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1 Role of Trimethoprim-Sulfamethoxazole Prophylaxis Against the Infectious Complications of Rituximab Treatment in Autoimmune Blistering Diseases a Retrospective Descriptive Study The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Williams, Ramone Role of Trimethoprim-Sulfamethoxazole Prophylaxis Against the Infectious Complications of Rituximab Treatment in Autoimmune Blistering Diseases a Retrospective Descriptive Study. Doctoral dissertation, Harvard Medical School. Citable link Terms of Use This article was downloaded from Harvard University s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at nrs.harvard.edu/urn-3:hul.instrepos:dash.current.terms-ofuse#laa

2 2 Abstract Background: Rituximab has emerged as a highly successful treatment in patients with severe-refractory autoimmune blistering diseases (AIBD) including pemphigus vulgaris. Marked clinical responses have been documented within just a few weeks of therapy and response rates as high as 97% have been reported. The B cell depleting action of rituximab occurs within 2 weeks of the first infusion and persists for an average of 11 months. Patients with AIBD on rituximab, often receive concurrent therapy with steroids and immunosuppressive agents. Thus, the B cell depleting action of rituximab occurs in the setting of glucocorticoid-mediated T cell suppression. Severe infection after rituximab treatment has been reported in up to 10% of patients, some fatal. This study investigates the rate and spectrum of infectious complications of rituximab in AIBD and potential role of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. Methods: We performed a retrospective cohort study of 30 patients with AIBD treated with rituximab. Patients received varying protocols of rituximab treatment. All patients received concurrent systemic corticosteroids and/ or immunosuppressive agents. We analyzed the incidence and spectrum of infectious complications as well as the use of antibiotic prophylaxis in the cohort. Results: Seventy-seven percent of patients received antibiotic prophylaxis. Thirty-three percent of patients developed at least one infectious complication of rituximab, 7% of infections were fatal. TMP- SMX was the agent of choice. The mean duration of antibiotic prophylaxis was 9 months. No adverse effects of TMP-SMX were observed. There was a decreased rate of infectious complications in patients who received antibiotic prophylaxis (RR 0.4; p 0.05), however this trend was not statistically significant. Conclusion: Even in the absence of published guidelines, physicians frequently administer TMP-SMX as prophylaxis for infections in patients with AIBD on rituximab, during treatment and in subsequent periods of B cell depletion. Based on our findings and existing guidelines for patients long term prednisone, we advocate for the use of TMP-SMX prophylaxis in patients with AIBD on rituximab and concurrent steroid-immunosuppressive therapy.

3 3 Table of Contents Section I: Introduction.4 Section II: Methods 9 Section III: Results 11 Section 4: Discussion, Conclusions, and Suggestions for Future Work 14 References.19 Tables and Figures 21

4 4 I. Introduction Autoimmune Blistering Diseases (AIBD) are potentially life threatening antibody-mediated diseases characterized by bullae and erosions on the skin and mucous membranes. Disease subtypes include pemphigus vulgaris (PV), bullous pemphigoid (BP), pemphigus foliaceus (PF), pemphigus vegetans and epidermolysis bullosa acquista. PV is characterized by an initial eruption of flaccid bullae. Primary lesions quickly rupture and leave behind painful erosions. PV can be further divided into two subgroups: patients with mucosal lesions only and patients with both mucosal and skin lesions. Mucosal lesions have predilection for the oropharynx, but also occur on the conjunctiva and genitalia. Skin lesions commonly affect the scalp, face, neck, trunk and groin. PF is characterized by small flaccid blisters on the skin that quickly rupture producing crusted erosions. Mucosal lesions are rare. BP is characterized by tense pruritic blisters over the trunk and flexure surfaces of the extremities. Blisters rupture after several days producing crusted erosions. Oral involvement is rare. It should be emphasized that AIBD is rare. BP is the most frequent form of AIBD, 6 to 7 cases per million are reported each year. PV is the most common form of pemphigus, worldwide incidence ranges from 0.7 to 5 new cases per million per year. There is not substantial data regarding the incidence of PF (1). IgG plays a pivotal role in pathogenesis of pemphigus as the main pathogenic agent targeting Desmoglein 1 (DSG1) (molecular weight (MW) 165 kd) in PV and Desmoglein 3 (DSG3) (MW 130 kd) in PF. These proteins are components of desmosomes and members of the cadherin family. DSG1 is expressed more superficially in the epidermis, while DSG3 is confined to the lower portions of the epidermis. In the mucosa, both DSG1 and DSG3 are expressed throughout the squamous layer. IgG autoantibodies damage cell-cell adhesion resulting in cell-cell detachment and intraepidermal blisters. In PV suprabasilar blisters form, in PF blisters form more superficially in the granular layer. In BP, BPAG1 (MW 230 kd) and BPAG2 or type XVII collagen (MW 180 kd) are target antigens. These proteins are components of hemidesmosmes and play a major role in cell-matrix adhesions. Autoantibody binding results in the disruption of the dermo-epidermal junction (1, 2). Conventional therapy for AIBD consists of combinations of systemic gluccocorticoids and immunosuppressive agents including mycophenolate motefil (MMF), azathioprine (AZA) and methotrexate (MTX) (3,4). However the consequences of conventional immunosuppressive therapy are

