it was found for the first time that IG% was the highest for diagnosing CoNS among the single inflammatory indicators. Taking 0.55% as the critical value, the sensitivity was 0.519, the specificity was 0.797, and the accuracy attained a value of 65.6%, all of which were higher than similar parameters reported for white blood cells and even CRP.
In our analysis, IG might be more closely related to systemic infection such as sepsis, while other inflammatory indicators will be increased due to local inflammation. Thus, the results of IG% in infected patients can assist in determining whether blood culture CoNS positivity is caused by an infection or from inadvertent cross-contamination. F I G U R E 1 ROC analysis of TTP and inflammatory parameters in identifying CoNS bloodstream infection from contamination. A, ROC curve analysis of single indicators including IG%, IG#, CRP, NE%, and NLR. The AUC of TTP was the highest, reaching a value of 0.913, the AUC of IG% was 0.712, and the AUC of IG#, CRP, NE%, and NLR were 0.679, 0.645, 0.595, and 0.605, respectively, which were all evidently lower than 0.7. B, ROC curve analysis of TTP combined with inflammatory parameters. When TTP was combined with IG%, IG#, CRP, or NLR, the AUC was 0.951, 0946, 0.945, and 0.923, respectively. When TTP was combined with IG%, CRP, and NLR, the AUC was the highest, with a value of up to 0.977 There is accruing evidence that neutrophil-to-lymphocyte ratio (NLR) is an easy-to-obtain inflammatory parameter and results a reliable predictor in a variety of disease conditions characterized by the involvement of immune cells and inflammatory processes 18,19 . When the patient's condition is serious, especially when the total number of white blood cells does not increase, the detection of the ratio is more meaningful than the white blood cell count. The results of this study showed that the NLR value of the CoNS infection group is higher than the contamination group, which can effectively identify bloodstream infections, and the identification efficiency was better than WBC and NE%.

TA B L E 3 Efficacy of TTP and inflammatory parameters in identifying CoNS blood stream infection from contamination
The results of this current study showed that the positive alarm time of children's blood culture, which was identified as co- This study has a limited sample size and the retrospective analysis of data collected at a single site may lead to bias. In the future, large-scale prospective studies should be conducted to obtain reproducible and effective estimates, and to determine the most predictable baseline cutoff points based on the characteristics of baseline patients.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
Lanlan Zeng, Shuhui Wang, and Minchun Lin performed the data analysis, made the picture, and wrote the article. Yaxing Chen, Qiulian Deng, and HuaminZhong conducted blood culture, bacterial identification, and typing. XiaoshanGuang and Shuwen Yao tested the laboratory inflammatory markers. Haiying Liu contributed to the article review and editing of the drafts. All authors read and approved the final draft of the article prior to its submission for external peer review.

E M PLOY M E NT O R LE A D E R S H I P
None declared.

H O N O R A R I U M
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated and/or analyzed during the current study are not publicly available since the medical records and data are confidential and respect or abide by the patient's legal rights to privacy; however, the data are available from the corresponding author on reasonable request under the consent from close relatives of the patient.