|Year : 2014 | Volume
| Issue : 2 | Page : 125-128
Autoantibody profile in a cohort of South Indian children with Kawasaki disease
Suchetha Rao1, Chaitanya Verma1, Rathika Shenoy2, Nutan Kamath1
1 Department of Pediatrics, Kasturba Medical College, Mangalore, Manipal University, Mangalore, Karnataka, India
2 Department of Pediatrics, KS Hegde Medical Academy, Deralkatte, Mangalore, Karnataka, India
|Date of Web Publication||1-Jul-2014|
Department of Pediatrics, Kasturba Medical College, Manipal University, Mangalore - 575 001, Karnataka
Source of Support: None, Conflict of Interest: None
Objective: There is no clear data on autoantibody levels in Kawasaki Disease (KD) especially from the Indian Subcontinent. Aim: To look for the presence of organ nonspecific and organ specific antibodies to strengthen the search for an autoimmune cause of KD. We tested the presence of antinuclear antibody (ANA) and antithyroid microsomal antibody (TMA) in children with KD, 6 months after the acute phase. Anti Neutrophil Cytoplasmic Antibody (pANCA, cANCA), Anti Endothelial Cytoplasm Antibody (AECA) and Anti Smooth Muscle Antibody (SMA) was additionally tested in those with elevated titers of ANA and/or TMA. Materials and Methods: Prospective case-control study of 24 children with KD on follow up and an equal number of age and sex matched controls. Historical data about acute phase of illness was obtained from the medical records. After obtaining institutional ethics committee clearance and informed consent from the parents, blood was tested for ANA and TMA by the indirect immunofluorescence method (IIF), using a kit developed by Euroimmun. Positive samples were additionally tested for pANCA, cANCA, AECA and SMA. Relationship of autoantibody elevation and clinical course in the cases was determined. Results: The age of the study group was 4 3.2 years. Incomplete KD was seen in 12.5% of the cases. Five cases (21%) had cardiac involvement. All but one with mitral and tricuspid regurgitation resolved after the acute phase of the disease. Only her ANA was elevated. Two children (8%) positive for TMA did not show any cardiac abnormalities. Further antibody testing was negative. All three children with elevated autoantibodies were females. (P value = 0.02: statistically significant). Conclusion: Elevated autoantibodies in three (12.5%) children after the acute phase may suggest the role of autoimmunity in the etiopathogenesis of KD, even though our observations were not statistically significant.
Keywords: Antinuclear antibody, anti-thyroid microsomal antibody, autoimmunity, indirect immunofloroscence
|How to cite this article:|
Rao S, Verma C, Shenoy R, Kamath N. Autoantibody profile in a cohort of South Indian children with Kawasaki disease. Muller J Med Sci Res 2014;5:125-8
|How to cite this URL:|
Rao S, Verma C, Shenoy R, Kamath N. Autoantibody profile in a cohort of South Indian children with Kawasaki disease. Muller J Med Sci Res [serial online] 2014 [cited 2021 Oct 16];5:125-8. Available from: https://www.mjmsr.net/text.asp?2014/5/2/125/135742
| Introduction|| |
Kawasaki Disease (KD) is a medium vessel panarteritis of unknown etiology and variable clinical presentation. It was first described by Tomisaku Kawasaki of Japan as Acute Mucocutaneous Lymph Node Syndrome in 1967.  KD has become the leading cause of acquired heart disease in children in the United States and Japan. 
KD incidence in India may vary from 18,000-1,80,000 new cases per year.  The KD burden is likely to pose a significant challenge to the health care system in India in the coming years due to the high cost of treatment and the potential for lifelong cardiovascular sequel. 
According to American Heart Association (AHA) guidelines, the diagnosis of KD is based on a typical temporal sequence of clinical features. It includes fever of a minimum of 5 days duration along with at least four of the following - bilateral bulbar conjunctival injection without exudates, cervical lymphadenopathy, polymorphous exanthem, erythema of the lips and oral cavity, or desquamation of the extremities. Some patients who do not fulfill the above criteria have been diagnosed as having incomplete KD, a diagnosis that often is based on echocardiographic (ECHO) findings of coronary artery abnormalities. Atypical Kawasaki disease should be reserved for patients who have manifestations, such as renal impairment, that generally is not seen in KD. 