5 5 now the major cause of death in patients with pemphigus vulgaris (5). In patients who fail conventional therapy, immune globulin (IVIG) has been utilized successfully as monotherapy. Massive doses of IgG neutralize and slow down the circulation of pemphigus-associated autoantibodies. There are several deterrents to the use of IVIG including high cost, slow onset of action and need for frequent infusions (4). Patients with severe-refractory disease rarely achieve sustained clinical remission with either conventional therapy or IVIG monotherapy. Rituximab has emerged as a promising treatment strategy in these patients. The efficacy of rituximab in pemphigus vulgaris was first established in Since that time numerous studies have confirmed its success response rates as high as 97% have been reported. Rituximab is a humanized chimeric monoclonal antibody targeted against the B-cell surface antigen CD- 20. The biologic was originally developed for the treatment of B cell non-hodgkin s lymphoma. Rituximab s selectively depletes pre-b cells and mature B cells, preventing transformation into autoantibody producing plasma cells. Due to the close interaction of B and T cells, rituximab affects both humoral and cell mediated responses (4). Within 2 weeks of the initiation of therapy, CD20+ B cells become undetectable. Clinical response to therapy is typically rapid, and is paralleled by a decrease in circulating anti-desmoglien antibodies. B cells levels remain depleted and do not normalize until 11 months after discontinuation of therapy (5). Side effects of rituximab include systemic infections (due to impaired humoral immunity), deep venous thrombosis, long-term hypogammaglobulinemia and neutropenia (4). The risk of infection in patients receiving rituximab is an important topic of discussion, as treatment leads to pronounced B cell depletion and hypogammaglobulinemia. Nissen et al. conducted a retrospective cohort study of 125 patients with hematologic malignancy treated with rituximab. The study concluded that rituximab, either as monotherapy or in combination with chemotherapy, did not increase the incidence of or change the spectrum of infections in patients with hematologic malignancy. However, these results should be interpreted with caution. The study compared patients on chemotherapeutic regimens containing rituximab to chemotherapeutic regimens without. Thus the immunosuppressive effect of rituximab may have been overshadowed by the profound neutropenic effect of chemotherapy. Furthermore 37% of patients in the study received antibiotic prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) (6). The study by Nissen et al. consisted of patients receiving induction, not maintenance therapy with rituximab. A sub-group analysis revealed that patients who developed infectious complications

6 6 received a significantly higher cumulative dose of concomitant prednisone (6). Corticosteroids are the mainstay of treatment in autoimmune disease patients typically receive substantial doses of prednisone prior to and during rituximab treatment. Furthermore, patients with autoimmune disease often receive higher cumulative doses of rituximab compared to doses administered for hematologic malignancy. As a result, infectious complications of rituximab are of greater concern in patients with autoimmune disease compared to those with hematologic malignancy. Most existing data suggest that infectious complications of rituximab therapy occur in a small proportion of patients with autoimmune diseases. The German Registry of Autoimmune Diseases (GRAID), a multicenter, non-interventional, retrospective study, examined the efficacy and clinical outcomes of rituximab in 370 patients with various autoimmune diseases including rheumatologic disease excluding rheumatoid arthritis (RA), dermatologic disease including pemphigus, renal and neurologic disease. Almost all patients received immunosuppressive agents and glucocorticoids prior to and during rituximab treatment. The overall rate of infection on rituximab treatment was 18.1 per 100 patient years. Most infections were mild (3.8% of the overall population) or moderate (3.2%). However severe infection was observed in 3.7% of patients. Bacterial infections were most frequent, followed by viral infections, fungal infections and infections caused by unknown pathogens. Overall opportunistic infections were rare occurring in only 5 of 370 patients. The risk of infection was highest 7 months from the first rituximab infusion. Very few patients had more than one infection. The mean cumulative dose of rituximab was 2,440 mg (7). Increased rates of infection during rituximab therapy have been reported by the EXPLORER trial (systemic lupus erythematousus), LUNAR trial (lupus nephritis) and RITUXIVAS trial (ANCA-associated vasculitis). Patients in these studies received adjuvant immunosuppressive agents (including MMF, AZA and MTX) in addition to high-dose corticosteroids. In all 3 trials, the risk of infection was elevated in both rituximab treatment groups and control groups. This finding suggests that infectious complications were related not only due to rituximab treatment, but also to concomitant immunosuppressive therapy and underlying disease (7). A study of patients with RA receiving rituximab reported a stable risk of infection over multiple treatment courses. Similar rates of infection and improved disease control were observed in patients retreated with rituximab at a fixed 6-month interval when compared to patients who received a single treatment course. Based on these findings, it is hypothesized that reduced disease activity may correlate with a reduced risk of infection (7).

7 7 The focus of this work is the risk of infection in patients with AIBDs treated with rituximab. Systemic steroids and immunosuppressive agents are the cornerstone of conventional therapy for AIBD. Due to the late onset of action of rituximab, patients with AIBD receive concurrent steroidimmunosuppressant therapy until adequate disease control is achieved (2). Consequently, patients are subject to both glucocorticoid-mediated T cell inactivation as well as rituximab-mediated B cell depletion. Furthermore, AIBD confers a risk of infection a priori unroofed bullae produce erosions that compromise the protective barrier function of the skin and mucosae, creating a conduit for pathogens. The literature on the risk of infection with rituximab treated AIBD is sparse. In most studies infectious complications are not uniformly described and are limited to the descriptions of severe infection. Schmidt et. al. reviewed published reports of 136 patients with severe pemphigus treated with rituximab, severe infection was reported in 13% of cases, of which 3% were fatal (8). In a study of rituximab in treatment resistant AIBD by Jensen et. al., infectious complications were reported in 5/10 (50%) of patients including sepsis, pneumonia and erysipelas (9). In a study of single cycle rituximab in severe pemphigus rituximab by Joly et. al., infectious complications were observed in 2/21 patients (10%) including pyelonephritis and fatal septicemia (10). A study of adjuvant rituximab in pemphigus by Lunardon et. al., reported infectious complications in 2/18 patients (11%) including 2 cases of osteomyelitis (11). Kanwar et. al studied the safety and efficacy of rituximab in pemphigus, infection was reported in 1/10 patients (10%), including one case of sepsis (12). These reports reveal a substantial risk of infectious complications from rituximab treatment in AIBD patients. We believe this risk of infection warrants evidence-based management with a prophylactic strategy. No guidelines exist to inform the use of antibiotic prophylaxis in patients with AIBDs on rituximab. Guidelines on antibiotic prophylaxis have been published regarding conventional treatments in AIBD. PCP prophylaxis with TMP-SMX is recommended in patients receiving > 20 mg of prednisone daily for more than one month with another cause of immunocompromise (13, 14, 15). Of note most patients with rituximab treated AIBD receive concurrent prednisone and warrant TMP-SMX prophylaxis based on these criteria. Alternative agents for PCP prophylaxis in patients on prednisone include atovaquone (for patients who cannot tolerate the hematologic toxicity of TMP-SMX) and dapsone. TMP-SMX prophylaxis is not indicated for AZA since infectious complications occur in the setting of dose-dependent bone marrow suppression and neutropenia. MTX is an immunomodulatory agent and is not significantly

