In febrile children for more than five days, serum concentrations of soluble urokinase plasminogen activator receptor fail to identify those with Kawasaki Disease - A Pilot Observational Cohort Study

Alsuwaidi AR, George JA and Narchi H

Published on: 2023-11-28

Abstract

Aim: To assess, in febrile children, the diagnostic effectiveness of the soluble form of urokinase plasminogen activator receptor (suPAR) to distinguish between Kawasaki disease (KD) and infections, and to also investigate its association with the development of coronary artery aneurysms (CAA) in KD.

Material and Methods: On admission of 17 children with fever lasting ≥ 5 days and lacking suggestive diagnostic signs, serum suPAR concentrations were compared between children later diagnosed with either KD or infections. We also calculated the sensitivity, specificity, Youden index, and generated a receiver operating characteristic (ROC) curve to determine the predictive ability of suPAR for diagnosing KD.

Results: KD was later confirmed in seven children, and febrile infections in 10. There was no significant difference in suPAR levels between these two groups: 5.35 ± 2.76 ng/mL in the former compared to 5.57 ± 1.69 ng/mL in the latter (p=0.84).The optimal cut-off value was ≥ 7.74 ng/mL, correctly classifying 64.7% of the cases, however, with a Youden index of 18.6% and an area under the curve of 60%, suPAR was unable to effectively differentiate between the two groups. Among the seven children with KD, the only child who developed CAA had a suPAR concentration of 4.69 ng/mL, whereas the average level was 5.47 ± 1.04 ng/mL in the others. However, the statistical significance could not be determined.

Conclusion: In febrile children for more than 5 days, serum suPAR concentrations cannot distinguish between KD and infections at hospital admission, nor can they predict the development of CAA in KD.

Keywords

Biomarker; Fever; Mucocutaneous lymph node syndrome; Coronary artery aneurysm

Introduction

Urokinase plasminogen activator receptor (uPAR) binds to the cell membrane of immunologically active cells, such as monocytes, activated T-lymphocytes, macrophages, endothelial cells, and other cells. It separates from the cell during inflammation, generating its soluble form, suPAR, which can be measured in blood and other body fluids. It facilitates the passage of inflammatory cells from the bloodstream into tissues and plays a role in the plasminogen-activating pathway, inflammation, and modulation of cell adhesion, migration, and proliferation. As the urokinase receptor system regulates the confluence of inflammatory, immune, coagulation, and fibrinolytic responses, suPAR has been used in the diagnosis and prognosis of multiple inflammatory, infectious, autoimmune, and neoplastic conditions, as well as coronary heart disease, heart failure, and COVID-19 infection [1–10]. Regardless of the underlying pathology, serum levels are raised in acutely ill patients and also have prognostic value as they predict high mortality [6]. As the biochemical and molecular mechanisms underlying these properties remain unclear, suPAR is currently considered to be a non-specific biomarker for disease presence, progression, and severity [6].

Kawasaki disease (KD) is an acute febrile vasculitis of unknown aetiology that mainly affects infants and young children, some of whom develop coronary artery aneurysms (CAA) if not treated early with intravenous immunoglobulin (IVIG) [11].

Activation of the innate immune system induces vasculitis and triggers the adaptive immune system to produce cytokines, chemokines, proteases, and reactive oxygen species [12]. Further recruitment of immune cells into the arterial wall occurs, where inflammation and oxidative stress interact and amplify each other, resulting in endothelial dysfunction [13]. CAA formation results from chronic inflammation and vascular wall remodelling related to suPAR. The latter activates immune cells and promotes the release of pro-inflammatory cytokines, leading to endothelial dysfunction and damage, which contributes to the weakening of the arterial wall and the development of aneurysms [13]. Moreover, suPAR activates matrix metalloproteinases (MMPs), which are enzymes that degrade the extracellular matrix of blood vessels, potentially contributing to aneurysm formation. KD needs to be differentiated from more common febrile illnesses, as early diagnosis is essential in preventing CAA; however, without pathognomonic diagnostic tests, its diagnosis remains essentially clinical [11]. Thus, laboratory investigations that help in diagnosis remain highly sought.  

We hypothesized that the complex inflammatory processes that underlie vasculitis in KD and the development of CAA may result in overexpression of suPAR, making it a potentially useful biomarker. To the best of our knowledge, this hypothesis has not yet been explored, and we compared, in febrile children, suPAR concentrations in those with KD to those with infectious causes. We also evaluated whether their concentrations differed between children with KD who developed CAA and those who did not.

Material and Methods

Study design

This retrospective observational cohort study was an extension of an earlier project [14]. It was performed on preserved residual sera from 17 children under six years of age who were consecutively admitted to XXX hospital with fever for ≥ 5 days and without any suggestive diagnostic signs on admission. Children with KD were diagnosed according to established criteria. Febrile illnesses were diagnosed by microbiology and/or radiology.