The etiology of KD is still not known. Both infectious and autoimmune etiologies have been proposed. Though the clinical and epidemiological aspects of the illness strongly suggest an infectious etiology, conventional bacterial and viral cultures and serological investigations have failed to yield an infectious cause.  A full understanding of the immunology of KD also awaits information about the etiology and pathogenesis of the illness. The reports on the presence of autoantibodies in KD are heterogeneous. ,,,,,,
The rationale for doing this study was that there is no clear cut data on autoantibody levels in KD, especially from the Indian subcontinent. This study attempts to look for the presence of organ nonspecific and organ specific antibodies to strengthen the search for an autoimmune cause of the disease. It would also enable us to decide whether these children with KD need to be monitored for the development of autoantibodies during follow-up.
| Materials and Methods|| |
This prospective case-control study consisted of 24 children with KD on follow-up in the Pediatric Rheumatology Clinic at Kasturba Medical College Hospital, Attavar, a tertiary referral hospital at Mangalore, Karnataka State, India and at least 6 months after the acute phase of illness and an equal number of age and sex matched controls. The study was carried out after the approval of the Institutional Ethics Committee. KD was diagnosed in our patients as per standard criteria [Table 1]. ,, The historical data about the acute phase was recorded from the child's medical records in a predesigned performa. Informed consent was obtained from parents. The blood for the investigations was drawn into a plain sample vacutainer after venupuncture, the serum was separated and stored at -80°C. A commercial kit developed by Euroimmun Medizinesche Labordiagnostika AG (Deutschland) was used for both Antinuclear antibodies (ANA) and Anti thyroid microsomal antibodies (TMA). The test kit was designed for the qualitative determination of antibodies in serum. For ANA test, if the reaction is positive in 1:100 dilutions, specific antibodies were stained with fluorescence labeled anti-human antibodies and made visible with the fluorescence microscope. These were then qualitatively read with the kit controls. For TMA, if the reaction is positive in initial dilution of 1:10, specific antibodies were stained with fluorescence labeled conjugate class anti-human IgG. The positive samples of ANA and/or TMA were further tested for Anti Neutrophil Cytoplasmic Antibody (pANCA, cANCA), Anti Endothelial Cytoplamic Antibody (AECA), and Anti Smooth Muscle Antibody (SMA). All interpretations were done under the direct supervision of a blinded microbiologist.
|Table 1: Kawasaki disease: Frequency and characteristics of diagnostic criteria|
Click here to view
Conventional statistics were used for calculating the mean and standard deviation (SD) of the parameters of the study. Fischer's Exact Test was applied to calculate the significance in this study.
| Results|| |
The clinical profile was analyzed in 24 children with KD who were diagnosed according to the criteria of AHA. 
Males constituted 67% and females 33% of the subjects. The male: The male : female sex ratio was 1.5:1. Majority of the children (79%) were below the age of 5 years. The youngest subject was 5 months. The age of the study group was 4 ± 3.2 years (5month-15years). Fever was the universal symptom. The frequency and characteristics of diagnostic criteria in the cases studied is depicted in [Table 1].
Incomplete KD was seen in three (12.5%) cases. Cardiac manifestations were present in five (21%) cases. Three (12.5%) cases had coronary artery dilatation, one (4%) case had coronary aneurysm which was resolved during follow-up phase. One (4%) case had mild mitral and tricuspid regurgitation which persisted during follow-up.
Among the laboratory investigations, mean ESR was 75.6 mm (range 10-192 mm) and mean CRP was 81.6 mg/dl (range 1.6-384 mg/dl). Thrombocytosis of more than 5 lac/cmm (mean 4.32, range 1.37-7.5 lac/cmm) and leucocytosis (mean 14,648, range 3800-34700 lac/cmm) was seen in 42% and 38% of the cases.
All the patients were advised intravenous immunoglobin (IVIG) but only 22 (92%) patients were treated with IVIG. Twenty (84%) subjects received IVIG at a dose of 2 g/kg. One child received two doses of IVIG. No child had any adverse effect to IVIG. All cases received high dose of aspirin (50-100 mg/kg/day) during the acute phase which was changed to low dose aspirin (3-5 mg/kg/day) and continued for an average of 6 weeks or till the acute phase reactants and coronary artery abnormalities returned to normal. 
The serum investigations for autoantibodies were done after at least 6 months of the acute illness. ANA was positive in one (4%) case at a dilution of 1:100 with the indirect immunofluoroscence (IIF), showing coarse speckling pattern for the nucleoli. The TMA was positive in two (8%) cases. These observations were not statistically significant. However, all three children with elevated autoantibodies were females, which was statistically significant (P = 0.02).