8 8 immunosuppressive, thus TMP-SMX prophylaxis is not warranted. Interestingly, MMF is thought to confer a protective effect against PCP infection and does not require prophylaxis. Authors have advocated for the consideration of TMP-SMX as prophylactic strategy in patient s receiving biologic agents based on the drugs efficacy in PCP prophylaxis in immunocompromised patients (16). Furthermore, TMP-SMX has a broad spectrum of activity against more common grampositive and gram-negative bacteria. Dapsone and atovaquone are commonly used alternative to TMP- SMX, however the spectrum of activity of each is limited to PCP, toxoplasmosis and malaria. A recent study by Gregoriou et. al. assessed the efficacy of rituximab in 17 patients with pemphigus vulgaris, 1 with pemphigus foliaceus and 1 with pemphigus vegetans. No serious infections were observed. Authors attributed the absence of infectious complications to the administration of prophylactic TMP-SMX and acyclovir. Prophylaxis was initiated prior to the first rituximab infusion and continued for 6 months after the completion of therapy (16). In the present study, we report our experience using rituximab for the treatment of AIBD. We examined the rate and spectrum of infectious complications of rituximab during treatment and in the subsequent period of B cell depletion as well as the use of antibiotic prophylaxis. This descriptive study will be the first to focus on the infectious complications of rituximab in AIBD and to examine the role antibiotic prophylaxis in these patients. Our findings will identify areas further research required to develop guidelines for an antibiotic prophylactic strategy in this patient population.

9 9 II. Methods With institutional review board approval, we conducted an automated search of the electronic medical records (EMR) of Brigham and Women s Hospital and Massachusetts General Hospital using the Research Patient Data Registry (RPDR) a centralized warehouse of clinical data. The RPDR Query Tool was used to determine the aggregate number of patients for patients with a diagnosis containing pemphigus or pemphigoid and treatment that included rituximab or rituxan. No restrictions were placed on the time period of the search. Patient data dating back to 2003 until present (February 2015) were procured. RPDR Data Acquisition Engine was used to collect patients demographic information. Eligible patients had a diagnosis of PV, PF or BP and received treatment with rituximab. Patients with paraneoplastic pemphigus were excluded from this study. Diagnoses of PV, PF or BP were confirmed by the documentation of clinical features, histopathology and/or immunofluorescence. Past medical history was reviewed. Co-morbidities known to influence the risk of infection, such as diabetes mellitus (DM) and chronic obstructive pulmonary disease (COPD) were included in our analyses. The type and dose of immunosuppressive therapy administered prior to and during rituximab treatment were recorded. Doses of prednisone were categorized as follows: high dose ( 100 mg daily), moderate dose (50-99 mg daily), and low dose ( 49 mg daily). The rituximab treatment strategy used in each patient was recorded, including the treatment protocol and total number of treatment courses. The cumulative dose of rituximab was calculated for each patient. A treatment course was defined as a discrete episode of treatment. The number of cycles/infusions of rituximab administered within a single course of treatment varied by the rituximab protocol used. According to the standard protocol, one course of treatment included 4 cycles of rituximab infused weekly. According to the intensive protocol described by Razzaque et al, one course of treatment included 10 cycles of rituximab administered over 6 months. Two or more courses of treatment were administered to patients who a experienced a clinical relapse a clinically visible change suggesting disease recurrence after a period of improvement. The primary outcome of this study was the development of infectious complications of rituximab treatment, defined as any infectious episode documented by microbiologic data, radiographic evidence or clinical signs requiring antibiotic treatment that occurred within 18 months of the first

10 10 rituximab infusion in a treatment cycle. Infections were categorized as the following: mild localized infection requiring oral treatment, moderate infection requiring hospitalization or IV antibiotics, severe life-threatening sepsis or septic shock. The efficacy of rituximab treatment was assessed by physical exam findings documented at follow up and was scored as the following: no response (little to no clinical change in lesions), partial response (resolution of up to 50% of lesions), clinical response (resolution of > 50% of lesions) or complete response (resolution of > 50% of lesions and the ability to discontinue all therapeutic agents). Statistical analyses were performed using Microsoft Excel and MedCalc Version When qualitative data (eg. causative pathogen of infectious complication) was unknown, it was reported accordingly. When quantitative data was not recorded (eg. rituximab dose or prednisone dose), the missing data was excluded from our analyses.

