Mol Biol Rep DOI 10.1007/s11033-013-2927-2

Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil Mayara Costa Mansur Tavares • Jamilly Lopes de Maceˆdo • Se´rgio Ferreira de Lima Ju´nior • Sandra de Andrade Hera´clio • Melaˆnia Maria Ramos Amorim • Maria de Mascena Diniz Maia Paulo Roberto Eleute´rio de Souza

•

Received: 12 November 2012 / Accepted: 18 December 2013 Ó Springer Science+Business Media Dordrecht 2014

Abstract Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. Highrisk human papillomavirus (HR-HPV) is considered the main etiological agent for cervical neoplasia. Evidences showed that the presence of co-infection of CT and HRHPV plays a central role in the etiology of cervical intraepithelial neoplasia (CIN) and cervical cancer. The goals of this study were: evaluate the human papillomavirus (HPV) and CT prevalence among Brazilian women with abnormal cytology and provide the effect of this association on the severity of cervical neoplasia. The population of this study was composed by 142 women with incident histological incidence of CIN grades I, II, III or cervical cancer from Recife, Northeast of Brazil. The polymerase chain reaction method on a cervical brush specimen was used to detect both agents and the automatic sequencing method was used

Electronic supplementary material The online version of this article (doi:10.1007/s11033-013-2927-2) contains supplementary material, which is available to authorized users. M. C. M. Tavares (&)  S. F. de Lima Ju´nior Master in Biology Applied to Health, Universidade Federal de Pernambuco (UFPE), Recife, PE, Brazil e-mail: [email protected] S. F. de Lima Ju´nior e-mail: [email protected] J. L. de Maceˆdo Master in Applied Molecular and Cellular Biology, Universidade de Pernambuco (UPE), Recife, PE, Brazil e-mail: [email protected] S. de Andrade Hera´clio Departament of Lower Genital Tract Pathology, Women’s Healthcare Center, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife, PE, Brazil e-mail: [email protected]

for HPV genotyping assay. The prevalence of HPV and CT was 100 and 24.65 %, respectively. Thirteen types of HPV were detected; HPV 16, 18, 31 and 33 were the most common. The most prevalent HPV types were HPV 16 and 18. A significant association between CT positive and HPV 16 infection was found (p \ 0.0106; OR = 5.31; 95 % IC 1.59–17.67). In the study population, there was diversity of HPV infections, with high-risk types being the most common. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection. Keywords Human papillomavirus  Abnormal cytology  Chlamydia trachomatis  Risk factors  Brazilian population Abbreviations CT Chlamydia trachomatis STDs Sexually transmitted diseases

M. M. R. Amorim Maternal and Child Healthcare Departament, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife, PE, Brazil e-mail: [email protected] M. de Mascena Diniz Maia  P. R. E. de Souza Biology Department, Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE, Brazil e-mail: [email protected] P. R. E. de Souza e-mail: [email protected]

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PID HPV LR-HPV HR-HPV CIN ICC PCR DNA EDTA

Pelvic inflammatory disease Human papillomavirus Low-risk HPV High-risk HPV Cervical intraepithelial neoplasia Invasive cervical cancer Polymerase chain reaction Deoxyribonucleic acid Ethylenediamine tetraacetic acid