Laboratory analysis

Plasma samples were collected within 24 hours of admission and immediately transported to the laboratory for storage at −80°C until further analysis. Plasma suPAR levels were measured using commercial Enzyme-Linked Immunosorbent Assay (ELISA) kits (suPARnostic™ assay, Virogates, Copenhagen, Denmark). Plasma (25 μL) was mixed with 225 μL of horseradish peroxidase (HRP)-labeled detection antibody. A total of 100 μL of this mix was then transferred to duplicate wells of a microwell plate precoated with the capture anti-suPAR antibody. The plates were washed after one hour of incubation at room temperature. To each well, 100 μL of substrate was added and incubated for 20 min in the dark before colour development was stopped by adding sulfuric acid. The optical absorbance was measured at 450 nm using a microplate reader. Plasma suPAR concentration (expressed as ng/mL) was determined by interpolation of the calibration curve prepared from the provided suPAR standard. The lower limit of detection of the assay was 0.1 ng/mL, as determined by the manufacturer.

Statistical methods

Serum suPAR concentrations, normally distributed continuous variables, were reported as mean ± standard deviation and were compared with the unpaired Student t-test. Sensitivity, specificity, Youden index (sensitivity + specificity -1), positive and negative likelihood ratios, as well as the receiver operating characteristic (ROC) of SuPAR concentrations to predict KD, were calculated. The cut-off concentration to differentiate between the two groups was that with a Youden index of 50%, with values below demonstrating failure to differentiate KD from febrile infections. All analyses were performed using the Stata software (Stata Statistical Software: Release 16; College Station, TX: StataCorp LLC), and a two-tailed p-value <0.05 defined statistical significance.

Results

There were 17 children (median age 25 months; interquartile range 18–53 months), including seven boys. KD was diagnosed in seven patients (of whom only one developed CAA), and ten had febrile infections (one bacterial pneumonia, one scarlet fever, three viral respiratory infections, and five viral gastroenteritis) (Fig. 1). Intravenous immunoglobulins were administered to all the children with KD. Their full details were published earlier [14].

Figure 1: Flowchart of the 17 children hospitalised with fever for ≥ 5 days without any suggestive signs.

There was no significant difference in suPAR concentrations between children with KD (5.35 ± 2.76 ng/mL) and febrile infections (5.57 ± 1.69 ng/mL) (p = 0.84) (Fig. 2). Additionally, SuPAR concentrations were not significantly different between bacterial and viral infections (4.75 ± 1.07 ng/mL vs. 5.78 ± 1.81 ng/mL) (p = 0.47), between sexes (p = 0.61), age groups (p = 0.47), or duration of fever before admission (p = 0.61). Similarly, they showed no correlation with the highest recorded temperature (p = 0.28), serum C-reactive protein level (p = 0.45), haematological parameters, or biochemical parameters, except for the serum sodium concentration (p = 0.02) (Fig. 3).

 

Figure 2: Soluble Urokinase Plasminogen Activator Receptor (suPAR) concentrations in 7 children with Kawasaki disease (KD) and 10 with febrile infections.

Horizontal lines represent the mean values ± standard deviation.

p values were calculated using the 2-sided unpaired Student’s t-test.

Figure 3: Linear regression analysis of temperature characteristics and blood test results in relation to serum Soluble Urokinase Plasminogen Activator Receptor (suPAR) concentrations in 17 febrile children for ≥ 5 days.

Panel A: temperature and fever duration before admission; Panel B: serum C-reactive protein (CRP) concentration; Panel C: white blood cell and neutrophil counts; Panel D: haemoglobin and platelet counts; Panel E: serum sodium and albumin concentrations.

The performance of suPAR concentrations in predicting KD included the calculation of sensitivity, specificity, Youden index, and positive and negative likelihood ratios (Table 1). The optimal cut-off level for suPAR concentration, with the highest Youden index of 18.6%, was ≥ 7.74 ng/mL, which correctly classified 64.7% of cases with a sensitivity of 28.6% and a specificity of 90%. However, the Youden index was < 50%, and the area under the curve was 60% (Fig. 4).

Figure 4: Receiver operating characteristic curve (ROC) of serum soluble urokinase plasminogen activator receptor (suPAR) concentrations to differentiate between Kawasaki disease and febrile infections.

One child with KD developed CAA and had a suPAR concentration of 4.69 ng/mL, compared to 5.47 ± 1.04 ng/mL in the other six children; hence, the p-value could not be computed.