11 11 III. Results Our search of the EMR yielded 34 patients with blistering diseases treated with rituximab; 4 patients with paraneoplastic disease were excluded from the study. Thirty patients with AIBDs were included in the study: 21 patients (70%) with pemphigus vulgaris, 5 (17%) with bullous pemphigoid and 4 (13%) with pemphigus foliaceus. Thirteen patients were male and 16 were female. The average age of the cohort was 54 years (range years). Disease severity ranged from mild (lesions limited to the oral mucosa) to severe (50% body surface area involvement requiring admission to the Burn Intensive Care Unit). The mean disease duration prior to rituximab treatment was 36 months (range months). All 30 patients received conventional therapy with glucocorticoids and/ or immunosuppressive agents prior to and during rituximab treatment. Twenty-five of 30 patients (83%) received concurrent prednisone at varying doses: 1 patient received high dose prednisone, 7 received moderate doses and 17 received low doses. Other co-therapies included: MMF in 17 of 30 patients (57%), MTX in 2/30 (7%), AZA in 1/30 (3%) and Tetracycline/Nicotinamide in 1/30 (3%). The most common immunosuppressive regimen was a combination of MMF and prednisone administered to 16 of 30 patients (53%) (Table 2). All 30 patients received at least one course of rituximab. Patients who experienced a clinical relapse received more than one course of treatment. In the cohort 30 patients, a total of 45 treatment courses were evaluated. Rituximab was administered according to various protocols including: rituximab 375 mg/ m 2 for 4 weekly cycles; rituximab 1000 mg for 2 weekly cycles; rituximab 375 mg/ m 2 for 10 cycles dispersed over 6 months. For all patients, the mean cumulative rituximab dose was 4417 mg (range 2000 mg 10,000 mg) and the mean number of treatment courses was 2 (range 2-5 courses). Nine of 30 patients (30%) received repeated infusions of high dose IVIG every 4 to 6 weeks in combination with rituximab treatment according to various protocols: 7/9 patients received rituximab followed by repeated cycles of IVIG (range 1-17 cycles), 2/9 patients received induction therapy with 2 weekly cycles of rituximab 1000 mg, followed by IVIG for 5 to 6 cycles, followed by consolidation therapy with 1 to 2 weekly cycles of rituximab 1000 mg (Table 1). Twenty-two of 30 patients (77%) received antibiotic prophylaxis during rituximab treatment and during the subsequent period of B cell depletion: 18 patients received TMP-SMX, 4 received atovaquone, and 1 received dapsone. TMP-SMX, the oral double strength table (TMP 160 mg/ SMX 800

12 12 mg), was the agent of choice. Patients with a contraindication to TMP-SMX received atovaquone or dapsone. Review of the EMR revealed physicians prescribed antibiotics for the explicit purpose of prophylaxis during the period of B cell depletion. No adverse reactions to antibiotic prophylaxis were reported. Antibiotic prophylaxis was initiated just prior to the first infusion with a mean duration of 9 months (range: 1-22 months) (Table 2). Nine of 30 patients (33%) did not receive antibiotic prophylaxis. Reasons listed in the EMR included: history of an adverse reaction to TMP-SMX and patient declined TMP-SMX due to cost. Patients who received antibiotic prophylaxis were compared to those who did not: the mean age, subtype of AIBD, proportion of patients with comorbidities including diabetes mellitus (DM) and chronic obstructive pulmonary disease (COPD), proportion of patients on adjuvant oral steroids and immunosuppressive agents of patients in both groups were similar. Mean disease duration prior to rituximab was longer in patients who did not receive antibiotic prophylaxis (60 months) compared to those who received prophylaxis (26 months) (Table 3). Thirty three percent of patients (10/30) developed at least one infectious complication of rituximab treatment, of which 7% (2/30) were fatal. Thirteen percent (4/30) patients developed more than one infection thus 18 infections complications were observed during the study. Sixty-seven percent of infections (12/18) were mild, 3/18 (17%) were moderate and 3/18 (17%) were severe. All observed infections were bacterial, based on microbiologic data or clinical response to antibiotic treatment. No opportunistic infections were observed. Time to first infection ranged between 5 and 486 days from the time of the first rituximab infusion, with a mean of 138 days. Of the 22 patients on antibiotic prophylaxis, 6 developed at least one infectious complication (27%). Patient 8 developed 3 infectious complications over two courses of treatment the first treatment course was complicated by sinusitis and pseudomonal paronychia, the second treatment course was complicated by a upper respiratory infection (URI). Patient 11 developed infectious pericarditis during the first course of treatment but did not experience infectious complications during the second course. Patient 12 developed bacterial pneumonia (PNA). Patient 15 completed one course of treatment without infectious complications, but developed two URIs during the second course of treatment. Patient 17 developed bronchitis during the first course of treatment but did not experience infectious complications during the second course. Patient 29 developed methicillin resistant staphylococcus aureus (MRSA) bacteremia resulting in death. The mean time to first infection in patients who received antibiotic prophylaxis was 137 days.

13 13 Of the 8 patients who did not receive antibiotic prophylaxis, 4 patients developed at least one infectious complication (50%). Patient 4 developed pseudomonal sepsis resulting in death. Patient 5 developed a URI. Patient 7 experienced 5 infectious complications over 2 courses of treatment the first course was complicated by 2 urinary tract infections (UTI), the second treatment course was complicated by 2 more UTIs as well bacteremia/urosepsis. Patient 13 completed one course of treatment without infectious complications, but developed a UTI during the second treatment course. The mean time to first infection in patients who did not receive antibiotic prophylaxis was 140 days (Table 3). We performed a sub-group analysis on patients who received antibiotic prophylaxis to determine the effect of other factors on the risk of developing infectious complications on rituximab. Of the 22 patients who received antibiotic prophylaxis 16 patients remained infection free and 6 developed infectious complications. We analyzed the effect of age 60, male sex, history of DM, high cumulative rituximab dose (defined as a dose greater than the mean cumulative dose of the cohort 4417 mg) and concurrent daily concurrent prednisone dose 50 mg (Table 6). All factors conferred an increased risk of infection; high cumulative rituximab dose was associated with the greatest risk of infection (RR 4.4, p 0.15) followed by high daily prednisone dose (RR 2.4, 0.23). However none of the relative risk values were statistically significant. In this study, the average duration of follow up was 30 months. The efficacy of rituximab treatment was graded by the ultimate response to treatment, as many of our patients received multiple treatment courses. Fifty-seven percent of patients (17/30) achieved a CR, however 4 of these patients subsequently relapsed. Seventeen percent of patients (5/30) achieved a CliR, however one of these patients subsequently relapsed. Ten percent (3/30) of patients achieved a PR and 17% (5/30) of patients had NR (Table 2).

