Arch Gynecol Obstet DOI 10.1007/s00404-014-3514-z

GENERAL GYNECOLOGY

Genital tract infection with Chlamydia trachomatis in women attended at a cervical cancer screening program in Northeastern from Brazil ´ rika Galva˜o Lima • Paulo Andre´ Freire Magalha˜es • Cleine Aglacy Nunes Miranda • E • • Raı´za Nara Cunha Moize´is Diego Breno Soares de Lima Ricardo Ney Oliveira Cobucci • Thales Allyrio Arau´jo de Medeiros Fernandes • Jenner Chrystian Verı´ssimo de Azevedo • Paulo Roberto Medeiros de Azevedo • Jose´lio Maria Galva˜o de Arau´jo • Jose´ Verı´ssimo Fernandes Received: 27 May 2014 / Accepted: 7 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose This cross-sectional study aimed to estimate the prevalence of Chlamydia trachomatis (CT) infection alone and in combination with human papillomavirus (HPV). Furthermore, the study investigates whether the CT infection increases the risk of contracting HPV and whether the presence of both pathogens is associated with a higher prevalence of cervical lesions. Methods Cervical samples of 1,134 asymptomatic women enrolled in a screening program for cervical cancer were analyzed. Two cervical specimens were collected from each patient, one for cytologic examination and the P. A. F. Magalha˜es  C. A. N. Miranda  E´. G. Lima  J. M. G. de Arau´jo  J. V. Fernandes Post-Graduate Program in Biological Sciences, Federal University of Rio Grande do Norte, Natal, RN, Brazil

other for detection of CT by polymerase chain reaction (PCR), using a primer pair which amplifies a specific sequence of the DNA plasmid. Results The overall prevalence rate infection was 10.9 %, being 10 % in the women with normal cytology, 13.8 % in those with atypical squamous cells of undetermined significance (ASC-US), and 25 % with low-grade squamous intraepithelial lesion (LSIL). The infection by CT did not increase the risk of acquiring HPV infection. The higher prevalence of LSIL in women co-infected with HPV and CT is possibly due to HPV. Conclusion CT infection was more prevalent in younger women aged up to 32 years, who had an early onset of reproductive activity and a history of having had multiple sexual partners lifelong may be at a greater risk of acquiring infection of the genital tract by C. trachomatis.

R. N. C. Moize´is  D. B. S. de Lima  J. M. G. de Arau´jo  J. V. Fernandes (&) Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Campus Universita´rio, Natal, RN 59072-910, Brazil e-mail: [email protected]; [email protected]

Keywords Chlamydia trachomatis  Human papillomavirus  Risk factors  Genital tract infection

R. N. O. Cobucci Post-Graduate Program in Health Sciences, Federal University of Rio Grande do Norte, Natal, RN, Brazil

Chlamydia trachomatis (CT) is an obligate intracellular gram-negative bacterium found only in humans, as tropism for squamous epithelial cells of the cervical mucosa [1]. During its developmental cycle, two different forms are observed: elementary bodies (EBs), which are infectious but not able to divide; and reticulate bodies (RBs), which are metabolically active and able to multiply. Persistent forms can be present under particular conditions. Currently, CT is divided into 19 serovars, according to the specificity of major outer membrane protein (MOMP) epitopes, being the serovars D-K, the most common sexually transmitted bacteria [1].

T. A. A. de Medeiros Fernandes Department of Biomedical Sciences, University of Rio Grande do Norte State, Mossoro´, RN, Brazil J. C. V. de Azevedo Onofre Lopes Hospital, Federal University of Rio Grande do Norte, Natal, RN, Brazil P. R. M. de Azevedo Department of Statistics, Federal University of Rio Grande do Norte, Natal, RN, Brazil

