Dermatology: Practical and Conceptual 262 Research | Dermatol Pract Concept 2018;8(4):3 DERMATOLOGY PRACTICAL & CONCEPTUAL www.derm101.com The potential roles of herpesvirus and cytomegalovirus in the exacerbation of pemphigus vulgaris Fariba Mohammadi1, Zahra Khalili1, Sayed Mahdi Marashi2, Amirhoushang Ehsani1, Maryam Daneshpazhooh1, Majid Teymoori-Rad2, Kamran Balighi1, Ahmad Nejati2, Shohreh Shahmahmoodi2, Shima Izadidakhrabadi2, Hamidreza Mahmoudi1, Pedarm Noormohammadpour1 1 Autoimmune Bullous Disease Research Center, Department of Dermatology, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran 2 Virology Department, School of Public Health (SPH), Tehran University of Medical Sciences (TUMS), Tehran, Iran Key words: pemphigus vulgaris, herpesvirus, cytomegalovirus Citation: Mohammadi F, Khalili Z, Mahdi Marashi S, Ehsani A, Daneshpazhooh M, Teymoori-Rad M, Balighi K, Nejati A, Shahmahmoodi S, Izadidakhrabadi S, Mahmoudi H, Noormohammadpour P. The potential roles of herpesvirus and cytomegalovirus in the exacerbation of pemphigus vulgaris. Dermatol Pract Concept. 2018;8(4):262-271. DOI: https://doi.org/10.5826/dpc.0804a03 Received: March 19, 2018; Accepted: July 18, 2018; Published: October 31, 2018 Copyright: ©2018 Mohammadi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by Tehran University of Medical Sciences (TUMS), Grant Numbers 32855-30-03-95 and 30976- 101-04-94. Competing interests: The authors have no conflicts of interest to disclose. All authors have contributed significantly to this publication. Corresponding author: Pedarm Noormohammadpour, MD, Autoimmune Bullous Disease Research Center, Razi Hospital, Vahdate-Eslami Square, 11996, Tehran, Iran. Email: normohamad@razi.tums.ac.ir Background: Among exogenous etiologies, the critical role of microbial agents such as herpesviruses (HSV1/2) and cytomegalovirus (CMV) in triggering and flaring autoimmune conditions such as pem- phigus vulgaris (PV) has been recently discovered. Objectives: The present study aimed to investigate the plausible role of these viruses in the exacerba- tion of PV using serological and molecular methods. Patients/Methods: Sixty patients with PV (30 with relapse type and 30 with remission type) were recruited for the purpose of this case-control study. Skin, mucosal, and throat specimens were obtained and examined for viruses by reverse transcriptase polymerase chain reaction. To determine the immu- noglobulin G (IgG) titer, enzyme-linked immunosorbent assay was used. Results: Desmoglein1-specific IgG was positive in  56.7% of patients with the relapse form and in  20.0% of those with the remission form indicating a significant difference across the  2  groups (P = 0.003), but the rate of positivity for desmoglein3-specific IgG in the relapse and remission types was  76.7% and  63.3%, respectively, with no significant difference (P = 0.260). There was no dif- ference in the mean levels of HSV-IgG and CMV-IgG in the relapse and remission groups. HSV and ABSTRACT Research | Dermatol Pract Concept 2018;8(4):3 263 Atypical HSV infections have been described in immu- nosuppressed patients [16]. Several studies have suggested the activation or exacerbation of pemphigus after HSV or CMV infections [17]. More recently, Japanese researchers have detected high levels of HSV in the saliva samples of PV patients [18]. In this regard, several studies have shown the benefits of adding antiviral therapies to an immunosuppres- sive therapy in recalcitrant disease [19,20]. Several studies also failed to detect herpesviruses in pemphigus patient speci- mens [21,22], suggesting that viruses may have only a tran- sient role for exacerbation of pemphigus disease, although yet to be further defined. The number of patients with PV in Iran appears to be increasing, and little information is available with regard to the role of HSV1/2 and CMV in triggering PV among the Iranian population. The present study aimed to investigate the plausible role of these viruses (HSV1/2 and CMV) in the exacerbation of PV using serological and molecular methods. Materials