14 14 IV. Discussion The results of this study show that even in the absence of published guidelines, physicians routinely prescribe antibiotic prophylaxis for patients with autoimmune diseases treated with rituximab. Seventy-seven percent of patients in our study received antibiotic prophylaxis during rituximab treatment and during the subsequent period of B cell depletion. TMP-SMX was the prophylactic agent of choice. Patients with documented allergies or adverse reactions to TMP-SMX, received atovaquone or dapsone (Table 2). Antibiotic prophylaxis was initiated just prior to the first infusion with a mean duration of 9 months. Thirty percent of patients developed at least one infectious complication of rituximab, of which 7% were fatal. Thirteen percent of patients developed more than one infectious complication, thus a total of 18 infections were observed during our study. Sixty-seven percent of observed infections were mild, 17% were moderate and 17% were severe. Opportunistic infections were not observed. There was a wide clinical spectrum of infections including URI, UTI, periodontal infection, bronchitis, pneumonia, infectious pericarditis, urosepsis and bacteremia/ sepsis leading to death. All observed infections were bacterial. The risk of infection was highest 4 to 5 months from the first infusion of rituximab. The rate of infection varied in patients receiving multiple courses of treatment (Table 3). The grade and spectrum of infection we observed are similar to prior studies. The GRAID trial, a multi-center retrospective study of 370 patients with various autoimmune diseases, observed mild or moderate infection in the majority of patients. Opportunistic infections were rare (7). However, we observed a higher incidence of infectious complications of rituximab treatment and a shorter time to infection In comparison to the existing literature. Schmidt et al. presented a metaanalysis of 136 patients with AIBD. Severe infectious complications of rituximab were reported in 13% of patients. Mild and moderate infections were not discussed in this study. This discrepancy may explain the higher rate of infectious complications we report our analyses included all types of infections. The GRAID trial reported infectious complications of rituximab in 12% of patients. The risk of infection was highest 7 months after the first infusion. We observed a higher rate of infection (33%) occurring within a shorter time frame (4 months from the first rituximab infusion). While repeated infections were infrequent in the GRAID trial, 13% of our patients developed more than one infectious complication. It is important to note that only 10% of the study population in the GRAID trial had pemphigus. Furthermore the mean cumulative doses of rituximab in both the GRAID trial and study by Schmidt et al. were

15 15 appreciably lower than we report in our study. The patients in our cohort received a mean cumulative dose of 4417 mg, in comparison to 2440 mg reported by Nissen et al., 3760 mg (all autoimmune diseases) reported in the GRAID trial (6,7). Based on the results of the GRAID, patient with pemphigus had lower rates of infectious complications of rituximab compared to patients with glomerunephritis, ANCA-associated granulomatous vasculitis, microscopic polyangitis, myasthenia gravis, SLE ad mixed connective tissue disease. However, authors reported an unusually low cumulative dose of rituximab in patients with pemphigus (1,755 mg) compared to the mean cumulative dose of 3760 mg reported in study population overall (7). This dose is not representative of doses administered according to the standard treatment protocol for pemphigus 4 weekly infusions of rituximab 375 mg/m 2 (3). For example, patient with 2 m 2 of body surface involvement, the cumulative dose for one course of treatment is 3000 mg. Furthermore many patients require more than one course of treatment to achieve sustained remission. We believe that patients with treatment refractory AIBD are at highest risk of infectious complications compared to other patient populations treated with rituximab, including those with hematologic malignancy and other autoimmune diseases. The high cumulative dose of rituximab, concurrent steroid-immunosuppressive regimen and inherent risk of infection conferred by AIBD are concerning risk factors unique to patients with AIBD. In general, the risk of infection with TMP-SMX susceptible organisms during rituximab treatment is difficult to quantify there is a paucity of existing data. The use of TMP-SMX was not consistently described in the methods of studies of rituximab in AIBD. Furthermore, reports of infectious complications were often limited to the term serious infections. The exception was a prospective study by Gregoriou et al. assessing the efficacy of rituximab in 19 patients with pemphigus. Authors attributed the absence of infectious complications to the administration of prophylactic TMP-SMX and acyclovir. Prophylaxis was initiated prior to the first rituximab infusion and continued for 6 months after the completion of therapy. Our results showed a decreased rate of infectious complications of rituximab in patients receiving antibiotic prophylaxis (Table 4). A small proportion of patients on antibiotic prophylaxis still developed infectious complications. A high cumulative rituximab dose (> 4417 mg), high daily prednisone dose (> 50 mg) and a history of diabetes mellitus emerged as other factors that increase the risk of infection in patients on antibiotics (Table 5). The trends that emerged from our study were not