Introduction Invasive cervical cancer (ICC) is the second most common malignancy in women worldwide, with an estimated incidence of 500,000 new cases per year and 250,000 deaths [1, 2]. In Brazil, ICC is the second most common type of cancer and the fourth cause of death by cancer among women. Data of National Institute of Cancer, for the year of 2012, estimate 17,540 new cases with associated risk of 17 cases/100,000 inhabitants for this type of cancer. In Pernambuco, the expected incidence for 2012 was 20.67 cases/100,000 inhabitants, higher than the Brazilian average [3]. Human papillomavirus (HPV) infection has now been determined to be involved in the development of cervical cancer and cervical dysplasia. There are approximately 45 HPV types that infect the genital mucosa, and they are classified into low-risk HPV (LR-HPV) and high-risk HPV (HR-HPV) on the basis of their association with premalignant and malignant lesions [4–6]. LR-HPVs are agents causing warts, papillomas and genital warts while HRHPVs are considered the main cause of cervical intraepithelial neoplasia (CIN) and ICC [7–12]. Most persisting infections are caused by HR-HPVs in approximately 99.7 % of the patients [4], and precedes the development of low- and high-grade squamous intraepithelial lesions [11, 13–15]. Although HPV is a prerequisite for ICC, only a small number of women exposed to this virus develops cancer, implying that other risk factors (environmental or exogenous) may be considered as cofactors rather than independent factors. Chlamydia trachomatis (CT) is the most common sexually transmitted pathogens in women. CT is an aerobic, obligate, immobile, coccoid, intracellular bacteria widely distributed in the animal kingdom. Its features are similar to the cell wall of gram-negative bacteria, due to the biphasic cell division cycle and the distinctive dimorphic life cycle [16]. They are responsible for more sexually transmitted diseases (STDs) than any other bacterial pathogen, making from these infections a large problem of public health in the world with approximately 90 million

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new cases per year [17, 18]. In most women (70–75 %) and in more than 50 % of men these infections occur in an asymptomatic way [19, 20]. Previous studies conducted in Brazil showed lower prevalence of genital CT infection, ranging from 12.2 to 22.2 % [21–23]. Differently from men, in which the infection by CT does not represent the most serious problems, women have a high-risk of severe complications. In women, undetected infections are highly associated with harmful and costly complications, such as pelvic inflammatory disease (PID), infertility, and ectopic pregnancy, and the incidence rate among female adolescents in urban areas is approximately greater than 30 % [20, 24, 25]. The annual cost of treatment and the monitoring only of patients with PID exceeds 10 billion dollars [24]. Some authors detected a high-risk for the development of ICC in patients with HPV infection and history of CT [26, 27]. Due to the importance of these infections to the public health, the goals of this study were to estimate the frequency of co-infection between HPV and CT in women with or without cervical lesions and investigate the most present types of HPV in the study population of the State of Pernambuco.

Materials and methods Study population: eligibility, identification, and recruitment A hospital-based cross-sectional prospective study was carried out in the outpatient clinics of the Lower Genital Tract Pathology Clinic at the Women’s Healthcare Center of the Prof. Fernando Figueira Institute of Integrated Medicine. The study population consisted of 142 sexually active women ranging between 16 and 75 years old, selected by spontaneous demand from October 2008 until December 2009. Information was collected from all women pertaining to their age, parity, socio-economic status, and smoking and contraception history. The inclusion criteria was women with oncotic cytology submitted to Papanicolaou test (cytological) according to Bethesda system terminology, performed on the state accredited networks and that presented diagnostic of CIN of lowgrade and high-grade or cervical cancer, confirmed by histological analysis. Subjects were evaluated for clinical features of other STIs on history and examination. Patients that were previously submitted to radiotherapy or chemotherapy to ICC were excluded. The Institute Ethics Committee approved the study protocol (n° 355/08). Informed written consent was taken from the women, informing them about the background of the study, risks and benefits and voluntary nature of participation.

Mol Biol Rep Table 1 Sequences of primers used in the study Name

Primers

Primer sequence

Human p-globinaa

PC04

50 -GGA GAG CCA AAG GAC AGG TAC-30

GH20

50 -GAA GAG CCA AGG ACA GGT AC-30

MY09

50 -CGT CCM ARR GGA WAC TA TC-30

b

MY

GP?c CTPd a

Size (bp)

0

Gene Position 54–73

268

195–176 7015–7034

0

MY11

5 -GCM CAG GGW CAT AAY AAT GG-3

GP05?

50 -TTT GTT ACT GTG GTA GAT ACT AC-30

GP06?

