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Serum procalcitonin concentration in patients with Kawasaki disease
Journal of Infection (2004) 48, 199–205
www.elsevierhealth.com/journals/jinf
Serum procalcitonin concentration in patients with Kawasaki disease Yasunori Okadaa,*, Hisanori Minakamib, Takeshi Tomomasaa, Masahiko Katoa, Yoshinari Inouea, Kunihisa Kozawac, Hirokazu Kimurac, Akihiro Morikawaa a
Department of Pediatrics, Gunma University School of Medicine, Showa-machi 3-39-22, Maebashi, Gunma 371-8511, Japan b Department of Reproductive and Development Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan c Gunma Prefectural Institute of Public Health and Environmental Sciences, Kamioki, Maebashi, Gunma 371-0052, Japan Accepted 7 August 2003
KEYWORDS Kawasaki disease; Procalcitonin; Coronary aneurysms; C-reactive protein
Summary Background. Procalcitonin (PCT) is a new parameter of inflammation, the clinical usefulness of which is currently being evaluated. Materials and methods. We determined simultaneously the serum concentrations of PCT and C-reactive protein (CRP) as well as the white blood cell (WBC) count in 25 patients with Kawasaki disease (KD), 17 with bacterial infections, 10 with systemic autoimmune diseases, 17 with viral infections and 18 healthy children. The optimal cut-off value of each parameter for predicting coronary aneurysms was determined using receiver operating characteristic curves. Results. Significantly higher serum concentrations of PCT were observed in patients with KD (2.3 ^ 3.0 ng/ml) and bacterial infections (2.2 ^ 2.9 ng/ml) than in patients with autoimmune diseases (0.4 ^ 0.4 ng/ml) or viral infections (0.4 ^ 0.3 ng/ml), or in healthy children (0.2 ^ 0.1 ng/ml). The serum PCT but not the WBC count or CRP, differentiated the KD patients from the patients with autoimmune diseases. The optimal cut-off value of 3.0 ng/ml of PCT increased the prediction rate of coronary aneurysms that subsequently occurred in 4 (16%) patients with KD. Conclusions. The serum PCT may be clinically useful for determining the severity of KD and for narrowing the differential diagnosis of patients with inflammatory diseases. Q 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction Abbreviations: PCT, procalcitonin; KD, Kawasaki disease; CRP, C-reactive protein; WBC, white blood cell. *Corresponding author. Tel.: þ81-27-220-8205; fax: þ81-27220-8215. E-mail address: [email protected]
Procalcitonin (PCT) is a 116 amino acid protein with a molecular weight of approximately 13 kD.1 PCT is a very stable protein in vivo and in vitro, and is not degraded to hormonally active calcitonin in plasma.2 Because an increased serum
0163-4453/$30.00 Q 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2003.08.002
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concentration of PCT is seen in patients with bacterial infection,3 – 5 PCT is thought to behave as an acute phase reactant and is now recognized as a new marker of bacterial infectious diseases.3,6 Kawasaki disease (KD), involves an acute systemic vasculitis and can induce serious coronary aneurysms.7 KD may be an infectious disease with abnormal immune responses to an unidentified bacterium or virus.8 Thus, KD involves an aspect of autoimmune disease in its pathogenesis.9,10 An acute phase reactant protein, C-reactive protein (CRP), in the serum and the white blood cell (WBC) count are more likely to increase in response to a bacterial infection and autoimmune diseases than in response to a viral infection.11,12 PCT, also an acute phase reactant, behaves in an unknown manner after the onset of KD. Accordingly, we determined simultaneously the serum concentration of PCT and CRP, and the WBC count in patients with several inflammatory diseases, including KD and autoimmune diseases.
Materials and methods Patients A total of 87 Japanese children were studied (Table 1), including 25 patients with KD, 17 patients with bacterial infections (eight with pneumonia, four with pyelonephritis, three with sepsis, and two with bacterial enterocolitis), 10 patients with autoimmune diseases (six with juvenile rheumatoid arthritis, two with systemic lupus erythematosus, one with mixed connective tissue disease, and one with Takayasu disease), 17 patients with viral infection (seven with influenza, three with chicken pox, three with exanthema subitum, three with rota, and one with mumps), and 18 healthy children with minor congenital cardiac defects. All patients or their parents gave informed consent to participate in this study.
KD was diagnosed based on the standard criteria.13 Echocardiography was performed on all patients with KD every 2 weeks for at least 3 months after disease onset. Because all 25 patients with KD were less than 5 years old, patients with coronary arteries greater than 3 mm diameter were defined as having coronary artery aneurysms.14 All patients with KD were treated with a high dose of g-globulin (400 mg/kg £ 5 days) and aspirin (30 mg/kg/day) within 7 days of disease onset. Bacterial infections were diagnosed based on the results of cultures of urine, stool, blood and/or sputum together with the clinical signs and chest X-ray. Viral infections were diagnosed based on changes in virus-specific antibodies in paired samples together with the clinical signs. The diagnosis in all 10 patients with autoimmune disease had been established by the time they had participated in this study. Blood samples for the measurement of the serum PCT and CRP concentrations and the WBC count were obtained simultaneously in all study subjects. In the 25 patients with KD, acute-phase (within 7 days after the disease onset) blood samples, subacute-phase (median 14 days, range 10 – 18 days) blood samples, and convalescence-phase (median 28 days; range 22 – 35 days) blood samples were available in 25, 13, and 12 patients, respectively. The first sample was obtained before any treatment in all the KD patients. In the patients with bacterial and viral infections, blood samples were obtained during the active stage of the diseases. In the patients with autoimmune diseases, the blood samples were obtained during an acute phase or flare. In the patients with minor congenital cardiac defects, the blood samples were obtained when they were afebrile, not taking any medications, and had normal physical findings.
