Evaluation of Papanicolaou smears in pemphigus vulgaris patients before and after rituximab

Evaluation of Papanicolaou smears in pemphigus vulgaris patients before and after rituximab | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Study protocol Evaluation of Papanicolaou smears in pemphigus vulgaris patients before and after rituximab Nabaa Al-Zubaidi, Reza M. Robati, Fahimeh Abdollahimajd, Sahar Dadkhahfar, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7912509/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous blistering disease. Rituximab (RTX) is a chimeric anti-CD20 monoclonal antibody that is increasingly being used and becoming the first-line therapy in the management of PV. Its impact on cervical cytology and human papillomavirus (HPV) infection risk in this population remains unclear. Objectives To prospectively evaluate cervical Papanicolaou ( Pap) smear results and HPV prevalence before and 6 months after rituximab therapy in women with PV. Methods Twenty-eight women with PV underwent Pap smear and HPV genotyping before and after two rituximab infusions (1 gram, 2 weeks apart) Results The mean age was 55.5 ± 12.1 years; 75% were postmenopausal. After treatment, the proportion of inflammatory smears increased (from 35.7% to 57.1%), though no high-grade lesions appeared ( p = 0.368). HPV DNA was detected in 25% of patients, predominantly high-risk genotypes. HPV positivity correlated with shorter disease duration (24.0 vs. 84.0 months, p = 0.005) and mucosal PV type (66.7%, p = 0.019). Conclusions Rituximab did not significantly alter Pap smear Bethesda categorization at 6 months, but coincided with increased inflammatory cytology and frequent high-risk HPV. Findings support regular Pap/HPV co-testing and vaccination in women with PV undergoing B-cell depletion therapy. Pemphigus vulgaris Rituximab Papanicolaou smear Cervix Human papillomavirus infection Figures Figure 1 Introduction Pemphigus vulgaris (PV) is a rare autoimmune blistering disease of the skin and mucous membranes( 1 ). The reported incidence is between 1 and 3.2 cases per 100,000 individuals annually, and those of Ashkenazi Jewish descent have a higher incidence. Males and females are equally affected( 2 ). PV is mediated by circulating autoantibodies targeting desmoglein (Dsg)3 in mucosal-dominant disease and to Dsg3 and Dsg1 in mucocutaneous disease, resulting in the loss of epidermal cell–cell adhesion and development of blisters( 3 , 4 , 5 , 6 ). The patients present with painful erosions, blisters, and ulcers on the mucosa and skin( 7 ). While the lesions are more common in the oral mucosa, they have also been reported in other mucosal areas, such as the pharynx, larynx, esophagus, conjunctiva, and anal mucosa( 8 ). Involvement of the female genital tract in PV was reported in previous studies and was the second-most frequently involved mucosal region after the oral mucosa( 1 , 8 , 9 ). Histologically, the characteristics of PV lesions are intraepidermal vesicles with acantholysis and an intact basal layer( 4 ). The disease has a chronic, severe, and potentially fatal course( 10 ). Mortality rates were approximately 90% before the introduction of systemic corticosteroids (CS), but with the use of systemic corticosteroids in high doses, the mortality rates reduced to less than 10%( 2 ). The introduction of adjuvant immunosuppressive agents has resulted in a better outcome for the disease and a decrease in the required corticosteroid dose and associated toxicities( 11 ). Patients may need long-term systemic treatment with high doses of corticosteroids and other steroid-sparing immunosuppressive drugs, such as azathioprine, mycophenolate mofetil (MMF), or cyclophosphamide. Rituximab (RTX) is a chimeric murine/human monoclonal antibody that targets the CD20 antigens present on B lymphocytes. Rituximab is thought to act through various mechanisms responsible for B-cell depletion, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity, and direct triggering of apoptosis( 10 ). Rituximab decreases the blood serum level of antidesmoglein autoantibodies rapidly, which results in healing of pemphigus lesions after a few weeks of infusion, and Complete remission is attained after 3–6 months( 4 ). In the management and treatment of various autoimmune conditions with rituximab or other immunosuppressive drugs, there was an increased risk of viral infections, such as cervical human papillomavirus (HPV) infection, which is strongly associated with cervical dysplasia( 12 ). Also, there was an increased risk of high-grade cervical dysplasia or cervical cancers( 13 ). The effect of this type of therapy on cervical dysplasia, cervical cancer, and cervical HPV infection in PV patients remains unknown. High-risk (HPV) infection is the principal etiologic factor for cervical cancer in women. The virus is highly prevalent, with up to 80% of sexually active women becoming infected at some point during their lifetime. One important risk factor for endocervical HPV acquisition is disruption of the mucosal barrier, which facilitates viral entry into basal epithelial cells. Therefore, HPV infection may theoretically be more common in disorders associated with mucocutaneous barrier disruption, such as PV( 14 , 15 ). In most individuals, HPV infection is transient and cleared by the host immune system. However, persistent infection with high-risk genotypes can lead to cervical intraepithelial neoplasia and, ultimately, carcinoma. Prolonged systemic glucocorticoid therapy has been suggested as a contributor to viral persistence and progression due to its broad immunosuppressive effects in patients with systemic lupus erythematosus (SLE). Data on the impact of B-cell–depleting therapies are more limited. Anti-CD20 monoclonal antibodies, including RTX, ocrelizumab, and ofatumumab, target CD20-positive B lymphocytes, which play a key role in antiviral and antitumor immune responses through antibody production, antigen presentation, and modulation of T-cell activation. Depletion of these cells may impair immune surveillance and the recognition of virally transformed or dysplastic cells( 16 ). Although studies in other autoimmune diseases, such as multiple sclerosis (MS) and SLE, have reported increased HPV infection rates in patients treated with anti-CD20 agents, data specific to PV remain lacking( 12 , 17 ). The purpose of this study was to evaluate female genital tract involvement and examine cervical Papanicolaou (Pap) smears to assess the risk of cervical dysplasia, cervical cancer, and cervical HPV infection in women with PV before and 6 months after the treatment with rituximab. Patients and methods 1. Study Design This cohort study was carried out at the Shohada-e-Tajrish and Loqhman-e-Hakim Hospitals, Shahid Beheshti University of Medical Sciences at Tehran, Iran. Approval was sought from the institutional ethics committee (code IR.SBMU.MSP.REC.1403.197), and informed consent was obtained from all participants after explaining the study’s purpose and procedures. 2. Patient selection and assessment In total, 28 women with pemphigus vulgaris (new and recurrent cases) who attended our clinics and received rituximab during the period from February 2023 to January 2025 were included in this study. In all patients, the diagnosis of PV was based on the presence of clinical features of the disease, including mucocutaneous bullae and erosions, along with histopathological examination with hematoxylin-eosin stain, direct immunofluorescence (DIF) findings of the biopsy material, and enzyme-linked immunosorbent assay (ELISA), based on revised diagnostic criteria.( 18 ). Only patients who were given a diagnosis of new-onset PV or major recurrences of the disease were enrolled in this study. Patients were eligible for the study if they were married or sexually active. To avoid potential confusion during genital examination and interpretation of Pap smear results, women who presented during menstruation were excluded. Additionally, patients were excluded if they were younger than age 18 years, pregnant, or had had a hysterectomy, cervical cancer, or a previous diagnosis of human papillomavirus infection. Other exclusion criteria for rituximab therapy were: breastfeeding, history of sensitization to murine protein, active and/or severe infections (including tuberculosis, sepsis, and viral hepatitis), and severe cardiac disease. None of the patients had been immunized against any HPV subtypes. Each participant underwent a detailed review of their clinical history. The following information was collected: age; duration of disease; PV type; affected anatomical sites (skin and mucous membranes); presence of vulvar, vaginal, and/or cervical involvement; and systemic treatment history. The cumulative dose of corticosteroids and immunosuppressive drugs (azathioprine, mycophenolate mofetil) and rituximab was recorded. A complete gynecological examination was performed by a single gynecologist, who examined the vulva, vagina, and cervix of each woman. Pap smears were collected using Ayre spatulas, spray-fixed with commercial fixative, and stained with the routine Pap stain, and reported by a cytopathologist who was aware of the history and clinical situation of the patients. In this study, Pap smear results were reported according to the 2001 Bethesda system:1. Negative for intraepithelial lesion or malignancy, which includes reports within normal limits, organisms, reactive cellular changes associated with inflammation (includes typical repair) and atrophy; 2. Squamous cell abnormalities: a. Atypical squamous cells of undetermined significance (ASC-US); b. Atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesion (HSIL); c. Low-grade squamous intraepithelial lesion (LSIL); d. HSIL; e. Squamous cell carcinoma; 3. Glandular epithelial cell abnormalities ( 19 ). In each patient, hemogram, routine biochemical investigations, electrocardiogram (ECG), Mantoux test, and serology for viral hepatitis and human immunodeficiency virus were performed. After premedication (hydrocortisone and pheniramine intravenously and paracetamol orally), patients received an intravenous (IV) infusion of 1 g rituximab on days 1 and 15 under strict monitoring over a period of 5–6 hours. After the last rituximab infusion, patients were evaluated at monthly intervals for six months; after that, each participant was re-evaluated and underwent a detailed review of their clinical history, a complete physical examination, including a gynecological examination, and the cumulative dose of the received medications. Pap smears were obtained from all the patients, and a sample was performed for detecting and identifying HPV DNA, using a spatula. 3. DNA extraction and HPV genotyping For DNA extraction, the QIAamp DNA Mini kit (Qiagen GmbH, Germany) was used according to the manufacturer’s directions, and the concentration of extracted DNA was assessed by spectrophotometry at 260 nm. The genomic DNA of the samples was used in polymerase chain reaction (PCR), using the MY09 and MY11 primers. A beta globin PCR assay was used to verify the quality of DNA in the samples. Detection of HPV DNA and genotypes was performed using the INNO-LiPA assay (Fujirebio Europe N.V., Belgium). INNO LiPA HPV genotyping determined 32 HPV genotypes: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and − 68 as high-risk HPV genotypes (HR HPV), HPV-26, -53, -66, -70, -73, and − 82 as possible high-risk HPV genotypes (pHR HPV), and HPV-6, -11, -40, -42, -43, -44, -54, -61, -62, -67, -81, 83, and − 89 as low-risk HPV genotypes (LR HPV). The basis of this HPV classification was the IARC Monographs( 20 ). Reverse hybridization is the principle behind INNO-LiPA HPV genotyping. Briefly, a part of the L1 region called SPF10 of the HPV genome was extended by specific primers; after that, the biotinylated amplimers were denatured and hybridized with specific oligonucleotide probes. Additionally, a primer pair of the human HLA-DPB1 gene was added to confirm the quality and extraction of DNA. Then, streptavidin-conjugated alkaline phosphatase was added. The results were visually interpreted after incubation with BCIP/NBT chromogen. 