Rheumatology

Rheumatol Int (2015) 35:459–463 DOI 10.1007/s00296-014-3157-1

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Human papillomavirus and chlamydia trachomatis infections in rheumatoid arthritis under anti-TNF therapy: an observational study Mariana G. Waisberg · Ana C. M. Ribeiro · Wellington M. Candido · Poliana B. Medeiros · Cezar N. Matsuzaki · Mariana C. Beldi · Maricy Tacla · Helio H. Caiaffa-Filho · Eloísa Bonfa · Clovis A. Silva 

Received: 22 June 2014 / Accepted: 8 October 2014 / Published online: 28 October 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  The objective of this study was to evaluate human papillomavirus (HPV) and Chlamydia trachomatis (CT) infections in RA patients pre- and post-TNF blocker. Fifty female RA patients (ACR criteria), who were eligible to anti-TNF therapy [n  = 50 at baseline (BL) and n  = 45 after 6 months of treatment (6 M)], and 50 age-matched healthy controls were prospectively enrolled. They were assessed for demographic data, gynecologic, sexual, cervical cytology and histological evaluations, disease parameters and current treatment. HPV DNA and CT DNA testing in cervical specimens were done using Hybrid Capture II assays. At BL, the median current age of RA patients and controls was 49 (18–74) versus 49 (18–74) years, p = 1.0. A trend of lower frequency of HPV infection was observed in AR patients pre-anti-TNF compared with controls (14 vs.

Eloísa Bonfa and Clovis A. Silva have contributed equally to this work. M. G. Waisberg · A. C. M. Ribeiro · W. M. Candido · P. B. Medeiros · E. Bonfa · C. A. Silva (*)  Disciplina de Reumatologia, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Arnaldo, nº 455, 3º andar, sala 3190 - Cerqueira César, São Paulo, SP 05403-010, Brazil e-mail: [email protected] e-mail: [email protected] C. N. Matsuzaki · M. C. Beldi · M. Tacla  Gynecology Department, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil H. H. Caiaffa-Filho  Laboratory Division, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil C. A. Silva  Pediatric Rheumatology Unit, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil

30 %, p  = 0.054). Further evaluation of AR patients with and without HPV infection before anti-TNF therapy showed that the former group had higher frequency of sexual intercourses (100 vs. 48 %, p  = 0.014), higher median number of sexual partners [1 (1–1) vs. 0 (0–1), p = 0.032] and higher frequency of abnormal cervical cytology (43 vs. 7 %, p = 0.029). Current age, disease duration, disease parameters and treatments were alike in both groups (p > 0.05). At 6 M after TNF blockage, HPV infection remained unchanged in five patients, whereas two became negative and one additional patient turned out to be positive (p = 1.0). CT infection was uniformly negative in RA patients pre- and postTNF blockage and in controls. Anti-TNF does not seem to increase short-term risk of exacerbation and/or progression of HPV and CT infections in RA patients. Keywords  HPV infection · Chlamydia trachomatis · Rheumatoid arthritis · TNF blockers

Introduction Cervical human papillomavirus (HPV) infection has been observed in 28 % of rheumatoid arthritis (RA) patients in a cross-sectional study [1] with no available data regarding Chlamydia trachomatis (CT) infection. We hypothesized that anti-TNF blockage may increase the risk of exacerbation and/or progression of these gynecologic complications reported in chronic inflammatory arthritis [2] and psoriasis [3] under this therapy. Therefore, the purpose of our study was to evaluate HPV and CT infections in RA patients pre-TNF and 6 months post-TNF blocker. The possible associations among these infections, demographic data, sexual function, disease parameters and treatment were also analyzed.

