Detection of herpes simplex viruses in the oral lesions of patients with pemphigus vulgaris: Is it diagnostic or predictive of disease severity?

Abstract Background Some studies emphasise the relationship between the herpes simplex virus (HSV) and pemphigus. Although the possible role of HSV in the pathogenesis of pemphigus and the severity of the disease is obscure, we aimed to evaluate the presence of herpes simplex viruses (HSV 1/2) in the oral lesions of patients with pemphigus vulgaris and also assess its association with disease severity and types of lesions. Methods A retrospective study was conducted on collected data in the form of collecting paraffin blocks, slides, and relevant pathology reports and referring to patients' medical records. A questionnaire containing details on the degree of skin, scalp, and mucosal involvement (Pemphigus Disease Area Index (PDAI)) was fulfiled. The immunoassay result was also collected to check the anti‐desmoglein 3 and 1 antibodies (using ELISA technique). Results In this study, 52 patients of pemphigus vulgaris with oral lesions (case) and 52 patients with oral lesions not related to the disease (control) were evaluated. HSV1 was detected in 13.5% of oral pemphigus lesions and 1.9% of the control group (p = 0.0598). There were no positive cases of HSV2 in either group. There was no significant association between the positivity of HSV1 and the site of lesions (p = 1.00) or disease severity (p = 0.28). However, we found a strong correlation between the PDAI disease severity score with the titre of the AntiDsg3 antibody (r = 0.487, p = 0.001) and AntiDsg1 antibody (r = 0.309, p = 0.026). Conclusion This study demonstrated a significant prevalence of HSV1 in oral pemphigus lesions, and acyclovir therapy may play a significant role in managing these patients. However, HSV's role in the pathogenesis of pemphigus vulgaris cannot be clearly determined.

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease that is clinically characterised by blisters that form erosions on skin and mucosa. 1 The characteristic histopathologic finding in PV is separation above the basal cell layer. 2 The primary pathophysiological mechanism for PV lesions is the formation of autoantibodies, including antibodies against desmoglein 3 (Dsg 3) and desmoglein 1 (Dsg 1). 3,4Dsg 3 and Dsg one involve in the cell-to-cell adhesion of keratinocytes; therefore, Dsg 1 and Dsg 3 antibodies can destroy intercellular desmosomal adhesive structures. 3,4In this regard, changes in the expression of genes encoding these proteins are thought to be the primary cause of the development of these lesions.Furthermore, some environmental and even infectious factors have also been suggested to be effective in the occurrence of these lesions.][7][8] Viral infections have been shown to stimulate disease-related autoimmunity.In this context, the central role of herpes viruses in the incidence and exacerbation of PV has been widely studied. 9,10For the first time, the close link between herpes simplex viruses (HSVs) and the risk for PV was described by Krain and colleagues in 1974.Subsequently, various studies have been conducted on the incidence and exacerbation of PV lesions following HSV infection.The possible relationship between HSV infection and PV lesions was suggested based on the elevated titres of HSV half IgG antibodies among PV patients. 11ased on the detection of HSV-specific DNA in swabs taken from PV oral lesions, it was hypothesised that antiviral therapeutic regimens could improve the disease-related lesions. 12,13Furthermore, HSV infection was found to be related to exacerbation of PV manifestations. 10,11][11][12][13][14] Since oral lesions are among the common findings in PV, identifying the presence of HSV infection in PV oral lesions can provide valuable justification for the pathogenesis of HSV infection in the formation of PV lesions.Therefore, the purpose of the present study was to evaluate the presence of HSV half in the oral lesions of patients with PV and to evaluate the diagnostic properties of HSV infection in oral lesions in the detection of PV.

| Study design
This case-control study was conducted on paraffin blocks of patients with mucosal/mucocutaneous PV who were referred to the dermatology clinic of Razi Hospital, Tehran, Iran from 2016 to 2021.This study was approved by the Research Ethics Committee of the Tehran University of Medical Sciences and Razi Hospital (code: IR.TUMS.MEDICINE.REC.1399.214).

| Study participants
The case group was selected among the PV patients who were referred to the mentioned hospital.The inclusion criterion for the case group was being documented new case of PV.The inclusion criteria for the control group were patients with oral lesions unrelated to PV, including oral erosion, oral lichen planus, margin of squamous cell carcinoma, and irritation fibroma.Patients in the control group were matched with the case group based on age and gender.Exclusion criteria for both the case and control groups were history of immunosuppressive therapy prior to diagnosis, or history of antiviral therapy.
Sample size was determined to be at least 50 participants in each group which considered a large sample size compared with similar previous studies.During the study period, 52 patients were included in each group.

| Instruments
A checklist was made to collect data from medical records, histopathological reports (suprabasal What is already known about this topic?� Controversial results exist upon the possible role of HSV in the pathogenesis of pemphigus and its severity.