4. Statistical analysis Descriptive statistics were computed for all study variables. Continuous data were assessed for normality using the Shapiro-Wilk test and are presented as mean ± standard deviation (SD) for normally distributed variables or median [interquartile range] for non-normally distributed variables. Categorical variables are summarized as frequencies and percentages. Group comparisons for continuous variables were performed using the Mann-Whitney U test (for two-group comparisons, such as HPV-positive versus HPV-negative status) or the Kruskal-Wallis test (for multi-group comparisons, such as Pap smear findings across three categories). Categorical variable associations were analyzed using Chi-square tests or Fisher's exact tests when expected cell counts were less than five. Changes in paired categorical data (pre- versus post-treatment Pap smear results) were assessed using the Stuart-Maxwell test for marginal homogeneity. All statistical tests were two-sided, and a p-value < 0.05 was considered statistically significant. Analyses were performed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R software version 4.5.1. Results 1. Cohort Demographics and Clinical Presentation The study cohort comprised 28 female patients with a confirmed diagnosis of pemphigus vulgaris. The mean age was 55.5 ± 12.1 years, with a pronounced postmenopausal predominance (21/28, 75.0%). The majority of patients presented with established disease (22/28, 78.6%) and had a median disease duration of 60.0 (36.0-114.0) months. The mucocutaneous form predominated (19/28, 67.9%), followed by mucosal (6/28, 21.4%) and cutaneous (3/28, 10.7%) variants. Oral mucosal involvement was nearly universal (96%), whereas genital symptoms (discharge, itching, burning and irritation) were present in 10 patients (35.7%). The most common sites of cutaneous involvement were the trunk (90.9%) and upper limbs (68.2%). Table 1 summarizes baseline demographic and clinical characteristics. Table 1 Baseline demographic and clinical characteristics of pemphigus vulgaris patients. Characteristic Value Age (years), mean ± SD 55.5 ± 12.1 Disease duration (months), median ( Q1-Q3 ) 60.0 (36.0-114.0) Number of pregnancies, median ( Q1-Q3 ) 2.5 (2.0–3.0) Menopausal status, n (%) Premenopausal 7 (25.0) Postmenopausal 21 (75.0) Disease status, n (%) New case 6 (21.4) Established case 22 (78.6) Disease type, n (%) Cutaneous 3 (10.7) Mucosal 6 (21.4) Mucocutaneous 19 (67.9) Genital symptoms at baseline, n (%) Present 10 (35.7) Absent 18 (64.3) Lesions Anatomical distribution, n (%) Oral cavity 24 (96.0%) Trunk 20 (90.9%) Upper limbs 15 (68.2%) Head and neck 12 (54.5%) Pharynx/Larynx 10 (40.0%) Nasal cavity 7 (28.0%) Lower limbs 6 (27.3%) Vulvar 2 (8.0%) Q1: first quantile, Q3: third quartile; SD: Standard deviation. 2. Clinical Outcomes Following Rituximab Therapy After rituximab therapy, clinical assessment at six months revealed that seven patients (25.0%) experienced clinical relapses, defined as the appearance of new mucocutaneous lesions after remission. Five recurrences involved the oral mucosa, and one each affected the leg and pharynx. Genital symptoms declined slightly from 10 (35.7%) to 8 (28.6%) patients. During the follow-up period, 23 patients received corticosteroid therapy (prednisolone) with a mean cumulative dose of 4.3 ± 6.7 g, administered over a mean duration of 4.5 ± 1.7 months (range: 1 to 6 months). Only one patient received additional treatment with azathioprine for a duration of 6 months. 3. Analysis of Cervical Cytology Before and After Treatment A comparative analysis of cervical cytology before and after rituximab administration is summarized in Table 2 and Fig. 1 A. Pre-treatment, the majority of Pap smears were interpreted as Normal (15/28, 53.6%), followed by inflammation (10/28, 35.7%) and Atypical Squamous Cells of Undetermined Significance (ASC-US) (3/28, 10.7%). Post-treatment, a shift in the distribution was observed: the proportion of normal results decreased to 35.7% (10/28), while smears indicating inflammation increased to 57.1% (16/28). The prevalence of ASC-US results remained low (2/28, 7.1%). No cases of low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma, atypical glandular cells (AGC), endocervical adenocarcinoma in situ (AIS), or adenocarcinoma were detected in any sample. Statistical comparison using the Stuart-Maxwell test confirmed that these longitudinal changes did not reach statistical significance (p = 0.368), indicating that rituximab did not induce a significant alteration in Pap smear categorization within this cohort. Table 2 Pap smear results before and after Rituximab administration. Pap smear before, n (%) Pap smear After, n (%) Total P-value* Normal Inflammatory ASC-US Normal 5(33.33) 10(66.67) 0(0.0) 15(53.6) Inflammatory 5(50) 4(40) 1( 10 ) 10 (35.7) 0.368 ASC-US 0(0.0) 2(66.67) 1(33.33) 3(10.7) Total 10(35.7) 16(57.1) 2(7.1) 28 *Stuart-Maxwell test; ASC-US: Atypical squamous cells of undetermined significance. 4. Genotype Distribution of Detected Human Papillomavirus HPV DNA was detected in 7 out of 28 patients (25%). Among these, high-risk (HR) oncogenic genotypes were predominant, identified in six patients (85.7% of positive cases). A notable finding was the high frequency of multiple HR-type co-infections, which were present in three patients (42.9% of positive cases). The specific co-infections identified were HPV-51 and HPV-56; HPV-31 and HPV-33; and HPV-16 and HPV-18. HPV-16 was the most prevalent individual genotype, detected in three patients (42.9% of positive cases). One patient carried a low-risk genotype (HPV-6) (14.3%). Figure 1 B illustrates genotype distribution. 5. Association between demographic/clinical Factors and Cytological and Virological Findings The relationships between demographic/clinical variables and post-treatment cytological (Pap smear) and virological (HPV) outcomes are delineated in Table 3 . Analysis revealed no significant associations between post-rituximab Pap smear results (Normal, Inflammatory, ASC-US) and any demographic variable, including age, parity, menopausal status, or clinical features such as disease status and baseline genital symptoms (p > 0.05 for all). Table 3 Association between demographic/clinical Characteristics and Post-Rituximab Pap Smear and HPV typing Results. Demographics/Clinical Characteristics Pap smear results HPV typing characteristic category Normal (n = 10) Inflammatory (n = 16) ASC-US (n = 2) P-value Negative (n = 21) positive (n = 7) P-value Age (years) Mean ± SD 55.4 ± 12.6 55.9 ± 12.2 53 ± 15.6 1.00 55.6 ± 10.8 55.3 ± 16.4 0.961 Pregnancies (n) Median(Q1-Q3) 2.0(2.0-2.8) 3.0(2.0–4.0) 2.5(2.2–2.8) 0.782 2(2.0–3.0) 3(1.5-3) 0.869 Duration (months) Median(Q1-Q3) 60(36–165) 60(36–108) 48(48–48) 1.00 84(48–165) 24(12–36) 0.005 Menopausal Yes No 7(33.3) 3(42.9) 13(61.9) 3(42.9) 1(4.8) 1(14.3) 0.511 5(83.3) 16(72.7) 1(16.7) 6(27.3) 1.00 Pemphigus vulgaris type Cutaneous Mucosal Mucocutaneous 2(66.7) 1(16.7) 7(36.8) 1(33.3) 4(66.7) 11(57.9) 0(0.0) 1(16.7) 1(5.3) 0.497 2(66.7) 2(33.3) 17(89.5) 1(33.3) 4(66.7) 2(10.5) 0.019 New case Yes No 4(66.7) 6(27.3) 1(16.7) 15(68.2) 1(16.7) 1(4.5) 0.070 5(83.3) 16(72.7) 1(16.7) 6(27.3) 1.00 Genital symptoms Present Absent 2( 20 ) 8(44.4) 8(80) 8(44.4) 0(0.0) 2(11.1) 0.249 7(70) 14(77.8) 3( 30 ) 4(22.2) 0.674 ASC-US: atypical squamous cells of undetermined significance; HPV: human papillomavirus; Q1: first quantile, Q3: third quartile; SD: standard deviation. In contrast, a robust and statistically significant association was identified between disease chronicity and HPV status. Patients positive for HPV had a markedly shorter median disease duration (24 (12–36) months) compared to their HPV-negative counterparts (84 (48–165) months) (p = 0.005). Furthermore, a significant association was observed between the clinical type of PV and HPV prevalence (p = 0.019). HPV positivity was highest in patients with solely mucosal disease (4/6, 66.7%), intermediate in those with cutaneous involvement (1/3, 33.3%), and lowest in the mucocutaneous group (2/19, 10.5%), suggesting a potential link between disease type and susceptibility to HPV infection. Of the 22 patients with recurrence, previous immunosuppressive therapies included corticosteroids (prednisolone or methylprednisolone pulse), azathioprine, rituximab, and mycophenolate mofetil. A comparative analysis using the Mann–Whitney U test was conducted to evaluate potential associations between the cumulative doses and treatment duration of these agents and Pap smear cytology and HPV typing results. No significant associations were identified between the cumulative dose or duration of any immunosuppressive drug and Pap smear or HPV outcomes (all p > 0.05). Discussion PV is an autoimmune blistering disease characterized by the production of IgG autoantibodies against desmoglein 1 and 3, key proteins for epidermal adhesion. This antibody-mediated attack results in the hallmark clinical presentation: painful flaccid blisters and erosions on the skin and mucous membranes( 21 , 22 , 23 ). The management of PV has been revolutionized by the introduction of RTX, a chimeric anti-CD20 monoclonal antibody that depletes B lymphocytes, leading to a rapid reduction in pathogenic autoantibodies and the induction of prolonged remission( 4 , 24 ). However, the profound immunosuppression required to control this autoimmune diathesis, along with mucocutaneous barrier disruption in PV, raises concerns about an increased susceptibility to cervical HPV infection and cervical dysplasia( 12 , 13 ). This cohort study provides a critical evaluation of this risk by analyzing cervical cytological changes and HPV prevalence in patients with PV before and after RTX therapy. Our primary finding is that, within a six-month follow-up period, RTX administration did not precipitate a statistically significant shift towards high-grade cervical cytological abnormalities, which is a reassuring short-term outcome. However, we also observed two clinically notable results: a substantial increase in inflammatory Pap smear findings and a high prevalence of oncogenic HPV infections (25%) in our cohort. Furthermore, our analysis revealed that this HPV positivity was strongly associated with a shorter duration of PV and a mucosal-dominant disease type. To our knowledge, this is the first prospective study to systematically evaluate longitudinal cervical cytological changes in women with pemphigus vulgaris before and after treatment with rituximab, coupled with analysis of HPV prevalence following therapy. While prior research has investigated genital involvement in PV and the general risk of HPV with immunosuppressants in other autoimmune conditions, our study uniquely focuses on the Pap smear changes following B-cell depletion therapy in this particular patient population. This study provides an observational perspective on the potential impact of both the underlying autoimmune process and its treatment on cervical cytologic findings. Our findings at baseline are consistent with earlier