Determination of the serum concentrations of PCT and CRP Blood samples were centrifuged and the sera were stored at 220 8C until use. The serum concentration
Table 1 Clinical and laboratory data in the 112 study subjects Disease
No. of patients
Age (year)
WBC ( £ 102/ml)
CRP (mg/dl)
PCT (ng/ml)
Kawasaki disease Acute ( % 7 days) Subacute (10–18 days) Convalescence (22–32 days) Bacterial infections Autoimmune diseases Viral infections Healthy controls
25 13 12 17 10 17 18
2.8 ^ 2.0 3.5 ^ 2.3 2.5 ^ 1.6 4.4 ^ 5.3 8.9 ^ 4.0 3.6 ^ 2.9 2.7 ^ 3.5
143 ^ 71 101 ^ 50 87 ^ 4.0 110 ^ 6.9 107 ^ 49 76 ^ 33** 87 ^ 22**
9.4 ^ 6.1 2.0 ^ 5.9 0.1 ^ 0.2 11.1 ^ 5.7 7.3 ^ 7.8 0.4 ^ 0.5** 0.0 ^ 0.0**
2.3 ^ 3.0 0.3 ^ 0.7 0.1 ^ 0.2 2.2 ^ 2.9 0.4 ^ 0.4* 0.4 ^ 0.3* 0.2 ^ 0.1**
WBC, white blood cell count; CRP, C-reactive protein; PCT, procalcitonin. * and **, p , 0:01 and p , 0:001 vs. acute KD, respectively.
Procalcitonin in Kawasaki disease
of PCT was measured using a specific and ultrasensitive commercial immunoluminometric assay (BRAHMS Diagnostika, Berlin, Germany), with a luminometer (LUMAT LB 9507, BERTHOLD, Berlin, Germany) according to the manufacturer’s instructions. This method uses two monoclonal antibodies that bind to two sites (katacalcin and calcitonin) of the PCT molecule, thus ruling out cross-reactivity with calcitonin. The limit of detection of this assay is 0.1 ng/ml. The normal reference values for the serum concentration of PCT range from 0.1 to 0.5 ng/ml.1 CRP was measured using a latex immunoassay on a CRP-LATEX ‘SEIKEN’ (DENKA, Tokyo). The limit of detection of this assay is 0.1 mg/dl. The normal reference value for the serum CRP is less than 0.5 mg/dl.
Statistical analysis Data are reported as the mean ^ SD. A receiver operating characteristic curve was constructed for the determination of the optimal cut-off values of PCT and CRP and the WBC count for predicting the development of coronary aneurysms. Results were analyzed using the Mann – Whitney U-test for unpaired samples, the unpaired t-test, and the Spearman correlation coefficient. A level of p , 0:05 was accepted as statistically significant.
Results Both the mean WBC count and the mean CRP were significantly greater in the patients with acute KD than in the patients with viral infections and the healthy controls, but did not differ from those in the patients with bacterial infections and autoimmune diseases (Table 1). The mean PCT was significantly higher in the patients with acute KD than in the patients with autoimmune diseases and viral infections and in the healthy controls, but did not differ from that in the patients with bacterial infections. Although the patients with autoimmune diseases had elevated WBC counts and serum CRP concentrations, they did not have an elevated PCT concentration. The individual patient PCT data are shown in Fig. 1. Patients with acute KD and bacterial infections exhibited a similar magnitude increase in the PCT concentration. However, patients with autoimmune diseases or viral infections did not exhibit a significant increase in the PCT concentration compared with the healthy controls. In the KD patients, PCT, WBC count, and CRP all decreased from the onset of disease (Table 1 and Fig. 2).
201
Figure 1 Serum procalcitonin concentration in the various study groups. Data were analyzed by the Mann– Whitney U-test.