16 16 statistically significant. However, we believe significant trends would emerge in a study with adequate power. For the interpretation of our study, it is important to realize that autoimmune blistering diseases are rare, and the use of rituximab treatment in these patients is exceedingly rare. Despite the inclusion of all the patients with autoimmune blistering diseases treated with rituximab ever, at two large institutions, our study was underpowered. Our findings underscore the fact that the infectious complications of rituximab are substantial and necessitate an evidence based prophylactic strategy. There are no published guidelines on the prevention of infection in patients receiving biologics, except for tuberculosis prevention. Recently authors have advocated for the use of TMP-SMX prophylaxis in patients receiving biologics. The protective effect of TMP-SMX against PCP infection has been well demonstrated in patients with HIV as well as recipients of hematopoetic stem cell and solid organ transplants (16). While other works have focused on the prevention of opportunistic infection, we focus this discussion on the prevention of more common but serious bacterial infections. TMP-SMX has a broad spectrum of activity against important organisms including Staphylococcus (including most community acquired MRSA), Streptococcus, Enterobacter, Escherichia coli, Shigella, Vribrio cholerae, Yersinia, Haemophilus influenzae, Klebsiella, Legionella pneumophila, Moraxella catarrhalis, Proteus, Salmonella, Serratia, Byrkholderia cepacia, Stenotrophomonas maltophilia, Acinetobacter and Citrobacter. In fact, mortality differences have been observed in HIV infected patients owing to the intrinsic in-vitro activity of TMP-SMX against these important pathogens. The potential risks of TMP-SMX prophylaxis in patients with AIBD on rituximab warrant discussion. The adverse reaction rate of TMP-SMX in HIV-infected patients is high, occurring in 25% to 50% of patients. Life threatening side effects such as neutropenia, anaphylaxis, Stevens-Johnson syndrome, toxic epidermal necrolysis and hyperkalemia are uncommon, but are more likely to occur in HIV-infected individuals and the elderly. Nonetheless, TMP-SMX is generally well tolerated. Adverse reactions occur in only 6% to 8% of non-hiv infected patients. Common adverse reactions include nausea, vomiting, rash, pruritus. Caution should be taken with co-administration of TMP-SMX and MTX, as there is a risk of blood cell drycrasias. TMP-SMX is also contraindicated in patients with folate deficiency, during pregnancy and in patients with known sulfonamide antimicrobial allergy. The risk of antibiotic resistance in patients on prolonged TMP-SMX is concerning. However, a recent study

17 17 concluded that TMP-SMX might in fact protect against the development of bacterial resistance to other antibiotic classes and colonization with multi-resistant organisms (16). We believe that antibiotic prophylaxis is beneficial in patients with AIBD on rituximab. We advocate for the use of TMP-SMX as a prophylactic strategy in patients with autoimmune blistering disease who receive concomitant corticosteroids and immunosuppressive agents. Antibiotic prophylaxis should be started prior to the first infusion of rituximab and during the subsequent period of B cell depletion. Clinicians should weigh the risks and benefits of this prophylactic strategy. Patients on high doses on concomitant prednisone, a history of diabetes or other co-morbidity with inherent risk of infection may be more susceptible to the infectious complications on rituximab. While our recommendation requires confirmation with prospective randomized trials, the availability of such evidence may be long awaited considering the rarity of this disease and treatment. Despite the absence of direct evidence, there is precedence for our recommendation. Clinicians may view our conclusions as a call for stricter implementation of existing guidelines. Most patients with AIBD on rituximab receive 20 mg of concomitant daily prednisone for greater than one month and warrant PCP prophylaxis with TMP-SMX based on current guidelines. Clinicians should adhere to these guidelines in patients with AIBD on rituximab due to their suppressed cell-mediated immunity in the setting of B cell depletion. Clinicians should be motivated by the additional protective effect of TMP- SMX against more common aerobic gram positive and gram-negative bacteria that cause infection in patients on rituximab. Limitations Our study was limited by its retrospective design and the limited size of the study population. Conclusions Even in the absence of published guidelines, physicians frequently administer TMP-SMX as prophylaxis for infections in patients with AIBD on rituximab, during treatment and in subsequent periods of B cell depletion. Based on our findings and existing guidelines for patients long term prednisone, we advocate

18 18 for the use of TMP-SMX prophylaxis in patients with AIBD on rituximab and concurrent steroidimmunosuppressive therapy. Suggestions for Future Work Due to the rarity of rituximab treatment for AIBD, our study was underpowered and the primary analysis was not significant. A logistic regression analysis using a generalized estimating equation has the ability to estimate the correlation between the infectious complications of rituximab and antibiotic prophylaxis in our small sample. This analysis could not be performed due to limitations in time and resources. Future work will utilize this statistical method in hopes of significant results. Additionally, the rate of infectious complications of rituximab and the potential benefit of antibiotic prophylaxis need to be confirmed by large multi-center prospective controlled trials. Such robust studies may be able to elucidate whether the infectious risk observed is attributed to rituximab itself, concomitant immunosuppressive agents, underlying blistering disease or a combination of these factors.

19 19 References 1. Kershenovich R, Hodak E, Mimouni D. Diagnosis and classification of pemphigus and bullous pemphigoid. Autoimmunity Reviews Apr;13(4-5): Ruocco V, Ruocco E, Schiavo Lo A, Brunetti G, Guerrera LP, Wolf R. Pemphigus: etiology, pathogenesis, and inducing or triggering factors: facts and controversies. Clinics in Dermatology Jul;31(4): Kasperkiewicz M, Schmidt E, Zillikens D. Current therapy of the pemphigus group. Clinics in Dermatology Jan;30(1): Ruocco E, Wolf R, Ruocco V, Brunetti G, Romano F, Schiavo Lo A. Pemphigus: associations and management guidelines: facts and controversies. Clinics in Dermatology Jul;31(4): Ahmed AR, Spigelman Z, Cavacini LA, Posner MR. Treatment of pemphigus vulgaris with rituximab and intravenous immune globulin. N Engl J Med Oct;355(17): Nissen JC, Hummel M, Brade J, Kruth J, Hofmann W-K, Buchheidt D, et al. The risk of infections in hematologic patients treated with rituximab is not influenced by cumulative rituximab dosage - a single center experience. BMC Infect Dis. 2014;14: Tony H-P, Burmester G, Schulze-Koops H, Grunke M, Henes J, Kötter I, et al. Safety and clinical outcomes of rituximab therapy in patients with different autoimmune diseases: experience from a national registry (GRAID). Arthritis Research & Therapy. 2011;13(3):R Schmidt E, Goebeler M, Zillikens D. Rituximab in severe pemphigus. Ann N Y Acad Sci Sep;1173: Jensen AØ, Møller BK, Vangkilde A, Mark B, Obitz ER, Kragballe K, et al. Treatment of treatment-resistant autoimmune blistering skin disorders with rituximab. Br J Dermatol Jun;160(6): Joly P, Mouquet H, Roujeau J-C, D'Incan M, Gilbert D, Jacquot S, et al. A single cycle of rituximab for the treatment of severe pemphigus. N Engl J Med Aug;357(6):