studies describing genital tract involvement and symptoms in PV ( 1 , 9 ). The predominance of mucosal lesions in our cohort echoes prior reports showing that mucosal surfaces are especially vulnerable to PV-associated erosions( 2 , 8 ). Such epithelial disruption may facilitate HPV acquisition or persistence. These findings confirm that PV itself commonly affects the genital mucosa and causes cytologic changes, even without biologic therapy. As expected, in our study, 18 cases (64.3%) at baseline and 20 cases (71.43%) after RTX administration were asymptomatic (no discharge, itching, spotting/bleeding, or pain). Based on previous studies, cervical involvement in PV is usually asymptomatic but can present with spotting, postcoital bleeding, discharge, or dyspareunia; a diagnosis can only be suspected if a prior history of PV is known( 7 , 25 , 26 , 27 ). The absence of progression to LSIL, HSIL, or malignancy following rituximab treatment represents a reassuring short-term outcome and is consistent with previous reports. Our results align with the large cohort study by Kim et al. (2016), which compared biologic (including rituximab) and non-biologic DMARDs in patients with rheumatoid arthritis and found no statistically significant increase in the incidence of high-grade cervical dysplasia or cancer among biologic users, despite a numerically higher rate. Similarly, the meta-analysis by García-Carrasco et al. (2018) demonstrated that, across autoimmune diseases, biologic and conventional immunosuppressive therapies were not associated with a significant rise in cervical neoplasia risk, although persistent high-risk HPV infection was more common in immunosuppressed women. Taken together, these findings support our observation that short-term rituximab therapy in PV does not appear to accelerate cervical cytologic progression, while emphasizing the importance of continued long-term monitoring for potential late effects ( 13 , 28 ). A population-based Danish cohort study on patients with autoimmune diseases, including pemphigus and pemphigoid, also showed no increased risk of cervical cancer associated with immunosuppressant use, except for high-dose users of azathioprine( 29 ). The increase in inflammatory smears post-treatment (35.7% to 57.1%) can be explained by the mucosal healing process after treatment with RTX and the recruitment of inflammatory cells to repair the mucosal erosions, which can appear cytologically as nonspecific inflammation. The prevalence of high-risk HPV infection in our cohort (25%) was markedly higher than expected for age-matched women in the general population. According to the global meta-analysis by Osmani et al. (2025, Lancet Microbe), the pooled prevalence of high-risk HPV among women aged 50 years and older is approximately 6.45% (95% CI 5.45–7.53), with regional estimates as low as 3.27% in western Asia( 30 ). Similarly, in the Iranian population-based study by Jamshidi et al. 2025, high-risk HPV was detected in only 4.5% of women aged 50 years and above, confirming a steep age-related decline in viral prevalence( 31 ). Given that most of our participants were in this age range, the 25% positivity observed here represents a substantially elevated rate, and is comparable to the 28–34% prevalence observed in studies on other autoimmune diseases such as SLE( 12 , 28 ). Both the underlying immune dysregulation inherent to autoimmune disorders and the use of immunosuppressive therapies have been implicated in this increased susceptibility. In the study by Mendoza-Pinto et al., a cross-sectional study on 148 women with SLE, the HPV prevalence was associated with the cumulative dose of glucocorticoids but not RTX. Interestingly, a large meta-analysis by García-Carrasco et al., 2018 on the prevalence of cervical HPV infection in women with SLE found that treatment with immunosuppressive agents, including glucocorticoids and biologics such as rituximab, did not independently increase the risk of high-risk HPV infection when compared with untreated patients, which is consistent and comparable to our findings that no significant associations were identified between the cumulative dose or duration of any immunosuppressive drug and Pap smear or HPV outcomes. This supports the hypothesis that the autoimmune disease itself, rather than the therapy alone, may predispose patients to viral persistence( 28 ). In PV, chronic mucosal erosions, disruption of the epithelial barrier, and impaired humoral as well as innate immune responses likely act synergistically to facilitate HPV acquisition and persistence. This excess risk supports the concept that mucosal barrier disruption and immune dysregulation in PV, particularly impaired humoral and innate antiviral responses, may facilitate persistence or reactivation of high-risk HPV infection. In the study by Mendoza-Pinto et al., a notably high proportion (72%) of HPV-positive patients with SLE were infected with high-risk genotypes. This finding closely parallels our observation that 85.7% of HPV-positive PV patients carried high-risk types. The predominance of HR-HPV genotypes (85.7%), including the highest-risk types 16 and 18, and the high frequency of multiple HR-HPV co-infections (42.9% of positive cases) are clinically important. This is a critical concern as these genotypes are responsible for approximately 70–75% of all cervical cancers worldwide. This risk is compounded by data showing that co-infection with multiple HPV genotypes can impair viral clearance and enhance persistence. Therefore, these findings mandate strict adherence to enhanced screening protocols as outlined in international guidelines( 19 , 32 ). An additional novel observation from our study is the inverse association between HPV positivity and disease duration. Patients who were HPV-positive had a significantly shorter median disease duration (24 months) than those who were HPV-negative (84 months; p = 0.005), and HPV infection was significantly more frequent in patients with the mucosal-predominant subtype of PV (66.7%; p = 0.019). These findings may indicate that recent-onset, mucosal-dominant PV represents a disease stage characterized by heightened epithelial activity and local immune dysregulation, which together may increase susceptibility to HPV infection( 33 ). We hypothesize that the intense, acute inflammatory milieu characteristic of this active, recent-onset mucosal PV type could disrupt the local epithelial barrier and immune surveillance, facilitating HPV entry or reactivation of latent infection. This is mechanistically plausible, as the erosive nature of active PV lesions may compromise the physical integrity of the mucosal epithelium, providing a direct portal of entry for HPV. This concept is supported by Akhyani et al. (2008), who demonstrated that genital tract involvement is present in 51% of their patients and manifests clinically as erosions providing direct evidence of the epithelial disruption required for such a mechanism( 8 ). Furthermore, as demonstrated by Eichhorn et al. (2022), this state involves caspase-dependent innate immune activation and elevated secretion of pro-inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6), which we propose that this state of local innate immune dysregulation could disrupt coordinated mucosal antiviral defenses, thereby facilitating initial HPV acquisition or reactivation of latent infection( 33 ). The strengths of this study include its prospective cohort design with paired cervical cytology assessments, consistent diagnostic methodology, and rigorous PCR-based HPV genotyping. However, several limitations must be acknowledged, including a relatively small sample size, a single-center design, and a short follow-up period, which may preclude detection of long-term cytological progression or malignancy risk. Consequently, larger multicenter studies with extended follow-up and serial HPV testing are warranted to validate these findings and further elucidate the long-term gynecological risks in PV patients. Despite these limitations, our results highlight the clinical significance of implementing vigilant cervical screening in patients with PV receiving rituximab. Integration of Pap smear and HPV co-testing into routine long-term monitoring protocols is strongly recommended, particularly for women with mucosal-dominant disease. In addition, consideration of prophylactic HPV vaccination prior to or early during immunosuppressive therapy represents a potentially valuable preventive strategy that merits further evaluation in this high-risk population. Conclusion In summary, rituximab therapy in women with PV did not produce significant cytological abnormalities within the six-month observation period, although a shift toward inflammatory smears was observed. A considerable proportion of patients were positive for high-risk HPV genotypes, particularly those with mucosal PV and shorter disease duration. These findings emphasize the need for ongoing gynecological surveillance and HPV testing in PV patients receiving rituximab. Larger, multicenter, longitudinal studies are needed to assess the risk of cervical dysplasia and cancer in this population. Incorporating HPV vaccination into the management of PV patients may help reduce oncogenic risk and improve long-term outcomes. Declarations Conflict of Interest Statement: The authors declare that there are no conflicts of interest regarding the publication of this article. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution N.A., N.M., and F.F. performed patient recruitment and data collection. N.Z assisted with the paraclinical assessment of patients. M.G. and F.A. supervised the project and critically revised the manuscript. S.D. and K.Z. contributed to data analysis and drafting of the first version. S.Z. and R.R designed the study, analyzed the data, and prepared the manuscript draft.All authors read and approved the final version of the manuscript. Acknowledgement This article is from the disease registry, titled “pemphigus disease”, and projected with code number "IR.SBMU.MSP.REC.1403.197" from the ethics committee supported by the skin research center, Shahid Beheshti University of Medical Sciences. References Fairbanks Barbosa ND, de Aguiar LM, Maruta CW, Aoki V, Sotto MN, Labinas GH et al (2012) Vulvo-cervico-vaginal manifestations and evaluation of Papanicolaou smears in pemphigus vulgaris and pemphigus foliaceus. J Am Acad Dermatol 67(3):409–416 Malik M, Ahmed AR (2005) Involvement of the female genital tract in pemphigus vulgaris. Obstet Gynecol 106(5 Pt 1):1005–1012 Sharma VK, Bhari N, Gupta S, Sahni K, Khanna N, Ramam M et al (2016) Clinical efficacy of rituximab in the treatment of pemphigus: A retrospective study. Indian J Dermatol Venereol Leprol 82(4):389–394 Beyzaee AM, Rahmatpour Rokni G, Patil A, Goldust M (2021) Rituximab as the treatment of pemphigus vulgaris in the COVID-19 pandemic era: A narrative review. Dermatol Ther 34(1):e14405 Chen DM, Odueyungbo A, Csinady E, Gearhart L, Lehane P, Cheu M et al (2020) Rituximab is an effective treatment in patients with pemphigus vulgaris and demonstrates a steroid-sparing effect. Br J Dermatol 182(5):1111–1119 Kamyab K, Daneshpazhooh M, Zaresharifi N, Ghanadan A, Shahsiah R, Mahmoudi HR et al (2023) Detection of herpes simplex viruses in the oral lesions of patients with pemphigus vulgaris: Is it diagnostic or predictive of disease severity? Skin Health Dis 3(5):e261 Wright C, Pipingas A, Grayson W, Leiman G (2000) Pemphigus vulgaris of the uterine cervix revisited: Case report and review of the literature. Diagn Cytopathol 22(5):304–307 Akhyani