50 -GAA AAA TAA ACT GTA AAT CAT ATT C-30

CTP1

50 -TAG TAA CTG CCA CTT CAT CA-30

CTP2

50 -TTC CCC TTG TAA TTC GTT GC-30

450

6533–6602

150

6624–6649

201

4761–4781

6719–6746 4941–4961

Bell et al. [28]

b

Manos et al. [29]

c

de Husman et al [30]

d

Gopalkrishna et al. [31]

Clinical samples Cervical smears were obtained using Cytobrushes. Each Cytobrush was packed in a TE buffer solution (Tris–HCl 10 mM and EDTA 1 mM pH 8.0) and conserved at -20 °C until analysis. DNA extraction Genomic DNA extraction was realized from 500 lL scraping the cells using the cervical region Nucleospin Tissue Òkit (Macherey–Nagel), following the manufacturer’s instructions. The analyses samples were realized in Laboratory of Genetic, Biochemistry and DNA Sequencing of Rural Federal University of Pernambuco. HPV detection by conventional polymerase chain reaction (PCR) HPV DNA detection in cervical scrapes was performed by PCR, using primers MY09 and MY11 (Table 1). To analyze the quality of target DNA for PCR purposes, we prescreened specimens by PCR with the use of b-globin gene-specific oligonucleotide primers (GH20 and PC04) (Table 1). For detection of the HPV a PCR mixture containing: 19 Taq DNA polymerase Buffer (PROMEGA), 1.5 mM MgCl2 (PROMEGA), 100 lM dNTP (PROMEGA), 1 pmol/lL of each specific primer (MY09 and MY11), 1U GoTaqÒ Hot Start Polymerase (PROMEGA) and about 200 ng of DNA genomic to a final volume of 15 lL was realized. The amplification conditions were as follows: 94 °C for 4 min; 40 cycles at 94 °C for 30 s; 55 °C for 30 s; and 72 °C for 30 s; and the final extension step at 72 °C for 8 min. The fragment size expected was 450 bp.

A positive control sample from DNA database previously detected by PCR and confirmed by sequencing was used. Furthermore, the negative samples to specific MY09 and MY11 primers and positive for b-globin specific primers were submitted to other reaction of PCR to determine the overall presence of HPV by the use of a general primer GP5?/6?-, which permits the detection of a broad spectrum of sequenced and still unsequenced genital HPV types at the subpicogram level. The PCR mixture was realized in a final volume of 15 lL containing: 19 Taq DNA polymerase Buffer (PROMEGA), 3.5 mM MgCl2 (PROMEGA), 200 lM dNTP (PROMEGA), 1 pmol/lL of each specific primer (GP5? and GP6?), 1U GoTaqÒ Hot Start Polymerase (PROMEGA) and about 200 ng of DNA genomic. The amplification conditions were as follows: 94 °C for 4 min; 35 cycles at 94 °C for 1 min; 45 °C for 2 min; and 72 °C for 1.5 min; and the final extension step at 72 °C for 4 min. The fragment size expected was 150 bp. The amplicons were fractionated on 1.5 % agarose gel. One positive sample, previously confirmed by sequencing, was used as positive control and one mix without DNA as negative control of PCR reaction. Sequencing The identification of the most frequent HPV types in the study population was realized by automatic DNA sequencing (MegaBACE 1000 DNA sequencer, GE Healthcare, USA). Forward and reverse sequencing reactions were carried out using 50–100 ng of template DNA from PCR products, 0.5 pmol of specific set primers (MY09/11 and GP5?/6?) and 8 lL of reagent DYEnamicTM ET Terminator Cycle Sequencing (GE Healthcare, USA) in a final volume of 20 lL. The sequencing rounds