Introduction

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Clinical and epidemiological studies reveal that CT is one of the most common causes of sexually transmitted diseases among the human populations worldwide, representing a serious global public health problem, especially among young women. The world health organization (WHO) estimates that more than 100 million new cases occur annually around the world [2]. Most of the infections are asymptomatic, but some women complain of mucopurulent vaginal discharge or post-coital bleeding [3]. In addition, such infections could cause pain during sexual intercourse, reducing sexual satisfaction and sexual quality of life in young sexually active women [4]. The most common clinical manifestations are urethritis, cervicitis, and endometritis. When bacteria ascend to the upper reproductive tract, this may result in salpingitis and pelvic inflammatory disease. Salpingitis may lead to tubal scarring, ectopic pregnancy with severe reproductive complications, and pelvic inflammatory disease, which can lead to infertility [1, 5]. In pregnant women, CT infection has been associated with an increased risk of ectopic pregnancy, spontaneous abortions, preterm delivery, low birth weight, premature rupture of membranes, and perinatal mortality [5]. The prevalence of infection with CT varies according to the characteristics of the population studied, as well as the detection method used. Prevalence rates varying from 2.9 to 14.9 % have been reported in South America [6–8]. In Brazil, studies conducted by different methodologies, involving groups of women treated by gynecology services, family planning programs, and prenatal clinics showed prevalence rates varying from 2.1 to 31.5 % for genital infection by CT [9–11]. Furthermore, research has demonstrated that the bacteria are circulating even among the indigenous populations of the Brazilian Amazon [12]. It has been shown that CT, mainly serotypes D-K, may induce cervical hypertrophy with inflammation, and squamous metaplasia, leading to the hypothesis that this bacteria can act as a cofactor in cervical HPV-related carcinogenesis. Chlamydial infection causes cervicitis, an infection of the endocervix cells of the transformation zone and this inflammation may predispose women to HPV infection by damaging epithelial integrity. Several epidemiological studies have reported a positive association involving this bacterium and cervical intraepithelial neoplasia of high-grade and/or cervical cancer [13, 14]. Several conditions including age, educational level, ethnicity, age at first sexual intercourse, number of sexual partners, oral contraceptive use, and smoking has been reported as possible risk factors for genital infections by CT [15, 16]. This study aimed to estimate the prevalence of CT infection alone and in combination with Human papillomavirus (HPV). Furthermore, the study investigates whether the CT infection increases the risk of contracting

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HPV and whether the presence of both pathogens is associated with a higher prevalence of cervical lesions.

Methods Selection of participants and sample collection This cross-sectional study included 1,134 sexually active women enrolled in a screening program for cervical cancer in municipalities of the different regions in Rio Grande do Norte State, Northeastern Brazil, during the period 2008–2012. The screening program of cervical cancer was chosen as the most viable way to recruit asymptomatic women for the study. In Brazil, the screening program for cervical cancer is accessible to the entire female population and establishes that all women aged between 25 and 60 years should perform the cytological examination annually or every 3 years after two consecutive negative test results. During the gynecological examination, the patients were questioned about the presence of common symptoms of chlamydia infection, such as abdominal or pelvic pain. Moreover, signs of cervicitis were surveyed and the bimanual exam was performed to diagnose cervical motion or adnexal tenderness. Women who did not present any of these signs and symptoms were considered asymptomatic. The inclusion criteria were as follows: agreeing to participate in the study and answer a standardized epidemiological questionnaire, in addition to agreeing to submit to a uterine cervix scraping to obtain a sample for cytopathological and molecular analysis. Excluded were pregnant women, those who had had an abortion or delivery less than 60 days before the collection, those who had undergone hysterectomy, and those with mental deficit that would compromise the understanding and/or the responses of the questionnaire. The women were informed about the methods and objectives of the research and those who agreed to participate signed an informed consent form and answered a standardized questionnaire about their marital status, ethnicity, sexual behavior, and reproductive activities. The patients’ ethnicity was defined based on self-reports according to the criterion of the Instituto Brasileiro de Geografia e Estatı´stica (IBGE), which classifies ethnicity into five categories: white, black, mulatto, Asian, and native. In this study, the black, mulatto, Asian, and native categories were combined into a nonwhite category. The study was approved by the Ethical Committee in Research of the Federal University of Rio Grande do Norte. Two cervical specimens containing exfoliated cells of the uterine cervix were collected from each patient using a cervical brush. One of these specimens was used to obtain a