What does this study add?
� HSV may be present in oral lesions of immune-suppressive therapy resistant Pemphigus.� Acyclovir therapy may play a significant role in managing Pemphigus patients with oral lesions.
acantholysis, cleft), and direct immunofluorescence (IgG/C3 deposition in intercellular space, fishnet pattern) of the patients.The Pemphigus Disease Area Index (PDAI) questionnaire was used to determine the degree of skin, scalp, and mucosal involvement.Disease activity is scored based on PDAI score in three domains scalp (10 points), skin (120 points) and mucosal activity (120 points) that sum up to the maximum 250 points.Higher PDAI scores indicate higher disease activity 15 with the cut-off values of 15 and 45, to distinguish moderate, significant, and extensive pemphigus forms. 16In this study, PDAI scores below and above 15 are defined as non-severe and severe PV, respectively.

| Laboratory assessments
The enzyme-linked immunosorbent assay (ELISA) technique was used to detect anti desmoglein 3 and 1 antibodies.The 20 RU/ml cut-off was set to define positive results for these serum markers.Samples were examined using real-time polymerase chain reaction (RT-PCR) technique according to the manufacturer's instructions to determine HSV 1/2 viruses.DNA extraction was performed by columnar method using tissue extraction kit (Favorgene, Taiwan).The presence of HSV 1/2 virus genome was investigated using Geneproof kit (made in Germany) based on RT-PCR method using the Lightcycler96 device.To evaluate the accuracy of tests in each run, the internal control (to check the presence of human cells and ensure the correct DNA extraction) positive control (to check the correct operation and sensitivity of the kits) non-template control (NTC), and negative control (to check the possibility of contamination) were used.

| Statistical analysis
Normality of the data was evaluated using the Kolmogorov-Smirnov test.The independent t-test or Mann-Whitney U tests were used to compare continuous variables between case and control groups for normally and non-normally distributed variables, respectively.Chi-Square or Fisher exact tests were used to evaluate the relationship between categorical variables.Pearson and Spearman correlation coefficients were used to evaluate the correlation between PDAI score and anti Dsg 1and anti Dsg 3 serum titres.p values less than 0.05 were considered statistically significant.Data analysis was performed using the statistical package for social sciences (SPSS) statistical software version 23.0 for windows (IBM, Armonk, New York).

| RESULTS
In this study, 52 patients with oral PV lesions (case) and 52 patients with oral lesions unrelated to PV (control) were evaluated.Comparison of the demographic characteristics of the case and control groups is presented in Table 1.There was no significant difference between case and control groups in terms of age (p = 0.349) and gender (p = 0.432).
Overall, seven samples (13.5%) of the 52 paraffin blocks in the case group and 1 sample (1.9%) of paraffin blocks in the control group had a positive result for HSV1 (p = 0.0598).There were no positive cases of HSV2 in the two groups.The details of baseline characteristics and diagnostic parameters in those with HSV positivity in study and control groups are summarised in Table 2.
The diagram of RT-PCR run results based on amplification curves are demonstrated in Figure 1 (case group) and Figure 2 (control group).
Relationship between PDAI scores and PV lesion location and HSV1 positivity is presented in Table 3.There was a significant relationship between disease severity (PDAI score) and the location of lesions (p = 0.040).Therefore, the likelihood of severe lesions was 4.08 times more in mucocutaneous than in mucosal sites.There was no significant relationship between PDAI score categories and HSV1 positivity (p = 0.285).
The median (IQR) for AntiDsg3 antibody titre in mucocutaneous and mucosal lesions were 200(0.0)and 200(73), respectively, which were not significantly different (p = 0.111).However, the median (IQR) level of AntiDsg1 antibody was significantly higher in  In general, the rate of HSV1 positivity was 13.6% in men and 13.3% in women indicating no difference between the two genders (p = 0.999).Also, the mean age of cases with and without HSV1 positivity was 47.85 � 4.06 years and 44.15 � 10.98 years, respectively, which were not significantly different (p = 0.112).

| DISCUSSION
Environmental factors, including viral infections, along with genetic predisposition have an undeniable role in the development and exacerbation of autoimmune diseases.Among the viral infections, the role of HSV in the development of PV has been highlighted.The reason for such claims was based on the high titre of serum anti-herpes antibodies as well as the detection of the virus genome in a number of disease-related The diagram presents the results of a real time-PCR run for the case group based on amplification curves.In this run, a positive example for HSV1 in cycle 24 (CT = 24) is observed (curves raised in cycles above CT 30 are related to positive and internal controls).