However, the WBC count did not decrease in some patients. Four (16%) of the 25 patients with KD eventually developed coronary aneurysms 10 ^ 2 days from disease onset. Patients with higher PCT concentrations, WBC counts, and CRP concentrations appeared to develop this complication (Fig. 2). The four patients who developed coronary aneurysms had higher acute-phase concentrations of PCT (7.2 ^ 3.8 ng/ml vs. 1.4 ^ 1.7 ng/ml, p , 0:01), WBC counts (223 ^ 145 £ 102/ml vs. 127 ^ 36 £ 102/ml, p , 0:05), and CRP concentrations (15.0 ^ 4.9 mg/dl vs. 8.4 ^ 5.9 mg/dl, p ¼ 0:05) than did the 21 patients who did not develop coronary aneurysms. Two of the four patients who developed coronary aneurysms exhibited a sustained, high PCT concentration and/or WBC count (Fig. 2). A receiver operating characteristic curve (ROC) was constructed to determine the optimal cut-off value for the PCT, WBC count, and CRP of the acute KD patients for predicting the subsequent development of coronary aneurysms (Fig. 3). The area under the ROC curve (AUC) was . 0.8 for all three parameters ðp , 0:001Þ: Thus, all the parameters were statistically significant predictors for coronary aneurysms. The optimal cut-off value for predicting coronary aneurysms was 3 ng/ml for PCT, 170 £ 102/ml for the WBC, and 12 mg/dl for CRP. We evaluated the predictive value of the optimal cut-off values for the PCT, the WBC count, and the CRP (Table 2). A serum PCT concentration of 3 ng/ ml gave a sensitivity of 100% (4/4), a specificity of 90% (19/21), and a positive predictive value (PPV) of 67% (4/6). A WBC of 170 £ 102/ml gave a sensitivity of 100% (4/4), a specificity of 86% (18/ 21), and a PPV of 57% (4/7). A serum CRP
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Figure 3 Receiver operating characteristic curves for predicting coronary aneurysms using the serum concentrations of procalcitonin (X), C-reactive protein (B), and the white blood cell count (O). The optimal cut-off value indicated by an arrow was 3 ng/ml for procalcitonin, 170 £ 102/ml for the white blood cell count, and 12 mg/dl for C-reactive protein.
Figure 2 Changes in the serum procalcitonin, and Creactive protein (CRP) concentrations, and the white blood cell counts (WBC) in patients with Kawasaki disease. (X), Patients who developed coronary aneurysms; (W), patients who did not develop coronary aneurysms. Data were analyzed by the Mann –Whitney U-test.
concentration of 12 mg/dl gave a sensitivity of 100% (4/4), a specificity of 81% (17/21), and a PPV of 50% (4/8). Thus, the optimal cut-off value of the PCT was superior to those of the WBC count and CRP with respect to specificity and PPV. We analyzed the correlation between the PCT, WBC count, and CRP (Fig. 4). There was no significant correlation between any two variables among the PCT, WBC, and CRP in the patients with acute KD (CRP and PCT; r ¼ 0:371; p ¼ 0:06); (WBC and PCT; r ¼ 0:19; p ¼ 0:35); and (WBC and CRP; r ¼ 0:21; p ¼ 0:32). This suggests that any combination of the two parameters better predicts the development of coronary aneurysms than does one parameter. Indeed, some of the patients who had only one parameter of ^ the optimal cut-off value were false negative with respect to the development of coronary aneurysms, while all patients who had at least two parameters . the optimal cut-off value developed coronary aneurysms (Fig. 4).
Table 2 Prediction of coronary aneurysms in patients with Kawasaki disease using the optimal cut-off values of PCT, WBC, and CRP
PCT WBC CRP
Cut-off value
Sensitivity
Specificity
PPV
NPV
3 ng/ml 170 £ 102/ml 12 mg/dl
4/4 (100%) 4/4 (100%) 4/4 (100%)
19/21 (90%) 18/21 (86%) 17/21 (81%)
4/6 (67%) 4/7 (57%) 4/8 (50%)
19/19 (100%) 18/18 (100%) 17/17 (100%)
PCT, procalcitonin; WBC, white blood cell count; CRP, C-reactive protein; PPV, positive predictive value; NPV, negative predictive value.
Procalcitonin in Kawasaki disease
203
Figure 4 Relationships among the procalcitonin and C-reactive protein (CRP) concentration and the white blood cell counts (WBC) in patients with acute-phase Kawasaki disease. (X), Patients who developed coronary aneurysms; (W), patients who did not develop coronary aneurysms. Horizontal and vertical bars indicate the optimal cut-off values determined by the operating characteristics curves in Fig. 3. The optimal cut-off value was 3 ng/ml for procalcitonin, 170 £ 102/ml for the white blood cell count, and 12 mg/dl for C-reactive protein.