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Methods Study design, setting and participants This is an observational study in a cohort of 50 female RA patients that fulfilled the disease classification criteria [4] and who were eligible to anti-TNF therapy. They were prospectively enrolled [n = 50 at baseline (BL) and n = 45 after 6 months treatment (6 M)] and were followed up at our University Hospital. Infliximab was administered in 36 RA patients (3 mg/kg body weight at 2 and 6 weeks and thereafter as recommended, every 6–8 weeks), etanercept in 11 (50 mg weekly) and adalimumab in 3 (40 mg every 2 weeks). The control group involved 50 healthy age-matched subjects followed routinely at the Primary Care Unit. All RA patients and controls were older than 18 years and had previous sexual activity. Exclusion criteria were as follows: current pregnancy, early postpartum period, diabetes mellitus, psychiatric diseases and cervical cancer. The protocol was approved by the local institutional review board, and all participants signed the informed consent. Variables Outcomes Determination of HPV and CT infections HPV DNA testing was performed using Hybrid Capture 2 (HC2 highrisk; Digene Corporation, currently QIAGEN, Gaithersburg, MD, USA) by DNA of oncogenic group of probes. These probes are designated to detect HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. CT DNA testing was performed using Hybrid Capture 2 (HC2 CT-ID DNA; Digene Corporation, currently QIAGEN, Gaithersburg, MD, USA). These HPV and CT tests were performed following strictly the manufacturer’s instructions, and the results were presented as the ratio of the relative light units, which was considered positive when a relative light unit was greater than 1.0. Cervical cytology and biopsy All RA patients and controls underwent colposcopy and cervical cytology. The Pap smears were collected with Cytobrush® and spatula Ayre, after insertion of the speculum [5]. The cervix was visualized, and the spatula Ayre was inserted in the ostium and rotated 360º under gentle pressure, after inserting the Cytobrush® for two-thirds in the endocervical canal and rotated 90°–180°. The material of the Cytobrush® was rolled over the outer third of the slide, and the material on the spatula Ayre was spread on a thin layer in one movement over the middle third of the slide. After the fixation by immersion in 95 % ethanol, the specimens were immediately transported to the laboratory. All Pap smears were

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evaluated by the same cytopathologist blinded to gynecology examination in our University Hospital. They were performed according to the Bethesda classification system in: (1) negative for intraepithelial lesions of malignancy with or without organisms (Trichomonas vaginalis, Candida spp., bacterial vaginosis, Actinomyces spp., herpes simplex virus), (2) other non-neoplastic findings (including inflammatory changes) and (3) epithelial cell abnormalities atypical [atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LGSIL) encompassing HPV/mild dysplasia/cervical intraepithelial neoplasia (CIN) 1, high-grade squamous intraepithelial lesion (HSIL) encompassing moderate and severe dysplasia, carcinoma in situ, CIN 1 and CIN 2] [6]. Colposcopically guided biopsies were obtained of the most severe visible lesions, and specimen slides were reviewed by a pathologist who had no knowledge of the other clinical or laboratory data [7]. Predictors Sexual function and gynecological exam Evaluation of the sexual function was based on a structured questionnaire and included: menarche age, age of the first sexual activity, number of sexual intercourse in the last month, number of sexual intercourse in the last year, number of sex partners in the last month, number of sex partners in the last year, presence of menopause and age of menopause [5]. The same gynecologist performed the clinical examination of the genitalia blinded to HPV and CT evaluations, and it included assessment of the vulva, hymen, vagina and uterine cervix. Disease parameters and treatment All patients were evaluated by physician global assessment of disease activity and patient global assessment of disease activity (10 cm visual analog scale), disease activity score of 28 joints (DAS 28) [8, 9], erythrocyte sedimentation rate (ESR) by modified Westergren method, C-reactive protein (CRP) by nephelometric method and health assessment questionnaire (HAQ) [10]. IgM rheumatoid factor was measured by latex and Waaler-Rose methods at baseline. Data on treatment were also recorded. Statistical analysis This is an exploratory study with a convenient sample. Results were presented as median (range) or mean ± standard deviation (SD) for continuous parameters and number (%) for categorical variables. Data were compared by Mann–Whitney test in continuous variables to evaluate differences between RA patients with and without HPV infections. Fisher’s exact test was used to analyze categorical parameters. McNemar’s test was used to evaluate differences between HPV infection before and after 6 months of TNF blockage. In all the statistical tests, the level of significance was set at 5 % (p 1.0

7 (14)

15 (30)

CT infection >1.0

0 (0)

0 (0)

Cytology Abnormal HSIL LSIL ASCUS

6 (12) 1/6 (17) 2/6 (33) 3/6 (50)

15 (30) 8/15 (53) 6/15 (40) 1/15 (7)

1.0 0.048 0.18 1.0 0.053

Sexually transmitted diseases Candida sp.