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scraping, which was stained by the Papanicolaou method and analyzed by cytopathological examination, based on the Bethesda system. The other was conditioned in a tube containing a preserving solution (PBS ? vancomycin ? nystatin) and sent to a laboratory, where it was processed for DNA extraction and analyzed for the presence HPV and CT. DNA extraction and C. trachomatis and HPV detection The tubes containing the samples were subjected to vigorous agitation and the brush was removed. Then, the content was centrifuged at 3,000g for 10 min and the supernatant was discarded. The remaining pellet was processed for DNA extraction, using a protocol rapid isolation of DNA of mammals, with proteinase K [17]. To check the quality of the obtained DNA, aliquots with about 30 ng of DNA samples were submitted to PCR analysis with specific b-globin primers [18]. In the same reaction, the presence of CT was analyzed using the primers CP24/CP27 [19], which amplify a specific sequence of a cryptic plasmid present in the bacterium. The products of PCR were submitted to electrophoresis on 8 % polyacrylamide gel, followed by silver staining [20]. A sample was considered positive for b-globin if it presented a band of 110 bp, and served as an internal control to evaluate the integrity and sufficiency of the DNA obtained. The patients were considered positive for CT if the sample analyzed presented a band of 207 pb. All positive samples for amplification of the segment of the human b-globin gene were checked for the presence or absence of HPV DNA using the generic primers GP5?/GP6? to amplify a fragment of the L1 viral gene [21]. The amplicons were submitted to electrophoresis on 8 % polyacrylamide gel, followed by silver staining [20]. The samples were considered positive for HPV if they presented a band with 140 pb. Each reaction tube for b-globin and CT contained 22.5 lL 109 Taq buffer, 50 mM MgCl2, 2 mM dNTP (Amershan Biosciences), 5 U/l Taq DNA Polymerase (Ivitrogen, Brazil), and 10 mM of primers PCO3/PCO4 ? and CP24/CP27. To this mixture was added 2.5 lL of DNA sample for a total volume of 25 lL of each reaction. The reaction conditions involving the amplification of both DNA segments consisted of the following: an initial step of 5 min at 95 °C for denaturation, followed by 35 cycles of 1 min at 95 °C for denaturation, 1 min at 58 °C for annealing, 1 min at 72° C for extension, and 10 min at 72° for final extension. In all reactions, samples known to be positive for both b-globin and CT were used as positive controls. Samples known to be negative for bacteria and a reaction containing only water and the mix served as negative controls.

Each reaction tube for HPV contained 22.5 lL 109 Taq buffer, 50 mM-KCl, 10 Mm-Tris–HCl pH 8.3, 200 lM of each dNTP (Amershan Biosciences), 3.5 Mm-MgCl2, 5 U/ l Taq DNA Polymerase (Ivitrogen, Brazil), and 50 pmol of each primer GP5?/GP6?. To this mixture was added 2.5 lL of DNA sample for a total volume of 25 lL of each reaction. The reaction conditions involving the amplification of HPV/DNA consisted of an initial step of 4 min at 95 °C for denaturation, followed by 40 cycles of 1 min at 95 °C for denaturation, 2 min at 50 °C for annealing, and 1 min at 72 °C for elongation, and the last cycle was extended by 4 min at 72 °C for final elongation. In all reactions, a sample of DNA from HeLa cells, known to be positive for HPV 18, was used as a positive control. A sample known to be negative for HPV and a reaction containing only water and the mix served as a negative control. The products of PCR were submitted to electrophoresis on 8 % polyacrylamide gel, followed by silver staining [16]. The patients were considered positive for HPV if they presented a band of 140 bp. Statistical analysis To verify the association between CT alone or in coinfection with HPV and occurrence of cervical changes, as well as to verify whether the considered variables could be considered risk factors for genital infection with CT, we used the v2 test, univariate and multivariate logistic regression analysis. All analyses were performed using the software SPSS, version 13.0 (SPSS, Chicago, IL, USA). p B 0.05 was considered statistically significant.

