Feasibility and acceptance of cervicovaginal self-sampling within the German National Cohort (Pretest 2)

This cross-sectional study took place in two study centers (Hamburg and Hanover) of the GNC during Pretest 2 (September 2012 to December 2012) (see Ahrens et al. in this issue) as an add-on module to the main program, which is described by Wichmann et al. [11]. Briefly, 18 study centers in Germany will interview and examine 200,000 men and women randomly selected from the population register. The examinations and interviews cover cardiovascular, metabolic, neurological and infectious diseases. In concordance with the GNC, the age structure of the study population in Pretest 2 was aimed at 10 % for 20–29 and 30–39-year-olds and 26.7 % for each of the age groups 40–49, 50–59 and 60–69 years [11].

The analysis of this feasibility study is limited to Pretest 2 participants randomly sampled through the population registration office. Study participation was offered to all female participants recruited for Pretest 2 (n = 162) and accessible for add-on modules. Pregnant women and women within 30 days after childbirth were excluded. Informed consent for this additional study on collecting a cervicovaginal lavage was obtained during the participant’s visit to the study center. After receiving the sampling kit at the study center, self-sampling was done at home. The tool applied (second generation Delphi Screener^TM, Delphi Bioscience, BV Scherpenzeel, The Netherlands) is a sterile instrument with 3 ml of physiological saline. After inserting the device into the vagina, followed by pushing a button at the end of the Screener, the lavage liquid is released into the vagina (near the cervix). The fluid is automatically reabsorbed by the device during removal from the vagina after the button is released. The participants then transferred the lavage into a transportation tube and mailed it to the laboratory (Charité, Berlin) where the HPV status of the samples was determined. Results were regularly reported back to the participants. After completion of recruitment, testing for further microbes was done at the German Cancer Research Center, Heidelberg, using sexually transmitted infection profiling (STIP). Participants whose samples indicated the presence of Neisseria gonorrhoeae infection were recommended to immediately contact a gynecologist for confirmation by clinical diagnosis. If sample analysis by STIP resulted in any other suspicious findings participants received a recommendation to see their gynecologist.

The questionnaire contained a question on sexual history (ever had sexual intercourse: yes/no) and assessed the acceptability of sampling as consent (i.e. yes/partly/no) to the following statements: “The acquisition of a vaginal lavage at home was acceptable for me.”, “It was easy to self-sample the specimen.”, “The sampling was user-friendly.”, “The sampling was physically uncomfortable.”, “I consider sampling of a vaginal lavage a violation of privacy.” and “It is more acceptable to sample a vaginal lavage at home than at a study center.”. Demographic data and medical history were collected during the main interview of the GNC.

Total DNA was extracted using the Qiagen DNA mini kit (Hilden, Germany) and a broad spectrum GP5 + /GP6 + -based polymerase chain reaction (PCR) including beta-globin was conducted as described [12] followed by Luminex^®-based HPV genotyping (multiplex HPV genotyping, Luminex, Austin TX [13]). For DNA sample validation a PCR for human beta-globin DNA was performed and products were separated and read on 2 % agarose gels. Only samples showing a specific product were considered to be DNA positive. The detection of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43, 45, 51, 52, 53, 54, 56, 57, 58, 59, 66, 68, 70, 72, 73, 82 and 90 was carried out using self-produced multiplex master mixes.

The STIP was carried out as previously described [10]. Briefly, STIP is a multiplex PCR for microbial target sequences followed by hybridization of the biotinylated amplimers to specific probes on fluorescent beads (xMAP technology, Luminex^®). The STIP is able to simultaneously identify Atopobium vaginae, Candida albicans, C. glabrata, C. krusei, Chlamydia trachomatis, Gardnerella vaginalis, herpes simplex virus (HSV) 1, HSV 2, Mycoplasma genitalium, M. hominis, M. pneumoniae, M. spermatophilum, Neisseria gonorrhoeae, Treponema pallidum, T richomonas vaginalis, Ureaplasma urealyticum and U. parvum, and the bacteria of the normal genital flora Lactobac i llus iners. Other Lactobacillus species, including L. gasseri, L. crispaticus, L. jensenii and L. vaginalis were detected together by a universal Lactobacillus probe. Additional Candida species, e.g. C. parapsilosis and C. tropicalis, were detected by a universal Candida probe. Presence of amplifiable human DNA was verified using the human DNA polymerase alpha (PolA) sequence as a target. The load of A. vaginae, G. vaginalis and Lactobacillus species was determined and a BV score using load ratios of either A. vaginae or G. vaginalis over Lactobacillus was calculated as previously described [10]. The score grades for BV were as follows: 0 absence of BV, 1 weak indication for BV, 2 some indications for BV, 3–4 strong indications for BV and 5 very strong indications for BV. Likewise, candidiasis was determined by a ratio of signals from several Candida spp. probes and Lactobacillus as previously described [10]. In cases where signal intensities of pathogens and Lactobacillus were too low, BV and candidiasis could not be computed.

