Journal club

March 15^th, 2021

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F1 Nutchayavaree/F2 Nuttanan

Introduction

Introduction

• Neonatal sepsis is a clinical syndrome described by systemic signs and symptoms of infection in the first month of life.
• Neonatal mortality due to sepsis is still 10–20% worldwide
• Risk factors include the need for invasive procedures combined with immunological immaturity.
• Early diagnosis of neonatal sepsis is still a challenge because of its non-specific clinical presentation
• Early identification and the use of broad-spectrum antibiotics remain the cornerstone of neonatal sepsis treatment

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Introduction

• A missed identification of sepsis delays treatment and increases the risk of death
• The overuse of antimicrobial agents in patients without sepsis causes antibiotic resistance.
• The rapid and accurate identification of sepsis is, therefore, crucial in improving the clinical outcomes and reducing the medical costs.
• The current gold standard test in diagnosis of neonatal sepsis is isolation of causative microorganisms by blood culture

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Introduction

• The sensitivity and specificity of blood culture is overall low, partly because it depends on the blood volume drawn, the timing of the blood draw, any prior treatment with antibiotics, and the presence of viable organisms
• A negative test result for bacteremia is common and non-informative as to whether the patient needs antibiotics
• False positives due to contamination are also common and problematic
• The delay in turnaround time for a blood culture result also often lead to unnecessarily long courses of empiric broad-spectrum antibiotics

Introduction

• Diagnostic biomarkers are urgently needed to improve the rapid and accurate diagnosis of neonatal sepsis
• An ideal diagnostic biomarker should display excellent sensitivity and negative predictive value as well as excellent specificity and positive predictive value
• Interleukin-6 (IL-6) and C-reactive protein (CRP) are the two most commonly used markers for the diagnosis of neonatal sepsis.
• In early stage of infection, the CRP level may be low, but serial measurements can give more helpful results and can be useful in deciding when to terminate antibiotic treatment

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Introduction

• Although studies have shown that IL-6 and CRP have diagnostic value, their cut-off values are inconstant and their diagnostic performances vary across the studies
• The aims of this study are to define the optimal cut-off values for IL-6 and CRP, and to investigate the diagnostic performance of these biomarkers both in combination and alone for the early diagnosis of neonatal sepsis using large number of culture-proven sepsis, clinical sepsis, and controls

Materials and Methods

Study Design and Period

• Retrospective cross-sectional study
• At the University Hospital of Leipzig, Germany
• From November 2012 to June 2020
• Data collected:
• Demographic characteristic: gender, age
• Laboratory test: blood culture, IL-6, CRP

Classification of Study Participants

• Inclusion

Sepsis-suspected neonates were classified as

1. Proven sepsis: CRP > 10 mg/L* and positive blood culture
2. Clinical sepsis: CRP > 10 mg/L* and negative blood culture
3. No sepsis (Controls): suspected sepsis with CRP < 10 mg/L, negative blood culture and had not started antibiotics

*at least one of the five serial measurements

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• Exclusion
• Neonates with positive blood cultures for coagulase negative staphylococci (CoNS) organisms and CRP < 10 mg/L 🡪 potential contamination

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Classification of Study Participants

• Definition
• Early onset sepsis: onset ≤ 72 hrs. after birth
• Late onset sepsis: onset > 72 hrs. after birth
• In case of suspicious of sepsis
• Day 1: CRP, IL-6, blood culture
• Day 2-5: CRP

Blood Culture

• 0.5-1 mL of blood was collected, inoculated into BacT/ALERT bottles and loaded into the automated BacT/ALERT 3D system.
• Bottles were incubated up to 7 days at 37°C.
• Once the positive signal was detected in the blood culture systems, 1 mL was drawn out for Gram staining and subculture

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IL-6 and CRP Determinations

• Blood samples were transported using automated tube mail system immediately after collection and centrifuged to separate the serum and processed for the test as soon as they arrived at the laboratory
• Serum IL-6 concentration was measured by ECLIA
• The detection limit of this method for IL-6 is 2.5 pg/mL
• Serial measurements of serum CRP concentrations were performed in the first five days after sepsis was suspected by Immunoturbidimetric assay, Tina-quant C-Reactive Protein method
• The detection limit of this method for CRP is 0.3 mg/L.

Statistical Analysis

• Data were entered and analysed using SPSS statistical package version 20 software
• Data that are normal distributed, Skewness and Kurtosis Z-values, the Shapiro–Wilk test p-value, and visual outputs including histograms, normal Q-Q plots, and Box plots were used
• Data that are not normally distributed, non-parametrical tests were used and median and range were presented

Statistical Analysis

• Descriptive statistics including frequency and percentages of culture proven sepsis cases, clinical sepsis, and no sepsis controls, and organisms isolated from culture positive neonates were calculated.
• The mean and SD: mean age of neonates.
• The median and range: concentrations of IL-6 and CRP.
• Receiver-operating characteristics (ROC) curve was drawn to calculate the area under the curve (AUC) and coordinates of the curve

Statistical Analysis

• The optimal cut-off levels of IL-6 and CRP were determined based on the results of sensitivity and 1-specificity of the coordinates of the ROC curve.
• Cross tabulation was used to calculate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for IL-6 and CRP tests in combination or alone.

