Pemphigus is a group of autoimmune blistering skin diseases. The target antigens are desmogleins (Dsgs), which are cadherins responsible for cell–cell adhesion 1,2. Pemphigus is mainly classified into pemphigus vulgaris (PV) and pemphigus foliaceus 3. PV is further subclassified into mucosal (mPV), mucocutaneous (mcPV), and cutaneous (cPV) 4. Pemphigus foliaceus targets Dsg1, mPV targets Dsg3, and mcPV and cPV target both Dsg3 and Dsg1. The diagnosis of pemphigus is based on clinical presentation, histopathology, immunofluorescence, and serology by using the Dsg enzyme-linked immunosorbent assay (ELISA) 5,6.
Pemphigus disease area index (PDAI) (Table 1) is a scoring system developed by the international pemphigus committee to standardize the description of disease severity 7,8. Quantifying disease severity allows evaluation and comparison of treatment efficacy among different centers 7–9. The PDAI comprises a total of 250 points: cutaneous, 120 points; mucosal, 120 points; and scalp, 10 points.
This work aims to determine the usefulness of PDAI and the Dsg ELISA in the follow-up of Egyptian PV patients. The severity of PV was determined using the PDAI scoring system. The Dsg3 and Dsg1 indices were measured throughout the disease time course of PV patients and then correlated with the PDAI.
Materials and methods
Twenty PV patients were included in this study. These patients were the first 20 patients diagnosed with PV in 2012 and who were followed up for 6 months. Sixteen patients did not receive treatment earlier. Four patients were treated previously in a private clinic. All the patients included in this study had typical clinical, pathological, and immunofluorescence features of PV. All the patients received systemic steroids 1 mg/kg/day and azathioprine 2 mg/kg/day or cyclophosphamide 3 mg/kg/day. One patient was resistant and she received additional therapy of mycophenolate mofetil 2 g/day. All the sera were collected and stored at −30°C until testing. This study was approved by the institution review board of Cairo University and was conducted according to the Declaration of Helsinki Principles.
PDAI (Table 1) was used to evaluate disease severity at the initial visit of the patients and during their follow-up. Seven patients were followed up every month. However, 13 patients were not compliant and they were followed up every 3 months. PDAI at least 25 was considered severe, PDAI between 24 and 14 was considered moderate, and PDAI less than 14 was considered mild.
The reactivity of the sera against human Dsgs was measured by Dsg3 and Dsg1 ELISAs (MBL, Nagoya, Japan) and was detected using horseradish peroxidase-conjugated anti-human IgG according to the manufacturer’s instructions. The index value was calculated by the following equation: (optical density−negative control)/(positive control−negative control)×100. Index value above 20 was considered positive; index value from 15 to 20 was considered the gray zone.
The correlation between PDAI and Dsg3 and Dsg1 indices was calculated using Spearman’s correlation with GraphPad prism 5 for windows (San Diego, California, USA). First, the PDAI was correlated with the Dsg index for all patients. Then the correlation between the PDAI and Dsg index for each patient was calculated. P values less than or equal to 0.05 were considered significant.
Fifteen patients were female and five were male. Their ages ranged from 20 to 76 years (mean age 44.4±15.4 years). Three patients were mPV and 17 were mcPV. At their initial visit, their PDAI ranged from 1 to 120 (mean PDAI 22.6±34). Five patients had PDAI greater than or equal to 25, four had PDAI between 24 and 14, and 11 patients had PDAI less than 14.
Dsg3 autoantibodies were detected in 100% of the tested sera
All of the tested PV sera possessed autoantibodies targeting the Dsg3. The 17 mcPV sera possessed anti-Dsg1 in addition to anti-Dsg3 antibodies. The initial anti-Dsg3 antibodies ranged from 23 to 300 (mean 145±85).
Initial PDAI correlated with both the Dsg3 and Dsg1 indices
The initial PDAI of all the patients was positively correlated with their Dsg3 indices (Fig. 1). The correlation coefficient was 0.76 (P=0.00008). Moreover, the initial PDAI of all the patients was found to be positively correlated with their Dsg1 indices. The correlation coefficient was 0.71 (P=0.0004). After 6 months of treatment, the Dsg3 and Dsg1 indices of the patients showed a tendency to decrease or increase with the PDAI. The correlation coefficient was 0.5 (P=0.01).