Results The epidemiological profile of the participants in this study was defined based on the analysis of the individual questionnaires and showed that the studied segment of the population was composed of 1,134 women aged 14–64 years, with a mean of 34.4 years. The majority were young women, up to 32 years of age and of nonwhite ethnicity, married or living in a stable relationship with her partner, having a low educational level, having used oral contraceptives, having only one sexual partner lifelong, having the first sexual intercourse and first pregnancy under 18 years of age, and having between one and two pregnancies. Of the total patients analyzed, 1,006 (88.7 %) had normal cytology, and of the 128 (11.3 %) with abnormal cytology, 80 (7.1 %) had atypical squamous cells of undetermined significance (ASC-US) and 48 (4.2 %) had squamous intraepithelial lesions of low grade (LSIL). The genital infection with CT was diagnosed in 124 patients,

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revealing an overall prevalence of 10.9 %. Of this total, 32 were also infected with HPV. The presence of HPV was detected in the samples of 324 patients, which corresponds to an overall prevalence rate of 28.6 %. Among the women with a positive HPV test, 229 (25.8 %) were infected with HPV alone and 32 (2.8 %) had simultaneous infection by HPV and CT. The prevalence of genital infection by CT and HPV, each one individually or both together, was analyzed in relation to the women´s health status based on the result of cytological examination. The CT infection was detected with prevalence rates of 10.0 % in the women with normal cytology, 13.8 % in those with ASC-US, and 25.0 % in those who had LSIL. The prevalence rate of this bacterium was significantly higher (p = 0.002) in the women with LSIL, compared with those who had normal cytology. HPV was detected with prevalence rates of 25.7 % in the women with normal cytology, 37.5 % in those who had ASC-US, and 72.9 % in the women with LSIL. The prevalence rate of HPV infection, was significantly higher (p = 0.022 and p \ 0.001) in women with ASC-US or LSIL, respectively, compared with those with normal cytology (Table 1). When analyzing the correlation between the presence of genital infection with CT and detection of HPV, it was found that the prevalence rates of HPV infection were 28.9 % in the women testing negative for CT and 25.8 % in those testing positive for the said bacterium, with no statistically significant difference between these two groups (OR = 0.86; CI 0.55–1.33). This shows that infection with CT did not significantly increase the risk of acquiring genital HPV infection (Table 2). We evaluated the presence of genital tract infection with HPV alone or in combination with CT and its correlation with the health status of the patients, related to the presence or absence of cervical abnormalities detected by cytological examination. The prevalence rates of HPV alone were 23.5 % in women with normal cytology, 32.5 % in those Table 1 Prevalence of C. trachomatis infection and by HPV, each alone stratified according to the women’s health status based on the cytological exam Health status

Normal cytology ASC-US LSIL Total

Total

C. trachomatis positive

HPV positive

N

%

p

N

%

p

1,006

101

10.0



259

25.7



80 48

11 12

13.8 25.0

0.390 0.002

1,134

124

10.9

Discussion

30 35 324

37.5 0.022 72.9 \0.001 28.6

ASC-US atypical squamous cells of undetermined significance, LSIL low-grade squamous intraepithelial lesion P value calculated by Chi-square test, considering significant value B0.05

