A Validation of ELISA Test for Detection of Desmoglein 1 & 3 Antibodies as a Diagnostic Test of Pemphigus

Teena, Mathew (2007) A Validation of ELISA Test for Detection of Desmoglein 1 & 3 Antibodies as a Diagnostic Test of Pemphigus. Masters thesis, Christian Medical College, Vellore.

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Abstract

INTRODUCTION : Pemphigus is the commonest autoimmune vesiculobullous disorder on the Indian subcontinent. A study on mortality among inpatients in dermatology from India found pemphigus vulgaris to be the commonestdisorder to cause death. Pemphigus is an autoimmune disease that results in blistering of the skin and oral cavity, which if left untreated is almost always fatal. It is caused by autoantibodies directed against cell-surface antigens on keratinocytes, which when targeted lose their cellular adhesion properties and separate from one another to form blisters within the epidermis. It is classified into two major subtypes: pemphigus vulgaris (PV) and pemphigus foliaceus (PF).4 Differences in the particular antigens targeted by the antibodies and in the distribution of these antigens in the different regions of the body and in the separate layers of the epidermis result in different clinical manifestations of the disease. The diagnosis of pemphigus rests upon the demonstration of these autoantibodies by immunofluorescence techniques whilst the differentiation of PV and PF relies upon the combination of clinical and histological features. Although Western immunoblotting and immunoprecipitation can be used to identify the target antigen in cases of pemphigus, these are both time consuming qualitative techniques which are impractical for routine screening of large number of serum samples. Pemphigus autoantibodies are mainly directed against the desmosomal adhesion molecules, desmoglein 1 (Dsg 1) and desmoglein 3 (Dsg 3). The production of recombinant Dsg 1 and Dsg 3 antigens has made possible the development of enzyme linked immunosorbent assays (ELISAs). Recently Dsg 1 and Dsg 3 ELISAs have been shown to provide a simple, sensitive and highly specific assay for the diagnosis of patients with PV and PF. Since precise diagnosis of these two diseases is important for determining treatment plans and prognosis, sensitive and specific testing for the pathogenic autoantibodies found in PV and PF would be helpful in the clinical management and laboratory investigation of these two severe autoimmune diseases. As there is a paucity of studies from India, the present study is an attempt to validate ELISA estimation of desmoglein antibodies as a diagnostic test for pemphigus and to study the prevalence of desmoglein antibodies in the various clinical types of pemphigus in Indian patients. AIMS OF THE STUDY : 1) To validate ELISA estimation of Dsg 1 and Dsg 3 antibodies in pemphigus as a diagnostic test in Indian patients. 2) To study the correlation of Dsg 1 and Dsg 3 antibody titres with a) mucosal and cutaneous involvement, b) severity of disease. 3) To investigate the prevalence of Dsg 1 and Dsg 3 antibodies in the different pemphigus variants. MATERIALS AND METHODS : Study Design : Cross sectional study. Setting: The study was conducted in the Outpatient Department of Dermatology, Venereology and Leprosy, Christian Medical College, a tertiary care hospital in South India. Duration of study: The study was conducted between March 2005 and July 2006. Study population: All patients with acquired immunobullous disorders, diseases targeting desmogleins or having conditions where antibodies mimic or show in vitro deposition in stratified squamous epithelium in the absence of pemphigus were eligible for inclusion into the study. Inclusion criteria: Patients : All consecutive patients with pemphigus who consented for the study and in whom the diagnosis had been confirmed by histopathology and direct immunoflourescence were included. Controls: 1) Patients with acquired immunobullous disorders other than pemphigus (eg. bullous pemphigoid, epidermolysis bullosa aquisita or chronic bullous dermatosis of childhood) who consented for the study and who had been diagnosed by clinical features, histopathology and DIF were included as in other studies. 2) Patients with diseases targeting desmogleins like bullous impetigo or Staphylococcal Scalded Skin Syndrome (SSSS) or having conditions where antibodies mimic or show in vitro deposition in stratified squamous epithelium in the absence of pemphigus like toxic epidermal necrolysis (TEN), who consented for the study and who had been diagnosed by clinical features, gram stain, histopathology or DIF as appropriate were included. Exclusion criteria: Patients : Patients not willing for the study. Controls : Patients not willing for the study Methodology: All pemphigus patients who conformed to the inclusion criteria were examined by the principal investigator after written informed consent was taken (annexure I). The details regarding duration of disease, sites of involvement, duration and type of treatment, relapses and extent of lesions were recorded in a proforma (annexure II). Details of diagnostic tests were recorded. Patients were categorized into the various clinical types based on the clinical features, histopathology and DIF. The severity of lesions were assessed clinically based on the scoring system used by Harman et al.81 Skin lesions and oral lesions were scored separately and recorded. RESULTS : Seventy five patients were enrolled into the study between March 2005 and July 2006. There were 39 (52%) pemphigus patients and 36 (48%) controls. The mean age of the study population was 37 years with a SD of ± 20.68. The oldest being 75 years and the youngest being 1 year old. There were 36 males (48%) and 39 females (52%). Male to female ratio was 1:1.08. CONCLUSIONS : 1. There were a total of 75 patients in the study consisting of 39 patients and 36 controls. The pemphigus patients had a mean age of 43.21 years and the male to female ratio was 1:1.43. Among the clinical variants of pemphigus there was a predominance of PV patients as seen in other studies. 2. ELISA estimation of Dsg 1 antibodies in pemphigus had a sensitivity and specificity of 100% and 97.2% respectively. Dsg 3 estimation had a sensitivity and specificity of 82.14% and 100% respectively. This is similar to the sensitivity and specificity reported from other studies. 3. The cut off values with the highest sum of sensitivity and specificity for Dsg 1 and Dsg 3 ELISAs in the population studied were 17.79 and 28.91 respectively. 4. Dsg 3 was positive in 23 (82.14%) of the 28 patients with PV, while Dsg 1 was postitive in 18 (64.28%) of the 28. Dsg 1 was posititve and Dsg 3 was negative in all patients with PF. 5. Using ELISA for Dsg 1 and Dsg 3 antibodies, it is possible to differentiate PV from PF. 6. There was a significant correlation between the presence of mucosal disease and positive Dsg 3 antibodies, and the presence of cutaneous disease and positive Dsg 1 antibodies. 7. There was a trend for increase in Dsg 1 titres with increasing skin severity scores but no such trend was seen between increasing oral severity scores and Dsg 3 antibody titres. 8. There was no significant difference in Dsg titres between patients with acute onset and chronic active disease. 9. There was no correlation between Dsg titres and duration of disease. 10. There was no correlation between Dsg titres and number of relapses.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Validation of ELISA Test ; Detection of Desmoglein 1 & 3 Antibodies ; Diagnostic Test ; Pemphigus.
Subjects: MEDICAL > Dermatology Venereology and Leprosy
Depositing User: Subramani R
Date Deposited: 02 Mar 2018 16:10
Last Modified: 03 Mar 2018 05:45
URI: http://repository-tnmgrmu.ac.in/id/eprint/5998

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