PDAI was useful in modifying the treatment of one patient
The PDAI and Dsg3 index of one patient remained the same (PDAI 10) after 1 month of treatment with prednisolone 1 mg/kg/day and azathioprine 2 mg/kg/day. After adding mycophenolate mofetil, her disease came under control and her PDAI decreased to 8. However, her Dsg3 index remained stable.
Dsg3 decreased gradually throughout the time course of controlled PV patients
After 6 months of therapy, the Dsg3 and Dsg1 indices decreased with PDAI in 18 of 20 patients. The average initial PDAI of the 18 patients was 127 (±86) and it decreased to 83 (±62). After 6 months of treatment, the Dsg3 was more than 20 U/ml (still positive) in 13 of 20 (65%) patients. Examples of patients are shown in Fig. 2 a–d. In contrast, Dsg1 was less than 15 (considered negative) in 16 of 17 mcPV cases.
Dsg3 increased before PDAI in two relapsing patients
Two patients relapsed during the treatment (Fig. 3). Their Dsg3 indices were elevated even before the clinical lesions started to develop and before the PDAI started to increase. An mPV patient developed skin lesions and her clinical phenotype shifted from mPV to mcPV during her relapse (Fig. 3b).
ELISA quantitatively measures the autoantibodies and is therefore considered a useful assay in autoimmune diseases. ELISA for pemphigus was developed by Ishii et al.5. Several research groups proved that ELISA is sensitive and specific for the diagnosis and follow-up of pemphigus patients 5,6,10–12. In this work 20 PV patients were followed up by PDAI and Dsg ELISA. To our knowledge this is the first work that evaluated ELISA in correlation with PDAI.
PDAI is an objective disease extent score developed by the international pemphigus committee to compare clinical trials and outcomes of treatments 7. When 19 patients started treatment, their PDAI decreased. In one patient, however, PDAI did not decrease initially and did so only after the addition of mycophenolate mofetil as an adjuvant treatment. Moreover, patients without clinical lesions scored 0. In contrast, relapsing patients showed an increase in their PDAI. Taken together, these results suggest that decreasing PDAI score is a useful indicator of therapeutic efficacy.
As the anti-Dsg3 antibodies are the main antibodies in PV patients, follow-up of PV patients is done by evaluating the anti-Dsg3 antibodies. The results showed that while controlling the PV disease the Dsg3 index decreased throughout the time course of the patients. However, longer follow-up for several years is necessary for the anti-Dsg3 index to become undetectable. Interestingly, in relapsing patients, the anti-Dsg3 antibodies increased before the PDAI. Therefore, the anti-Dsg3 antibodies are useful for the management of PV patients. Measuring autoantibodies should be done before tapering the systemic steroids. If the anti-Dsg3 antibodies are decreasing, it is safe to taper the systemic steroids. However, if the anti-Dsg3 antibodies are increasing, adjuvant therapy should be added.
In contrast, mcPV possesses anti-Dsg1 in addition to anti-Dsg3 antibodies. The results showed that the anti-Dsg1 antibodies decreased once the disease was under control. Moreover, in a relapsing mPV patient, acquiring the anti-Dsg1 antibodies shifted the clinical phenotype from mPV to mcPV. The results confirmed the previous work of other research groups that stated that anti-Dsg1 antibodies are linked to the skin lesions 13,14.
Although this work and that of others demonstrated a relationship between severity and autoantibody levels 5,15–18, others showed that the autoantibodies did not correlate with the disease activity 19. Moreover, few patients remained with high index even in remission 20. This was explained by the fact that those autoantibodies were nonpathogenic. Therefore, efforts have been made to develop ELISAs that detect only pathogenic antibodies 21,22.
In active PV patients, PDAI is valuable in evaluating the treatment efficacy and modifying the treatment if necessary. However, after PDAI reaches 0, the Dsg3 index is valuable for determining either disease control or relapse. Although routine monthly follow-up of PV patients with ELISA is expensive, it is recommended for detecting early relapse.
Conflicts of interest
There are no conflicts of interest.
1. Amagai M.Autoimmunity against desmosomal cadherins in pemphigus.J Dermatol Sci1999;20:92–102.
2. Eyre RW, Stanley JR.Identification of pemphigus vulgaris
antigen extracted from normal human epidermis and comparison with pemphigus foliaceus antigen.J Clin Invest1988;81:807–812.
3. Stanley JR, Amagai M.Pemphigus, bullous impetigo, and the staphylococcal scalded-skin syndrome.N Engl J Med2006;355:1800–1810.