and Methods Disease Definitions in PV Patients PV patients were categorized as follows: relapse (develop- ment of more than 3 new lesions/month that do not heal within a week without treatment, or development of estab- lished lesions in a patient whose disease was controlled) and complete remission (absence of new or established lesions in a patient whose all-systemic therapy is discontinued for ≥2 months, or while the patient is receiving minimal therapy) [23]. Scoring of disease was recorded based on the pemphigus disease area index. Study Population Sixty patients with PV (27 male, 33 female; mean age 46.3 ± 12.7 years) were recruited for the purpose of this case-control study. Patients were selected among inpatients and outpa- tients who were referred to the pemphigus clinic of Razi Hospital (Tehran, Iran). The diagnosis of pemphigus had been confirmed by histology and direct immunofluorescence. Among patient subjects, 30 were in complete remission as in the control group, whereas the rest (n = 30) were in relapse as in the case group. None of the patients had overt labial herpetic lesions at the time of sampling. All study participants Introduction Pemphigus disease is an autoimmune disease that is character- ized by secretion of autoantibodies that act against surface glycoproteins of epithelial cells [1]. The immunological basis for pemphigus disease includes autoantibodies targeted against keratinocyte surface antigens desmoglein1 and des- moglein3 (DSG1, DSG3) [2]. These proteins are primarily involved in intercellular cell-to-cell adhesion structures. Pem- phigus vulgaris (PV) is a common type of pemphigus disease with painful blistering on the skin and mucous membranes [3]. Overall, the close link between autoimmune diseases and both endogenous (genetic) and exogenous (environmen- tal) factors has been clearly understood. Exogenous factors include thiol drugs, physical trauma such as burn, ultraviolet exposure, X-ray, neoplasm, hormones and pregnancy, nutri- tional factors, and emotional stress [4-7]. Among exogenous etiologies, the critical role of microbial agents such as viruses in triggering and flaring autoimmune conditions has been recently discovered. In this regard, the causative role of herpesviruses (HSVs) as the most important human pathogens in the pathogenesis of PV has been recently suggested [8]. Thus, efforts have been made to clarify viral etiologies for PV and in this way the role of HSVs and cyto- megalovirus (CMV) is taken into consideration [9,10]. The main common features of both pointed viruses include their ability to survive in the host body for a long time as well as activating periodically [11]. The human HSVs including her- pesviruses 1 and 2 (HSV1/2) and CMV infect between 60% and 90% of the adult population worldwide [12,13]. After primary infection, the virus establishes lifelong latency, with periodic reactivations that are effectively controlled by a robust immune response in most infected individuals despite the virus-producing proteins that interfere with adaptive and innate immunity. The activation of T cells is the important aspect of adaptive immunity. Tissue-resident memory (Trm) T cells are a subtype of memory lymphocytes that enter nonlymphoid tissues such as skin and become permanently established without recirculating. Local immune control of viruses can be mediated by Trm T cells through direct killing of infected cells and recruiting circulating memory CD8+ to the skin. Also, Trm T cells maintain HSV1 latency by secret- ing granzyme B, which degrades the early protein ICP4 that is important in viral replication [14,15]. CMV positivity in PV patients was independent of the site of the samples. Using the multivariable linear regression model, the level of CMV-IgG in PV patients was directly affected by female sex and advanced ages. Conclusions: Our study could not demonstrate the role of HSV1/2  and CMV as triggering factors for PV exacerbation. Further studies are needed to evaluate the potential role of these viruses in PV exacerbation especially considering demographic