Prevalence of human papillomavirus infection and phylogenetic analysis of HPV-16 E6 variants among infected women from Northern Brazil

Background The main cause of cervical cancer in the world is high risks human papillomavirus infection (mainly represented by HPV-16 and HPV-18), that are associated to the development of malign transformation of the epithelium. HPV prevalence exhibits a wide geographical variability and HPV-16 variants have been related to an increased risk of developing cervical intraepithelial lesion. The aim of this study was to describe DNA-HPV prevalence and HPV-16 variants among a women population from Northern Brazil. Methods One hundred and forty three women, during routine cervical cancer screening, at Juruti Project, fulfilled an epidemiological inquiry and were screened through a molecular HPV test. HPV-16 variants were determined by sequencing the HPV-16 E6 open reading frame. Results Forty two samples were considered HPV positive (29.4%). None of those had abnormal cytology results. HPV prevalence varied between different age groups (Z(U) = 14.62; p = <0.0001) and high-risk HPVs were more frequent among younger ages. The most prevalent type was HPV-16 (14%) and it variants were classified, predominantly, as European (87.5%). Conclusions HPV prevalence in our population was higher than described by others and the most prevalent HPV types were high-risk HPVs. The European HPV-16 variant was the most prevalent among HPV-16 positive samples. Our study reinforces the fact that women with normal cytology and a positive molecular test for high-risk HPVs should be submitted to continuous follow up, in order to verify persistence of infection, promoting an early diagnosis of cervical cancer and/or its precursors.


Background
Human papillomavirus (HPV) is a large group of epitheliotropic viruses of more than 160 different types. HPVs are very successful infectious agentsinducing chronic infections that have no systemic sequelae, rarely killing the host and shedding large amounts of infectious particles for transmission [1]. However, a dozen of the 40 HPV types that infect the human body are associated with probable or definite oncogenic risk [2].
High-risk HPVs (hrHPV), which includes HPV type 16 (HPV- 16), are etiological agents of cervical cancer [3][4][5], being the persistent of hrHPV infection, known as the integrated HPV DNA into host genome, in the presence of other environmental and host factors, a necessary step for the development of high-grade cervical intraepithelial neoplasia and invasive cervical cancer [6][7][8][9]. Cervical cancer is the third most common cancer in women and more than 85% of the global burden occurred in developing countries, causing 275,000 deaths in 2008, with 31,400 of those deaths occurring in Latin America [10].
HPV molecular variants are defined taxonomically based on L1 coding region of the DNA sequence [11]. The comparative nucleotide sequence analysis, that includes ORF E6, have shown that HPV-16 variants, which differ in nucleotide sequence by no more than 2% in the coding regions and until a 5% in the non coding region, evolved into six major phylogenetic branches: European (E), Asian (As), Asian-American (AA), African-1 (Af1), African-2 (Af2) and North-American (NA1) [5,12,13]. Instead only a small portion of HPV-16 infections persist, several studies from Europe and the Americas, based principally on the sequencing of E6 and/or the LCR, had suggested that HPV-16 variants can influence viral persistence and the development of cervical cancer [14,15].
The aim of this study was to describe HPV infection prevalence and related epidemiological variables, as well as, the HPV-16 variants circulating in a population of women from Northern Brazil.

Results
Socio-demographic data was obtained for a total of 143 women. Data analysis revealed that these women had 34 years median age (SD ± 13.74 y; 95% CI 32-36), were married (48%), had basic education as the main school level (58%), had housewife occupation (35%), were not exposed to alcohol (58%) or to tobacco (86%), had no history of STD's (Sexual Transmitted Diseases) (84%), had 16 years old (SD ± 2.9 y; 95% CI 15.5-16.5) as the median age of the first sexual intercourse and had a median of 10 sexual partners (SD ± 32.3 partners; 95% CI 4.3-15.3). The higher median observed on the variable number of sexual partners was probably influenced by data related to women that referred to be sex workers (n = 7). When these data was removed from analysis, observed media of sexual partners was 5.5 partners (SD ± 8.6 partners; 95% CI 4-7). There was no observed statistical association between the described variables and the occurrence of HPV infection in our population (p > 0.05) ( Table 1).
Phylogenetic analysis was performed for a total of 8 samples among the HPV-16 positive samples (73%). The phylogenetic tree segregated into four major branches that could be recognized as European (EUR), Asian (As), African (including Afr1 and Afr2) and American (including Asian-American and North-American) lineages. The HPV-16 variants were classified as European (87.5%) and American (12.5%) ( Figure 2).