Discussion This may be the first report in which the serum PCT concentration was determined in patients with KD. In this study, the serum concentration of PCT increased in the acute-phase KD patients to a similar extent to that seen in patients during the acute phase of a bacterial infection. Although the extent of the increase in both the WBC count and the CRP was similar between the patients with KD and autoimmune diseases, the increase in the PCT differed between them: the PCT increased in the patients with KD, but not in the patient with autoimmune diseases, suggesting, that the serum PCT concentration differentiates KD patients from patients with autoimmune diseases. Although the number of study subjects was small, the serum PCT concentration appeared to be a better predictor of the development of coronary aneurysms than either the WBC count or the CRP. Use of the PCT concentration in conjunction with the WBC or the CRP concentration enhanced the accuracy of the prediction of coronary aneurysms, suggesting the clinical usefulness of PCT in patients with KD. The present study confirmed the results of earlier studies on PCT in patients with bacterial infections, viral infections, and autoimmune diseases.6,15 A systemic generalized bacterial infection is associated with an increase in the serum PCT concentration.1 Localized bacterial infections without systemic manifestations and viral infection are associated with a small increase in the PCT concentration (0.3 – 1.5 ng/ml).1 The PCT concentration does not increase (, 0.5 ng/ml) in adult patients with systemic lupus erythematosus and
systemic antineutrophil cytoplasmic antibody associated vasculitis.15 Although the microorganism responsible for KD is still not determined, abnormal immune responses to bacterial infection are thought to play a role in the pathogenesis of KD.16 In addition, viruses such as Epstein – Barr and adenoviruses have been considered responsible for KD.17,18 However, the patients with acute KD were similar to the patients with bacterial infections with respect to the increase in the PCT in this study. Since elevated autoantibodies are seen in KD patients, autoimmunity also is thought to be involved in the pathogenesis of KD.9 It may be interesting to note that the WBC and CRP concentration did not differentiate acute KD patients from patients with autoimmune diseases, while the PCT concentration differentiated the acute KD patients from the patients with autoimmune diseases. Thus, the determination of PCT may be clinically useful in differential diagnosis of febrile patients with skin rashes caused by some bacterial infections, viral infections, autoimmune diseases, or KD. KD, an acute febrile illness, is the most common cause of systemic vasculitis in children.19 Coronary aneurysms which occur in 10 – 20% of KD patients, are the most serious complication of KD.20 Therefore, the counselling of patients with KD is an important task for clinicians whether coronary aneurysms develop. Parameters such as the WBC and the CRP, TNFa, and IL-6 concentrations have been reported to predict the later development of coronary aneurysms.21 – 24 The serum PCT concentration appeared to be the best single parameter for the prediction of this complication in this study. Since the PCT did not significantly correlate with the WBC count or the CRP concentration, a combination of the PCT with the WBC count or the
204
CRP concentration enhanced the accuracy of predicting coronary aneurysms. The sensitivity and specificity were 100% and 100%, respectively using the two optimal cut-off values of the PCT and WBC or CRP. However, the combination of PCT and the WBC or CRP was not superior to that of the WBC and the CRP in predicting coronary aneurysms in this study, perhaps due to the small number of study subjects. Differences in the induction time and half-life in the serum between PCT and CRP25 may partly explain why the PCT concentration did not significantly correlate with the CRP concentration. The physiologic role of PCT has not been extensively studied. TNFa is massively released in response to bacterial toxins and plays a role in septic shock.26 PCT has recently been demonstrated to inhibit lipopolysaccharide-induced TNFa release from human whole blood.27 It is possible that an increased PCT in response to a bacterial infection may have a role in the prevention of excessive inflammation through suppressing TNFa. Since an increased serum concentration of TNFa may be associated with the pathogenesis of generalized vasculitis in patients with KD,23 the increased PCT in the acute KD patients may play a physiologic role in suppressing the vasculitis caused by KD, although the KD patients with a high PCT actually tended to develop coronary aneurysms. In conclusion, the serum concentration of PCT was increased in acute KD patients. The PCT predicted the later development of coronary aneurysms, suggesting that it might be clinically useful to measure the serum PCT concentration in patients with KD.
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9.
10.
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13. 14.
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18. 19.
References 1. Assicot M, Gendrel D, Carsin H. High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 1993;341:515—582. 2. Le Moullec JM, Jullienne A, Chenais J, et al. The complete sequence of human preprocalcitonin. FEBS Lett 1984;13: 93—97. 3. Gendrel D, Bouhoun C. Procalcitonin, a marker of bacterial infection. Infection 1997;25:133—134. 4. Dandona P, Nix D, Wilson MF. Procalcitonin increase after endotoxin injection in normal subjects. J Clin Endocrinol Metab 1994;79:1605—1608. 5. Nylen ES, Whang KT, Snider Jr. RH. Mortality is increased by procalcitonin and decreased by antiserum reactive to procalcitonin in experimental sepsis. Crit Care Med 1998; 26:1001—1006. 6. Gendrel D, Raymond J, Coste J, et al. Comparison of procalcitonin with C-reactive protein, interleukin 6 and interferon-alpha for differentiation of bacterial vs. viral infections. Pediatr Infect Dis J 1999;18:875—881. 7. Kawasaki T, Kosaki F, Okawa S, Shigematsu I, Yanagawa H. A new infantile acute febrile mucocutaneous lymph node