variables. ABSTRACT 264 Research | Dermatol Pract Concept 2018;8(4):3 Statistical Analysis The results were presented as mean ± standard deviation for quantitative variables and were summarized by absolute frequencies and percentages for categorical variables. Nor- mality of data was analyzed using the Kolmogorov-Smirnoff test. Categorical variables were compared using chi-squared test or Fisher’s exact test when more than 20% of cells with an expected count of less than 5 were observed. Quantitative variables were also compared with t test or Mann-Whitney U test. The association between quantitative variables was assessed using the Pearson correlation test. To determine the main correlates of IgG antibodies against viruses, multivari- able linear regression analysis was used. For the statistical analysis, the statistical software SPSS version 16.0 for Win- dows (SPSS Inc., Chicago, IL) was used. P values of 0.05 or less were considered statistically significant. We used Graph- Pad Prism software (GraphPad Software, La Jolla, CA) for plotting graphs. Results In total, 30 patients with the relapse type of PV and 30 patients with the remission type were assessed. The demographic and clinical features of patients are presented in Table 1. Compari- son of the baseline characteristics of the 2 groups (Table 2) showed no difference in gender, mean age, subtypes of disease (mucous or mucocutaneous), or disease duration and oral medications. Regarding medical history, hypertension and hyperlipidemia were more prevalent in those with relapse PV than in the remission group, while no difference was revealed in other underlying comorbidities including diabetes mel- litus, history of smoking, coronary artery disease, or thyroid abnormalities. With regard to DSG-specific IgG positivity in the relapse and remission forms of PV, DSG1-specific IgG was positive in 56.7% of patients with the relapse form and in 20.0% of those with the remission form, indicating a significant difference across the 2 groups (P = 0.003), but the rate of positivity for DSG3-specific IgG in the relapse and remission types was 76.7% and 63.3%, respectively, with no signifi- cant difference (P = 0.260). The mean HSV-IgG in the relapse and remission groups was 154.47 ± 66.80 and 151.17 ± 62.73, respectively, with no significant difference (P = 0.844). Moreover, the mean CMV-IgG in the subgroups was 130.30 ± 47.88 and 127.93 ± 53.56, respectively, with no meaningful difference (P = 0.857). There was no correla- tion between prednisolone dose and HSV-IgG (P = 0.373) or CMV-IgG (P = 0.647). As shown in Table 3, HSV and CMV positivity in PV patients was independent of the source of the samples. gave informed consent to provide samples for the study, which was approved by the Local Research Ethics Commit- tee of Tehran University of Medical Sciences (approval num- ber: IR.TUMS.MEDICINE.REC.1395.1017 and IR.TUMS. MEDICINE.REC.1395.2472). Sample Preparation For viral detection and determination of specific antibod- ies, we used blood, skin biopsy, and throat swab samples from patients. After patient consent forms were obtained, one skin specimen or mucosal specimens were obtained by punch biopsy (3 mm) and blood samples were taken from all patients. In addition, a sample from the throat was collected by swabbing. All samples were transferred in cold chain con- ditions to the virology laboratory. As soon as samples were received, preparation and storage were carried out. Sera (for serology) and buffy coat (for virus detection) were isolated from whole blood and stored at −20°C. DNA extraction from buffy coat, skin biopsy, and throat swab were performed using DNA Isolation Kit for Cells and Tissues according to the manufacturers’ instructions (Roche, Berlin, Germany). After DNA extraction, it was eluted in 50 µL of buffer and then adjusted to a definitive concentration of 500 ng/µL. Virus-Specific Antibody Responses HSV1/2- and CMV-immunoglobulin G (IgG) antibodies were determined using enzyme-linked immunosorbent assay (ELISA). Quantification of IgG antibodies was determined