Prior studies have investigated the role of microorganisms like Ct that cause a chronic inflammation as a potential risk factor in the transmission and persistence of HPV, as well as contributing to the progression of cervical carcinogenesis [11, 12]. Our study population had a 15.6% frequency of Ct infection, as assessed by analysis of the liquid preservative media utilized for cervical cancer screening. However, we did not detect an association between p16/Ki-67 expression and Ct in women with CIN.

In Brazil, four studies conducted in different states, one in Goiás, two in Paraná and one in Rio Grande do Norte showed a Ct prevalence of 10.9, 10.7, 12.7 and 10.9%, respectively, also compatible with results in the present study. A study conducted in the city of Manaus had a higher prevalence of 20.7%, and may be explained by regional or socioeconomic reasons [4, 13–15].

Ct is not an infection subjected to mandatory reporting in Brazil. Additionally, there are no specific programs implemented by the Ministry of Health to encourage screening for this infection. Because it is asymptomatic in most women, it is likely to be under-diagnosed. As such, its true prevalence in this country remains ambiguous. Asymptomatic untested women are obviously not treated. This may account for the high number of positive women in our study. The Ct prevalence estimates must be interpreted within the context of national and cultural differences, of sexual behavior and the health system. Brazil is a very large country whose population has different cultural and socioeconomic development levels, which most likely explains the reported regional differences [16–18].

Age was the only demographic variable in this study that was associated with Ct infection. Women aged 35 to 45 years had the highest likelihood of being Ct positive. This observation differs from studies conducted in the United States, where the highest prevalence was among women aged 15 to 24 years. In the United States screening programs for Ct are available for women aged 25 years or younger, and for women at risk for this infection. Increased testing and treatment may explain the overall lower prevalence, as well as the lower rate found among women aged 25 years and older in the United States as compared to Brazil [12].

When assessing other demographic factors, such as years of schooling, marital status and smoking, there was no positive association with Ct presence, similar to what has been reported in other national studies [13].

The analysis of sexual and obstetric history reveals that only the number of lifetime partners was associated with being positive for Ct. Women with more than 10 partners had a four fold higher rate of positivity than did women with 1–4 partners. This is explained by the fact that Ct is a sexually transmitted disease, and infection is associated with high-risk sexual behavior. This association was also demonstrated in a prior Brazilian study [13].

Because it is an infection in which 90% of infected women are asymptomatic but, nevertheless, can have serious consequences for both reproductive and fetal health, Ct has been considered the non-viral STD with the highest burden for public health. Several lines of evidence suggest that screening for Ct is cost effective when its prevalence is above 3%. The few Brazilian studies conducted until now clearly justify the implementation of systematic screening of women for this infection [18].

We did not observe an association between abnormal cervical cytology and the presence of Ct. An analysis of women with abnormal cytological results revealed a higher prevalence of Ct infection in women with atypical lesions (ASCUS), followed by low-grade lesions (LSIL), in which almost 20% were Ct positive. These observed rates were similar to other Brazilian studies with women in the same age group [14].

In women who underwent a colposcopy examination a greater prevalence of Ct positivity was found in women with CIN1. However, the lack of statistical significance may be due to the low number of biopsies conducted after the cytology results. No prior Brazilian studies have assessed this association.

Our study was limited by the lack of HPV testing, due to the limited amount of residual material available. It is necessary, however, to take into consideration the “status” of HPV infection and the natural history of cervical cancer when determining the role of Ct as a co-factor in CIN or invasive cancer. HPV infection is a highly prevalent sexually transmitted viral infection, whereas Ct is the most commonly sexually transmitted bacterial infection, and co-infections with both microorganisms are quite common. No association was found in this study between Ct infection and cytology and positive biopsies for CIN, presumed positive for HPV. These results differ from other studies that found a positive association between HPV and Ct detected by serology [19, 20],. However it was similar to other studies using the same methodology that did not find any association between Ct DNA detection and subsequent risk of CIN2/3 [21, 22].

This study utilized p16/Ki-67 expression as a marker of transforming HPV infections. The study was designed to explore the possible association between a Ct infection and consequences of a persistent HPV infection by the use of p16/Ki-67, instead of testing for HPV DNA. In most cases, HPV DNA is unable to distinguish between persistent and transient infection. We conclude that there is no association between Ct and p16/Ki-67 positivity in women with abnormal cytology [22].

Most women with cytological findings were negative for p16/Ki-67 expression. However, approximately one fourth of the abnormal cytological results were positive for p16/Ki-67 double staining, a finding compatible with publications demonstrating that women with p16/Ki-67 positivity were also the women infected with HPV. Women with HSIL had a higher positivity prevalence (37%) than did women with LSIL. p16/Ki-67 positivity is therefore associated with a threefold higher prevalence in women with a high-grade lesion. These results were comparable to a recent study assessing the use of p16/Ki-67 in a sample from women referred for colposcopy and finding that this marker indeed had a higher positivity in women with high-grade lesions [23, 24].

Evaluation of the association of p16/Ki-67 expression with histopathological results also revealed a higher rate of p16/Ki-67 positivity in women with CIN2 or more advanced lesions. p16/Ki-67 positivity increased with the seriousness of the lesion. When the biopsy result revealed chronic cervicitis, approximately 20% of the cases were positive; positivity was higher with CIN1 (25%) and, in women with CIN2 or more advanced lesions, almost 70% of the cytological preparations were positive for the double stain. The results are consistent with other studies in which p16/Ki-67 activity in a colposcopy population had similar associations [24].

A possible relationship between Ct and either cytological or histopathological alterations characteristic of HPV infection remains to be clarified. An association between Ct and a persistent HPV infection may occur preferentially in high-risk women, while a Ct infection may be associated with the clearance of an HPV infection in women who develop an intense inflammatory response to Ct. The identification of women in whom a concurrent Ct infection may be beneficial or detrimental to HPV persistence and progression remains to be clarified [21, 25, 26].