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syndrome (MLNS) prevailing in Japan. Pediatrics 1974;54: 271—276. Rowley AH, Wolinsky SM, Relman DA, et al. Search for highly conserved viral and bacterial nucleic acid sequences corresponding to an etiologic agent of Kawasaki disease. Pediatr Res 1994;36:567—571. Cunningham MW, Meissner HC, Heuser JS, Pietra BA, Kurahara DK, Leung DY. Anti-human cardiac myosin autoantibodies in Kawasaki syndrome. J Immunol 1999;163: 1060—1065. Falcini F, Trapani S, Turchini S, et al. Immunological findings in Kawasaki disease: an evaluation in a cohort of Italian children. Clin Exp Rheumatol 1997;15:685—689. Hatherill M, Tibby SM, Sykes K, Turner C, Murdoch IA. Diagnostic markers of infection: comparison of procalcitonin with C reactive protein and leucocyte count. Arch Dis Child 1999;81:417—421. Mangge H, Kenzian H, Gallistl S, et al. Serum cytokines in juvenile rheumatoid arthritis. Correlation with conventional inflammation parameters and clinical subtypes. Arthritis Rheum 1995;38:211—220. Dajani AS, Bisno AL, Chung KJ, et al. Diagnostic guidelines for Kawasaki disease. Am J Dis Child 1990;144:1218—1219. Emmanouilides GC, Riemenschneider TA, Allen HD, Gutgesell HP. Moss and Adames’ Heart Disease in Infants, Children, and Adolescents: Including the Fetus and Young Adult, 5th ed. Baltimore: Williams and Wilkins; 1995. p. 1390—1399. Eberhard OK, Haubitz M, Brunkhorst FM. Usefulness of procalcitonin for differentiation between activity of systemic autoimmune disease (systemic lupus erythematosus/ systemic antineutrophil cytoplasmic antibody-associated vasculitis) and invasive bacterial infection. Arthritis Rheum 1997;40:1250—1269. Barron KS, Shulman ST, Rowley A, et al. Report of the National Institutes of Health Workshop on Kawasaki disease. J Rheumatol 1999;261:170—190. Okano M, Thiele GM, Sakiyama Y, Matsumoto S, Purtilo DT. Adenovirus infection in patients with Kawasaki disease. J Med Virol 1990;32:53—57. Okano M. Kawasaki disease and human lymphotropic virus infection. Curr Med Res Opin 1999;15:129—134. Leung DY. Kawasaki disease. Curr Opin Rheumatol 1993;5: 41—50. Dhillon R, Clarkson P, Donald AE, et al. Endothelial dysfunction late after Kawasaki disease. Circulation 1996; 94:2103—2106. Mori M, Imagawa T, Yasui K, Kanaya A, Yokota S. Predictors of coronary artery lesions after intravenous gamma-globulin treatment in Kawasaki disease. J Pediatr 2000;137: 177—180. Beiser AS, Takahashi M, Baker AL, Sundel RP, Newburger JW. A predictive instrument for coronary artery aneurysms in Kawasaki disease. US Multicenter Kawasaki Disease Study Group. Am J Cardiol 1998;81:1116—1120. Lin CY, Lin CC, Hwang B, Chiang B. Serial changes of serum interleukin-6, interleukin-8, and tumor necrosis factor alpha among patients with Kawasaki disease. J Pediatr 1992;121: 924—926. Koyanagi H, Yanagawa H, Nakamura Y, Yashiro M. Serum Creactive protein levels in patients with Kawasaki disease: from the results of nation-wide surveys of Kawasaki disease in Japan. Acta Paediatr 1997;86:613—619. Nijsten MW, Olinga P, The TH, et al. Procalcitonin behaves as a fast responding acute phase protein in vivo and in vitro. Crit Care Med 2000;28:458—461. de Werra I, Jaccard C, Corradin SB. Cytokines, nitrite/
Procalcitonin in Kawasaki disease
nitrate, soluble tumor necrosis factor receptor, and procalcitonin concentrations: comparisons in patients with septic shock, cardiogenic shock, and bacterial pneumonia. Crit Care Med 1997;25:607—613.
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27. Monneret G, Pachot A, Laroche B, Picollet J, Bienvenu J. Procalcitonin and calcitonin gene-related peptide decrease LPS-induced TNF production by human circulating blood cells. Cytokine 1999;12:762—764.
www.elsevierhealth.com/journals/jinf
Serum procalcitonin concentration in patients with Kawasaki disease Yasunori Okadaa,*, Hisanori Minakamib, Takeshi Tomomasaa, Masahiko Katoa, Yoshinari Inouea, Kunihisa Kozawac, Hirokazu Kimurac, Akihiro Morikawaa a
Department of Pediatrics, Gunma University School of Medicine, Showa-machi 3-39-22, Maebashi, Gunma 371-8511, Japan b Department of Reproductive and Development Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan c Gunma Prefectural Institute of Public Health and Environmental Sciences, Kamioki, Maebashi, Gunma 371-0052, Japan Accepted 7 August 2003
KEYWORDS Kawasaki disease; Procalcitonin; Coronary aneurysms; C-reactive protein
Summary Background. Procalcitonin (PCT) is a new parameter of inflammation, the clinical usefulness of which is currently being evaluated. Materials and methods. We determined simultaneously the serum concentrations of PCT and C-reactive protein (CRP) as well as the white blood cell (WBC) count in 25 patients with Kawasaki disease (KD), 17 with bacterial infections, 10 with systemic autoimmune diseases, 17 with viral infections and 18 healthy children. The optimal cut-off value of each parameter for predicting coronary aneurysms was determined using receiver operating characteristic curves. Results. Significantly higher serum concentrations of PCT were observed in patients with KD (2.3 ^ 3.0 ng/ml) and bacterial infections (2.2 ^ 2.9 ng/ml) than in patients with autoimmune diseases (0.4 ^ 0.4 ng/ml) or viral infections (0.4 ^ 0.3 ng/ml), or in healthy children (0.2 ^ 0.1 ng/ml). The serum PCT but not the WBC count or CRP, differentiated the KD patients from the patients with autoimmune diseases. The optimal cut-off value of 3.0 ng/ml of PCT increased the prediction rate of coronary aneurysms that subsequently occurred in 4 (16%) patients with KD. Conclusions. The serum PCT may be clinically useful for determining the severity of KD and for narrowing the differential diagnosis of patients with inflammatory diseases. Q 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction Abbreviations: PCT, procalcitonin; KD, Kawasaki disease; CRP, C-reactive protein; WBC, white blood cell. *Corresponding author. Tel.: þ81-27-220-8205; fax: þ81-27220-8215. E-mail address: [email protected]
Procalcitonin (PCT) is a 116 amino acid protein with a molecular weight of approximately 13 kD.1 PCT is a very stable protein in vivo and in vitro, and is not degraded to hormonally active calcitonin in plasma.2 Because an increased serum
0163-4453/$30.00 Q 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2003.08.002
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concentration of PCT is seen in patients with bacterial infection,3 – 5 PCT is thought to behave as an acute phase reactant and is now recognized as a new marker of bacterial infectious diseases.3,6 Kawasaki disease (KD), involves an acute systemic vasculitis and can induce serious coronary aneurysms.7 KD may be an infectious disease with abnormal immune responses to an unidentified bacterium or virus.8 Thus, KD involves an aspect of autoimmune disease in its pathogenesis.9,10 An acute phase reactant protein, C-reactive protein (CRP), in the serum and the white blood cell (WBC) count are more likely to increase in response to a bacterial infection and autoimmune diseases than in response to a viral infection.11,12 PCT, also an acute phase reactant, behaves in an unknown manner after the onset of KD. Accordingly, we determined simultaneously the serum concentration of PCT and CRP, and the WBC count in patients with several inflammatory diseases, including KD and autoimmune diseases.