by standard curve as RU/mL according to the manufacturer (values greater than 22 RU/mL were defined as seropositive). DSG-Specific IgG The levels of autoantibody to DSG1 and DSG3 were mea- sured by ELISA. The cutoff values were 20 U/mL for both. Determination of Viral Load The absolute quantification of HSV1/2 and CMV-specific DNA was performed by real-time polymerase chain reaction (PCR) using the RealStar HSV PCR Kit 1.0 and RealStar CMV PCR Kit 1.0, respectively (Altona Diagnostics; Ham- burg, Germany). All stages of the amplification of the viruses’ specific target sequences for the detection and quantification of the amplified DNA were carried out according to the manufacturer’s instructions. Ten microliters of the DNA extracted from samples in distilled water, and the 4 controls were included in each experiment as a template and a negative control and quantification standards, respectively. Moreover, we performed internal control to identify possible PCR’s inhibitor and to confirm the reliability of all reagents of the kit used for each sample. Finally, the concentration of the samples was calculated in copies per microliter and copies per milliliter. Research | Dermatol Pract Concept 2018;8(4):3 265 TABLE 1. Demographic and clinical characteristics of pemphigus vulgaris patients ID Sex Age (Years) Type Duration (Years) Activation PDN Dose (mg/d) Drug History DSG1 DSG3 PDAI P1 F 72 MCPV 2 Relapse 15 Azathioprine Positive Positive 94 P2 F 41 MPV 2.5 Remission 5 Azathioprine Negative Positive 0 P3 F 47 MCPV 4 Remission 5 Mycophenolic acid, Azathioprine Negative Positive 0 P4 F 48 MCPV 1.5 Remission 5 Mycophenolic acid Negative Positive 0 P5 F 31 MCPV 2 Remission 5 Azathioprine, rituximab Positive Positive 0 P6 F 28 MCPV 5 Relapse 10 Mycophenolic acid, Azathioprine Positive Positive 12 P7 M 61 MCPV 4 Remission 5 Azathioprine, Mycophenolic acid, rituximab, Mtx Negative Positive 0 P8 M 67 MCPV 10 Relapse 30 Cyclophosphamide, Mycophenolic acid, rituximab Negative Positive 34 P9 F 63 MCPV 3.5 Remission 0 Mycophenolic acid monotherapy Negative Negative 0 P10 M 41 MCPV 3 Remission 0 Mycophenolic acid Positive Negative 0 P11 M 60 MCPV 2.5 Remission 5 Azathioprine Negative Negative 0 P12 M 45 MCPV 7 Remission 5 Mycophenolic acid, Azathioprine Negative Negative 0 P13 M 32 MCPV 10 Remission 7.5 Azathioprine Negative Positive 0 P14 M 41 MCPV 1.5 Remission 5 Azathioprine, rituximab Negative Negative 0 P15 M 38 MCPV 4 Remission 10 Azathioprine, mycophenolic acid, rituximab Positive Negative 0 P16 M 55 MPV 2 Remission 7.5 Azathioprine Negative Positive 0 P17 M 65 MCPV 2 Relapse 5 Azathioprine Negative Positive 14 P18 F 45 MCPV 2 Relapse 10 Azathioprine Positive Positive 12 P19 F 58 MCPV 2 Remission 7.5 Azathioprine Negative Positive 0 P20 M 39 MPV 2 Remission 1.25 Azathioprine Negative Negative 0 P21 F 45 MPV 2 Relapse 7.5 Mycophenolic acid Negative Positive 5 P22 M 21 MCPV 1 Relapse 15 Azathioprine Positive Positive 34 P23 F 65 MCPV 12 Remission 5 None Negative Positive 0 P24 F 45 MCPV 10 Remission 5 Azathioprine Negative Positive 0 P25 F 33 MCPV 5 Relapse 0 Azathioprine 15 P26 M 44 MPV 19 Remission 7.5 Mycophenolic acid, Azathioprine Negative Positive 0 P27 F 54 MCPV 2.5 Remission 5 Mycophenolic acid, rituximab Positive Positive 0 P28 F 62 MCPV 3 Remission 2.5 Azathioprine Negative Negative 0 P29 F 55 MCPV 1.5 Remission 2.5 Mycophenolic acid, Mtx Negative Negative 0 P30 M 38 MCPV 1.5 Remission 7.5 Rituximab Positive Positive 0 P31 F 67 MCPV 11 Remission 5 Azathioprine, mycophenolic acid, rituximab, Mtx Negative Positive 0 P32 F 46 MCPV 0.5 Remission 7.5 Rituximab Negative Negative 0 (Continued next page) 266 Research | Dermatol Pract Concept 2018;8(4):3 ID Sex Age (Years) Type Duration (Years) Activation PDN Dose (mg/d) Drug History DSG1 DSG3 PDAI P33 F 40 MCPV 12 Remission 10 Azathioprine Negative Positive 0 P34 M 40 MCPV 3 Relapse 25 Azathioprine, rituximab Negative Positive 5 P35 M 55 MCPV 6 Remission 7.5 Azathioprine Negative Negative 0 P36 M 35 MCPV 14 Remission 7.5 Mycophenolic acid, Azathioprine, Mtx, rituximab, IVIG Negative