M, Chams-Davatchi C, Naraghi Z, Daneshpazhooh M, Toosi S, Asgari M et al (2008) Cervicovaginal involvement in pemphigus vulgaris: a clinical study of 77 cases. Br J Dermatol 158(3):478–482 Kavala M, Topaloglu Demir F, Zindanci I, Can B, Turkoglu Z, Zemheri E et al (2015) Genital involvement in pemphigus vulgaris (PV): correlation with clinical and cervicovaginal Pap smear findings. J Am Acad Dermatol 73(4):655–659 Hassan I, Rehman F, Sultan SJ, Aslam A, Tasaduq I, Reyaz S (2022) Rituximab in Pemphigus - An Observational Study from a Tertiary Care Center of North India. Indian Dermatol Online J 13(5):620–624 Nafiseh E, Cheyda C-D, Maryam D, Maryam G, Robabe A, Hossein M et al (2012) Successful treatment of major pemphigus vulgaris relapse with mycophenolate mofetil and high-potent topical corticosteroid. Iran J Dermatology 15(2):33–37 Mendoza-Pinto C, Garcia-Carrasco M, Vallejo-Ruiz V, Taboada-Cole A, Munoz-Guarneros M, Solis-Poblano JC et al (2013) The impact of glucocorticoids and anti-cd20 therapy on cervical human papillomavirus infection risk in women with systemic lupus erythematosus. Clin (Sao Paulo) 68(12):1475–1480 Kim SC, Schneeweiss S, Liu J, Karlson EW, Katz JN, Feldman S et al (2016) Biologic Disease-Modifying Antirheumatic Drugs and Risk of High-Grade Cervical Dysplasia and Cervical Cancer in Rheumatoid Arthritis: A Cohort Study. Arthritis Rheumatol 68(9):2106–2113 Alizadeh N, Golchai J, Ghanbari A, Golchai B, Zaresharifi S (2021) Genotype distribution of human papillomavirus in anogenital warts in Guilan province, northern Iran. Iran J Dermatology 24(2):117–120 Wang R, Pan W, Jin L, Huang W, Li Y, Wu D et al (2020) Human papillomavirus vaccine against cervical cancer: Opportunity and challenge. Cancer Lett 471:88–102 Melamed E, Lee MW (2019) Multiple Sclerosis and Cancer: The Ying-Yang Effect of Disease Modifying Therapies. Front Immunol 10:2954 Bridge F, Brotherton JML, Foong Y, Butzkueven H, Jokubaitis VG, Van der Walt A (2023) Risk of cervical pre-cancer and cancer in women with multiple sclerosis exposed to high efficacy disease modifying therapies. Front Neurol 14:1119660 Ikeda S, Imamura S, Hashimoto I, Morioka S, Sakuma M, Ogawa H (2003) History of the establishment and revision of diagnostic criteria, severity index and therapeutic guidelines for pemphigus in Japan. Arch Dermatol Res 295(Suppl 1):S12–S16 Solomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M et al (2002) The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 287(16):2114–2119 Biological agents. IARC Monogr Eval Carcinog Risks Hum (2012) ;100(Pt B):1-441 Joly P, Maho-Vaillant M, Prost-Squarcioni C, Hebert V, Houivet E, Calbo S et al (2017) First-line rituximab combined with short-term prednisone versus prednisone alone for the treatment of pemphigus (Ritux 3): a prospective, multicentre, parallel-group, open-label randomised trial. Lancet 389(10083):2031–2040 Joly P, Litrowski N (2011) Pemphigus group (vulgaris, vegetans, foliaceus, herpetiformis, brasiliensis). Clin Dermatol 29(4):432–436 Tavakolpour S, Mahmoudi H, Balighi K, Abedini R, Daneshpazhooh M (2018) Sixteen-year history of rituximab therapy for 1085 pemphigus vulgaris patients: A systematic review. Int Immunopharmacol 54:131–138 Kasperkiewicz M, Ellebrecht CT, Takahashi H, Yamagami J, Zillikens D, Payne AS et al (2017) Pemphigus Nat Rev Dis Primers 3:17026 Karanjgaokar V, Khan A, Ramachandra S, Pisal N (2008) HR-HPV DNA to differentiate between cervical pemphigus and CIN: a case report. Arch Gynecol Obstet 277(4):349–351 Lonsdale RN, Gibbs S (1998) Pemphigus vulgaris with involvement of the cervix. Br J Dermatol 138(2):363–365 Mikhail GR, Drukker BH, Chow C (1967) Pemphigus vulgaris involving the cervix uteri. Arch Dermatol 95(5):496–498 García-Carrasco M, Mendoza-Pinto C, Rojas-Villarraga A, Molano-González N, Vallejo-Ruiz V, Munguía-Realpozo P et al (2019) Prevalence of cervical HPV infection in women with systemic lupus erythematosus: A systematic review and meta-analysis. Autoimmun rev 18(2):184–191 Dugue PA, Rebolj M, Hallas J, Garred P, Lynge E (2015) Risk of cervical cancer in women with autoimmune diseases, in relation with their use of immunosuppressants and screening: population-based cohort study. Int J Cancer 136(6):E711–E719 Osmani V, Hörner L, Nkurunziza T, Rank S, Tanaka LF, Klug SJ (2025) Global prevalence of cervical human papillomavirus in women aged 50 years and older with normal cytology: a systematic review and meta-analysis. Lancet Microbe 6(1):100955 Jamshidi M, Zare Karizi S, Baghizadeh H, Ezzatizadeh V, Shasti Karimi F, Khazaee G et al (2025) Determination of Prevalence and Genotype Distribution of High-Risk Human Papillomavirus in Varamin (Iran). Diagn Cytopathol 53(7):325–332 Arbyn M, Weiderpass E, Bruni L, de Sanjose S, Saraiya M, Ferlay J et al (2020) Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Glob Health 8(2):e191–e203 Eichkorn RA, Schmidt MF, Walter E, Hertl M, Baron JM, Waschke J et al (2022) Innate immune activation as cofactor in pemphigus disease manifestation. Front Immunol 13:898819 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 17 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers invited by journal 08 Jan, 2026 Editor assigned by journal 22 Oct, 2025 Submission checks completed at journal 22 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Robati","email":"","orcid":"","institution":"Skin Research Center, Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"M.","lastName":"Robati","suffix":""},{"id":572902284,"identity":"d01750a2-b03d-4613-88ae-c3b3f94f3cef","order_by":2,"name":"Fahimeh Abdollahimajd","email":"","orcid":"","institution":"Clinical Research Development Unit, Shohada-e Tajrish Hospital, Shahid Beheshti University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Fahimeh","middleName":"","lastName":"Abdollahimajd","suffix":""},{"id":572902285,"identity":"dee298e5-f68a-4b2f-9c40-602f11366db4","order_by":3,"name":"Sahar Dadkhahfar","email":"","orcid":"","institution":"Skin Research Center, Shahid Beheshti University of Medical 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08:14:37","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110613,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7912509/v1/91a46b3cff11f6be1915547b.html"},{"id":100343291,"identity":"a6e7930a-93c7-42e7-aa7d-1b2bed358f4b","added_by":"auto","created_at":"2026-01-16 00:09:15","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52164,"visible":true,"origin":"","legend":"\u003cp\u003eCervical cytology and HPV typing results in women with pemphigus vulgaris treated with rituximab. (A) Distribution of Papanicolaou (Pap) smear interpretations before and 6 months after rituximab therapy (N=28). The proportion of smears reported as normal decreased post-treatment, while inflammatory smears increased. The prevalence of atypical squamous cells of undetermined significance (ASC-US) remained low.\u003cstrong\u003e \u003c/strong\u003eP-value was calculated using the Stuart–Maxwell test for marginal homogeneity; NS, not significant. (B) Human papillomavirus (HPV) typing results following rituximab therapy. HPV DNA was detected in 25% of patients (7/28), with high-risk oncogenic genotypes predominating (21.4%).\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7912509/v1/390120db10ea68de6117938f.jpeg"},{"id":100383795,"identity":"55ff9c31-2eaa-4bc1-aa73-7af567b43ffa","added_by":"auto","created_at":"2026-01-16 10:48:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":947080,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7912509/v1/a4affcd9-bbbc-498a-8a27-d8bc6fc55902.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Papanicolaou smears in pemphigus vulgaris patients before and after rituximab","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePemphigus vulgaris (PV) is a rare autoimmune blistering disease of the skin and mucous membranes(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The reported incidence is between 1 and 3.2 cases per 100,000 individuals annually, and those of Ashkenazi Jewish descent have a higher incidence. Males and females are equally affected(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). PV is mediated by circulating autoantibodies targeting desmoglein (Dsg)3 in mucosal-dominant disease and to Dsg3 and Dsg1 in mucocutaneous disease, resulting in the loss of epidermal cell\u0026ndash;cell adhesion and development of blisters(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The patients present with painful erosions, blisters, and ulcers on the mucosa and skin(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). While the lesions are more common in the oral mucosa, they have also been reported in other mucosal areas, such as the pharynx, larynx, esophagus, conjunctiva, and anal mucosa(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Involvement of the female genital tract in PV was reported in previous studies and was the second-most frequently involved mucosal region after the oral mucosa(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Histologically, the characteristics of PV lesions are intraepidermal vesicles with acantholysis and an intact basal layer(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The disease has a chronic, severe, and potentially fatal course(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Mortality rates were approximately 90% before the introduction of systemic corticosteroids (CS), but with the use of systemic corticosteroids in high doses, the mortality rates reduced to less than 10%(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The introduction of adjuvant immunosuppressive agents has resulted in a better outcome for the disease and a decrease in the required corticosteroid dose and associated toxicities(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Patients may need long-term systemic treatment with high doses of corticosteroids and other steroid-sparing immunosuppressive drugs, such as azathioprine, mycophenolate mofetil (MMF), or cyclophosphamide.\u003c/p\u003e \u003cp\u003eRituximab (RTX) is a chimeric murine/human monoclonal antibody that targets the CD20 antigens present on B lymphocytes. Rituximab is thought to act through various mechanisms responsible for B-cell depletion, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity, and direct triggering of apoptosis(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Rituximab decreases the blood serum level of antidesmoglein autoantibodies rapidly, which results in healing of pemphigus lesions after a few weeks of infusion, and Complete remission is attained after 3\u0026ndash;6 months(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In the management and treatment of various autoimmune conditions with rituximab or other immunosuppressive drugs, there was an increased risk of viral infections, such as cervical human papillomavirus (HPV) infection, which is strongly associated with cervical dysplasia(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Also, there was an increased risk of high-grade cervical dysplasia or\u003c/p\u003e \u003cp\u003ecervical cancers(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The effect of this type of therapy on cervical dysplasia, cervical cancer, and cervical HPV infection in PV patients remains unknown.