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Fig. 1 Amplification curve for detection of positive samples for Chlamydia trachomatis based on the technique of real-time PCR

were performed in the thermocycler Veriti 96 (Applied Biosystems Inc., Norwalk, CT, USA) with the initial step of 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s; 50 °C for 15 s; and 60 °C for 4 min. The samples were purified by ethanol/EDTA/sodium acetate protocol and the precipitated DNA products were diluted in 10 lL of formamide Hi-Di, denatured (95 °C for 2 min) and injected in the automated DNA MegaBACE 1000 DNA Sequencer (GE HEALTHCARE). The sequence data were collected using the Data Collection program v1.0.1 with the parameters Dye Set ‘‘Z’’. Quality analysis was performed using the Sequencing Analysis v.5.3.1 software by evaluating the main technical parameters as raw data, electropherogram and quality value of sequenced bases (MegaBACE 1000 DNA sequencer). The nucleotide sequences of the same amplicon (primers sense and antisense) were edited and assembled using SeqMan software (DNAStar, Madison, Wisconsin, USA). The nucleotide sequences of the samples were aligned using the MegAlign program (DNAStar, Madison, Wisconsin, USA) and types were deduced from the phylogenies generated by the program itself. Further, data obtained was compared with sequences available in GenBank database (www.ncbi.nlm.nih.gov) using BLAST tool. Chlamydia trachomatis detection by real time PCR The detection of CT infection was realized by the methodology of Real Time PCR using the apparatus Line-gene K Real-time PCR Detection System (BIOER TECHNOLOGY).The reaction was prepared at final volume of 15 lL,

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containing approximately 100 ng DNA sample, 1X Sybr Green Rox Plus (LGC) and 1 pmol/lL of each primer (Table 1). The reaction conditions were: 95 °C for 4 min, followed for 40 cycles of 95 °C for 1 s and 60 °C for 60 s, adapted of Oliveira et al. [32]. Posteriorly, a melting temperature curve was obtained by slow denaturation from 95 °C to 60 °C for 20 s, with a temperature variation of 0.2 °C/s. This phase was important to identify unspecific products of reaction (Figs. 1, 2). Two CT DNA positive samples, previously assayed using the Hybrid Capture 2TM (HC2) assay (Qiagen Gaithersburg Inc., MD, USA) were used as positive control. In addition, a mix without DNA was used as negative control. A melting temperature assay of CT DNA positive control was compared with negative control without DNA to avoid false positive results. Statistical analysis Statistical analysis was done using the BioEstat 5.0 software program. Cytology was reported according to the Bethesda System. Cases were stratified as normal, low and high-grade CIN and ICC based on results of colposcopy and directed biopsy. Data are reported as number (percentage) and median (range). Association of outcome and exposure was tested with Fischer exact test and exposure to more than two categories with the non parametric trend v2-test. p \ 0.05 was considered significant. The associations of specific HPV genotypes and phylogenetic groups with the final diagnosis were assessed using odds ratios (OR) with their respective 95 % confidence intervals (95 %

Mol Biol Rep

Fig. 2 Melting peak of 79.70 C for the amplification product of Chlamydia trachomatis based on the technique of real-time PCR

Table 2 HPV genotypes prevalence and histologic diagnosis Total n (%)

CIN I n (%)

HPV 16

12 (36.36)

40 (39.22)

3 (37.5)

55 (38.73)

HPV 18

8 (24.24)

23 (22.55)

2 (25)

33 (23.24)

Others HR (31, 33, 45, 56, 58, 67)

3 (9.09)

15 (14.71)

1 (12.5)

19 (13.38)

LR (6, 11, 61, 70, 83)

4 (12.12)

2 (1.96)

Co-infection (HPV 16/18)

3 (9.09)

18 (17.65)

Co-infection (18/31, 31/6, 16/18/56, 31/67) Co-infection (16/6, 18/6, 18/61)

2 (6.06)

2 (1.96)

0

4 (2.82)

1 (3.03)

2 (1.96)

0

3 (2.11)

33 (100)

102 (100)

Total

CIN II/III n (%)

ICCa n (%)

Types

0 2 (25)

8 (100)

6 (4.23) 23 (16.20)

142 (100)

HPV human papillomavirus a

Invasive cervical cancer CIN indicates cervical intraepithelial neoplasia

IC). The reference for the calculation of OR was HPV 16 and/or HPV 18 cases.