Positive 0 P37 M 65 MCPV 5 Relapse 0 Mycophenolic acid, Azathioprine Positive Negative 23 P38 F 74 MCPV 2 Relapse 7.5 Azathioprine Positive Positive 24 P39 F 32 MCPV 2 Relapse 10 Rituximab Negative Positive 14 P40 F 39 MCPV 8 Relapse 2.5 Mycophenolic acid, rituximab Positive Positive 3 P41 F 36 MCPV 5 Relapse 5 Mycophenolic acid, Azathioprine Positive Positive 34 P42 F 54 MCPV 2 Relapse 5 Mycophenolic acid, Azathioprine, Mtx Positive Positive 12 P43 F 54 MCPV 6 Relapse 25 Rituximab, mycophenolic acid Negative Positive 11 P44 M 20 MCPV 1 Relapse 15 Rituximab Negative Negative 5 P45 F 42 MPV 1 Remission 2.5 Rituximab Negative Positive 0 P46 M 38 MPV 3 Relapse 15 None Negative Positive 27 P47 F 46 MCPV 15 Relapse 20 Mycophenolic acid, Azathioprine, Mtx, rituximab, IVIG, cyclophosphamide Positive Positive 0 P48 M 27 MCPV 1.5 Relapse 30 Mycophenolic acid, Azathioprine Positive Positive 0 P49 M 42 MCPV 0.5 Relapse 35 None Negative Negative 17 P50 F 34 MPV 1 Relapse 10 Mycophenolic acid, Azathioprine, Mtx, rituximab, IVIG, cyclophosphamide 10 P51 F 34 MCPV 1 Relapse 6.25 Rituximab Positive Negative 38 P52 F 37 MCPV 2 Relapse 25 Azathioprine, rituximab Positive Positive 19 P53 M 57 MCPV 3 Relapse 15 Azathioprine Positive Positive 84 P54 M 54 MCPV 5.5 Relapse 2.5 Azathioprine, mycophenolic acid, rituximab, Mtx Positive Negative 12 P55 F 54 MCPV 2 Relapse 12.5 Rituximab Negative Positive 8 P56 F 48 MCPV 1 Relapse 10 Azathioprine Positive Positive 23 P57 F 60 MCPV 10 Remission 10 Azathioprine Negative Positive 0 P58 M 29 MCPV 3 Relapse 5 Mycophenolic acid, rituximab Positive Positive 14 P59 M 45 MCPV 1 Relapse 15 Azathioprine Positive Positive 21 P60 F 39 MCPV 1.5 Relapse 35 Mycophenolic acid, Azathioprine Negative Positive 15 Abbreviations: Mtx, methotrexate; DSG, desmoglein; MCPV, mucocutaneous PV; MPV, mucous PV; PDN dose, oral prednisolone dose at the time of the study; PDAI, pemphigus disease area index TABLE 1. Demographic and clinical characteristics of pemphigus vulgaris patients (continued) Research | Dermatol Pract Concept 2018;8(4):3 267 46.60 vs 112.00 ± 50.86, P = 0.020). Also, a direct correlation was revealed between patients’ age and the level of CMV-IgG (r coefficient = 0.404, P = 0.001). Discussion Several studies have investigated the relationship between viral infections and the initiation or exacerbation of autoimmune As indicated in Table 4 and Figure 1 and using the multi- variable linear regression model, the level of HSV-IgG was not associated with the baseline characteristics including demo- graphics, underlying comorbidities, disease duration, and form of PV. However, as shown in Table 5 and Figure 2, the level of CMV-IgG in PV patients was affected by the gender and age factors. The mean CMV-IgG was significantly higher in female than in male patients suffering from PV (142.21 ± TABLE 2. Comparing baseline characteristics between the patients with relapse and those in remission Relapse Type (n = 30) Remission Type (n = 30) P Value Male 12 (40.0) 14 (46.7) 0.602 Mean Age, Years (±SD) 44.90 ± 14.30 47.83 ± 11.04 0.377 Disease Duration, Years (±SD) 3.42 ± 3.14 5.23 ± 4.75 0.086 Medical History History of hypertension 9 (30.0) 2 (6.7) 0.020 History of hyperlipidemia 5 (16.7) 0 (0.0) 0.050 History of diabetes mellitus 5 (16.7) 2 (6.7) 0.424 History of smoking 1 (3.3) 1 (3.3) 1.000 History of coronary disease 2 (6.7) 1 (3.3) 0.998 History of AVN 2 (6.7) 0 (0.0) 0.492 History of hypothyroidism 2 (6.7) 0 (0.0) 0.492 Form of Disease 0.706 Mucous 3 (10.0) 5 (16.7) Mucocutaneous 27 (90.0) 25 (83.3) Medication Azathioprine 19 (63.3) 20 (66.7) 0.787 Mycophenolic acid 13 (43.3) 13 (43.3) 1.000 Rituximab 12 (40.0) 10 (33.3) 0.592 Methotrexate 4 (13.3) 4 (13.3) 1.000 IVIG 2 (6.7) 1 (3.3) 0.999 Cyclophosphamide 3 (10.0) 0 (0.0) 0.237 Unless noted otherwise, values represent number (percent). TABLE 3. Comparison of HSV and CMV detection based on reverse transcrip- tase polymerase chain reaction techniques according to the source of sam- ples between the patients with relapse and those in remission Relapse Type (n = 