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: mascen[email protected] P. R. E. de Souza e-mail: [email protected]


Mol Biol Rep


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


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


Primer sequence

Human p-globinaa









GP?c CTPd a

Size (bp)


Gene Position 54–73


195–176 7015–7034


















6719–6746 4941–4961

Bell et al. [28]


Manos et al. [29]


de Husman et al [30]


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


Mol Biol Rep

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 ( 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,


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)


4 (2.82)

1 (3.03)

2 (1.96)


3 (2.11)

33 (100)

102 (100)


CIN II/III n (%)

ICCa n (%)


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


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




Total b

CT? n (%)

CTn (%)

CT? n (%)

CTn (%)




7 (63.64)

5 (33.33)

7 (29.17)

33 (48.53)



HPV 16/18

2 (18.18)

1 (6.67)

6 (25.0)

12 (17.65)



HPV 16/Others Total

– 9

– 6

– 13

1 46

– 5

1 79


8 (53.33)

5 (20.83)

18 (26.47)



HPV 18/31




HPV 18/Others











1 (9.09)

1 (6.67)

1 (4.17)

1 (1.47)


HPV 31/67

2 (8.33)










4 (5.88)







1 (9.09)

1 (4.17)






1 (4.17)


Low risk




11 (100)

22 (100)

24 (100)

77 (100)

8 (100)

142 (100)


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


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


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.


Mol Biol Rep

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 %


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.

References 1. Stanley M (2008) Immunobiology of HPV and HPV vaccines. Gynecol Oncol 109:15–21. doi:10.1016/j.ygyno.2008.02.003 2. Simonetti AC, Melo JHL, Souza PRE, Bruneska D, Filho JLL (2009) Immunological’s host profile for HPV and Chlamydia trachomatis, a cervical cancer cofactor. Microbes Infect 11:435–442. doi:10.1016/j.micinf.2009.01.004 3. Brasil (2012) Ministe´rio da Sau´de. Instituto Nacional do Caˆncer. Accessed 12 June 2012 4. Burd EM (2003) Human papillomavirus and cervical cancer. Clin Microbiol Rev Jan 16:1–17. doi:10.1128/CMR.16.1.1-17.2003 5. Kirkpatrick A, Bidwell J, Van den Brule AJC, Meijer CJLM, Pawade J, Glew S (2004) TNFa polymorphism frequencies in HPV-associated cervical dysplasia. Gynecol Oncol 92:675–679. doi:10.1016/j.ygyno.2003.11.025 6. Van Ranst M, Kaplan JB, Burke RD (1992) Phylogenetic classification of human papillomaviruses: correlation with manifestations. J Gen Virol 73:2653–2660 7. Albring L, Brentano JE, Vargas VRA (2006) O caˆncer do colo do u´tero, o Papilomavı´rus Humano (HPV) e seus fatores de risco e as mulheres indı´genas Guarani: estudo de revisa˜o. RBAC 38:87–90