Results One hundred and forty-two eligible women were enrolled in the study. Complete information for analysis was available for 125 women. The median age was 34.0 years old (range 16–74 years old), with 73.6 % (92/125) women in the age group of 20–39; 6.4 % (8/125) younger than 20 years old; and 20 % (25/125) over 40 years old. Regarding to risk factors for cervical neoplasia: More than

a half (n = 90, 72 %) belonged to the lower socio-economic strata, 39.2 % (49/125) were smokers and about half (n = 60, 48 %) reported irregular condom use. Distribution of HPV genotypes The distribution of HPV genotypes and histologic diagnosis is shown in Table 2. Once the 142 (100 %) women from this study all presented abnormal Pap smear ([ASCUS), the overall prevalence of HPV cervical smear was 100 % (142 of 142), and 95.77 % (136 of 142) consisted of high-risk oncogenic types. The prevalence of single infection was 71.83 % (102 of 142) and multiple infections represented 25.35 % (36 of 142) of all cases. Of the total population of HPV-positive women, 55.63 % (79 of 142) were infected with HPV 16 either as a single or multiple-type infection and 26.06 % (37 of 142) exhibited HPV 18 either as a single or multiple-type infection. For all samples analyzed, 16.20 % (23 of 142) presented both HPV 16 and 18, and 13.38 % (19 of 142) presented other HR-HPV types (HPV 31, 33, 45, 56, 58, 67 and 70). Furthermore, 4.23 % (6 of 142) were diagnosed as LRHPV as single infection and 4.93 % (7 of 142) were diagnosed with simultaneous co-infection between HPV 16 and/or 18 and other HR and LR-HPV types, in the same patient. When all the 142 clinical samples were stratified according to the severity of cervical lesions (CIN, grades I–III or ICC), 23.24 % (33 of 142) exhibited low-grade lesion (CIN I), 71.83 % (102 of 142) high-grade lesion (CIN II/III) and 5.63 % (8 of 142) ICC (Table 2). The prevalence of HPV 16 in the women with a histologic diagnosis of CIN I was 36.36 % (12 of 33). Regarding to the most severe lesions, HPV 16 was present in 39.22 % of cases (40 of 102) of CIN II and CIN III and 37.5 % of cases (3 of 8) of invasive carcinoma. The HPV 18, either as a single or multiple infections, was detected in

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Mol Biol Rep Table 3 Prevalence of HPV types in positive C. trachomatis women in relation to histologic diagnosis HPV types

CIN I

ICCa

CIN II/III

Total b

CT? n (%)

CTn (%)

CT? n (%)

CTn (%)

CTn

n

16

7 (63.64)

5 (33.33)

7 (29.17)

33 (48.53)

3

55

HPV 16/18

2 (18.18)

1 (6.67)

6 (25.0)

12 (17.65)

2

23

HPV 16/Others Total

– 9

– 6

– 13

1 46

– 5

1 79

18

–

8 (53.33)

5 (20.83)

18 (26.47)

2

33

HPV 18/31

–

1

–

1

–

2

HPV 18/Others

–

1

–

1

–

2

Total

–

10

5

20

2

37

31

1 (9.09)

1 (6.67)

1 (4.17)

1 (1.47)

–

4

HPV 31/67

–

–

2 (8.33)

–

–

2

Total

1

1

3

1

–

6

33

–

–

1(4.17)

4 (5.88)

1

6

45

–

–

–

1

–

1

56

1 (9.09)

–

1 (4.17)

–

–

2

58

–

–

–

3

–

3

67

–

–

1 (4.17)

–

–

1

Low risk

–

–

–

2

–

6

Total

11 (100)

22 (100)

24 (100)

77 (100)

8 (100)