4. Yoshida K, Takae Y, Saito H, Oka H, Tanikawa A, Amagai M, Nishikawa T.Cutaneous type pemphigus vulgaris
: a rare clinical phenotype of pemphigus.J Am Acad Dermatol2005;52:839–845.
5. Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, Gamou S, et al..Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins.J Immunol1997;159:2010–2017.
6. Amagai M, Komai A, Hashimoto T, Shirakata Y, Hashimoto K, Yamada T, et al..Usefulness of enzyme-linked immunosorbent assay
using recombinant desmogleins 1 and 3 for serodiagnosis of pemphigus.Br J Dermatol1999;140:351–357.
7. Murrell DF, Dick S, Ahmed AR, Amagai M, Barnadas MA, Borradori L, et al..Consensus statement on definitions of disease, end points, and therapeutic response for pemphigus.J Am Acad Dermatol2008;58:1043–1046.
8. Daniel BS, Hertl M, Werth VP, Eming R, Murrell DF.Severity score indexes for blistering diseases.Clin Dermatol2012;30:108–113.
9. Rosenbach M, Murrell DF, Bystryn J-C, Dulay S, Dick S, Fakharzadeh S, et al..Reliability and convergent validity of two outcome instruments for pemphigus.J Invest Dermatol2009;129:2404–2410.
10. Anand V, Khandpur S, Sharma VK, Sharma A.Utility of desmoglein
ELISA in the clinical correlation and disease monitoring of pemphigus vulgaris
.J Eur Acad Dermatol Venereol2012;26:1377–1383.
11. Tampoia M, Giavarina D, Di Giorgio C, Bizzaro N.Diagnostic accuracy of enzyme-linked immunosorbent assays (ELISA) to detect anti-skin autoantibodies in autoimmune blistering skin diseases: a systematic review and meta-analysis.Autoimmun Rev2012;12:121–126.
12. Harman KE, Gratian MJ, Seed PT, Bhogal BS, Challacombe SJ, Black MM.Diagnosis of pemphigus by ELISA: a critical evaluation of two ELISAs for the detection of antibodies to the major pemphigus antigens, desmoglein
1 and 3.Clin Exp Dermatol2000;25:236–240.
13. Harman KE, Gratian MJ, Bhogal BS, Challacombe SJ, Black MM.A study of desmoglein
1 autoantibodies in pemphigus vulgaris
: racial differences in frequency and the association with a more severe phenotype.Br J Dermatol2000;143:343–348.
14. Amagai M, Tsunoda K, Zillikens D, Nagai T, Nishikawa T.The clinical phenotype of pemphigus is defined by the anti-desmoglein
autoantibody profile.J Am Acad Dermatol1999;402 I167–170.
15. Chorzelski TP, Von Weiss JF, Lever WF.Clinical significance of autoantibodies in pemphigus.Arch Dermatol1966;93:570–576.
16. Harman KE, Seed PT, Gratian MJ, Bhogal BS, Challacombe SJ, Black MM.The severity of cutaneous and oral pemphigus is related to desmoglein
1 and 3 antibody levels.Br J Dermatol2001;144:775–780.
17. Herrero-Gonzälez JE, Iranzo P, Benítez D, Lozano F, Herrero C, Mascaró JM Jr.Correlation of immunological profile with phenotype and disease outcome in pemphigus.Acta Derm Venereol2010;90:401–405.
18. Schmidt E, Dähnrich C, Rosemann A, Probst C, Komorowski L, Saschenbrecker S, et al..Novel ELISA systems for antibodies to desmoglein
1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients.Exp Dermatol2010;19:458–463.
19. Fitzpatrick RE, Newcomer VD.The correlation of disease activity and antibody titers in pemphigus.Arch Dermatol1980;116:285–290.
20. Kwon EJ, Yamagami J, Nishikawa T, Amagai M.Anti-desmoglein
IgG autoantibodies in patients with pemphigus in remission.J Eur Acad Dermatol Venereol2008;22:1070–1075.
21. Kamiya K, Aoyama Y, Shirafuji Y, Hamada T, Morizane S, Fujii K, et al..Detection of antibodies against the non-calcium-dependent epitopes of desmoglein
3 in pemphigus vulgaris
and their pathogenic significance.Br J Dermatol2012;167:252–261.
22. Sharma PM, Choi EJ, Kuroda K, Hachiya T, Ishii K, Payne AS.Pathogenic anti-desmoglein
MAbs show variable ELISA activity because of preferential binding of mature versus proprotein isoforms of desmoglein
3.J Invest Dermatol2009;129:2309–2312.