30) Remission Type (n = 30) P Value HSV Skin 1 (3.3) 0 (0.0) 0.998 PBMC 1 (3.3) 2 (6.7) 0.898 Throat 0 (0.0) 0 (0.0) 1.000 CMV Skin 3 (10.0) 0 (0.0) 0.237 PBMC 5 (16.7) 0 (0.0) 0.052 Throat 3 (10.0) 0 (0.0) 0.237 Values represent number (percent). Abbreviation: PBMC, peripheral blood mononuclear cell. 268 Research | Dermatol Pract Concept 2018;8(4):3 TABLE 4. Multivariable linear regression model to assess the main corre- lates of HSV-IgG antibody in patients with pemphigus vulgaris Unstandardized Coefficients Standardized Coefficients t P Value (Constant) B Std. Error Beta Sex −35.727 164.984 −0.217 0.829 Age −3.982 18.674 −0.031 −0.213 0.832 Type 0.492 0.809 0.098 0.608 0.546 Duration −10.558 26.124 −0.056 −0.404 0.688 Form 0.825 2.345 0.053 0.352 0.726 Hypertension −15.421 19.720 −0.121 −0.782 0.438 Hyperlipidemia 0.185 34.852 0.001 0.005 0.996 Diabetes 58.505 46.066 0.254 1.270 0.210 Coronary Disease −14.900 33.394 −0.075 −0.446 0.657 Smoking −2.827 45.347 −0.010 −0.062 0.951 (Constant) 67.297 54.454 0.190 1.236 0.222 Figure 1. Level of HSV-IgG antibody (cut-off value: 22 RU/mL) according to baseline variables. Research | Dermatol Pract Concept 2018;8(4):3 269 TABLE 5. Multivariable linear regression model to assess the main corre- lates of CMV-IgG antibody in patients with pemphigus vulgaris Unstandardized Coefficients Standardized Coefficients t P Value (Constant) B Std. Error Beta Sex −62.825 114.201 −0.550 0.585 Age 28.334 12.926 0.281 2.192 0.033 Type 1.196 0.560 0.303 2.135 0.038 Duration 24.026 18.083 0.163 1.329 0.190 Form −0.515 1.623 −0.042 −0.317 0.752 Hypertension −2.244 13.650 −0.022 −0.164 0.870 hyperlipidemia −14.238 24.124 −0.110 −0.590 0.558 Diabetes 12.675 31.886 0.070 0.397 0.693 Coronary dis. 35.510 23.115 0.228 1.536 0.131 Smoking −46.372 31.389 −0.202 −1.477 0.146 (Constant) 39.650 37.693 0.142 1.052 0.298 Figure 2. Level of CMV-IgG antibody (cut-off value: 22 RU/mL) according to baseline variables. 270 Research | Dermatol Pract Concept 2018;8(4):3 mati-Roodsari et al, similar to our study, only 1 patient had a positive HSV PCR result for skin samples [32,33]. These values are less than the findings of the study by Tufano et al, reporting positive HSV PCR in 5 of 7 skin specimens [29]. Other PCR results show evidence of HSV1 infection in 10% of all blood samples and CMV infection in 16.6% of all blood samples, 10% of all skin samples, and 10% of all throat samples. On the other hand, several studies failed to detect herpes DNA viruses in pemphigus patient specimens [26,27]. Senger et al showed the role of HSV1 in exacerbation of pemphigus by using the ELISA technique [31]. However, we found no cor- relation between HSV and CMV PCR results or IgG antibody levels and disease activity (relapse or remission). These findings cannot rule out the role of these viruses in exacerbation of the disease; improvement in some patients, especially those with the mucosal form, after receiving antiviral therapy as well as exacerbation of pemphigus after HSV infection are clinical evidence of the possible role of viruses in disease exacerbation [22]. Therefore, the role of viruses in exacerbation of pemphi- gus remains a hypothesis and requires further study. Our study is unique for several reasons. First, we used both ELISA and PCR techniques to find evidence of viruses, whereas most previous studies used only one technique. Second, the samples were sourced from several sites includ- ing skin, throat, and blood. Third, we evaluated virus status in relapse and remission PV patients to determine the role of viruses in the exacerbation of the disease, whereas most previous studies examined the status of viruses in newly diagnosed patients and healthy controls to determine the etiological role of viruses. However, this study had some limitations. We could not evaluate other types of pemphigus disease due to their lower prevalence. Because of high costs of the techniques, we were able to investigate only the role of HSV1/2 and CMV in the exacerbation of disease. Conclusions In conclusion, our study could not demonstrate the role of HSV1/2 and CMV as triggering factors for PV exacerbation. Further studies are needed to evaluate the potential role of these viruses in PV exacerbation. References 1. Kasperkiewicz M, Ellebrecht CT, Takahashi H, et al. Pemphigus. Nat Rev Dis Primers. 