20 Lunardon L, Tsai KJ, Propert KJ, Fett N, Stanley JR, Werth VP, et al. Adjuvant rituximab therapy of pemphigus: a single-center experience with 31 patients. Arch Dermatol Sep;148(9): Kanwar AJ, Tsuruta D, Vinay K, Koga H, Ishii N, Dainichi T, et al. Efficacy and safety of rituximab treatment in Indian pemphigus patients. J Eur Acad Dermatol Venereol Jan;27(1):e Huang L, Morris A, Limper AH, Beck JM. An Official ATS Workshop Summary: Recent advances and future directions in pneumocystis pneumonia (PCP). Proc Am Thorac Soc Nov;3(8): Sepkowitz KA. Opportunistic infections in patients with and patients without Acquired Immunodeficiency Syndrome. Clinical Infectious Diseases Apr;34(8): Limper AH, Knox KS, Sarosi GA, Ampel NM, Bennett JE, Catanzaro A, et al. An official American Thoracic Society statement: Treatment of fungal infections in adult pulmonary and critical care patients. Am J Respir Crit Care Med Jan;183(1): Bodro M, Paterson DL. Has the time come for routine trimethoprim-sulfamethoxazole prophylaxis in patients taking biologic therapies? Clinical Infectious Diseases Jun;56(11):

21 21 Tables and Figures Table 1. Patient characteristics, treatment regimen, infectious complications and efficacy (Table included below) Legend: PV pemphigus vulgaris, PF pemphigus foliaceus, BP bullous pemphigoid, ext extremities, TMP-SMX trimethoprim sulfamethoxazole, PNA pneumonia, UTI urinary tract infection, URI upper respiratory tract infection, NR no response, PR partial response, CliR- clinical response, CR complete response. Rituximab was infused weekly, IVIG was infused very 4 to 6 weeks,

22 Table 1. Patient characteristics, treatment regimen, infectious complications and efficacy Patient Age/Sex Dx Clinical Features Co Morbities Prior Disease Duration (mo.) Rituximab Protocol No. of Courses Cumulative Dose of Rituximab (mg) Concurrent Therapy Antibiotic Prophylaxis Infectious Complications Response Patient 1 74 F BP Face, trunk, ext., conjunctival and oral mucosa DM, COPD mg/m2 x 4 cycles MMF TMP SMX PR, relapse Patient 2 41 M PV Trunk, ext. and penis, desqumation of the scalp mg/m2 x 4 cycles Pred (high), AZA TMP SMX CR Patient 3 47 F PV Scalp, trunk and ext., vaginal and anal mucosae. Uknown mg/m2 x 10 cycles + IVIG X 18 cycles MTX CliR, relapse mg/m2 x 4 cycles + IVIG x 17 cycles MMF, Pred (mod) Atovaquone CR, relapse Patient 4 68 F BP Diffuse invovlement (60% BSA) DM 30 Unknown 2 Uknown Tet/NAD, MMF, Pred 1. Bacteremia, death NR Patient 5 36 F PF Face and scalp mg/m2 x 4 cycles MMF 1. URI CR, relapse Patient 6 75 M PV Oral, pharyngeal, laryngeal mucosa DM mg/m2 x 4 cycles Pred (low) TMP SMX CliR Patient 7 80 F PF Scalp invovlement mg/m2 x 4 cycles MTX 1. UTI, 2. UTI NR mg/m2 x 4 cycles MTX 3. UTI, 4. Bactermis, 5. UTI NR Patient 8 71 M PV Oral mucosa mg x 4 cycles + IVIG x 2 cycles MMF, Pred (low) Atovaquone 1. Sinusitis, 2. SSTI, 3. Infx Colitis CliR, relapse mg/m2 x 4 cycles MMF, Pred (low) Atovaquone 4. URI CliR Patient 9 40 F PV Oral and vaginal mucosae mg/m2 x 4 cycles MMF, Pred (low) TMP SMX CR Patient F PV Oral and vaginal mucosae mg/m2 x 4 cycles Pred (low) TMP SMX CR Patient M PV Oral, pharyngeal and larygneal mucosa mg/m2 x 4 cycles + IVIG x 1 cycle MMF, Pred (mod) TMP SMX 1. Infx pericarditits CR, relapse mg/m2 x 4 cycles MMF, Pred (low) TMP SMX NR Patient F PV Scalp, trunk, ext., buttocks, oral and vaginal mucosa DM mg/m2 x 3 cycles + IVIG x 6 cycles Pred (low) TMP SMX 1. PNA CR, relapse Patient M PF Face mg/m2 x 4 cycles MMF, Pred (low) TMP SMX CR, relapse mg/m2 x 4 cycles 1. UTI CR Patient F PV Trunk, exremities, anal mucosa mg/m2 x 4 cycles MMF, Pred (mod) TMP SMX CR Patient F PV Ocular, oral, vaginal, anal mucosae COPD mg/m2 x 4 cycles MMF, Pred (low) TMP SMX CliR, relapse mg/m2 x 4 cycles MMF, Pred (low) TMP SMX 1. URI 2. URI CR Patient M PV Oral mucosa mg/m2 x 4 cycles MMF, Pred (mod) TMP SMX PR mg/m2 x 4 cycles MMF, Pred (low) TMP SMX PR Patient M BP Trunk and ext. with widespread milia mg/m2 x 4 cycles MMF, Pred (mod) TMP SMX 1. Bronchitis CliR, relapse mg/m2 x 4 cycles MMF, Pred (mod) TMP SMX CliR Patient M PV Scalp, nasal and oral mucosal DM mg/m2 x 4 cycles MMF, Pred (low) Dapsone CliR mg/m2 x 4 cycles MMF, Pred (low) Dapsone CR Patient M PV Oral, pharyngeal and larygneal mucosa DM mg/m2 x 4 cycles Pred (low) Atovaquone CR Patient20 19 F PV Vaginal mucosa, trunk and ext mg/m2 x 4 cycles MMF, Pred (low) TMP SMX NR mg/m2 x 4 cycles MMF, Pred (low) TMP SMX CR Patient M MMP Oral, pharyngeal and larygneal mucosa mg x 2 cycles Pred (mod) TMP SMX CliR Patient M PV Scalp, trunk, ext., oral and penile mucsoa mg/m2 x 4 cycles MMF, Pred (low) CR Patient F PV Sclap, oral mucos, vulvar mucosa mg x 2 cycles + IVIG x 9 cycles MMF, Pred (low) TMP SMX NR mg x 2 cycles + IVIG x 1 cycles TMP SMX PR mg x 2 cycles + IVIG x 4 cycles TMP SMX CliR mg x 2 cycles + IVIG x 5 cycles TMP SMX CliR relapse mg x 2 cycles + IVIG x 2 cycles MMF, Pred (low) TMP SMX CliR, relapse Patient F PF Trunk, ext. and scalp mg x 2 cycles + IVIG x 6 cycles mg x 2 cycles MMF, Pred (low) TMP SMX CR Patient F PV Scalp, conjunctival and oral mucosa mg/m2 x 4 cycles MMF, Predisone (low) Atovaquone CR Patient F PV Oral and nasal mucosa mg x 2 cycles Pred (low) CR, relapse mg x 7 cycles Pred (low) Patient M PV Oral mucosa mg x 2 cycles + IVIG x 5 cycles mg x 1 cycle MMF TMP SMX PR Patient F PV Trunk and ext mg x 2 cycles + IVIG x 14 cycles Pred (low) CR Patient M PV Diffuse invovlement (60% BSA) mg/m2 x 2 cycles 1 Uknown MMF, Pred (mod) TMP SMX 1. Bacteremia, death NR Patient F PV Oral and pharyngeal mucosa mg/m2 x 2 cycles + IVIG x 2 cycles Pred (low) NR