\u003c/p\u003e \u003cp\u003eHigh-risk (HPV) infection is the principal etiologic factor for cervical cancer in women. The virus is highly prevalent, with up to 80% of sexually active women becoming infected at some point during their lifetime. One important risk factor for endocervical HPV acquisition is disruption of the mucosal barrier, which facilitates viral entry into basal epithelial cells. Therefore, HPV infection may theoretically be more common in disorders associated with mucocutaneous barrier disruption, such as PV(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In most individuals, HPV infection is transient and cleared by the host immune system. However, persistent infection with high-risk genotypes can lead to cervical intraepithelial neoplasia and, ultimately, carcinoma. Prolonged systemic glucocorticoid therapy has been suggested as a contributor to viral persistence and progression due to its broad immunosuppressive effects in patients with systemic lupus erythematosus (SLE). Data on the impact of B-cell\u0026ndash;depleting therapies are more limited. Anti-CD20 monoclonal antibodies, including RTX, ocrelizumab, and ofatumumab, target CD20-positive B lymphocytes, which play a key role in antiviral and antitumor immune responses through antibody production, antigen presentation, and modulation of T-cell activation. Depletion of these cells may impair immune surveillance and the recognition of virally transformed or dysplastic cells(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Although studies in other autoimmune diseases, such as multiple sclerosis (MS) and SLE, have reported increased HPV infection rates in patients treated with anti-CD20 agents, data specific to PV remain lacking(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The purpose of this study was to evaluate female genital tract involvement and examine cervical Papanicolaou (Pap) smears to assess the risk of cervical dysplasia, cervical cancer, and cervical HPV infection in women with PV before and 6 months after the treatment with rituximab.\u003c/p\u003e "},{"header":"Patients and methods","content":"\n\u003ch3\u003e1. Study Design\u003c/h3\u003e\n\u003cp\u003eThis cohort study was carried out at the Shohada-e-Tajrish and Loqhman-e-Hakim Hospitals, Shahid Beheshti University of Medical Sciences at Tehran, Iran. Approval was sought from the institutional ethics committee (code IR.SBMU.MSP.REC.1403.197), and informed consent was obtained from all participants after explaining the study\u0026rsquo;s purpose and procedures.\u003c/p\u003e\n\u003ch3\u003e2. Patient selection and assessment\u003c/h3\u003e\n\u003cp\u003eIn total, 28 women with pemphigus vulgaris (new and recurrent cases) who attended our clinics and received rituximab during the period from February 2023 to January 2025 were included in this study. In all patients, the diagnosis of PV was based on the presence of clinical features of the disease, including mucocutaneous bullae and erosions, along with histopathological examination with hematoxylin-eosin stain, direct immunofluorescence (DIF) findings of the biopsy material, and enzyme-linked immunosorbent assay (ELISA), based on revised diagnostic criteria.(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnly patients who were given a diagnosis of new-onset PV or major recurrences of the disease were enrolled in this study. Patients were eligible for the study if they were married or sexually active. To avoid potential confusion during genital examination and interpretation of Pap smear results, women who presented during menstruation were excluded. Additionally, patients were excluded if they were younger than age 18 years, pregnant, or had had a hysterectomy, cervical cancer, or a previous diagnosis of human papillomavirus infection. Other exclusion criteria for rituximab therapy were: breastfeeding, history of sensitization to murine protein, active and/or severe infections (including tuberculosis, sepsis, and viral hepatitis), and severe cardiac disease. None of the patients had been immunized against any HPV subtypes. Each participant underwent a detailed review of their clinical history. The following information was collected: age; duration of disease; PV type; affected anatomical sites (skin and mucous membranes); presence of vulvar, vaginal, and/or cervical involvement; and systemic treatment history. The cumulative dose of corticosteroids and immunosuppressive drugs (azathioprine, mycophenolate mofetil) and rituximab was recorded.\u003c/p\u003e \u003cp\u003eA complete gynecological examination was performed by a single gynecologist, who examined the vulva, vagina, and cervix of each woman. Pap smears were collected using Ayre spatulas, spray-fixed with commercial fixative, and stained with the routine Pap stain, and reported by a cytopathologist who was aware of the history and clinical situation of the patients. In this study, Pap smear results were reported according to the 2001 Bethesda system:1. Negative for intraepithelial lesion or malignancy, which includes reports within normal limits, organisms, reactive cellular changes associated with inflammation (includes typical repair) and atrophy; 2. Squamous cell abnormalities: a. Atypical squamous cells of undetermined significance (ASC-US); b. Atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesion (HSIL); c. Low-grade squamous intraepithelial lesion (LSIL); d. HSIL; e. Squamous cell carcinoma; 3. Glandular epithelial cell abnormalities (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In each patient, hemogram, routine biochemical investigations, electrocardiogram (ECG), Mantoux test, and serology for viral hepatitis and human immunodeficiency virus were performed. After premedication (hydrocortisone and pheniramine intravenously and paracetamol orally), patients received an intravenous (IV) infusion of 1 g rituximab on days 1 and 15 under strict monitoring over a period of 5\u0026ndash;6 hours. After the last rituximab infusion, patients were evaluated at monthly intervals for six months; after that, each participant was re-evaluated and underwent a detailed review of their clinical history, a complete physical examination, including a gynecological examination, and the cumulative dose of the received medications. Pap smears were obtained from all the patients, and a sample was performed for detecting and identifying HPV DNA, using a spatula.\u003c/p\u003e\n\u003ch3\u003e3. DNA extraction and HPV genotyping\u003c/h3\u003e\n\u003cp\u003eFor DNA extraction, the QIAamp DNA Mini kit (Qiagen GmbH, Germany) was used according to the manufacturer\u0026rsquo;s directions, and the concentration of extracted DNA was assessed by spectrophotometry at 260 nm. The genomic DNA of the samples was used in polymerase chain reaction (PCR), using the MY09 and MY11 primers. A beta globin PCR assay was used to verify the quality of DNA in the samples. Detection of HPV DNA and genotypes was performed using the INNO-LiPA assay (Fujirebio Europe N.V., Belgium). INNO LiPA HPV genotyping determined 32 HPV genotypes: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and \u0026minus;\u0026thinsp;68 as high-risk HPV genotypes (HR HPV), HPV-26, -53, -66, -70, -73, and \u0026minus;\u0026thinsp;82 as possible high-risk HPV genotypes (pHR HPV), and HPV-6, -11, -40, -42, -43, -44, -54, -61, -62, -67, -81, 83, and \u0026minus;\u0026thinsp;89 as low-risk HPV genotypes (LR HPV). The basis of this HPV classification was the IARC Monographs(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Reverse hybridization is the principle behind INNO-LiPA HPV genotyping. Briefly, a part of the L1 region called SPF10 of the HPV genome was extended by specific primers; after that, the biotinylated amplimers were denatured and hybridized with specific oligonucleotide probes. Additionally, a primer pair of the human HLA-DPB1 gene was added to confirm the quality and extraction of DNA. Then, streptavidin-conjugated alkaline phosphatase was added. The results were visually interpreted after incubation with BCIP/NBT chromogen.\u003c/p\u003e\n\u003ch3\u003e4. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eDescriptive statistics were computed for all study variables. Continuous data were assessed for normality using the Shapiro-Wilk test and are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) for normally distributed variables or median [interquartile range] for non-normally distributed variables. Categorical variables are summarized as frequencies and percentages. Group comparisons for continuous variables were performed using the Mann-Whitney U test (for two-group comparisons, such as HPV-positive versus HPV-negative status) or the Kruskal-Wallis test (for multi-group comparisons, such as Pap smear findings across three categories). Categorical variable associations were analyzed using Chi-square tests or Fisher's exact tests when expected cell counts were less than five. Changes in paired categorical data (pre- versus post-treatment Pap smear results) were assessed using the Stuart-Maxwell test for marginal homogeneity. All statistical tests were two-sided, and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Analyses were performed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R software version 4.5.1.\u003c/p\u003e"},{"header":"Results","content":"\n\u003ch3\u003e1. Cohort Demographics and Clinical Presentation\u003c/h3\u003e\n\u003cp\u003eThe study cohort comprised 28 female patients with a confirmed diagnosis of pemphigus vulgaris. The mean age was 55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1 years, with a pronounced postmenopausal predominance (21/28, 75.0%). The majority of patients presented with established disease (22/28, 78.6%) and had a median disease duration of 60.0 (36.0-114.0) months. The mucocutaneous form predominated (19/28, 67.9%), followed by mucosal (6/28, 21.4%) and cutaneous (3/28, 10.7%) variants. Oral mucosal involvement was nearly universal (96%), whereas genital symptoms (discharge, itching, burning and irritation) were present in 10 patients (35.7%). The most common sites of cutaneous involvement were the trunk (90.9%) and upper limbs (68.2%). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes baseline demographic and clinical characteristics.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026nbsp;Baseline demographic and clinical characteristics of pemphigus vulgaris patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (years), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease duration (months), median\u003c/b\u003e(\u003cb\u003eQ1-Q3\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.0 (36.0-114.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of pregnancies, median\u003c/b\u003e(\u003cb\u003eQ1-Q3\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 (2.0\u0026ndash;3.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMenopausal status, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremenopausal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (25.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePostmenopausal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (75.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease status, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNew case\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (21.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstablished case\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (78.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease type, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCutaneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (10.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMucosal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (21.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMucocutaneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19 (67.