142 (100)

a

CIN cervical intraephitelial neoplasia, ICC invasive cervical cancer, HPV Human Papillomavirus

b

CT Chlamydia trachomatis

24.24 % (8 of 33) of CIN I cases, 22.55 % (23 of 102) of CIN II or CIN III cases and 25 % (2 of 8) of invasive carcinoma cases. In the overall positivity for HPV, including both single and multiple-type infections, no statistically significant association was found with a diagnosis of cervical neoplasia (p = 0.3176). Furthermore, our results did not find association when compared: HPV 16 and 18 either as a single or multiple-type infection (p [ 0.05); co-infection HPV 16 and 18 versus other-HPVs (p = 0.7019); or HPV 16 and 18 versus ICC (p = 0.9213). CT DNA positive was identified in 24.65 % (35 of 142) of the samples (Table 3). CT prevalence was the highest in the age group of 20–39 years old (23.91 %, 22/92), followed by the group over 40 years old (16 %, 4/25). All CT DNA positive exhibited HR-HPV co-infection. From these, 40 % (14 of 35) contained HPV 16, 14.28 % (5 of 35) HPV 18, 5.71 % (2 of 35) HPV31, 2.86 % (1 of 35) HPV 33, 5.71 % (2 of 35) HPV 56 and 2.86 % (1 of 35) HPV 67 as a single infection and 22.86 % (8 of 35) contained HPV 16 and 18, 5.71 % (2 of 35) HPV 31 and 67. However, from 107 negative samples of CT infection 94.39 % (101 of 107) exhibited HR-HPV and 5.61 % (6 of 107) LR-HPV (Table 3). Regarding to the most severe lesions (CIN II/III), 23.76 % (24 of 101) of the patients

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exhibited CT infection and 76.24 % (77 of 101) were CT negative. A significant association was found when CT positive/High-grade lesion with HPV 16 was compared with CT negative/low grade lesion with HPV 16 (p \ 0.0106; OR = 0.1884; 95 % IC = 0.06–0.63). On the other hand, no significant difference was observed in both co-infection HPV 16 and 18/CT positive versus HPV 16 and 18/CT negative and other-HPV/CT positive versus other-HPV/CT negative (p = 0.7274 and 0.8738, respectively). In the CT negative group, 96.26 % (103 of 107) of patients showed HR-HPV. One more time, the types 16 and 18 were the most frequent 53.27 % (57 of 107) and 29.9 % (32 of 107), respectively.

Discussion HPV genital infection is highly frequent worldwide and its prevalence in the general population ranges from 2 to 44 % depending on the region of the world [10]. So far, more than 100 different HPV types have been identified, of which more than 40 have been detected in the anogenital area and some types were associated with cancer, precancerous lesions and genital warts [12]. The presence of HPV associated with CIN in more than 90 % of cases make