Results

916 neonates

899 neonates

104 proven sepsis

160 clinical sepsis

635 controls

17 culture positive neonates with CoNS and CRP < 10 mg/L

9 EOS

95 LOS

87 EOS

73 LOS

Exclude

549 EOS

86 LOS

Results

• The median concentration of IL-6 among proven sepsis (1612 pg/mL) was significantly higher than that of the clinical sepsis (138 pg/mL) and control groups (57 pg/mL) (p < 0.001)
• The median concentration of IL-6 among clinical sepsis group was significantly higher than that of the control group (p < 0.001)
• The median CRP1 concentration among proven (12 mg/L) and clinical sepsis (21 mg/L) groups was significantly higher than that of the control group (0.6 mg/L) (p < 0.001)
• However, the median CRP1 concentration among proven sepsis group were not significantly different from that of the clinical sepsis group (data not shown)

Discussion

Discussion

• This study is to define the optimal cut-off values of serum IL-6 and CRP using ROC curves, and evaluate their performance for early diagnosis of neonatal sepsis.
• The ideal diagnostic marker is desired to have about 100% sensitivity and NPV.
• To minimize the unnecessary use of antibiotics in false-positive cases, a diagnostic marker also needs to have reasonably high, preferably better than 85% specificity, and a good PPV.

Discussion

• In the previous studies, the optimal cut-off values of IL-6 and CRP are defined inconsistently, and their diagnostic performance for identification of neonatal sepsis varies
• Our study included large number of neonates with culture proven sepsis, clinical sepsis, and controls to correctly define the optimal cut-off values and measure the diagnostic performances of IL-6 and CRP
• The large sample size and use of automated blood culture system are the foremost strengths of our study.

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Discussion

• The results of the present study showed a significant increase (<0.001) in IL-6 and CRP concentrations among culture proven sepsis and clinical sepsis cases compared to controls
• The optimal cut-off level for IL-6 in neonates with culture proven sepsis was 313.5 pg/mL. At this cut-off level, we observed 73.1% sensitivity, 80.2% specificity, 37.6% PPV, and 94.8% NPV
• In our study, five different optimal cut-off levels were observed for CRP measured in the first five days of serial measurements among proven sepsis.

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Discussion

• The highest performance of CRP for the diagnosis of neonatal sepsis was observed in the second day measurement with 89.4% sensitivity, 97.3% specificity, 84.5% PPV, and 98.3% NPV.
• We recommend the combination of IL-6 (313.5 pg/mL) and CRP1 (2.15 mg/L) or CRP2 (8.01 mg/L) for early and accurate diagnosis of neonatal sepsis

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Discussion

• Variations in the cut-off levels and the diagnostic performance of IL-6 and CRP among the previous studies and this study might be due to the variations in the number of neonates enrolled in the study, the type of patient categories (culture proven sepsis, clinical sepsis or both) used for cut-off level calculations, the age of the neonates and the number of low birth weight infants included in the study
• Sampling time is also another important issue in the correct diagnosis of neonatal sepsis.

Discussion

• This issue was clearly demonstrated in our study with marked differences in CRP concentrations, differences in optimal cut-off values and diagnostic performance in the five days serial measurements. The peak time of CRP concentration was observed in the second day of inflammatory response and afterwards the concentration showed a declining trend in the third, fourth, and fifth days of measurements. In most other previous studies optimal cut-off values calculated only based on a single measurement of CRP.
• There might also be differences for IL-6 and CRP results between different laboratories, especially if they use different assays.

Limitations

• Lack of clinical data such as signs and symptoms of neonates, gestational age, and mode of delivery.
• Other useful data to more accurately define clinical sepsis and control groups such as the data from automethod blood culture, enzymology, PCR, and ethnicity are not available in the laboratory records
• These limitations are due to the retrospective design of our study, however, the findings are strengthen by relatively large number of investigated neonates, the use of automated blood culture system for all neonates, and the use of culture proven neonates to define the optimal cut-off values for IL-6 and CRP.

Conclusion

• This study defines the optimal cut-off values for IL-6 and CRP in the first five days of serial measurements using large number of culture-proven sepsis cases.
• Of the five days serial measurements of CRP, the CRP measured in the second day showed the highest median concentration and the highest diagnostic performance. CRP1 at low optimal cut-off value showed good diagnostic performance
• The combination of IL-6 and CRP demonstrated increased sensitivity. The CRP 2 at cut-off 8.01 mg/L showed the highest diagnostic performance for identification of culture negative clinical sepsis cases.

Conclusion

• We recommend the combination of IL-6 (313.5 pg/mL) and CRP1 (2.15 mg/L) or IL-6 (313.5 pg/mL) and CRP2 (8.01 mg/L) for early and accurate diagnosis of neonatal sepsis.
• The recommendation is based on increased sensitivity, that is, to minimize the risk of missing any cases of sepsis.
• The CRP2 alone at cut-off 8.01 mg/L might be used to identify clinical sepsis cases among culture negative sepsis-suspected neonates in hospital settings

Appraise journal

Population : All neonates that was born in University of Leipzig

Test : CRP / IL-6 or combination

Reference : CRP > 10 + blood culture positive

Outcome : sensitivity, specificity, NPV, PPV

จะมีผู้ป่วยบางกลุ่ม ถูกexclude ไป เช่น true CONS sepsis แต่ CRP ยังน้อยกว่า 10

มีการ report sensitivity, specificity, NPV, PPV

โดย test มี high NPV ซึ่งสามารถนำไปใช้ ในการ Rule-out diseaseได้

High cost