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenital symptoms at baseline, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e Present\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (35.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18 (64.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLesions Anatomical distribution, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOral cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 (96.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrunk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (90.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUpper limbs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (68.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHead and neck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (54.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePharynx/Larynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNasal cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (28.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLower limbs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (27.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVulvar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (8.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eQ1: first quantile, Q3: third quartile; SD: Standard deviation.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2. Clinical Outcomes Following Rituximab Therapy\u003c/h3\u003e\n\u003cp\u003eAfter rituximab therapy, clinical assessment at six months revealed that seven patients (25.0%) experienced clinical relapses, defined as the appearance of new mucocutaneous lesions after remission. Five recurrences involved the oral mucosa, and one each affected the leg and pharynx. Genital symptoms declined slightly from 10 (35.7%) to 8 (28.6%) patients. During the follow-up period, 23 patients received corticosteroid therapy (prednisolone) with a mean cumulative dose of 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7 g, administered over a mean duration of 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 months (range: 1 to 6 months). Only one patient received additional treatment with azathioprine for a duration of 6 months.\u003c/p\u003e\n\u003ch3\u003e3. Analysis of Cervical Cytology Before and After Treatment\u003c/h3\u003e\n\u003cp\u003eA comparative analysis of cervical cytology before and after rituximab administration is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Pre-treatment, the majority of Pap smears were interpreted as Normal (15/28, 53.6%), followed by inflammation (10/28, 35.7%) and Atypical Squamous Cells of Undetermined Significance (ASC-US) (3/28, 10.7%). Post-treatment, a shift in the distribution was observed: the proportion of normal results decreased to 35.7% (10/28), while smears indicating inflammation increased to 57.1% (16/28). The prevalence of ASC-US results remained low (2/28, 7.1%). No cases of low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma, atypical glandular cells (AGC), endocervical adenocarcinoma in situ (AIS), or adenocarcinoma were detected in any sample. Statistical comparison using the Stuart-Maxwell test confirmed that these longitudinal changes did not reach statistical significance (p\u0026thinsp;=\u0026thinsp;0.368), indicating that rituximab did not induce a significant alteration in Pap smear categorization within this cohort.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026nbsp;Pap smear results before and after Rituximab administration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePap smear before, n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003ePap smear After, n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP-value*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInflammatory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(33.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10(66.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0(0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15(53.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflammatory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4(40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (35.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.368\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(66.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(33.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3(10.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10(35.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16(57.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(7.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003e*Stuart-Maxwell test; ASC-US: Atypical squamous cells of undetermined significance.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e4. Genotype Distribution of Detected Human Papillomavirus\u003c/h3\u003e\n\u003cp\u003eHPV DNA was detected in 7 out of 28 patients (25%). Among these, high-risk (HR) oncogenic genotypes were predominant, identified in six patients (85.7% of positive cases). A notable finding was the high frequency of multiple HR-type co-infections, which were present in three patients (42.9% of positive cases). The specific co-infections identified were HPV-51 and HPV-56; HPV-31 and HPV-33; and HPV-16 and HPV-18. HPV-16 was the most prevalent individual genotype, detected in three patients (42.9% of positive cases). One patient carried a low-risk genotype (HPV-6) (14.3%). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB illustrates genotype distribution.\u003c/p\u003e\n\u003ch3\u003e5. Association between demographic/clinical Factors and Cytological and Virological Findings\u003c/h3\u003e\n\u003cp\u003eThe relationships between demographic/clinical variables and post-treatment cytological (Pap smear) and virological (HPV) outcomes are delineated in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Analysis revealed no significant associations between post-rituximab Pap smear results (Normal, Inflammatory, ASC-US) and any demographic variable, including age, parity, menopausal status, or clinical features such as disease status and baseline genital symptoms (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation between demographic/clinical Characteristics and Post-Rituximab Pap Smear and HPV typing Results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDemographics/Clinical Characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003ePap smear results\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eHPV typing\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echaracteristic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecategory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInflammatory (n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eASC-US (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative (n\u0026thinsp;=\u0026thinsp;21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epositive (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.4\u0026thinsp;\u0026plusmn;\u0026thinsp;12.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.961\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePregnancies (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian(Q1-Q3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0(2.0-2.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0(2.0\u0026ndash;4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5(2.2\u0026ndash;2.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2(2.0\u0026ndash;3.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3(1.5-3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.869\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration (months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian(Q1-Q3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60(36\u0026ndash;165)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60(36\u0026ndash;108)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48(48\u0026ndash;48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e84(48\u0026ndash;165)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24(12\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMenopausal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7(33.3)\u003c/p\u003e \u003cp\u003e3(42.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13(61.9)\u003c/p\u003e \u003cp\u003e3(42.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(4.8)\u003c/p\u003e \u003cp\u003e1(14.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5(83.3)\u003c/p\u003e \u003cp\u003e16(72.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1(16.7)\u003c/p\u003e \u003cp\u003e6(27.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePemphigus vulgaris type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCutaneous\u003c/p\u003e \u003cp\u003eMucosal\u003c/p\u003e \u003cp\u003eMucocutaneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(66.7)\u003c/p\u003e \u003cp\u003e1(16.7)\u003c/p\u003e \u003cp\u003e7(36.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(33.3)\u003c/p\u003e \u003cp\u003e4(66.7)\u003c/p\u003e \u003cp\u003e11(57.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0(0.0)\u003c/p\u003e \u003cp\u003e1(16.7)\u003c/p\u003e \u003cp\u003e1(5.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2(66.7)\u003c/p\u003e \u003cp\u003e2(33.3)\u003c/p\u003e \u003cp\u003e17(89.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1(33.3)\u003c/p\u003e \u003cp\u003e4(66.7)\u003c/p\u003e \u003cp\u003e2(10.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNew case\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4(66.7)\u003c/p\u003e \u003cp\u003e6(27.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(16.7)\u003c/p\u003e \u003cp\u003e15(68.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(16.7)\u003c/p\u003e \u003cp\u003e1(4.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5(83.3)\u003c/p\u003e \u003cp\u003e16(72.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1(16.7)\u003c/p\u003e \u003cp\u003e6(27.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenital symptoms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e8(44.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8(80)\u003c/p\u003e \u003cp\u003e8(44.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0(0.0)\u003c/p\u003e \u003cp\u003e2(11.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7(70)\u003c/p\u003e \u003cp\u003e14(77.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e4(22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.674\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eASC-US: atypical squamous cells of undetermined significance; HPV: human papillomavirus; Q1: first quantile, Q3: third quartile; SD: standard deviation.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn contrast, a robust and statistically significant association was identified between disease chronicity and HPV status. Patients positive for HPV had a markedly shorter median disease duration (24 (12\u0026ndash;36) months) compared to their HPV-negative counterparts (84 (48\u0026ndash;165) months) (p\u0026thinsp;=\u0026thinsp;0.005). Furthermore, a significant association was observed between the clinical type of PV and HPV prevalence (p\u0026thinsp;=\u0026thinsp;0.019). HPV positivity was highest in patients with solely mucosal disease (4/6, 66.7%), intermediate in those with cutaneous involvement (1/3, 33.3%), and lowest in the mucocutaneous group (2/19, 10.5%), suggesting a potential link between disease type and susceptibility to HPV infection.