Materials and methods Patients A total of 87 Japanese children were studied (Table 1), including 25 patients with KD, 17 patients with bacterial infections (eight with pneumonia, four with pyelonephritis, three with sepsis, and two with bacterial enterocolitis), 10 patients with autoimmune diseases (six with juvenile rheumatoid arthritis, two with systemic lupus erythematosus, one with mixed connective tissue disease, and one with Takayasu disease), 17 patients with viral infection (seven with influenza, three with chicken pox, three with exanthema subitum, three with rota, and one with mumps), and 18 healthy children with minor congenital cardiac defects. All patients or their parents gave informed consent to participate in this study.
KD was diagnosed based on the standard criteria.13 Echocardiography was performed on all patients with KD every 2 weeks for at least 3 months after disease onset. Because all 25 patients with KD were less than 5 years old, patients with coronary arteries greater than 3 mm diameter were defined as having coronary artery aneurysms.14 All patients with KD were treated with a high dose of g-globulin (400 mg/kg £ 5 days) and aspirin (30 mg/kg/day) within 7 days of disease onset. Bacterial infections were diagnosed based on the results of cultures of urine, stool, blood and/or sputum together with the clinical signs and chest X-ray. Viral infections were diagnosed based on changes in virus-specific antibodies in paired samples together with the clinical signs. The diagnosis in all 10 patients with autoimmune disease had been established by the time they had participated in this study. Blood samples for the measurement of the serum PCT and CRP concentrations and the WBC count were obtained simultaneously in all study subjects. In the 25 patients with KD, acute-phase (within 7 days after the disease onset) blood samples, subacute-phase (median 14 days, range 10 – 18 days) blood samples, and convalescence-phase (median 28 days; range 22 – 35 days) blood samples were available in 25, 13, and 12 patients, respectively. The first sample was obtained before any treatment in all the KD patients. In the patients with bacterial and viral infections, blood samples were obtained during the active stage of the diseases. In the patients with autoimmune diseases, the blood samples were obtained during an acute phase or flare. In the patients with minor congenital cardiac defects, the blood samples were obtained when they were afebrile, not taking any medications, and had normal physical findings.
Determination of the serum concentrations of PCT and CRP Blood samples were centrifuged and the sera were stored at 220 8C until use. The serum concentration
Table 1 Clinical and laboratory data in the 112 study subjects Disease
No. of patients
Age (year)
WBC ( £ 102/ml)
CRP (mg/dl)
PCT (ng/ml)
Kawasaki disease Acute ( % 7 days) Subacute (10–18 days) Convalescence (22–32 days) Bacterial infections Autoimmune diseases Viral infections Healthy controls
25 13 12 17 10 17 18
2.8 ^ 2.0 3.5 ^ 2.3 2.5 ^ 1.6 4.4 ^ 5.3 8.9 ^ 4.0 3.6 ^ 2.9 2.7 ^ 3.5
143 ^ 71 101 ^ 50 87 ^ 4.0 110 ^ 6.9 107 ^ 49 76 ^ 33** 87 ^ 22**
9.4 ^ 6.1 2.0 ^ 5.9 0.1 ^ 0.2 11.1 ^ 5.7 7.3 ^ 7.8 0.4 ^ 0.5** 0.0 ^ 0.0**
2.3 ^ 3.0 0.3 ^ 0.7 0.1 ^ 0.2 2.2 ^ 2.9 0.4 ^ 0.4* 0.4 ^ 0.3* 0.2 ^ 0.1**
WBC, white blood cell count; CRP, C-reactive protein; PCT, procalcitonin. * and **, p , 0:01 and p , 0:001 vs. acute KD, respectively.
Procalcitonin in Kawasaki disease
of PCT was measured using a specific and ultrasensitive commercial immunoluminometric assay (BRAHMS Diagnostika, Berlin, Germany), with a luminometer (LUMAT LB 9507, BERTHOLD, Berlin, Germany) according to the manufacturer’s instructions. This method uses two monoclonal antibodies that bind to two sites (katacalcin and calcitonin) of the PCT molecule, thus ruling out cross-reactivity with calcitonin. The limit of detection of this assay is 0.1 ng/ml. The normal reference values for the serum concentration of PCT range from 0.1 to 0.5 ng/ml.1 CRP was measured using a latex immunoassay on a CRP-LATEX ‘SEIKEN’ (DENKA, Tokyo). The limit of detection of this assay is 0.1 mg/dl. The normal reference value for the serum CRP is less than 0.5 mg/dl.