2 (4)

1 (2)

1.0

Trichomonas vaginallis

1 (2)

0 (0)

1.0

Histological findings Chronic cervicitis CIN 1 CIN 2

5/5 (100) 0/5 (0) 0/5 (0)

CIN 3

0/5 (0)

10/17 (59) 3/17 (18) 1/17 (5)

0.14 1.0 1.0

3/17 (18)

1.0

The results are presented in n (%) and median (range),epithelial cell abnormalities atypical [atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LGSIL) encompassing HPV/mild dysplasia/cervical intraepithelial neoplasia (CIN) 1, high-grade squamous intraepithelial lesion (HSIL) encompassing moderate and severe dysplasia, carcinoma in situ, CIN 1, CIN 2 and CIN 3]

Results At BL, the median current age of RA patients and controls was 49 (18–74) versus 49 (18–74) years, p = 1.0 with comparable sexual function (p > 0.05). A trend of lower frequency of HPV infection was observed in RA patients pre-anti-TNF (14 vs. 30 %, p  = 0.054) and of abnormal

cervical cytology (12 vs. 30 %; p = 0.048) compared with controls (Table 1). None of patients had genital warts. Further evaluation of AR patients with and without HPV infection before anti-TNF therapy (BL) showed that the former group had a high frequency of sexual intercourses in the last months (100 vs. 48 %, p  = 0.014), a higher median number of sexual intercourses and sexual partners in the last month [3 (1–5) vs. 0 (0–19), p = 0.057 and 1 (1–1) vs. 0 (0–1), p  = 0.032, respectively] as well as a higher frequency of abnormal cervical cytology (43 vs. 7 %, p = 0.029). All other sexual function parameters were alike (p > 0.05). In these patients, the median current age, disease duration, physician and patient visual analog scales, DAS 28, ESR and CRP were alike in both groups (p > 0.05). No differences were evidenced in the frequencies and/or median of current dose of prednisone, methotrexate, leflunomide, chloroquine, azathioprine, sulfasalazine and cyclosporin in both groups (p > 0.05) (Table 2). At 6 M after TNF blockage, HPV infection remained unchanged in five patients, whereas two became negative and one additional patient turn out to be positive (McNemar’s test, p  = 1.0). None of them had cervical cancer in Pap smear. CT infection was uniformly negative in RA patients preand post-TNF blockage and in controls.

Discussion To our knowledge, this was the first study to assess prospectively HPV and CT infections in RA patients under TNF blockage therapy, and it clearly demonstrated no short-term risk of exacerbation and/or progression of these genital infections. The strength of our study was the systematical evaluation of HPV and CT infections in AR patients refractory to DMARDs. The inclusion of sexually active age-matched healthy control group is important because age is a risk factor of persistent HPV [5, 11] and CT infections [12]. In addition, we used a routine HPV test with high sensitivity and high specificity, and it is gold standard to clinical practice diagnosis [13]. Data regarding the prospective incidence of HPV and CT infections are lacking in the literature preventing an accurate estimate of the likelihood of getting infected. We cannot exclude therefore that the present study is underpowered to determine the incidence of these two conditions in the short period of assessment after TNF blockage therapy. In addition, the non-identification of viral types and subtypes, particularly oncogenic ones, precludes a definitive conclusion regarding cancer risk. HPV infection identified was subclinical, mild and associated with cervical dysplasia in RA patients. An increased

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Table 2  Demographic data, sexual function, disease parameters and treatment in rheumatoid arthritis (RA) patients with and without cervical human papillomavirus (HPV) before anti-TNF therapy Variables Demographic data Current age (years) Disease duration (years)

HPV+ (n = 7) HPV− (n = 43) p

49 (18–74) 11 (3–56)

0.24 0.59

13 (11–15) 19 (15–26)

13 (10–19) 18 (12–30)

0.51 0.20

7 (100)

21 (48)

0.014

3 (1–5)

0 (0–19)

0.057

1 (1–1)

0 (0–1)

0.032

1 (1–1)

1 (0–2)

0.05

3 (43) 45 (35–47)

26 (60) 47 (27–58)

0.43 0.39

3 (43) 1 (14) 1 (14) 1 (14)

3 (7) 0 (0) 1 (2) 2 (4)

0.029 0.14 0.26 0.37

Sexually transmitted diseases 0 (0) Candida sp.

2 (4)

1.0

Trichomonas vaginallis

0 (0)

1 (2)

1.0

Histological findings Chronic cervicitis CIN 1 CIN 2 CIN 3 CA in situ

1 (14) 0 (0) 0 (0) 0 (0) 0 (0)

4 (9) 0 (0) 0 (0) 0 (0) 0 (0)

0.55 1.0 1.0 1.0 1.0

Disease parameters Physician VAS Patient VAS DAS 28 ESR (mm/1st hour) CRP (mg/dL) HAQ Rheumatoid factor+

5.4 (0.1–6.7) 5.5 (0.1–8) 5.2 (2.7–6.7) 31 (2–61) 3 (1.2–70) 1.0 (0–1.75) 6 (86)

5.9 (0.5–10) 6.3 (0–10) 5.3 (2.7–8.0) 20 (4–124) 5.8 (0.1–84.2) 1.38 (0–2.75) 31 (72)