Migration status was defined according to Schenk et al. [14] as either both parents not born in Germany or one parent not born in Germany and interviewee not living in Germany since birth or German not being native language. Household net equivalent income per month was calculated using midpoint estimates of group levels; the highest group (≥ 8000 €) was set at 10,000 €. To account for household size, weighting was done according to the German demographic standards [15] using the modified Organisation for Economic Co-operation and Development (OECD) equivalence scale. Women were categorized according to their menstruation status as premenopausal versus perimenopausal/postmenopausal. For bacteria detected by STIP, a participant was included into the statistical analysis if at least one species of microbe was detected and amplified human DNA (PolA) was present. For samples without detection of any microbial agent, potential treatment with antibiotics was assumed [10] and these specimens were therefore excluded from the respective statistical analysis (bacteria). For the analysis regarding viruses, all samples were included as viruses are not affected by antibiotics. The age of participants was grouped in 10-year increments (e.g. 20–29 and 30–39 years). To test differences between groups for statistical significance (comparisons of participants and non-participants, of age groups and categories of acceptability, and of time elapsed until arrival of samples at the laboratory depending on HPV status), for nominal or ordinal variables χ ²-test and Fisher’s exact test were used and for continuous variables the Mann-Whitney test after evaluating observed data regarding normal distribution using graphical tools and the Shapiro-Wilk test. All statistical tests were 2-sided and a p-value < 0.05 was considered statistically significant. Proportions were calculated with missing or unspecified values as an additional category, whereas statistical tests were performed without this category. Approximate binomial confidence intervals (CI) were calculated based on the formula given in [16] using Excel (Microsoft, Redmond WA). For all other statistical analyses STATA 12 (StataCorp LP, College Station TX) was used.

Of the 162 women initially recruited for Pretest 2 from a random registration office sample, 1 woman was excluded due to pregnancy and 126 (77.8 %) agreed to participate in the additional module on cervicovaginal lavage, from whom 109 (86.5 %) samples and 108 (85.7 %) completed questionnaires were received (Fig. 1). The participation in the add-on module within Pretest 2 of the GNC was thus 67.3 % (109 out of 162, 95 % CI 59.7; 74.0).

Characteristics of participating and non-participating women are presented in Table 1. A significant difference between both groups was detected with respect to hysterectomy. The median age of participating women was 5 years lower than that of non-participating women (no significant difference) (Table 1).

The median time from the visit at the study center to sampling was 7 days (n = 107, interquartile range (IQR) 3–16 days, range 0–90 days). Median reported duration of sampling was 5 min (n = 105, IQR 3–6 min, range 1–30 min). It took a median time of 2 days (n = 102, IQR 2–4 days, range 1–140 days) after self-sampling for the specimens to arrive at the laboratory. There was no significant difference in this respect between HPV positive and negative samples (identical median, p = 0.13).

A total of 98.1 % (106/108, 95 % CI 93.5; 99.5) judged the sampling of a cervicovaginal lavage at home to be acceptable, 88.9 % (96/108, 95 % CI 81.6; 93.5) as easy and 96.3 % (104/108, 95 % CI 90.9; 98.6) as user-friendly. Only 6.5 % reported a physically uncomfortable feeling during sampling (7/108, 95 % CI 3.2; 12.8) and 10.2 % (11/108, 95 % CI 5.8; 17.3) stated “partly” for this item. Self-sampling of a cervicovaginal lavage was seen as no violation of privacy by 82.4 % (89/108, 95 % CI 74.2; 88.4) and was preferably done at home instead of at the study center in 78.7 % of cases (85/108, 95 % CI 70.1; 85.4) (Fig. 2).

No statistically significant differences could be detected between age groups and any of the aforementioned categories of acceptability with the exception of the item on privacy violation (here p = 0.02, n = 108); other results of Fisher’s exact test for comparison of each category and age groups: acceptance p = 0.2 (n = 108), easiness p = 0.86 (n = 107), user-friendliness p = 0.87 (n = 108), physical discomfort p = 0.26 (n = 107) and preferred place of sampling p = 0.63 (n = 108).

With respect to age categories, sampling a cervicovaginal lavage was viewed as no violation of privacy by 57.1 % (4/7) of 20–29-year-olds, by 60.0 % (9/15) of 30–39-year-olds, 82.1 % (23/28) of 40–49-year-olds, 87.1 % (27/31) of 50–59-year-olds and 96.3 % (26/27) of 60–69-year-olds.

Two minor adverse events without the necessity to contact a doctor were reported (i.e. slightly painful sensation in lower abdomen after sampling). Three women aged between 32 and 65 years stated no previous sexual intercourse; all of whom assessed the sampling as acceptable and user-friendly, 2 as easy, physically not uncomfortable and as no violation of privacy.

Two samples arrived too late to be included in the STIP (n = 107). PolA DNA quality was sufficient for analysis of all samples analyzed by STIP; five samples were excluded from statistical analyses of bacterial occurrence because no microbes could be detected by STIP which was interpreted as potential antibiotic use. The bacteria A. vaginae, U. parvum, Candida spp., C. albicans, G. vaginalis, M. genitalium, M. hominis, M. spermatophilum, C. krusei, and N. gonorrhoeae were detected (Table 2). In the case of pathogen and Lactobacillus intensities being too low, BV (n = 7) and candidiasis (n = 8) could not be computed. Evidence for BV was present in 14 out of 95 of specimens, of which 9 had a BV score of 2 (i.e. some indications for BV) and 5 a score of 4–5 (strong and very strong indications for BV). Candidiasis was detected in 13 out of 94 samples and BV, U. parvum; and candidiasis occurred in all age groups (data not shown).