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who had ASC-US, and 62.5 % in the women with LSIL. We found a value of p very close to statistical significance (p = 0.069) for the women with ASC-US and significant (p 0.001) for those with LSIL, when compared to women with normal cytology. The simultaneous infection by HPV and CT was detected with prevalence rates of 2.3 % in women with normal cytology, 5.0 % in those who had ASC-US, and 10.4 % in the women with LSIL, observing a significant difference only for women with LSIL (p = 0.001), compared with those who had normal cytology (Table 3). Also analyzed was the correlation between the presence of simultaneous infection by the both pathogens in the same sample and the status of the patient’s health, based on the result of cytological examination. We found an overall prevalence rate of co-infection of 2.8 %, being 2.3 % in the women with normal cytology, 5.0 % in those with ASCUS, and 10.4 % in the women with LSIL. These results show that the patients simultaneously infected with HPV and CT presented a higher incidence of LSIL, but not of ASC-US, taking as reference the women with normal cytology (Table 4). When we analyzed the correlation between the presence of CT genital infection and the variables considered, using univariate logistic regression analysis, we observed a positive association with chronologic age, ethnicity, education, age at first sexual intercourse, number of sexual partners, age at first pregnancy, and number of pregnancies. However, after multivariate logistic regression analysis, only the variables chronological age, number of sexual partners over lifetime, and age of first pregnancy were significantly associated with genital infection by CT. The higher rates of prevalence of CT infection were found in young women, up to 32 years of age, who had two or more sexual partners over their lifetime and who had become pregnant for the first time when less than 18 years of age. No significant association was observed between CT infection with ethnicity, education, marital status, oral contraceptive use, age of first sexual intercourse, and number of pregnancies (Table 5).

Currently, infection with CT is considered one of the most prevalent sexually transmitted infections, surpassing in number of cases syphilis and gonorrhea. The primarily asymptomatic course of infection with this bacterium in both, men and women, serves as a basis for forming new reservoirs. In this study, genital infection with CT was detected in 10.9 % of the women surveyed. This overall prevalence rate is similar to those reported in two studies conducted in

Arch Gynecol Obstet Table 2 Prevalence of genital infection by human papillomavirus in women with positive and negative test for C. trachomatis C. trachomatis

PCR for HPV N

Negative

HPV (-)

HPV (?)

1,010

718

292

28.9

1

Reference

124

92

32

25.8

0.86

0.55–1.33

1,134

810

324

28.6

Positive Total

%

OR

95 % CI

p

0.470

OR odds ratio, CI confidence interval Table 3 Prevalence of genital infection for HPV alone, or in combination with C. trachomatis, stratified according to the women’s health status based on the cytological exam Health status

Normal

Total

Positive HPV alone

Positive HPV ? C. trachomatis

N

N

%

p

1,006

236

23.5



80

26

32.5

0.069

62.5 \0.001

ASC-US LSIL

48

30

Total

1,134

292

25.7

%

p

23

2.3



4

5.0

0.134

5

10.4

0.001

32

2.8

ASC-US atypical squamous cells of undetermined significance, LSIL low-grade squamous intraepithelial lesion P value calculated by Chi-square test, considering significant value B0.05

Brazil, one in Rio Grande do Sul (12.6 %) [15] and the other in Minas Gerais (9.3 %) [22], in the south and southeast regions of the country. However, this is far below the prevalence rates found in the states of Amazonas (20.7 %) [10] and Pernambuco (24.7 %) [11], located in the northern and northeastern regions of Brazil. The prevalence found in the present study is slightly above those described for women of Venezuela (7.7 %) [6], the USA (6.7 %) [16], and Portugal (6.9 %) [23]. These variations in prevalence rates found in different studies conducted in Brazil can be explained by differences in the health status of participants, as well as characteristics of the populations studied, including socio-economic level, cultural factors, customs, and behavioral attitudes.

We found a prevalence rate of 10.0 % for genital infection with CT in women with normal cytology, similar to rates reported for women in Rio Grande do Sul and Minas Gerais [15, 22]. Contrary to what was reported by these two researchers, we found a significantly higher prevalence rate of genital infection with CT in women who had LSIL, but not in those who had ASC-US, when compared to women with normal cytology. However, this may have been due to concomitant infection by CT and HPV. Although some studies [23, 24] have reported a positive association between genital infection with CT and an increased risk of acquiring HPV infection, we did not find such correlation in the present study. When we analyzed the presence of cervical abnormalities in women with genital infection with CT or HPV, each one individually and the two together in the same patient, it was found that HPV infection alone showed to be associated with the occurrence of both ASC-US and LSIL. On the other hand, the simultaneous infection by both pathogens was associated only with the occurrence of LSIL. These results suggest that there is no synergistic action between CT and HPV in the development of low-grade cervical lesions. Our results are consistent with those reported in a study conducted in India [25], where no association was found between HPV and CT co-infection and the occurrence of cervical intraepithelial neoplasia (CIN). However, our results are, to some extent, discordant with those reported for women in Portugal, Brazil, and the Philippines, where evidence was found to support that CT could play a relevant role as a cofactor, acting together with HPV for