\u003c/p\u003e \u003cp\u003eOf the 22 patients with recurrence, previous immunosuppressive therapies included corticosteroids (prednisolone or methylprednisolone pulse), azathioprine, rituximab, and mycophenolate mofetil. A comparative analysis using the Mann\u0026ndash;Whitney U test was conducted to evaluate potential associations between the cumulative doses and treatment duration of these agents and Pap smear cytology and HPV typing results. No significant associations were identified between the cumulative dose or duration of any immunosuppressive drug and Pap smear or HPV outcomes (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePV is an autoimmune blistering disease characterized by the production of IgG autoantibodies against desmoglein 1 and 3, key proteins for epidermal adhesion. This antibody-mediated attack results in the hallmark clinical presentation: painful flaccid blisters and erosions on the skin and mucous membranes(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The management of PV has been revolutionized by the introduction of RTX, a chimeric anti-CD20 monoclonal antibody that depletes B lymphocytes, leading to a rapid reduction in pathogenic autoantibodies and the induction of prolonged remission(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). However, the profound immunosuppression required to control this autoimmune diathesis, along with mucocutaneous barrier disruption in PV, raises concerns about an increased susceptibility to cervical HPV infection and cervical dysplasia(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This cohort study provides a critical evaluation of this risk by analyzing cervical cytological changes and HPV prevalence in patients with PV before and after RTX therapy. Our primary finding is that, within a six-month follow-up period, RTX administration did not precipitate a statistically significant shift towards high-grade cervical cytological abnormalities, which is a reassuring short-term outcome. However, we also observed two clinically notable results: a substantial increase in inflammatory Pap smear findings and a high prevalence of oncogenic HPV infections (25%) in our cohort. Furthermore, our analysis revealed that this HPV positivity was strongly associated with a shorter duration of PV and a mucosal-dominant disease type.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first prospective study to systematically evaluate longitudinal cervical cytological changes in women with pemphigus vulgaris before and after treatment with rituximab, coupled with analysis of HPV prevalence following therapy. While prior research has investigated genital involvement in PV and the general risk of HPV with immunosuppressants in other autoimmune conditions, our study uniquely focuses on the Pap smear changes following B-cell depletion therapy in this particular patient population. This study provides an observational perspective on the potential impact of both the underlying autoimmune process and its treatment on cervical cytologic findings. Our findings at baseline are consistent with earlier studies describing genital tract involvement and symptoms in PV (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The predominance of mucosal lesions in our cohort echoes prior reports showing that mucosal surfaces are especially vulnerable to PV-associated erosions(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Such epithelial disruption may facilitate HPV acquisition or persistence. These findings confirm that PV itself commonly affects the genital mucosa and causes cytologic changes, even without biologic therapy. As expected, in our study, 18 cases (64.3%) at baseline and 20 cases (71.43%) after RTX administration were asymptomatic (no discharge, itching, spotting/bleeding, or pain). Based on previous studies, cervical involvement in PV is usually asymptomatic but can present with spotting, postcoital bleeding, discharge, or dyspareunia; a diagnosis can only be suspected if a prior history of PV is known(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The absence of progression to LSIL, HSIL, or malignancy following rituximab treatment represents a reassuring short-term outcome and is consistent with previous reports. Our results align with the large cohort study by Kim et al. (2016), which compared biologic (including rituximab) and non-biologic DMARDs in patients with rheumatoid arthritis and found no statistically significant increase in the incidence of high-grade cervical dysplasia or cancer among biologic users, despite a numerically higher rate. Similarly, the meta-analysis by Garc\u0026iacute;a-Carrasco et al. (2018) demonstrated that, across autoimmune diseases, biologic and conventional immunosuppressive therapies were not associated with a significant rise in cervical neoplasia risk, although persistent high-risk HPV infection was more common in immunosuppressed women. Taken together, these findings support our observation that short-term rituximab therapy in PV does not appear to accelerate cervical cytologic progression, while emphasizing the importance of continued long-term monitoring for potential late effects (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). A population-based Danish cohort study on patients with autoimmune diseases, including pemphigus and pemphigoid, also showed no increased risk of cervical cancer associated with immunosuppressant use, except for high-dose users of azathioprine(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe increase in inflammatory smears post-treatment (35.7% to 57.1%) can be explained by the mucosal healing process after treatment with RTX and the recruitment of inflammatory cells to repair the mucosal erosions, which can appear cytologically as nonspecific inflammation.\u003c/p\u003e \u003cp\u003eThe prevalence of high-risk HPV infection in our cohort (25%) was markedly higher than expected for age-matched women in the general population. According to the global meta-analysis by Osmani et al. (2025, Lancet Microbe), the pooled prevalence of high-risk HPV among women aged 50 years and older is approximately 6.45% (95% CI 5.45\u0026ndash;7.53), with regional estimates as low as 3.27% in western Asia(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Similarly, in the Iranian population-based study by Jamshidi et al. 2025, high-risk HPV was detected in only 4.5% of women aged 50 years and above, confirming a steep age-related decline in viral prevalence(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Given that most of our participants were in this age range, the 25% positivity observed here represents a substantially elevated rate, and is comparable to the 28\u0026ndash;34% prevalence observed in studies on other autoimmune diseases such as SLE(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Both the underlying immune dysregulation inherent to autoimmune disorders and the use of immunosuppressive therapies have been implicated in this increased susceptibility. In the study by Mendoza-Pinto et al., a cross-sectional study on 148 women with SLE, the HPV prevalence was associated with the cumulative dose of glucocorticoids but not RTX. Interestingly, a large meta-analysis by Garc\u0026iacute;a-Carrasco et al., 2018 on the prevalence of cervical HPV infection in women with SLE found that treatment with immunosuppressive agents, including glucocorticoids and biologics such as rituximab, did not independently increase the risk of high-risk HPV infection when compared with untreated patients, which is consistent and comparable to our findings that no significant associations were identified between the cumulative dose or duration of any immunosuppressive drug and Pap smear or HPV outcomes. This supports the hypothesis that the autoimmune disease itself, rather than the therapy alone, may predispose patients to viral persistence(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In PV, chronic mucosal erosions, disruption of the epithelial barrier, and impaired humoral as well as innate immune responses likely act synergistically to facilitate HPV acquisition and persistence. This excess risk supports the concept that mucosal barrier disruption and immune dysregulation in PV, particularly impaired humoral and innate antiviral responses, may facilitate persistence or reactivation of high-risk HPV infection. In the study by Mendoza-Pinto et al., a notably high proportion (72%) of HPV-positive patients with SLE were infected with high-risk genotypes. This finding closely parallels our observation that 85.7% of HPV-positive PV patients carried high-risk types. The predominance of HR-HPV genotypes (85.7%), including the highest-risk types 16 and 18, and the high frequency of multiple HR-HPV co-infections (42.9% of positive cases) are clinically important. This is a critical concern as these genotypes are responsible for approximately 70\u0026ndash;75% of all cervical cancers worldwide. This risk is compounded by data showing that co-infection with multiple HPV genotypes can impair viral clearance and enhance persistence. Therefore, these findings mandate strict adherence to enhanced screening protocols as outlined in international guidelines(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn additional novel observation from our study is the inverse association between HPV positivity and disease duration. Patients who were HPV-positive had a significantly shorter median disease duration (24 months) than those who were HPV-negative (84 months; p\u0026thinsp;=\u0026thinsp;0.005), and HPV infection was significantly more frequent in patients with the mucosal-predominant subtype of PV (66.7%; p\u0026thinsp;=\u0026thinsp;0.019). These findings may indicate that recent-onset, mucosal-dominant PV represents a disease stage characterized by heightened epithelial activity and local immune dysregulation, which together may increase susceptibility to HPV infection(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe hypothesize that the intense, acute inflammatory milieu characteristic of this active, recent-onset mucosal PV type could disrupt the local epithelial barrier and immune surveillance, facilitating HPV entry or reactivation of latent infection. This is mechanistically plausible, as the erosive nature of active PV lesions may compromise the physical integrity of the mucosal epithelium, providing a direct portal of entry for HPV. This concept is supported by Akhyani et al. (2008), who demonstrated that genital tract involvement is present in 51% of their patients and manifests clinically as erosions providing direct evidence of the epithelial disruption required for such a mechanism(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Furthermore, as demonstrated by Eichhorn et al. (2022), this state involves caspase-dependent innate immune activation and elevated secretion of pro-inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6), which we propose that this state of local innate immune dysregulation could disrupt coordinated mucosal antiviral defenses, thereby facilitating initial HPV acquisition or reactivation of latent infection(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe strengths of this study include its prospective cohort design with paired cervical cytology assessments, consistent diagnostic methodology, and rigorous PCR-based HPV genotyping. However, several limitations must be acknowledged, including a relatively small sample size, a single-center design, and a short follow-up period, which may preclude detection of long-term cytological progression or malignancy risk. Consequently, larger multicenter studies with extended follow-up and serial HPV testing are warranted to validate these findings and further elucidate the long-term gynecological risks in PV patients.