A persistent mitral and tricuspid regurgitation was seen in the child who had positive ANA result. Children with positive TMA result did not show any cardiac abnormalities. Thyroid function tests (Tri-iodothyronine, Thyroxine, and Thyroid Stimulating Hormone) in TMA positive children were normal. Tests done for pANCA, cANCA, SMA, AECA on all positive samples for ANA or TMA did not show any positive results [Table 2].
| Discussion|| |
Autoantibodies in KD to prove an autoimmune etiology is yet to be ascertained. Previous reports on the subject from North India and the developed world are controversial. ,,,,,,, Presentation of pediatric rheumatologic disease differ in our population when compared to Western literatures.  We therefore studied KD children in coastal Karnataka for their autoantibodies to look for variations, if any, in their profile within our country. ,
Nineteen out of 24 of our patients (79%) were younger than 5 years and youngest subject was 5 months, consistent with the age groups in other studies. 
Typical features [Table 1] were compared. Our study had higher percentage of nonpurulent bulbar conjunctivitis than the study from North India.  Three patients (12.5%) had diarrhea; an atypical feature of KD. Narayanan et al., observed diarrhea as a significant feature. 
Cardiac manifestations were found in 21% of the cases (5/24). Three (12.5%) cases had mild coronary artery dilatation, one (4%) case had coronary aneurysm, and all of which resolved during follow up. One case (4%) had mild mitral regurgitation (MR) and mild tricuspid regurgitation (TR) which persisted during follow up. Singh et al., showed ECG abnormalities in 17% of the cases. Western literatures reports coronary artery aneurysm to be less than 1% in patients who are treated with IVIG.  Twenty-two (92%) patients received IVIG within 10-days of fever and none of our subjects had coronary artery aneurysm in the convalescent phase. The two subjects who did not receive IVIG did not have coronary artery involvement.
Our case control study was designed to study the presence of one organ specific (TMA) and one organ nonspecific (ANA) autoantibody in KD patients at least 6 months after the acute episode. Antibodies can be passively acquired through IVIG given in the acute phase of illness. These antibodies are likely to disappear by about 3 months (after four half-lives of IVIG as each half-life is of 20-22 days)  AECA mediated-injury can play a major role in vascular lesions in collagen diseases. However, few are expressed on the cell surface and majority are directed against the intracellular molecules.  We could not study the autoantibodies like pANCA, cANCA, SMA and AECA described in the previous studies in all of our cases and controls due to restricted resources. These auto-antibodies were tested only if ANA and/or TMA were positive.
The gold standard screening method for the detection of ANA is the IIF method on human epithelial (HEp-2) cells. The characteristic fluorescence pattern and titers of autoantibodies can be identified on this substrate with high sensitivity. When the antigen is characterized, enzyme linked immunosorbent assay (ELISA) is believed to be a more sensitive test. However, this assay can have false negative screening results due to the limited number of displayed antigens. 
ANA was elevated in one case (4%). In earlier done studies higher prevalence of ANA has been demonstrated in the acute phase of KD when compared to the convalescent phase.  None of our controls was ANA positive. ANA positivity ranged from 1-2% in normal adult population. 
The child with ANA positivity has persistent MR and TR. No coronary artery aneurysm was demonstrable by ECHO. Thallium scan could not be done. Singh et al., demonstrated abnormal Thallium scan and normal ECHO in two patients with ANA positivity. 
Two subjects (8%) were positive for TMA. Their thyroid profile was normal. None of them had demonstrable coronary artery aneurysm as determined by ECHO. To the best of our knowledge, the first study on the role of TMA in KD was done by Singh et al., in North India, which demonstrated TMA positivity in 24%.  and our study may be the second. None of our controls was positive for TMA. The reported prevalence of TMA in the normal adult population is 2.17%.  The three cases who were ANA/TMA positive did not have antibodies to neutrophil cytoplasmic components. In a study comprising of 39 children (23 boys and 16 girls; median age 2.0 years) with KD, 14 (36%) had antibodies to neutrophil cytoplasmic components. 