Table 1: Characteristics of serum suPAR concentrations in Kawasaki disease and febrile infections in 17 children with fever.

suPAR concentration cutpoint (ng/mL)

Sensitivity

Specificity

Correctly classified

LR+

LR-

Youden index

>= 3.8

71.43%

10.00%

35.29%

0.7937

2.8571

-18.57%

>= 3.99

57.14%

10.00%

29.41%

0.6349

4.2857

-32.86%

>= 4.04

57.14%

20.00%

35.29%

0.7143

2.1429

-22.86%

>= 4.34

57.14%

30.00%

41.18%

0.8163

1.4286

-12.86%

>= 4.69

42.86%

30.00%

35.29%

0.6122

1.9048

-27.14%

>= 5.34

28.57%

30.00%

29.41%

0.4082

2.381

-41.43%

>= 5.51

28.57%

40.00%

35.29%

0.4762

1.7857

-31.43%

>= 5.8

28.57%

50.00%

41.18%

0.5714

1.4286

-21.43%

>= 6.46

28.57%

60.00%

47.06%

0.7143

1.1905

-11.43%

>= 6.78

28.57%

70.00%

52.94%

0.9524

1.0204

-1.43%

>= 7.07

28.57%

80.00%

58.82%

1.4286

0.8929

8.57%

>= 7.74

28.57%

90.00%

64.71%

2.8571

0.7937

18.57%

>= 8.24

14.29%

90.00%

58.82%

1.4286

0.9524

4.29%

LR+ and  LR- : positive and negative likelihood ratio; Youden index= sensitivity + specificity -1 The cut-off point of serum suPAR concentrations ≥ 7.74 ng/mL (highlighted in bold), had the highest Youden index (18.6%) and was the best to correctly classify 64.7% of the cases with a sensitivity of 28.6% and specificity of 90%.

Discussion

To the best of our knowledge, this study is the first to investigate suPAR concentration in children with KD. Although serum levels were elevated in both KD and febrile infections, the difference was not statistically significant. Furthermore, the performance of SuPAR in diagnosing KD was poor, with an optimal cut-off value of ≥ 7.74 ng/mL and the highest Youden index of only 18.6%, well below 50%. Additionally, the area under the curve was only 60%. The similarity in suPAR levels between children with KD and those with febrile infections is not surprising since suPAR is a nonspecific marker of inflammation, which can be caused by both conditions [3, 5, 15]. Various factors, including age, sex, stress, genetics, lifestyle, obesity, chronic pain, and underlying conditions, can affect serum suPAR concentrations [16–18]. Furthermore, since there are no universally accepted reference ranges and suPAR levels can vary throughout different stages of an illness, they may not always be elevated to the same extent throughout the course of an illness. Comparing suPAR concentrations between the two conditions was difficult because they were measured at various points during febrile illnesses in our study.

There were no differences in suPAR levels between patients with KD with and without CAA. This could be attributed to the small sample size of the cohort and the fact that CAA developed in only one child. Moreover, since suPAR levels increase in vasculitic conditions such as KD, they are expected to be elevated in KD-associated CAA and coronary microvascular dysfunction, which involves monocyte and macrophage infiltration and structural damage to the artery with arterial dilation [13, 19]. Increased suPAR levels have been observed in adults with coronary artery disease and slow coronary flow on angiography, likely because of underlying inflammation [20].

The limitations of this study include the small sample size and retrospective design, which restrict the generalisability of the results. Other limitations include the opportunistic measurement of suPAR upon hospital admission, which may have occurred at different stages of febrile episodes. Serial measurements were not performed, as only a single suPAR concentration measurement was performed upon admission. This precluded an analysis of suPAR dynamics in children with KD. The study intentionally did not include a control group of healthy children because the primary focus was to differentiate between KD and infections in febrile children.

Larger prospective multicenter studies with larger sample sizes are required to validate these findings. Serial suPAR measurements should be conducted throughout the course of the illness, with comparisons made between similar stages of fever. In addition, exploring the potential of combining suPAR measurements with other biomarkers or clinical factors to enhance the diagnostic accuracy of KD is warranted.

Conclusion

In febrile children for more than 5 days, serum suPAR concentrations cannot distinguish between KD and infections at hospital admission, nor can they predict the development of CAA in KD.

Contributors: HN and ARA conceptualized and designed the study, and JG performed the laboratory analyses. HN analyzed the data and drafted the initial manuscript. All authors critically reviewed and revised the manuscript for important intellectual content, approved the final manuscript as submitted, and agreed to be accountable for all aspects of this work.

Ethics approval: The study was performed in accordance with the Declaration of Helsinki of the World Medical Association and was approved by the XXX Research Ethics Committee under registry number ERH-2021-7392. Signed parental consent was obtained for all participants.

Funding: This research was funded by a grant from the College of Medicine and Health Sciences, United Arab Emirates University (NP/09/11).

Conflict of interest: None

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