T A B L E 2
The details of baseline characteristics and diagnostic parameters in those with HSV-1 positivity in case and control groups.samples including tissue (mostly skin biopsies) and body fluid (saliva, …) specimens.However, this hypothesis has not yet been fully documented by evaluating HSV infection in immunocompetent PV patients using reliable gold standard methods in a large sample size and control group.1][12][13] In the present study, we evaluated HSV positivity in oral mucosal lesions of patients with new onset PV using the molecular gold standard method (PCR) in comparison with the control group consisting of a wide range of PV-unrelated oral mucosal lesions.We also evaluated the relationship between HSV and disease severity in the present study.

Group
Although the association between the oral lesions severity and HSV positivity was not statistically significant, considering the low power of the test (0.273), it can be estimated that this relationship may not stay non-significant if the sample size is increased.Similar to the findings of the present study, Konda et al. reported HSV infection in 38.3% of PV patients and reported that HSV was associated with clinical and pathological features of PV. 11 In the mentioned study, the presence of fissures, haemorrhagic crusts, erosions with angulated margins, linear erosions, and raised ESR were found to be significantly associated with HSV infection. 11In another study, PV patients carried significantly higher levels of anti-HSV1 antibodies than healthy controls, and this effect was more pronounced in the active phase of the disease when compared to remission. 17Patients in the mentioned study were treated with immunosuppressive drugs, and the treatment may have a role in the activation of virus and high antibody titres due to previous infections. 17In another study, HSV infection and cessation of smoking were found to be correlated in oral pemphigus vulgaris lesions. 5In contrast to the findings of the present study, Mohammadi et al. did not find any association between HSV infection and PV. 14 In this study, the location of the lesions (mucocutaneous vs. mucosal) and PDAI score, as two important indicators for disease severity, were not significantly  related to HSV1 positivity.Therefore, the role of this virus in the disease severity cannot be confirmed.
Considering the contradictory findings of previous studies, [9][10][11][12] it is not yet clear whether tracking HSV genome in tissue or evaluating serum levels of HSV antibodies can predict PV severity.Nevertheless, what we found in our study was that the existence of HSV genome in oral lesions could not be a predictor of PV severity.Furthermore, it can be hypothesised that the presence of HSV in PV oral lesions might be the result of HSV activation due to tissue trauma caused by PV erosions.Regardless, as indicated in previous studies 18 immunosuppressive therapy-unresponsive pemphigus patients may benefit from oral acyclovir therapy.

| Strengths and limitations
To the best of our knowledge this study was among the few studies that evaluated the presence of HSV genome in oral mucosal lesions of patients with PV, in particular, based on DNA extraction and polymerase chain reaction method.Although, using this gold standard technique, rather low prevalence of PV, and financial limitations prevented us from increasing the sample size, to the best of our knowledge, this study was the largest study on its kind that has been conducted on the oral lesions of PV patients and HSV.One of the limitations of this study might be the small number of positive HSV patients in our sample, which might have resulted in the non-significant relationship between HSV positivity and PV severity.Although considering the low power of the test and the nonsignificant result, it can be hypothesised that there is a high probability that the relationship will become significant by increasing the sample size.Therefore, conducting a study with larger sample size might result in a significant relationship between HSV one positivity and PV severity.Furthermore, considering the higher sensitivity of RT-PCR in detecting HSV infection in fresh samples compared to formalin-fixed paraffinembedded blocks, it can be suggested that further studies use fresh samples to evaluate the presence of HSV genome in oral PV lesions.Since all of the case samples were new cases with no prior history of treatment, immunosuppressive therapy cannot be suggested as a reason for virus activation.However, the history of viral infections along with other risk factors for PV should be included in discussing the findings.

| CONCLUSION
Although HSV traces were present in a considerable number of oral PV lesions, no statistically significant relationship was observed between PV severity and HSV positivity.However, this finding can still indicate that HSV may be a risk factor for PV.More studies are needed to prove the causal relationship between HSV and oral lesions in PV.Although the role of HSV-2/1 in the pathogenesis of PV is still debated, HSV1/2 may be present in oral lesions that are resistant to immunesuppressive therapy.

F I G U R E 2
The chart shows the results of a real-time-PCR run for the control group based on amplification curves in which there was no positive HSV result.T A B L E 3Relationship between PDAI score categories and PV lesion dissemination and HSV1 positivity.
Comparison of demographic characteristics between case and control groups.
T A B L E 1 aThe independent t-test was used to compare mean and standard deviation between groups.b The chi-square test was used to compare the distribution pattern between case and control groups.