\u003c/p\u003e \u003cp\u003eDespite these limitations, our results highlight the clinical significance of implementing vigilant cervical screening in patients with PV receiving rituximab. Integration of Pap smear and HPV co-testing into routine long-term monitoring protocols is strongly recommended, particularly for women with mucosal-dominant disease. In addition, consideration of prophylactic HPV vaccination prior to or early during immunosuppressive therapy represents a potentially valuable preventive strategy that merits further evaluation in this high-risk population.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, rituximab therapy in women with PV did not produce significant cytological abnormalities within the six-month observation period, although a shift toward inflammatory smears was observed. A considerable proportion of patients were positive for high-risk HPV genotypes, particularly those with mucosal PV and shorter disease duration. These findings emphasize the need for ongoing gynecological surveillance and HPV testing in PV patients receiving rituximab. Larger, multicenter, longitudinal studies are needed to assess the risk of cervical dysplasia and cancer in this population. Incorporating HPV vaccination into the management of PV patients may help reduce oncogenic risk and improve long-term outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement:\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest regarding the publication of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.A., N.M., and F.F. performed patient recruitment and data collection. N.Z assisted with the paraclinical assessment of patients. M.G. and F.A. supervised the project and critically revised the manuscript. S.D. and K.Z. contributed to data analysis and drafting of the first version. S.Z. and R.R designed the study, analyzed the data, and prepared the manuscript draft.All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis article is from the disease registry, titled \u0026ldquo;pemphigus disease\u0026rdquo;, and projected with code number \"IR.SBMU.MSP.REC.1403.197\" from the ethics committee supported by the skin research center, Shahid Beheshti University of Medical Sciences.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFairbanks Barbosa ND, de Aguiar LM, Maruta CW, Aoki V, Sotto MN, Labinas GH et al (2012) Vulvo-cervico-vaginal manifestations and evaluation of Papanicolaou smears in pemphigus vulgaris and pemphigus foliaceus. J Am Acad Dermatol 67(3):409\u0026ndash;416\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik M, Ahmed AR (2005) Involvement of the female genital tract in pemphigus vulgaris. Obstet Gynecol 106(5 Pt 1):1005\u0026ndash;1012\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma VK, Bhari N, Gupta S, Sahni K, Khanna N, Ramam M et al (2016) Clinical efficacy of rituximab in the treatment of pemphigus: A retrospective study. Indian J Dermatol Venereol Leprol 82(4):389\u0026ndash;394\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeyzaee AM, Rahmatpour Rokni G, Patil A, Goldust M (2021) Rituximab as the treatment of pemphigus vulgaris in the COVID-19 pandemic era: A narrative review. Dermatol Ther 34(1):e14405\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen DM, Odueyungbo A, Csinady E, Gearhart L, Lehane P, Cheu M et al (2020) Rituximab is an effective treatment in patients with pemphigus vulgaris and demonstrates a steroid-sparing effect. Br J Dermatol 182(5):1111\u0026ndash;1119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamyab K, Daneshpazhooh M, Zaresharifi N, Ghanadan A, Shahsiah R, Mahmoudi HR et al (2023) Detection of herpes simplex viruses in the oral lesions of patients with pemphigus vulgaris: Is it diagnostic or predictive of disease severity? Skin Health Dis 3(5):e261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright C, Pipingas A, Grayson W, Leiman G (2000) Pemphigus vulgaris of the uterine cervix revisited: Case report and review of the literature. Diagn Cytopathol 22(5):304\u0026ndash;307\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkhyani M, Chams-Davatchi C, Naraghi Z, Daneshpazhooh M, Toosi S, Asgari M et al (2008) Cervicovaginal involvement in pemphigus vulgaris: a clinical study of 77 cases. Br J Dermatol 158(3):478\u0026ndash;482\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKavala M, Topaloglu Demir F, Zindanci I, Can B, Turkoglu Z, Zemheri E et al (2015) Genital involvement in pemphigus vulgaris (PV): correlation with clinical and cervicovaginal Pap smear findings. J Am Acad Dermatol 73(4):655\u0026ndash;659\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassan I, Rehman F, Sultan SJ, Aslam A, Tasaduq I, Reyaz S (2022) Rituximab in Pemphigus - An Observational Study from a Tertiary Care Center of North India. Indian Dermatol Online J 13(5):620\u0026ndash;624\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNafiseh E, Cheyda C-D, Maryam D, Maryam G, Robabe A, Hossein M et al (2012) Successful treatment of major pemphigus vulgaris relapse with mycophenolate mofetil and high-potent topical corticosteroid. Iran J Dermatology 15(2):33\u0026ndash;37\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendoza-Pinto C, Garcia-Carrasco M, Vallejo-Ruiz V, Taboada-Cole A, Munoz-Guarneros M, Solis-Poblano JC et al (2013) The impact of glucocorticoids and anti-cd20 therapy on cervical human papillomavirus infection risk in women with systemic lupus erythematosus. Clin (Sao Paulo) 68(12):1475\u0026ndash;1480\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SC, Schneeweiss S, Liu J, Karlson EW, Katz JN, Feldman S et al (2016) Biologic Disease-Modifying Antirheumatic Drugs and Risk of High-Grade Cervical Dysplasia and Cervical Cancer in Rheumatoid Arthritis: A Cohort Study. Arthritis Rheumatol 68(9):2106\u0026ndash;2113\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlizadeh N, Golchai J, Ghanbari A, Golchai B, Zaresharifi S (2021) Genotype distribution of human papillomavirus in anogenital warts in Guilan province, northern Iran. Iran J Dermatology 24(2):117\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Pan W, Jin L, Huang W, Li Y, Wu D et al (2020) Human papillomavirus vaccine against cervical cancer: Opportunity and challenge. Cancer Lett 471:88\u0026ndash;102\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelamed E, Lee MW (2019) Multiple Sclerosis and Cancer: The Ying-Yang Effect of Disease Modifying Therapies. Front Immunol 10:2954\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBridge F, Brotherton JML, Foong Y, Butzkueven H, Jokubaitis VG, Van der Walt A (2023) Risk of cervical pre-cancer and cancer in women with multiple sclerosis exposed to high efficacy disease modifying therapies. Front Neurol 14:1119660\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkeda S, Imamura S, Hashimoto I, Morioka S, Sakuma M, Ogawa H (2003) History of the establishment and revision of diagnostic criteria, severity index and therapeutic guidelines for pemphigus in Japan. Arch Dermatol Res 295(Suppl 1):S12\u0026ndash;S16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSolomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M et al (2002) The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 287(16):2114\u0026ndash;2119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiological agents. IARC Monogr Eval Carcinog Risks Hum (2012) ;100(Pt B):1-441\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoly P, Maho-Vaillant M, Prost-Squarcioni C, Hebert V, Houivet E, Calbo S et al (2017) First-line rituximab combined with short-term prednisone versus prednisone alone for the treatment of pemphigus (Ritux 3): a prospective, multicentre, parallel-group, open-label randomised trial. Lancet 389(10083):2031\u0026ndash;2040\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoly P, Litrowski N (2011) Pemphigus group (vulgaris, vegetans, foliaceus, herpetiformis, brasiliensis). Clin Dermatol 29(4):432\u0026ndash;436\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavakolpour S, Mahmoudi H, Balighi K, Abedini R, Daneshpazhooh M (2018) Sixteen-year history of rituximab therapy for 1085 pemphigus vulgaris patients: A systematic review. 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Lancet Glob Health 8(2):e191\u0026ndash;e203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEichkorn RA, Schmidt MF, Walter E, Hertl M, Baron JM, Waschke J et al (2022) Innate immune activation as cofactor in pemphigus disease manifestation. Front Immunol 13:898819\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pemphigus vulgaris, Rituximab, Papanicolaou smear, Cervix, Human papillomavirus infection","lastPublishedDoi":"10.21203/rs.3.rs-7912509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7912509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous blistering disease. Rituximab (RTX) is a chimeric anti-CD20 monoclonal antibody that is increasingly being used and becoming the first-line therapy in the management of PV. Its impact on cervical cytology and human papillomavirus (HPV) infection risk in this population remains unclear.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTo prospectively evaluate cervical Papanicolaou \u003cb\u003e(\u003c/b\u003ePap) smear results and HPV prevalence before and 6 months after rituximab therapy in women with PV.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwenty-eight women with PV underwent Pap smear and HPV genotyping before and after two rituximab infusions (1 gram, 2 weeks apart)\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe mean age was 55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1 years; 75% were postmenopausal. After treatment, the proportion of inflammatory smears increased (from 35.7% to 57.1%), though no high-grade lesions appeared (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.368). HPV DNA was detected in 25% of patients, predominantly high-risk genotypes. HPV positivity correlated with shorter disease duration (24.0 vs. 84.0 months, p\u0026thinsp;=\u0026thinsp;0.005) and mucosal PV type (66.7%, p\u0026thinsp;=\u0026thinsp;0.019).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eRituximab did not significantly alter Pap smear Bethesda categorization at 6 months, but coincided with increased inflammatory cytology and frequent high-risk HPV. Findings support regular Pap/HPV co-testing and vaccination in women with PV undergoing B-cell depletion therapy.\u003c/p\u003e","manuscriptTitle":"Evaluation of Papanicolaou smears in pemphigus vulgaris patients before and after rituximab","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 00:09:09","doi":"10.21203/rs.3.rs-7912509/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-01-17T14:04:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56681047888514800142188028271741681888","date":"2026-01-09T05:44:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34895800095612699621916650456778037219","date":"2026-01-09T01:17:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-08T21:59:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-22T12:08:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T12:06:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Dermatological Research","date":"2025-10-21T08:41:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"30e1d27c-34f4-4660-bb48-13e5762a2427","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-16T00:09:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 00:09:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7912509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7912509","identity":"rs-7912509","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}