Table 4 Association between co-infection with C. trachomatis ? HPV and the occurrence of cervical changes detected by the cytologic examination Health status

C. trachomatis ? HPV Positive

Normal cytology ASC-US LSIL Total

%

Negative

%

OR

95 % CI

p

23

2.3

983

97.7

1

Reference



4

5.0

76

84.9

2.3

0.66–7.31

0.120

5 32

10.4 2.8

43 1,102

89.6 97.2

5.0

1.57–14.71

0.001

ASC-US atypical squamous cells of undetermined significance, LSIL low-grade squamous intraepithelial lesion, OR odds ratio, CI confidence interval

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Arch Gynecol Obstet Table 5 Prevalence of genital infection with C. trachomatis and distribution according to socio-demographic characteristics, sexual activity and reproductive

Variables

C. trachomatis

Univariate analysis

Multivariate analysis

Positive/negative

%

OR

Up to 32

513/65

11.2

2.45

1.23–5.03*

4.57

2.31–9.05*

33–45

316/16

4.8

0.98

0.42–2.31

1.42

0.68–2.98

C46

213/11

4.9

1.0

Reference

1.0

Reference

Nonwhite

656/70

9.6

1.89

1.12–3.20*

1.46

0.96–2.24

White

386/22

5.4

1.0

Reference

1.0

Reference

High

243/30

11.0

1.59

0.98–2.58*

1.37

0.88–2.12

Low

799/62

7.2

1.0

Reference

1.0

Reference

OR

95 % CI

95 % CI

Age (years)

Ethnicity

Education

Marital status Single

303/34

10.1

1.43

0.90–2.28

0.56

0.35–0.90

739/58

7.3

1.0

Reference

1.0

Reference

No

286/24

8.4

1.0

Reference

1.0

Reference

Yes

756/68

9.0

1.07

0.64–1.79

1.13

0.71–1.82

\18 years

596/64

9.7

1.71

1.06–2.78*

0.92

0.58–1.44

C18 years

446/28

5.9

1.0

Reference

1.0

Reference

2 or more

414/51

26.5

1.89

1.20–2.96*

1.72

1.15–2.56*

Only one

628/41

6.1

1.0

Reference

1.0

Reference

\18 years

334/42

11.3

2.33

1.06–5.28*

3.22

1.30–8.00*

C18 years

541/41

7.0

1.41

0.64–3.18

1.69

0.66–4.34

Never been pregnant

167/9

5.1

1.0

Reference

1.0

Reference

Number of pregnancies 3 or more

0.35–1.92

Married or accompanied Use oral contraceptive

Age of 1o sexual intercourse

Number of sexual partners

Age of first pregnancy

OR odds ratio, CI confidence interval * Statistically significant

305/41

11.8

2.49

1.13–5.66*

0.82

1–2

570/42

6.9

1.37

0.63–3.08

10.81

0.74–4.38

Never been pregnant

167/9

5.1

1.0

Reference

1.0

Reference

development of cervical high-grade lesions, including cervical cancer [23, 26]. Clinical and epidemiological studies have provided evidence for the existence of an interaction between CT and HPV, contributing in some way to the development of CIN and cervical cancer [11, 13, 23, 26]. Although the nature of this interaction is not yet well understood, it has been shown that CT may induce local effects on the cervicovaginal epithelium, such as hypertrophy, metaplasia, and chronic inflammation, providing a microenvironment that can favor the immune evasion of pathogens, including HPV. This mechanism may facilitate infection and/or the persistence of the virus [13, 14, 23]. The analysis of immune parameters in the mucosa of the uterine cervix has revealed that these are potentially influenced by several factors, including CT infection, once the chronic inflammation amends the regulatory mechanisms involved in the