2017;3:17026. 2. Spindler V, Waschke J. Pemphigus: a disease of desmosome dysfunction caused by multiple mechanisms. Front Immunol. 2018;9:136. 3. Kridin K. Pemphigus group: overview, epidemiology, mortality, and comorbidities. Immunol Res. 2018;66(2):255-270. 4. Tur E, Brenner S. Contributing exogenous factors in pemphigus. Int J Dermatol. 1997;36(12):888-893. diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and diabetes mellitus [24]. In this regard, efforts have been made to detect viral pathogens in blood samples and skin specimens of patients with pemphi- gus by techniques such as immunochemistry and PCR for DNA particles. The association between onset or reactivation of pemphigus disease and viral infections may be causal, or as a result of drug-induced immunosuppression which can increase the likelihood of viral infections, or established on the pathogenic connection between viral infection and immune dysregulation leading to autoimmunity. The mechanism of viral induction of autoimmunity can be explained in several ways, including molecular mimicry (due to cross-reactions between pathogen-derived epitopes and self-derived epitopes); bystander activation (due to activation of preexisting autore- active T cells as a result of the inflammatory environment); epitope spreading (when antigen-presenting cells recognize self-antigens which were not initially directed by the immune system); polyclonal activation (due to infection of B cells by lymphocytotropic viruses which lead to B cell proliferation and increased antibody production); and viral superantigens (encoded by some viruses, are able to activate polyclonal T cells) [25]. Molecular mimicry is possibly the most accept- able explanation for the association of viral infections and pemphigus. Further explanation is that viruses upregulate production of interferon and interleukins. High levels of inter- feron gamma lead to increased expression of HLA2 on kerati- nocytes, making the pemphigus antigen structural site active. Also, overproduction of IL4 and IL10 causes a shift of TH1 to TH2 response which potentiates antibody response [26,27]. The role of viruses in the pathogenesis of pemphigus has been assessed in some previous studies. ‘The importance of viral infections as an exacerbating factor of PV is neglected by some clinicians. Concerning this, in our current case-control study, we evaluated the probable role of HSV1/2 and CMV in the exacerbation of PV. In our study, a significantly higher number of PV patients (regardless of disease activity) were positive for HSV- and CMV-IgG antibodies, similar to the studies by Tufano et al and Sagi et al [28,29]. This may show a history of HSV and CMV exposure in most patients and may indicate more frequent recrudescence of latent viruses [30]. A significant increase in IgG antibody levels, discovered by comparing relapse and remission samples, indicates an active infection. According to a general consensus, relatively higher IgG levels are associated with a higher probability of having an active viral infection and relatively lower IgG levels represent a previous viral infection rather than an active viral infection. In this regard, Senger et al showed higher levels of HSV1 antibody in patients in the active phase of disease than in remission patients [31]. In contrast to our study, there was no significant difference in antibody levels between relapse and remission patients. In the studies of Marzano et al and Rah- Research | Dermatol Pract Concept 2018;8(4):3 271 20. Oliveira-Batista DP, Janini ME, Fernandes NC, Santos N. Labora- tory diagnosis of herpesvirus infections in patients with pemphi- gus vulgaris lesions. Intervirology. 2013;56(4):231-236. 21. Cohen SS, Blauvelt A, Weinstein MD, Herndier BG, Anhalt GJ. 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