Study group
The examined group consisted of 197 Polish female participants and their matched partners. Women were qualified to the group based on two premises: current or previous pathology of the cervix (including cancer) and HPV detected in the smear taken during gynecological examination. If a woman fulfilled these criteria, then her partner was included automatically to the study group. Women were diagnosed during routine cervical cytology or in the framework of preventive screening programs. The examinations were performed by specialists (gynecologists and oncologists) in “Boramed” Medical Center in Warsaw and two Clinics of Poznan University of Medical Sciences: Department of Gynecology and Department of Gynecological Oncology in Poznan, between years 2014–2016. The brush smears were taken with the use of Cervex-Brush® (Rovers, Oss, Holand) from the endocervix, ectocervix or cervical transformation-zone from women with CIN I–II or from the vaginal vault in patients after hysterectomy, performed previously to treat cervical cancer. From each patient’s partner the swab from the foreskin was taken with the use of FLOQSwabs (Copan, Brescia, Italy). Ultimately, 71 women with previous history of cervical cancer were included, out of which 18 had cancer detected in the year of material collection and 53 had cancer detected in the past (24 - one year before sampling, 22 – from two to nine years earlier and 7 – more than 10 years earlier). All of them were successfully treated for cervical cancer and during the study they were free of disease, with no symptoms of either recurrence or other malignancy. The remaining, 126 women had CIN I or CIN II detected (Additional file 1: Table S1).
In parallel, from each woman and man, the oropharyngeal swabs from tonsillar or tonsillar-lingual region were obtained with the use of FLOQSwabs. Oropharyngeal HPV positive patients were consulted at the Department of Otolaryngology, Head and Neck Surgery, Poznan University of Medical Sciences, Poznan, Poland. None of them presented any suspicious lesions in this anatomical site at the moment of the examination.
All samples were collected on ThinPrep liquid medium (Hologic Inc., Marlborough, USA). Each sample was designated for HPV genotyping with the same method. The experiments were performed in accordance with relevant guidelines and regulations, approved by the Ethics Review Board of Poznan University of Medical Sciences (decisions no. 21/13 and 75/15) and the written, informed consent was obtained from all participants.
DNA isolation & HPV genotyping
DNA was isolated from 1 ml of hypercellular suspension containing gynecological or laryngological smear collected on ThinPrep liquid medium. The suspension was first centrifuged for 15 min at 13.000 rpm in order to separate cellular material from the brush or swab. Then, the supernatant was discarded and the pellet was suspended in 200 μl of 1 x PBS. Further, cell lysis, DNA precipitation, binding to the membrane and elution were performed according to procedure of NucleoSpin® Blood Kit (Macherey Nagel, Duren, Germany).
The HPV genotyping was performed with the use of Anyplex™ II HPV28 Detection system (Seegene, Seoul, Korea), which detects simultaneously 19 high-risk HPVs (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9 low-risk HPVs (6, 11, 40, 42, 43, 44, 54, 61, 70) in a single specimen. This system, targeting L1 gene, is based on multiplex real-time PCR with high sensitivity and specificity due to the utilization of DPO™ (Dual-Priming Oligonucleotides) and TOCE™ (Tagging Oligonucleotide Cleavage and Extension) technologies. The reaction mixture of 20 μl included 1 x HPV28 primer mix A or B and 1 x Anyplex Master Mix. The real time PCR reaction was performed on a CFX96 Real-Time PCR thermocycler (Bio-Rad, Hercules, USA) with the following steps: 50 °C/4 min., 95 °C/15 min. and 50 cycles of 95 °C/30 s., 60 °C/60 s., 72 °C/30 s. Melting curve analysis was performed from 55 to 85 °C. The internal control - Beta-Globin (HBB) gene - was simultaneously amplified to ensure that the DNA amount is sufficient and that the PCR reaction is efficient. The positive (for each HPV type) and negative controls were included during each PCR run. The results were analyzed using the Seegene Viewer v. 2.0 program and were considered as valid, when internal control was present in each sample. A positive result (+++/++/+) indicated the presence of HPV DNA, while the negative result (−) indicated the absence of the HPV virus. The detection limit of the applied test is 50 copies of HPV per reaction.
The statistical analysis
Chi - square exact test was used to evaluate the significance of given HPV/s in relation to women’s health status. It was performed with the application of free, online tool and the p value < 0.05 was considered as statistically significant.