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production of cytokine and chemokine and, consequently, the local immune response [27]. Several studies reinforce the hypothesis that the genital infection by CT is an important risk factor for invasive cervical cancer, possibly by inducing a chronic irritation and inflammation with the release of molecules of the innate immune response, such as selectins, cytokines, chemokines, prostaglandins, and their receptors with decreased apoptosis, and of the local immune surveillance. These conditions seem to favor the HPV persistence, as well as the expression of their carcinogenic potential [23, 27, 28]. On the other hand, it has also been described that chlamydial infection induces centrosome amplification, multipolar spindles, and early anaphase onset leading to multinucleation in replicating cells, in addition to inducing anchorage independence in 3T3 fibroblasts. It was proven that such cellular changes occur even in the absence of

Arch Gynecol Obstet

expression of the E6 and E7 HPV genes, or of any other specific oncogene. Thus, it is possible that the presence of these defects within infected dividing cells is a likely mechanism for explaining the role of CT as a cofactor of HPV in the development of cervical lesions, including cancer [29–31]. Predictors of genital infection with CT have been investigated, establishing correlations between the presence of this pathogen and the socio-demographic characteristics and the cultural and behavioral aspects of the studied population, as well as sexual and reproductive activity. Most of these studies revealed that this bacterium behaves with a classic sexually transmitted agent, presenting risk factors similar to other agents that are transmitted by sexual contact. In the women of the present study, infection of the genital tract with CT was associated with chronological age, number of sexual partners lifelong, and age at first pregnancy. There was no association with ethnicity, education, marital status, contraceptive oral use, age of first sexual intercourse, and number of pregnancies. Significantly higher prevalent rates of infection were found among younger women up to 32 years of age, who had two or more sexual partners lifelong and who initiated reproductive activity when less than 18 years of age. This suggests that these variables are risk factors to CT genital infection in the local population. The association between genital infection by CT and multiple sexual partners was already expected, since CT is a sexually transmitted agent. The greatest risk of infection with CT presented by younger women who started early reproductive activity could be due to the immaturity and metaplasia of the cervical epithelium as well as of the local immunity. Our results are concordant with those reported for women of Rio Grande do Sul, Brazil [15], with regard to chronological age, number of sexual partners, marital status, education level, and oral contraceptive use, but divergent in relation to ethnicity and early onset of the reproductive activity. Furthermore, our results are discordant with those obtained for women in the USA [16], with regard to ethnicity and chronological age. Considering the evidence of the involvement of CT as a cofactor in cervical HPV-induced carcinogenesis and the high prevalence rates of infection by this pathogen in the different regions of the country, we suggest a better qualification of prevention and control of genital infection by CT. This could be done by researching CT by PCR in patients with biopsy confirming HPV-induced lesions, which could provide detection of CT-infected women and the specific treatment of this sexually transmitted disease. The CT infection cure probably facilitates the control of progression to cancer of HPV-induced lesions.

In conclusion, the infection by CT did not increase the risk of acquiring HPV infection. The higher prevalence of LSIL in women co-infected with HPV and CT is possibly due to HPV. In addition, our results suggest that younger women aged up to 32 years, who had an early onset of reproductive activity and a history of having had multiple sexual partners lifelong may be at a greater risk of acquiring infection of the genital tract by C. trachomatis. Acknowledgments The research described in this publication was made possible by support from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico—CNPq and Fundac¸a˜o de Amparo a Pesquisa do estado do Rio Grande do Norte, FAPERN (grant PPSUS-III). Conflict of interest

No actual or potential conflict of interest.

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Genital tract infection with Chlamydia trachomatis in women attended at a cervical cancer screening program in Northeastern from Brazil.

This cross-sectional study aimed to estimate the prevalence of Chlamydia trachomatis (CT) infection alone and in combination with human papillomavirus...
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