Mol Biol Rep 8. Magi JC, Brito SEM, Grecco ETO et al (2006) Prevaleˆncia de papilomavirus humano (HPV) anal, genital e oral, em ambulato´rio geral de coloproctologia. Rev Bras Coloproct 26:233–238. doi:10.1590/S0101-98802006000300001 9. Magi JC, Magi DAS, Reche LMC et al (2002) Anuscopia com exacerbac¸a˜o para diagno´stico de Papilomavirus humano ano-retal na forma subclı´nica. Rev bras Coloproct 22:178–183 10. Bosch FX, Burchell AN, Schiffman M et al (2008) Epidemiology and natural history of human papillomavirus infections and typespecific implications in cervical neoplasia. Vaccine 26(Suppl 10):K1–K16. doi:10.1016/j.vaccine.2008.05.064 11. Hausen HZ (2000) Papillomaviruses causing cancer: evasion from host-cell host in early events in carcinogenesis. J Natl Cancer Inst 92:690–698. doi:10.1093/jnci/92.9.690 12. Mun˜oz N, International Agency for Research on Cancer Multicenter Cervical et al (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518–527. doi:10.1056/NEJMoa021641348/6/ 518 13. Kjaer SK, Van Der Brule AJ, Paull G et al (2002) Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ 325:5722. doi:10.1136/bmj.325.7364.572 14. Josefsson AM, Magnusson PKE, Ylitalo N, Sorensen P, Qwarforth-Tubbin P, Anderson PK et al (2000) Viral load of human papilloma virus 16 as a determinant for development of cervical carcinoma in situ: a nested case-control study. Lancet 355: 2189–2193 15. Sellors JW, Mahony JB, Kaczorowski J, Lytwyn A, Bangura H, Chong S et al (2000) Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group. CMAJ 163:503–508 16. Manavi K (2006) A review on infection with Chlamydia trachomatis. Best Pract Res Clin Obstet Gynaecol 20:941–951. doi:10.1016/j.bpobgyn.2006.06.003 17. Giraldo PC, Simo˜es JA (2000) Clamı´dia e micoplasmas. In: Halbe HW (ed) Tratado de ginecologia, 3rd edn. Roca, Sa˜o Paulo, pp 1047–1058 18. Seadi CF, Oravec R, Poser BV et al (2002) Diagno´stico laboratorial da infecc¸a˜o pela Chlamydia trachomatis: vantagens e desvantagens das te´cnicas. J Bras Patol Med Lab 38:125–133. doi:10.1590/S1676-24442002000200009 19. World Health Organization (2005) Sexually transmitted and other reproductive tract infections. Geneva, World Health Organization. Accessed 25 Jan 2011 20. Machado ACS, Guimara˜es EMB, Sakurai E et al (2007) High titers of Chlamydia trachomatis antibodies in Brazilian women with tubal occlusion or previous ectopic pregnancy. Infect Dis Obstet Gynecol. doi:10.1155/2007/24816 21. Miller WC, Ford CA, Morris M et al (2004) Prevalence of chlamydial and gonococcal infections among young adults in the United States. JAMA 291:2229–2236 22. Araujo RSC, Guimaraes EMB, Alves MFC et al (2006) Prevalence and risk factors for Chlamydia trachomatis infection in adolescent females and young women in central Brazil. Eur J Clin Microbiol Infect Dis 25:397–400. doi:10.1007/s10096-0060142-y 23. Miranda AE, Szwarcwald CL, Peres RL, Page-Shafer K (2004) Prevalence and risk behaviors for chlamydial infection in a population-based study of female adolescents in Brazil. Sex Transm Dis 31:542–546 24. WHO (2007) Global strategy for intervention and control of sexually transmitted infections 2006-2015. Geneva, World Health Organization.