23 22 Table 2. Summary of patient characteristics, treatment regimen, infectious complications and efficacy Characteristics Patients (n = 30) Male (no.) 13 Female (no.) 15 Age (yrs.) Mean Range Diagnosis (no.) PV BP PF 21 (70%) 5 (17%) 4 (13%) Disease duration prior to rituximab (mo.) Mean Range Concurrent Therapy Pred MMF MTX AZA (83%) 20 (67%) 2 (7%) 1 (3%) Concurrent IVIG (no.) 9 (30%) Antibiotic Prophylaxis TMP-SMX Atovaquone Dapsone 22 (77%) Infectious complications, at least one (no.) 10 (30%) Response to rituximab (no.) NR PR CliR CR (17%) 3 (10%) 5 (17%)* 17 (57%)** Legend: PV pemphigus vulgaris, PF pemphigus foliaceus, BP bullous pemphigoid, TMP-SMX trimethoprim sulfamethoxazole, Pred Prednisone, MMF Mycophenolate Mofetil, MTX Methotrexate, AZA Azathioprine, NR no response, PR partial response, CliR- clinical response, CR complete response. *Four patients who achieved CR subsequently relapsed, **1 patient who achieved CliR subsequently relapsed.

24 23 Table 3. Comparison of patients who received antibiotic prophylaxis to those who did not Characteristics Antibiotic Prophylaxis (n = 22) No Antibiotics (n = 9) Male (no.) 11 (50%) 11 (50%) Female (no.) 3 (33%) 5 (67%) Age (yrs.) Mean Range Diagnosis (no.) PV 18 (82%) 4 (44%) BP 3 (14%) 2 (22%) PF 1 (5%) 3 (33%) Disease duration prior to rituximab (mo.) Mean Range Comorbidities (no.) DM 5 (23%) 2 (22%) COPD 2 (9%) 0 Concurrent Therapy (no.) Pred 20 (91%) 6 (67%) MMF 16 (73%) 4 (44%) MTX 1 (5%) 0 AZA 1 (5%) 1 (11%) Legend: PV pemphigus vulgaris, PF pemphigus foliaceus, BP bullous pemphigoid, Pred Prednisone, MMF Mycophenolate Mofetil, MTX Methotrexate, AZA Azathioprine.

25 24 Table 4. Rate and Spectrum of Infectious Complications of Rituximab Treatment Patient Treatment Course Antibiotic Prophylaxis Infectious Complications (Pathogen) Grade of Infection Time to Infection (days) Patient 4 1 None Bacteremia/sepsis leading to death (Pseudomonas) Severe 8 Patient 5 1 None URI Mild 206 Patient 7 1 None UTI Mild 144 UTI (Proteus) Mild None UTI (Enterococcus) Mild 187 Bactermia/urosepsis(E. Coli) Severe 218 UTI Mild 324 Patient 8 1 Atovaquone Sinusitis Mild 185 Paronychia (Pseudomonas) Mild 284 Infectious Colitis Moderate Atovaquone URI Mild 78 Patient 11 1 TMP-SMX Infectious pericarditits Moderate TMP-SMX None - - Patient 12 1 TMP-SMX PNA Moderate 5 Patient 13 1 TMP-SMX None None UTI Mild 202 Patient 15 1 TMP-SMX None TMP-SMX URI Mild 95 URI Mild 217 Patient 17 1 TMP-SMX Bronchitis Mild 97 2 TMP-SMX None - - Patient 29 1 TMP-SMX Bacteremia/ sepsis leading to death (MRSA) Severe 12 Legend: TMP-SMX trimethoprim sulfamethoxazole, PNA pneumonia, UTI urinary tract infection, URI upper respiratory tract infection.