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

Human papillomavirus (HPV) is a large group of epitheliotropic viruses of more than 160 different types. HPVs are very successful infectious agents – inducing 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[3–5], 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[6–9]. 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.

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%)[19–21].

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].

HPV-16 was the most prevalent type observed in our study (14.3%), followed by HPV-52 and HPV-45. The most prevalent HPV types described in women with normal cytology are HPV-16, -58, -31, -18, -45 and -52, however, it is well documented that this prevalence is lower than that observed among women with HSIL (high-grade lesions) and SCC (squamous-cell carcinoma)[26]. HPV-16 prevalence in our population was higher than that described in Natal (Northeast Brazil) (12.7%)[27] and lower than that observed in Rio Grande do Sul (19.6%), Rio de Janeiro (19%), São Paulo (18.2%) and Amazonas (31.8%)[18–21].

Phylogenetic analysis based on E6 sequences of eight HPV-16 isolates allowed the identification of seven European and only one non-European variants. A previous study realized in Belém (Northern Brazil) exhibited a higher prevalence of Asian-American (43.2%) and European (42%) variants among women with cervical cancer[28]. Previous data suggests that women harboring non-European HPV-16 variants have an 2- to 9- fold increased risk of cervical cancer[29, 30], however, it has been reported that HPV-16 European variants persists longer in white women[31–33].

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 follow-up 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.

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.