Global_Strategy_for_Prevention_and_Control_of_STIs_20062015_Key_Messages.pdf. Accessed 15 Feb 2012 Krech T, Castriciano S, Jang D, Smieja M, Enders G, Chernesky M (2009) Detection of high-risk HPV and Chlamydia trachomatis in vaginal and cervical samples collected with flocked nylon and wrapped rayon dual swabs transported in dry tubes. J Virol Methods 162:29–291. doi:10.1016/j.jviromet.2009.08.011 Wallin KL, Wiklund F, Luostarinen T et al (2002) A populationbased prospective study of Chlamydia trachomatis infection and cervical cancer carcinoma. Int J Cancer 101:371–374 Smith JS, Mun˜oz N, Herrero R, Eluf-Neto J, Ngelangel C, Franceschi S et al (2002) Evidence for Chlamydia trachomatis as a human papillomavirus cofactor in the etiology of invasive cervical cancer in Brazil and the Philippines. J Infect Dis 185:324–331 Bell DA, Taylor JA, Paulson DF, Robertson CN, Mohler JL, Lucier GV (1993) Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J Natl Cancer Inst 85:1159–1164. doi:10. 1093/jnci/85.14.1159 Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM (1989) Use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. In: Furth M, Greaves M (eds) Molecular diagnostics of human cancer. Cold Spring Harbor, Cold Spring Harbor Laboratory, pp 209–214 (Cancer Cells Series, 7) de Husman AMR, Walboomers JM, Van Den Brule AJ, Meijer CJ, Snijders PJ (1995) The use of general primers GP5 and GP6 elongated at their 30 ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 76(Suppl 4):1057–1062 Gopalkrishna V, Aggarwal N, Malhotra VL, Koranne RV, Mohan VP, Mittal A, Das BC (2000) Chlamydia trachomatis and human papillomavirus infection in Indian women with sexually transmitted diseases and cervical precancerous and cancerous lesions. Clin Microbiol Infect 6:88–93. doi:10.1046/j.1469-0691.2000. 00024.x Oliveira ML, Amorim MMR, Souza PRE, Albuquerque LCB, Branda˜o LAC, Guimara˜es RLG (2008) Chlamydia infection in patients with and without cervical intra-epithelial lesions tested by real-time PCR vs. direct immunofluorescence. Braz J Infect Dis 12:324–328 Mun˜oz N, Castellsagu0 e X, de Gonz0 alez AB, Gissmann L (2006) HPV in the etiology of human cancer. Vaccine 24(Suppl l3):1–10. doi:10.1016/j.vaccine.2006.05.115 Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S (2007) Human papillomavirus and cervical cancer. Lancet 370:890–907 de Lima Soares V, de Mesquita AM, Cavalcante FG, Silva ZP, Hora V, Diedrich T, de Carvalho Silva P, de Melo PG, Dacal AR, de Carvalho EM, Feldmeier H (2003) Sexually transmitted infections in a female population in rural north-east Brazil: prevalence, morbidity and risk factors. Trop Med Int Health 8:595–603 Nonnemacher MR, Hogan TH, Quiterio S, Wigdahl B, Henderson A, Krebs FC (2003) Identification of binding sites for members of the CCAAT/enhancer binding protein transcription factor family in the simian immunodeficiency virus long terminal repeat. Biomed Pharmacother 57:34–40 Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, Clifford GM (2007) Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a metaanalysis update. Int J Cancer 121:621–632. doi:10.1002/ijc.22527 Clifford G, Fraceschi S, Diaz M, Mun˜oz N, Villa LL (2006) HPV type-distribution in women with and without cervical neoplastic disease. Vaccine 24:26–34. doi:10.1016/j.vaccine.2006.05.026