Mol Biol Rep

the virus one of the main risk factors for the development of ICC. However, only a small amount of women infected with a HR-HPV infection will develop ICC [33, 34]. In Brazil, the prevalence of HPV infection ranges from 15 to 27 % [35, 36]. The HPV 16 and 18 types are most frequently reported in the world population, accounting for approximately 70 % of all cervical cancers [37]. Nevertheless, the distribution and prevalence of HR-HPV types have been showing variation among populations worldwide [38, 39] and also in Brazil [40–44]. In the present study, we analyzed 142 HPV positive women with cervical lesion and found the following distribution among frequencies of HPVs: 16, 18, 31 and 33 types (55.63, 42.25, 5.63 and 4.22 %, respectively). Baldez et al. [45] also conducted in Recife a study analyzing 213 HPV positive women and found a higher frequency of HPV 16 (78 %) and HPV 31 (15.5 %) and lower frequency of HPV18 (2.8 %). By the other hand, Lorenzato et al. [43] analyzing 214 HPV positive women, found a higher frequency of HPV 31 (21.4 %) and a lower frequency of HPV 18 (2.4 %). Furthermore, Oliveira-Silva et al. [46] in a study performed in two municipalities with low socioeconomic status in the state of Rio de Janeiro, at the Southeast region of Brazil, analyzing 132 HPV positive women reported a high frequency of HPV 16 (28 %), followed by HPV 18 (14.4 %), HPV 31 (3.8 %) and HPV 33 (3 %). A possible explanation for HPV prevalence values reported in previous studies vary considerably—probably as a function of the methods used, the specific populations studied and how it is obtained. Different of the previous studies, in this study, for HPV detection two sets of primers (MY09/11 and GP05?/GP06?, respectively) were used before HPV typing. Further, our population was constituted exclusively by hospital patients with cervical lesions who had been referred for HPV testing by a gynaecologist or cytopathologist as part of their standard medical care. In one meta-analysis realized by Clifford et al. [38], the distribution for HPV 16, 18, 33 and 31 in 24,558 samples of patients with cervical lesions was 54.6, 15.8, 4.4 and 3.5 %, respectively. On the other hand, when Mun˜oz [47] described the distribution of frequencies, a change in the order only to frequencies 31 and 33 types was observed, as follows: HPV16 (53 %), HPV18 (15 %), HPV31 (6 %) and HPV33 (3 %). It is worth noting that HPV detection rates depend on the study population, the type of specimen and how it is obtained. Our results are in agreement with the trend worldwide for a higher proportion of the 16 and 18 types in women infected by HR-HPV types, followed by 31 and 33 types. These are very important baseline data to take into account for future post-vaccination epidemiological surveillance. In the present study, we found among the 55 cases diagnosed with CIN I, 36.36 % of the samples presenting

HPV16. The frequency of this type of HPV was higher than another study performed in Brazil by Freitas et al. [44] in which HPV16 was detected in 20 % among the cases of low grade squamous intraepithelial lesion (LSIL). Lorenzato et al. [43] also detected low frequency of HPV16 (19.4 %) in samples diagnosed with CIN I. On the other hand, Van den Brule et al. reported an association of HPV16 and HPV18 with CIN I/II and III [48]. They observed that low-risk (LR-HPV) oncogenic types were predominant in samples with LSIL/intraepithelial lesion and malignancy (NILM) cytology [48]. These results suggest that factors as socioeconomic status and life-style of the host could be contributing to this high frequency of HPV 16 in CIN I in the study population. Levi et al. [49] reported that 45 % of the patients were infected with more than two HPV types, in a HIV-infected population from Sa˜o Paulo, SP, Brazil; Similarly, Gonc¸alves et al. [50] observed multiple genotypes in 45 % of the HPV positive patients. In this study, 21.13 % (30/142) of the HPV positive patients were diagnosed with two genotypes. This fact may be due to behavioral, socioeconomic characteristics and the frequency of exposure of these patients to unprotected sexual contacts. In the infection by HPV it is not yet fully understood the way some cofactors favor the persistence of the virus in the epithelium and contribute to the development of the disease [51, 52]. According to literature, CT acts as a cofactor that promotes the penetration of HPV and damages the mucous facilitating the progress of cervical lesions, interfering in the immunological response and viral release [2, 53–55]. Although epidemiological data have not yet provided consistent evidence about its real implication in ICC. In developing countries, the prevalence of CT infections range from 4.5 to 22 % in the general population [56] and can reach peaks as high as 80 % in patients with intraepithelial cervical lesions [32, 57]. In a study with 227 women from the Northeast of Argentina, the overall prevalence was 26.4 % for CT, 46.7 % for HPV and 16.3 % for concurrent infection [55]. The prevalence of CT infection was significantly higher among women with HPV DNA positive (34.9 %). Gopalkrishna et al. [31] studying an Indian population, found low frequency of CT, both women with cancer (22 %) and pre-cancer lesions (12 %). Our data are in agreement with these studies, once from the HPV positive women with cervical lesion recruited for the research, the prevalence of CT was 24.65 % (35/142). Our data showed that 24.65 % of the women were positive for CT infection (Table 3) and all of them exhibited HR-HPV co-infection. Since LR-HPV group was little representative in this population with only 5.61 % (6 of 107) of the cases, it could explain why we did not find CT positive women in this group.