A similar study showed that antibodies to myeloperoxidase (MPO), the dominant antigen responsible for pANCA reactivity, were detected by ELISA in 73% of acute phase and 89% of convalescent phase of KD subjects, in contrast to 4% of normal adult control subjects (P < 0.002 and P < 0.001, respectively).  Antibodies to myocardial muscle, cardiac perimysial connective tissue, nuclear antigens, and smooth muscle were also detected in some KD patients, but titers did not differ significantly from control patients. Autoantibody results were not predictive of patients with ECHO abnormalities.  It is also reported to have ANCA, AECA positivity in KD patients who are ANA negative. 
| Conclusion|| |
Autoantibodies were demonstrated in 12% of KD children on follow up. Prevalence of ANA was similar to values seen in earlier studies. , Prevalence of TMA was lower than the previously published data on North Indian children.  ANA profile, AECA, pANCA, cANCA and SMA were negative in our ANA/TMA positive children. Although a variety of autoantibodies are produced in patients with KD their specificities in many instances are controversial and their role in disease pathogenesis is undetermined.
| References|| |
|1.||T. Acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children. Arerugi 1967;16:178-222. |
|2.||Son MB, Newburger JW. Kawasaki disease. In: Nelson Textbook of Pediatrics. 19 th ed. Kliegman. Stanton, St Geme, Schor, Behrman, editors. Philadelphia: Saunders; 2011. p. 862-67. |
|3.||Kushner HI, Macnee R, Burns JC. Impressions of Kawasaki syndrome in India. Indian Pediatr 2006;43:939-42. |
|4.||Newburger JW, Takahashi M, Gerber MA, Gewitz MH, Tani LY, Burns JC, et al. Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association. Diagnosis, treatment, and long-term management of Kawasaki disease: A statement for health professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association. Pediatrics 2004;114:1708-33. |
|5.||Soppi E, Salo E, Pelkonen P. Antibodies against neutrophil cytoplasmic components in Kawasaki disease. APMIS 1992;100:269-72. |
|6.||Savage CO, Tizard J, Jayne D, Lockwood CM, Dillon MJ. Antineutrophilic cytoplasmic antibodies in Kawasaki disease. Arch Dis Child 1989;64:360-3. |
|7.||Birdi N, Laxer RM, Silverman ED. Antineutrophil cytoplasmic antibodies in Kawasaki disease. Arthritis Rheum 1991;34:70. |
|8.||Falcini F, Trapani S, Turchini S, Farsi A, Ermini M, Kesser G, et al. Immunological findings in Kawasaki disease: An evaluation in a cohort of Italian children. Clin Exp Rheumatol 1997;15:685-9. |
|9.||Nash MC, Shah V, Reader JA, Dillon MJ. Anti-neutrophil cytoplasmic antibodies and anti-endothelial cell antibodies are not increased in Kawasaki disease. Br J Rheumatol 1995;34:882-7. |
|10.||Rider LG, Wener MH, French J, Sherry DD, Mendelman PM. Autoantibody production in Kawasaki syndrome. Clin Exp Rheumatol 1993;11:445-9. |
|11.||Venkatraman R, Singh S, Minz RW. Study of the auto antibody profile after the acute phase of Kawasaki Disease in a cohort of children from North India. Rheumatol Int 2006;26:693-6. |
|12.||Sundel RP, Petty RE. Kawasaki disease. In: Textbook of Pediatric Rheumatology. 6 th ed. Cassidy, Petty, Laxer, Lindsley, editors. Philadelphia: Saunders; 2011. p. 505-20. |
|13.||Singh S, Bansal A, Gupta A, Kumar RM, Mittal BR. Kawasaki disease: A decade of experience from North India. Int Heart J 2005;46:679-89. |
|14.||Narayanan SN, Krishna V, Sabarinathan K. Kawasaki disease. Indian Pediatr 1997;34:139-43. |
|15.||Newburger JW, Sanders SP, Burns JC, Parmess IA, Beiser AS Colan SD. Left ventricular contractility and function in Kawasaki syndrome. Effect of intravenous gamma-globulin. Circulation 1989;79:1237-46. |
|16.||Shirai T, Fujii H, Ono M, Watanabe R, Ishii T, Harigae H. An innovative method to identify autoantigens expressed on the endothelial cell surface: Serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). Clin Dev Immunol 2013;2013:453058. |
|17.||Voigt J, Krause C, Rohwäder E, Saschenbrecker S, Hahn M, Danckwardt M, et al. Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells. Clin Dev Immunol 2012;2012:651058. |
|18.||Krasovec S, Bezrodnik L, Gaillard MI, Carabajal P, Ginaca A, Vainstein E. Kawasaki disease. Immunological evaluation of 26 cases. Medicina (B Aires) 2001;61:8-14. |
|19.||Sehgal S, Aikat BK, Pasricha N. Diagnostic significance of antinuclear antibodies. Indian J Med Res 1978;67:116-23. |
[Table 1], [Table 2]