Discussion
In the present study we found a 29.4% HPV prevalence between women without abnormal cytology screened by Pap Smear Test, at Juruti, Northern Brazil. This prevalence was higher than that described by SanJosé et al [16], in meta-analysis of a systematic literature review of HPV prevalence among women with normal cervical cytology worldwide, where global and South America prevalence's were 10.4% and 12.3%, respectively. As well as, that described by Bruni et al [17], in meta-analysis of one million women with normal cytological findings, where global and South America prevalence's were 11.7% and 15.3%, respectively. A broader range of HPV prevalence (from 10.4% to 24.5%) has been described in Brazilian studies including women with normal cytology. Our result was similar to that described by the Latin America Screening Study (24.5%) [8] and to data related to Amazonas State (29.2%) [18]. However, it was lower than described in others Brazilian studies including women populations from Rio Grande do Sul (34%), São Paulo (58.4%), Distrito Federal (62%) and Rio de Janeiro (63.6%) [19][20][21].
A marked variation of HPV prevalence among age groups was observed in our population. Women with less than 25 years and comprehend between 25-34 years were more predisposal to have a positive HPV-DNA test, including for hrHPV types. Some studies in Central and South America have demonstrated a first peak prevalence of HPV infection in women below age 25 and a second peak after age 55 [22,23] which corroborates our data. The first peak suggest that women acquire infection in the first few years after become sexually active and the second peak suggest that middle-age women harbor long-term HPV persistent infections or new incident infections, which might also reflect changes in hormonal or immunological status [24,25].
Genital HPV infection is one of the most common sexually transmitted infections worldwide. It has been estimated that approximately 10% of women worldwide with normal cytological findings carry a detectable cervical HPV infection, depending on the HPV testing technology, study size, and the age groups and geographical region studied [34]. It is necessary to emphasize that women with one positive DNA test for HPV-16, as well as, for others hrHPV, need to be followed-up to confirm persistent infection, to monitor and to avoid late diagnosis of possible cervical cancer and its precursors, since it is well documented that HPV-16 and HPV-18 account for 70% of all cervical cancer cases worldwide. This will be only achieved through consistent cervical screening associated with alternative screening tools as molecular techniques.
A major limitation of the present study is represented by the modest sample size and by the absence of a followup period to confirm persistence of HPV infection or clearance through timeline, as well as, the lack of ethnicity data, which was expected to be associated with HPV-16 variants. Another limitation was related with no amplification of internal control (β-globin gene) between 23 cervical samples on the Linear Array HPV Genotyping Test®. Those results were probably caused by inadequate sample cellularity (yield DNA specimens), as the DNA extraction technique was held as a constant, with DNA isolated using the same commercial kit.

Conclusion
HPV prevalence (29.4%) in a population of women with normal cytology from Juruti (Northern Brazil) is higher than described by others with the most prevalent HPV types being hrHPVs (HPV-16, -52 and -45). The European HPV-16 variant (87.5%) is the most prevalent among  HPV-16 positive samples. HPV-16 is defined as a human carcinogenic agent and is associated with cervical cancer among carriers. Despite being descriptive, the present study reinforces the necessity to follow up women with normal cytology, meanwhile, with a positive molecular test for hrHPVs, to determine persistence of infection and to promote early diagnosis of cervical cancer and its precursors.

HPV cytological and molecular analysis
After samples collection cytobrush was immersed into 15 ml falcon tubes with 3ml of phosphate buffer saline (PBS: 137 mM NaCl, 10 mM Phosphate buffer, 2,7 mM KCl, pH 7.4) and kept at 4ºC according to the manufacturer's instructions. At the HPV laboratory the falcon tubes were submitted to manual agitation, followed by cytobrush discard and centrifugation of the liquid per fifteen minutes (1200 rpm). Supernatant was discarded and cell pellet was resuspended in 3 mL of PBS solution.
The conventional cytology results were classified according to the recommendations of Brazilian Ministry of Health and Brazilian Society of Cytology which is based on Bethesda's definition [35] and were performed at Juruti by the local routine professionals. Cervical specimen DNA extraction was performed according to the Linear Array HPV Genotyping Test® kit protocol (Roche Molecular Systems, Alameda, CA), which can detect until thirty seven distinct HPV types in a unique reaction, which includes high and low risk and multiple infections. Only the strips that had an internal positive control test (β-globin gene) were considered proper to genotyping analysis.

Statistical analysis
Data were analyzed with the software BioEstat 5.3 [36]. Fisher test was used to compare categorical variables between two groups (HPV-infected and non-infected women). Wilcoxon-Mann-Whitney test and G test were used to verify a possible association between age and HPV prevalence. After plotting HPV prevalence and hrHPV prevalence among HPV-infected women, in each age-group, a polynomial fit was assessed as the best model for logistic regression.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions BPTL performed the genotyping of HPV variants, data and statistical analysis and lead drafting of the manuscript. ESCJ participated in the HPV variant analysis and performed phylogenetic analysis. WAM is member of the HPV at Instituto Evandro Chagas and is one of the researchers of the Juruti Project. FP participated in the study design and data interpretation. CGMS participated in the study design and data interpretation. RVDS was responsible for study design, for the initial HPV-DNA investigation, is a researcher and member of the HPV group at Instituto Evandro Chagas. All authors read and approved the final manuscript.