Mol Biol Rep 39. Bao YP, Li N, Smith JS, Qiao YL (2008) Human papillomavirus type distribution in women from Asia: a meta-analysis. Int J Gynecol Cancer 18:71–79 40. Lippman SA, Sucupira MC, Jones HE, Luppi CG, Palefsky J, Van de Wijgert JH, Oliveira RL, Diaz RS (2010) Prevalence, distribution and correlates of endocervical human papillomavirus types in Brazilian women. Int J STD AIDS 21:105–109. doi:10. 1258/ijsa.2009.008436 41. Fernandes JV, Meissner RV, Carvalho MG, Fernandes TA, Azevedo PR, Sobrinho JS, Prado JC, Villa LL (2010) Prevalence of human papillomavirus in archival samples obtained from patients with cervical pre-malignant and malignant lesions from Northeast Brazil. BMC Res Notes 3:96. doi:10.1186/1756-0500-3-96 42. Oliveira LH, Ferreira MD, Augusto EF, Melgaco FG, Santos LS, Cavalcanti SM, Rosa ML (2010) Human papillomavirus genotypes in asymptomatic young women from public schools in Rio de Janeiro, Brazil. Rev Soc Bras Med Trop 43:4–8. doi:10.1590/ S0037-86822010000100002 43. Lorenzato F, Ho L, Terry G, Singer A, Santos LC, Lucena BR et al (2000) The use of human papillomavirus typing in detection of cervical neoplasia in Recife (Brazil). Int J Gynecol Cancer 10:143–150 44. Freitas TP, Carmo BB, Paula FD, Rodrigues LF, Fernandes AP, Fernandes PA (2007) Molecular detection of hpv 16 and 18 in cervical samples of patients from Belo Horizonte, Minas Gerais, Brazil. Rev Inst Med Trop 49:297–301. doi:10.1590/S003646652007000500005 45. Baldez da Silva MF, Chagas BS, Guimaraes V, Katz LM, Felix PM, Miranda PM, Lima AA, Arraes LC, Martins DB, Lima Filho JL et al (2009) HPV31 and HPV33 incidence in cervical samples from women in Recife, Brazil. Genet Mol Res 8:1437–1443 46. Oliveira-Silva M, Lordello CX, Zardo LMG, Bonvicino CR, Moreira MAM (2011) Human papillomavirus in Brazilian women with and without cervical lesions. Virol J 1:4–9. doi:10. 1186/1743-422X-8-4 47. Mun˜oz N (2000) Human papillomavirus and cancer: the epidemiological evidence. J Clin Virol 19:1–5. doi:10.1016/S13866532(00)00125-6 48. Van Den Brule AJC, Pol R, Fransen-Daalmeijer N et al (2002) GP5?/6? PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes. J Clin Micorbiol 40(3):779–787 49. Levi JE, Kleter B, Quint WGV et al (2002) High prevalence of human papillomavirus (HPV) infections and high frequency of multiple HPV genotypes in human immunodeficiency virusinfected women in Brazil. J Clin Microbiol 40:3341–3345. doi:10.1128/JCM.40.9.3341-3345.2002 50. Gonc¸alves MA, Massad E, Burattni MN, Villa LL (1999) Relationship between human papillomavirus (HPV) genotyping and genital neoplasia in HIV-positive patients of Santos City, Sa˜o Paulo. Brazil Int J STD AIDS 10:803–807. doi:10.1258/0956462991913583 51. Almonte M, Albero G, Molano M, Carcamo C, Garcı´a PJ, Pe´rez G (2008) Risk factors for human papillomavirus exposure and cofactors for cervical cancer in Latin America and the Caribbean Vaccine. 26(Suppl 11):L16–L36. doi:10.1016/j.vaccine.2008.06.008 52. Drain PK, Holmes KK, Hughes JP et al (2002) Determinants of cervical cancer rates in developing countries. Int J Cancer 100:199–205. doi:10.1002/ijc.10453 53. Samoff E, Koumans EH, Markowitz LE, Sternberg M, Sawyer MK, Swan D et al (2005) Association of Chlamydia trachomatis with persistence of high-risk types of human papillomavirus in a cohort of female adolescents. Am J Epidemiol 162:668–675. doi:10.1093/aje/kwi262 54. Silins I, Ryd W, Strand A, Wadell G, To¨rnberg S, Hansson BG et al (2005) Chlamydia trachomatis infection and persistence of



56. 57.