Statistical analysis Data are reported as the mean ^ SD. A receiver operating characteristic curve was constructed for the determination of the optimal cut-off values of PCT and CRP and the WBC count for predicting the development of coronary aneurysms. Results were analyzed using the Mann – Whitney U-test for unpaired samples, the unpaired t-test, and the Spearman correlation coefficient. A level of p , 0:05 was accepted as statistically significant.
Results Both the mean WBC count and the mean CRP were significantly greater in the patients with acute KD than in the patients with viral infections and the healthy controls, but did not differ from those in the patients with bacterial infections and autoimmune diseases (Table 1). The mean PCT was significantly higher in the patients with acute KD than in the patients with autoimmune diseases and viral infections and in the healthy controls, but did not differ from that in the patients with bacterial infections. Although the patients with autoimmune diseases had elevated WBC counts and serum CRP concentrations, they did not have an elevated PCT concentration. The individual patient PCT data are shown in Fig. 1. Patients with acute KD and bacterial infections exhibited a similar magnitude increase in the PCT concentration. However, patients with autoimmune diseases or viral infections did not exhibit a significant increase in the PCT concentration compared with the healthy controls. In the KD patients, PCT, WBC count, and CRP all decreased from the onset of disease (Table 1 and Fig. 2).
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Figure 1 Serum procalcitonin concentration in the various study groups. Data were analyzed by the Mann– Whitney U-test.
However, the WBC count did not decrease in some patients. Four (16%) of the 25 patients with KD eventually developed coronary aneurysms 10 ^ 2 days from disease onset. Patients with higher PCT concentrations, WBC counts, and CRP concentrations appeared to develop this complication (Fig. 2). The four patients who developed coronary aneurysms had higher acute-phase concentrations of PCT (7.2 ^ 3.8 ng/ml vs. 1.4 ^ 1.7 ng/ml, p , 0:01), WBC counts (223 ^ 145 £ 102/ml vs. 127 ^ 36 £ 102/ml, p , 0:05), and CRP concentrations (15.0 ^ 4.9 mg/dl vs. 8.4 ^ 5.9 mg/dl, p ¼ 0:05) than did the 21 patients who did not develop coronary aneurysms. Two of the four patients who developed coronary aneurysms exhibited a sustained, high PCT concentration and/or WBC count (Fig. 2). A receiver operating characteristic curve (ROC) was constructed to determine the optimal cut-off value for the PCT, WBC count, and CRP of the acute KD patients for predicting the subsequent development of coronary aneurysms (Fig. 3). The area under the ROC curve (AUC) was . 0.8 for all three parameters ðp , 0:001Þ: Thus, all the parameters were statistically significant predictors for coronary aneurysms. The optimal cut-off value for predicting coronary aneurysms was 3 ng/ml for PCT, 170 £ 102/ml for the WBC, and 12 mg/dl for CRP. We evaluated the predictive value of the optimal cut-off values for the PCT, the WBC count, and the CRP (Table 2). A serum PCT concentration of 3 ng/ ml gave a sensitivity of 100% (4/4), a specificity of 90% (19/21), and a positive predictive value (PPV) of 67% (4/6). A WBC of 170 £ 102/ml gave a sensitivity of 100% (4/4), a specificity of 86% (18/ 21), and a PPV of 57% (4/7). A serum CRP
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Figure 3 Receiver operating characteristic curves for predicting coronary aneurysms using the serum concentrations of procalcitonin (X), C-reactive protein (B), and the white blood cell count (O). The optimal cut-off value indicated by an arrow was 3 ng/ml for procalcitonin, 170 £ 102/ml for the white blood cell count, and 12 mg/dl for C-reactive protein.
Figure 2 Changes in the serum procalcitonin, and Creactive protein (CRP) concentrations, and the white blood cell counts (WBC) in patients with Kawasaki disease. (X), Patients who developed coronary aneurysms; (W), patients who did not develop coronary aneurysms. Data were analyzed by the Mann –Whitney U-test.
concentration of 12 mg/dl gave a sensitivity of 100% (4/4), a specificity of 81% (17/21), and a PPV of 50% (4/8). Thus, the optimal cut-off value of the PCT was superior to those of the WBC count and CRP with respect to specificity and PPV. We analyzed the correlation between the PCT, WBC count, and CRP (Fig. 4). There was no significant correlation between any two variables among the PCT, WBC, and CRP in the patients with acute KD (CRP and PCT; r ¼ 0:371; p ¼ 0:06); (WBC and PCT; r ¼ 0:19; p ¼ 0:35); and (WBC and CRP; r ¼ 0:21; p ¼ 0:32). This suggests that any combination of the two parameters better predicts the development of coronary aneurysms than does one parameter. Indeed, some of the patients who had only one parameter of ^ the optimal cut-off value were false negative with respect to the development of coronary aneurysms, while all patients who had at least two parameters . the optimal cut-off value developed coronary aneurysms (Fig. 4).