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Although most women are infected with high risk HPV at some time, a few will progress to invasive disease [58], because most HPV infections could regress within 2 years and only a minority of women will develop persistent HPV infection that could eventually cause CIN [59]. When we stratified the samples according to grade of cervical lesion, we verified that the percentage of women with high-grade of lesion (CIN II and III) was fairly more representative in CT negative infection 76.24 % (77/101) than CT positive group with 23.76 % (24/101). In this way, although CT behaves as an important factor in the HPV infection, our results do not suggest association with viral persistence and lesion progress. Some articles associated the presence of HPV16 with lesion and cancer [60–62]. Tamim et al. [63] verifies that 67.35 % (33/49) of positive HPV women that were genotyped exhibited HPV16. Furthermore, Wu et al. [64] found high frequency (79.6 %) of HPV16 in cervical cancer. In their work, it was found that, in Chinese women, the lesions of grades II and III showed frequency of HR-HPV between 80 and 90 %. Chopjitt et al. [65] observed that 38.9 % (14/36) of patients with CIN II and III were infected by this type of HPV. In our study, the frequency for this most frequent type of HPV (HPV16) was 41.42 % (41/99) for patients that had lesions of grades II and/or III and the overall CT positive patients exhibited HR-HPV. For HPV18 the frequency was 23.23 % in this group. In this way, we can suggest the presence of HPV as a determinant factor to the development of CINs and ICC, especially HPVs 16 and 18 types, with large oncogenic potential and quite often in the world population. In our study, we found patients that were CT positive, co-infected with LR-HPV infection and presented high grade of lesion (CIN II or III). Since infection with HPV or CT has been linked with the development of cervical cancer, a number of studies investigated the relationship between past infection with HPV or CT and subsequent infection with other agents [66]. This fact was exemplified by the demonstration that CT was found in samples from the cervix of HPV positive women. The levels of CT DNA or IgG antibodies were also higher in HPV positive compared to HPV negative women. It is possible that CT infection may be an independent factor or cofactor for HPV in the development of invasive cervical carcinoma. However, the exact relationship between CT and HPV infection is still not completely understood [53, 67]. Bhatia et al. [68] studied 618 women coinfected by HPV and CT and found 62.3 % women in the age group of 30–39 years old presenting CIN and 59.2 % were younger than 18 at the time of first coitus. Authors identified yet that 50.3 % belonged to the lower socio-economic strata, 10.2 % were HR-HPV and 5.2 % were CT positive. In the present study, regarding to socio-economic strata 63.38 %

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of women were from the lower socio-economic strata. For the presence of HR-HPV, the prevalence was higher in the age-group 20–39 years old (73.24 %). Even if CT acts as a cofactor in the steps of progression from premalignancy to ICC, we cannot confirm this by the results of our study because this study was not sufficiently powered to evaluate the role of CT in ICC. Similar results were found by Safeian et al. [69] in a study from the large population-based Costa Rica. On the other hand, our findings are contrary of International Agency for Research on Cancer (IARC) [70].

Conclusion In the study population, there was diversity of HPV infections, with high-risk types being the most common detected. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection. In the present context, we suggest that this association seems more related with potentiating mutual than with common way of transmission. Acknowledgments We thank the patients and their families, whose collaboration and understanding have made this work possible. This study was supported by the Brazilian funding agency FACEPE (Fundac¸a˜o de Amparo a` Cieˆncia e Tecnologia do Estado de Pernambuco). We are also grateful for the facilities of the Laborato´rio de Gene´tica, Bioquı´mica e Sequenciamento de DNA Profa Tania Falca˜o of Universidade Federal Rural de Pernambuco, where the experiments were performed. Conflict of interest

No conflict of interest to declare.

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