human papillomavirus. Int J Cancer 116:110–115. doi:10.1002/ ijc.20970 Deluca GD, Basiletti J, Schelover E, Va´squez ND, Alonso JM, Marı´n HM, Lucero RH, Picconi MA (2011) Chlamydia trachomatis as a probable cofactor in human papillomavirus infection in aboriginal women from northeastern Argentina. Braz J Infect Dis 15(Suppl 6):567–572. doi:10.1016/S1413-8670(11)70252-5 Franceschi S, Smith JS, Van Den Brule A et al (2007) A crosssectional study. Sex Transm Dis 34:563–569 Rodrigues E, Rodrigues L, Portugal V, Rodrigues N, Na´poles S, Casanova C (2011) Anogenital warts in children: the importance of a multidisciplinary approach. Acta Med Port 24:367–370 Cricca M, Venturoli S, Leo E et al (2009) Disruption of HPV 16 E1 and E2 genes in precancerous cervical lesions. J Virol Methods 158:180–183 Ramanakumar AV, Goncalves O, Richardson H et al (2010) Human papillomavirus (HPV) types 16, 18, 31, 45 DNA loads and HPV-16 integration in persistent and transient infections in young women. BMC Infect Dis 10:326 Fernandes APM, Gonc¸alves MAG, Duarte G, Cunha FQ, Simo˜es RT, Donadi EA (2005) HPV16, HPV18, and HIV infection may influence cervical cytokine intralesional levels. Virology 334:294–298. doi:10.1016/j.virol.2005.01.029 Fontaine J, Hankins C, Money D, Rachlis A, Pourreaux K, Ferenczy A, Coutle´e F (2008) Human papillomavirus type 16 (HPV16) viral load and persistence of HPV-16 infection in women infected or at risk for HIV. J Clin Virol 43:307–312. doi:10.1016/ j.jcv.2008.07.013 Sjoeborg KD, Trope´ A, Lie AK, Jonassen CM, Steinbakk M, Hansen M, Jacobsen MB, Cuschieri K, Eskild A (2010) HPV genotype distribution according to severity of cervical neoplasia. Gynecol Oncol 118:29–31. doi:10.1016/j.ygyno.2010.03.007 Tamim H, Finan RR, Sharida HE, Rashid M, Almawi WY (2002) Cervicovaginal coinfections with human papillomavirus and Chlamydia trachomatis. Diagn Microbiol Infect Dis 43:277–281 Wu Y, Chen Y, Li L, Yu G, Zhang Y, He Y (2006) Associations of high-risk HPV types and viral load with cervical cancer in China. J Clin Virol 35(3):264–269 Chopjitt P, Ekalaksananan T, Pientong C, Kongyingyoes B, Kleebkaowc P, Charoensri N (2009) Prevalence of human papillomavirus type 16 and its variants in abnormal squamous cervical cells in Northeast Thailand. Int J Infect Dis 13:212–219. doi:10.1016/j.ijid.2008.06.017 Syrjanen K, Hakama M, Saarikoski S, Vayrynen M, Yliskoski M, Syrjanen S, Kataja V, Castren O (1985) Natural history of cervical human papillomavirus (HPV) infections based on prospective follow-up. Br J Obstet Gynaecol 92:1086–1092 Smith JS, Mun˜oz N, Herrero R, Eluf-Neto J, Ngelangel C, Franceschi S, Bosch FX, Walboomers JM, Peeling RW (2002) Evidence for Chlamydia trachomatis as a human papillomavirus cofactor in the etiology of invasive cervical cancer in Brazil and the Philippines. Infect Dis 185:324–331 Bhatla N, Puri K, Joseph E, Kriplani A, Venkateswaran KI, Sreenivas V (2013) Association of Chlamydia trachomatis infection with human papillomavuris (HPV) & cervical intraepithelial neoplasia—a pilot study. Indian J Med Res 137:533–539 Safaeian M, Quint K, Schiffman M, Rodriguez AC, Wacholder S, Herrero R, Hildesheim A, Viscidi RP, Quint W, Burk RD (2010) Chlamydia trachomatis and risk of prevalent and incident cervical premalignancy in a population-based cohort. J Natl Cancer Inst 102:1794–1804. doi:10.1093/jnci/djq436 Di Felice V, David S, Cappello F, Farina F, Zummo G (2005) Is Chlamydia l heat shock protein 60 a risk factor for oncogenesis? Cell Mol Life Sci 62:4–9

Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil.

Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. High-risk human papillomavirus (HR-HPV) is considered t...
402KB Sizes 0 Downloads 0 Views