Setting
This study was conducted in the Central Denmark Region (CDR), which covers 23% of the Danish population [12]. Like the rest of the country, this region has been covered by a nationwide, organized, free-of-charge cervical cancer screening program since the late 1990s [13, 14].
Currently, 23–49-year-old women are invited for cervical cancer screening every third year, 50–64-year-old women every fifth year. A liquid-based cervical cytology sample is taken at their GP (GP-collected sample). The primary screening method is microscopic examination of the cytology sample for 23–59-year-old women; a primary HPV-DNA check-out test for 60–64-year-old women [13].
In the CDR, the Department of Pathology at Randers Regional Hospital handles all cytology and HPV analyses. As per routine, GPs obtain the sample using a cervical brush and the brush head is placed in 10 ml SurePath medium (BD Diagnostics, Burlington, NC) and mailed to the Pathology Department for further processing and testing according to guidelines, which for women aged 30–59 is microscopic examination. Women with high-grade cytological lesions (threshold ASC-H or HSIL or, AGC, or AIS or malignant tumor cells) are referred directly for colposcopy. Women diagnosed with ASC-US undergo routine reflex HPV DNA triage testing using the cell pellet from 1 mL SurePath medium. ASC-US/HPV-positive women are referred for colposcopy within 3 months. ASCUS/HPV-negative women are referred back to the routine screening program. Women with LSIL are monitored by repeated cytology testing [13].
For HPV DNA analyses, the Cobas 4800 assay (Roche Diagnostic, Switzerland) is used. This test is a real-time PCR fully automated method separately detecting HPV16, HPV18, and 12 other high-risk HPV types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) using the β-globin gene as an extraction and amplification control [15].
Inclusion of participants
Paired GP-collected samples and cervico-vaginal self-samples were obtained from 30 to 59-year-old women diagnosed with low-grade cytological lesions (ASC-US) within the screening program. As per routine, GP-collected samples from these women are all analyzed by both microscopy and HPV, which is not the case for younger and older women, or women with other cytological diagnoses [13].
Between June 2015 and December 2016, eligible women were consecutively identified daily through patient lists provided by the Department of Pathology. They received written information about the study and then contacted the investigator for oral information if they wanted to participate. A written and signed informed consent form had to be mailed back to the investigator before inclusion.
The exclusion criteria were pregnancy, giving birth less than < 3 months previously, and collecting the self-sample after colposcopy.
Self-sampling collection, storage, and analysis
The women were sent a self-sampling kit comprising a brush device (Evalyn® Brush, Rovers Medical Devices B.V., Oss, Netherlands), written and picture-based user instructions showing how to collect the cervico-vaginal sample using the device, a questionnaire, and a pre-addressed return envelope.
Upon arrival at the laboratory, the brush head was placed in 10 ml of SurePath medium (BD Diagnostics, Burlington, NC), stored overnight at 4 °C, and then vortexed for 5 min. A 6.4 ml volume of the self-sample material was centrifuged at 3000 x RPM for 20 min at room temperature. After centrifugation, with supernatant removed, the cell pellet was placed in 1 mL 25% ethanol-buffered (TRIS) and stored at -80 °C, until further processing. A volume of 6.4 ml was chosen to adjust for the material volume used for cytology examination performed on the GP-collected samples.
The self-samples were thawed overnight at 4 °C before the day of analysis. For analysis, the self-sample (1 ml volume) was vortexed for 15 s before being placed in test tubes, being the starting point for the HPV testing. Each run included four water samples to measure contamination. All HPV testing was performed following the manufacturers’ instructions using a protocol without a sample pre-heating step which was also the case for the GP-collected samples. The investigator and the laboratory personnel performing the HPV testing were blinded to the HPV results of the GP-collected samples.
Questionnaire
The questionnaire included three questions on self-sampling experience and one on the clarity of the user instructions. To avoid low frequencies, the responses were grouped into three groups “Agree” (totally agree or agree), “Disagree” (disagree or totally disagree), and “Do not know”. Multiple response answers were not allowed. The women were asked to record the date they had taken the self-sample, whether they had had sexual intercourse in between the two samplings, and their partner status (i.e., regular partner). Open feedback was possible. The data were double-entered into REDCap [16].
Data on test results
Information on the HPV self-sample test results was obtained through patient lists provided by the Department of Pathology. Data on the GP-collected HPV test results and the histological results were retrieved from the nationwide Danish Pathology Data Bank (DPDB), which has been complete since the late 2000s [17]. Histological results were available only for women who had tested HPV-positive in their GP-collected sample and were classified using the CIN classification, which was grouped into normal, CIN, CIN1, and CIN2+ (including CIN2, CIN3/AIS, and carcinoma). To ascertain the histological result, the most severe diagnosis was used if more were available.
Sample size
The study was designed to estimate a 95% confidence interval (CI) with a width of +/− 5%. With an expected 86% sensitivity and 85% specificity of HPV detection (high-risk HPV types) using the Evalyn Brush and a PCR-based HPV DNA test, a minimum of 198 women had to be included [10].
Statistical analyses
The HPV concordance between the paired samples was assessed using the Kappa statistic (Cohen’s Kappa, κ) and defined as “Poor” (κ ≤ 0.20), “Fair” (0.21 ≤ κ ≤ 0.40), “Moderate” (0.41 ≤ κ ≤ 0.60), “Good” (0.61 ≤ κ ≤ 0.80), or “Very good” (κ ≥ 0.81) [18]. The overall percentage of agreement between the paired samples was calculated as the proportion of concordant sample sets divided by the total number of samples. We calculated the sensitivity and specificity of HPV detection in the self-samples with corresponding 95% CIs based on the binomial distribution using the GP-collected samples as reference standard. When assessing the HPV concordance regarding specific genotypes (HPV16/18 and HPV other), the genotypes were defined as “HPV16/18” (HPV16 and/or HPV18 including those having co-infections with other HPV types) and “HPV other” (HPV of other types including those having co-infections with HPV16/18). Concordance was determined as at least one identical genotype in both samples; discordance was determined as no genotype similarities. For continuous data, medians and interquartile ranges (IQR) were calculated; the Mann Whitney rank sum test was used to test for differences. Descriptive statistics (proportions and 95% CIs) were used to measure the women’s accept of self-sampling. The χ2-test was used to test for differences in categorical data.
P-values < 0.05 were considered statistically significant. All statistical analyses were performed using STATA, version 14 (STATA College).
Ethical approval
The study was approved by the local Ethical Committee of the Central Denmark Region (journal no.: 1–16–02-209-15) and by the Danish Data Protection Agency (journal no.:1–10–72-69-15).