0.25 0.25 0.57 0.58 0.13 0.21 0.66

Treatment Prednisone Current dose (mg/day) Methotrexate

7 (100) 10 (5–20) 5 (71)

41 (95) 10 (5–20) 28 (65)

1.0 0.8 1.0

current dose (mg/week) Leflunomide Chloroquine Azathioprine Sulfasalazine

25 (15–25) 5 (71) 2 (29) 1 (14) 2 (29)

25 (12.5–25) 30 (70) 23 (53) 3 (7) 6 (14)

0.51 1.0 0.42 0.46 0.31

Cytology Abnormal HSIL LSIL ASCUS

13

Variables

HPV+ (n = 7) HPV− (n = 43) p

Cyclosporin

0 (0)

2 (5)

1.0

The results are presented in n (%) and median (range) 43 (28–66) 16 (1–26)

Sexual function Menarche age (years) First sexual activity age (years) Sexual intercourses in last month Sexual intercourses in last month, number Sexual partners in last month, number Sexual partners in last year, number Menopause Menopause age (years)

Table 2  continued

risk of cervical dysplasia and persistent HPV infection was also observed after stem cell, lung and kidney transplantation [11] and in systemic lupus erythematosus patients [14, 15]. The prevalence of HPV was, however, lower than the reported in a recent Mexican study [12], and the most likely explanation for this discrepant finding is the higher current age of patients evaluated herein. Additionally, a very low frequency of HPV was also observed in Sjögren syndrome, a disease affecting more frequently older adults [16]. The most important risk factors associated with HPV infection was the number of lifetime sexual partners and number of sexual intercourses, as previously described [17, 18]. TNF-∝ appears to be critical for protection against infection, and TNF-∝ blockage may increase the risk of virus infection reactivation [11]. In fact, HPV lesions of the genital and perianal areas were reported in the first months of anti-TNF agent use [2, 3]. This finding contrasts with the uniform absence of condyloma acuminatum in our patients treated with TNF blockage. CT infection was also not observed, and older age of AR patients may account for this result. Indeed, CT infection remains epidemic among sexually active young women [12]. In conclusion, anti-TNF does not seem to increase shortterm risk of exacerbation and/or progression genital infections in RA patients. Acknowledgments  This study was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP#2009/51897-5 to EB 11/12471-2 to C.A.S.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ #301411/2009-3 to E.B. and #302724/2011-7 to C.A.S.), Federico Foundation (to E.B. and C.A.S.) and Núcleo de Apoio à Pesquisa “Saúde da Criança e do Adolescente” da USP (NAP-CriAd-SP) to C.A.S. We thank Elaine P. Leon and Vilma T. Viana for their technical support and Dr. Ulysses Doria Filho for the statistical analysis.

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Rheumatol Int (2015) 35:459–463 3. Georgala S, Katoulis AC, Kanelleas A, Befon A, Georgala C (2012) Human papilloma virus and molluscum contagiosum lesions related to infliximab therapy for psoriasis: a case series. Dermatol Online J 18:9 4. Arnett FC, Edworthy SM, Bloch DA et al (1998) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–324 5. Febronio MV, Pereira RM, Bonfa E, Takiuti AD, Pereyra EA, Silva CA (2007) Inflammatory cervicovaginal cytology is associated with disease activity in juvenile systemic lupus erythematosus. Lupus 16:430–435 6. Solomon D, Davey D, Kurman R et al (2002) The 2001 Bethesda system: terminology for reporting results of cervical cytology. JAMA 287:2114–2119 7. Beldi MC, Tacla M, Caiaffa-Filho H et al (2012) Implementing human papillomavirus testing in a public health hospital: challenges and opportunities. Acta Cytol 56:160–165 8. van der Heijde DM, van ‘t Hof MA, van Riel PL et al (1990) Judging disease activity in clinical practice in rheumatoid arthritis: first step in the development of a disease activity score. Ann Rheum Dis 49:916–920 9. Prevoo ML, van’t Hof MA, Kuper HH et al (1995) Modified disease activity scores that include twenty-eight joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 38:44–48 10. da Mota Falcao D, Ciconelli RM, Ferraz MB (2003) Translation and cultural adaptation of quality of life questionnaires: an evaluation of methodology. J Rheumatol 30:379–385

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Human papillomavirus and chlamydia trachomatis infections in rheumatoid arthritis under anti-TNF therapy: an observational study.

The objective of this study was to evaluate human papillomavirus (HPV) and Chlamydia trachomatis (CT) infections in RA patients pre- and post-TNF bloc...
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