Table 2 Prediction of coronary aneurysms in patients with Kawasaki disease using the optimal cut-off values of PCT, WBC, and CRP
PCT WBC CRP
Cut-off value
Sensitivity
Specificity
PPV
NPV
3 ng/ml 170 £ 102/ml 12 mg/dl
4/4 (100%) 4/4 (100%) 4/4 (100%)
19/21 (90%) 18/21 (86%) 17/21 (81%)
4/6 (67%) 4/7 (57%) 4/8 (50%)
19/19 (100%) 18/18 (100%) 17/17 (100%)
PCT, procalcitonin; WBC, white blood cell count; CRP, C-reactive protein; PPV, positive predictive value; NPV, negative predictive value.
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Figure 4 Relationships among the procalcitonin and C-reactive protein (CRP) concentration and the white blood cell counts (WBC) in patients with acute-phase Kawasaki disease. (X), Patients who developed coronary aneurysms; (W), patients who did not develop coronary aneurysms. Horizontal and vertical bars indicate the optimal cut-off values determined by the operating characteristics curves in Fig. 3. The optimal cut-off value was 3 ng/ml for procalcitonin, 170 £ 102/ml for the white blood cell count, and 12 mg/dl for C-reactive protein.
Discussion This may be the first report in which the serum PCT concentration was determined in patients with KD. In this study, the serum concentration of PCT increased in the acute-phase KD patients to a similar extent to that seen in patients during the acute phase of a bacterial infection. Although the extent of the increase in both the WBC count and the CRP was similar between the patients with KD and autoimmune diseases, the increase in the PCT differed between them: the PCT increased in the patients with KD, but not in the patient with autoimmune diseases, suggesting, that the serum PCT concentration differentiates KD patients from patients with autoimmune diseases. Although the number of study subjects was small, the serum PCT concentration appeared to be a better predictor of the development of coronary aneurysms than either the WBC count or the CRP. Use of the PCT concentration in conjunction with the WBC or the CRP concentration enhanced the accuracy of the prediction of coronary aneurysms, suggesting the clinical usefulness of PCT in patients with KD. The present study confirmed the results of earlier studies on PCT in patients with bacterial infections, viral infections, and autoimmune diseases.6,15 A systemic generalized bacterial infection is associated with an increase in the serum PCT concentration.1 Localized bacterial infections without systemic manifestations and viral infection are associated with a small increase in the PCT concentration (0.3 – 1.5 ng/ml).1 The PCT concentration does not increase (, 0.5 ng/ml) in adult patients with systemic lupus erythematosus and
systemic antineutrophil cytoplasmic antibody associated vasculitis.15 Although the microorganism responsible for KD is still not determined, abnormal immune responses to bacterial infection are thought to play a role in the pathogenesis of KD.16 In addition, viruses such as Epstein – Barr and adenoviruses have been considered responsible for KD.17,18 However, the patients with acute KD were similar to the patients with bacterial infections with respect to the increase in the PCT in this study. Since elevated autoantibodies are seen in KD patients, autoimmunity also is thought to be involved in the pathogenesis of KD.9 It may be interesting to note that the WBC and CRP concentration did not differentiate acute KD patients from patients with autoimmune diseases, while the PCT concentration differentiated the acute KD patients from the patients with autoimmune diseases. Thus, the determination of PCT may be clinically useful in differential diagnosis of febrile patients with skin rashes caused by some bacterial infections, viral infections, autoimmune diseases, or KD. KD, an acute febrile illness, is the most common cause of systemic vasculitis in children.19 Coronary aneurysms which occur in 10 – 20% of KD patients, are the most serious complication of KD.20 Therefore, the counselling of patients with KD is an important task for clinicians whether coronary aneurysms develop. Parameters such as the WBC and the CRP, TNFa, and IL-6 concentrations have been reported to predict the later development of coronary aneurysms.21 – 24 The serum PCT concentration appeared to be the best single parameter for the prediction of this complication in this study. Since the PCT did not significantly correlate with the WBC count or the CRP concentration, a combination of the PCT with the WBC count or the
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CRP concentration enhanced the accuracy of predicting coronary aneurysms. The sensitivity and specificity were 100% and 100%, respectively using the two optimal cut-off values of the PCT and WBC or CRP. However, the combination of PCT and the WBC or CRP was not superior to that of the WBC and the CRP in predicting coronary aneurysms in this study, perhaps due to the small number of study subjects. Differences in the induction time and half-life in the serum between PCT and CRP25 may partly explain why the PCT concentration did not significantly correlate with the CRP concentration. The physiologic role of PCT has not been extensively studied. TNFa is massively released in response to bacterial toxins and plays a role in septic shock.26 PCT has recently been demonstrated to inhibit lipopolysaccharide-induced TNFa release from human whole blood.27 It is possible that an increased PCT in response to a bacterial infection may have a role in the prevention of excessive inflammation through suppressing TNFa. Since an increased serum concentration of TNFa may be associated with the pathogenesis of generalized vasculitis in patients with KD,23 the increased PCT in the acute KD patients may play a physiologic role in suppressing the vasculitis caused by KD, although the KD patients with a high PCT actually tended to develop coronary aneurysms. In conclusion, the serum concentration of PCT was increased in acute KD patients. The PCT predicted the later development of coronary aneurysms, suggesting that it might be clinically useful to measure the serum PCT concentration in patients with KD.
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