Temporal changes in the vaginal microbiota in self-samples and its association with persistent HPV16 infection and CIN2.

BACKGROUND: The vaginal microbiota has been reported to be associated with HPV infection and cervical cancer. This study was performed to compare the vaginal microbiota at two timepoints in women performing self-sampling and had a persistent or transient HPV16 infection. The women were tested for 12 high-risk HPV (hrHPV) types but only women with single type (HPV16) were included to reduce confounding variables. METHODS: In total 96 women were included in this study. Of these, 26 were single positive for HPV16 in the baseline test and HPV negative in the follow-up test and 38 were single positive for HPV16 in both tests and diagnosed with CIN2+ in histology. In addition, 32 women that were negative for all 12 HPV tested were included. The samples of vaginal fluid were analyzed with the Ion 16S™ Metagenomics Kit and Ion 16S™ metagenomics module within the Ion Reporter™ software. RESULTS: K-means clustering resulted in two Lactobacillus-dominated groups, one with Lactobacillus sp. and the other specifically with Lactobacillus iners. The two remaining clusters were dominated by a mixed non-Lactobacillus microbiota. HPV negative women had lower prevalence (28%) of the non-Lactobacill dominant cluster in the baseline test, as compared to women with HPV16 infection (42%) (p value = 0.0173). Transition between clusters were more frequent in women with persistent HPV16 infection (34%) as compared in women who cleared the HPV16 infection (19%) (p value = 0.036). CONCLUSIONS: The vaginal microbiota showed a higher rate of transitioning between bacterial profiles in women with persistent HPV16 infection as compared to women with transient infection. This indicate an instability in the microenvironment in women with persistent HPV infection and development of CIN2+.

HPV viral load in self-collected vaginal fluid samples as predictor for presence of cervical intraepithelial neoplasia.

OBJECTIVE: This study was performed to evaluate the use of high-risk HPV (hrHPV) viral load in screening tests for cervical cancer to predict persistent infection and presence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+). METHODS: We followed women between 30 and 60 years of age who performed self-sampling of vaginal fluid and subsequently a hrHPV test. Women who were hrHPV positive in their screening test repeated the hrHPV test 3-6 months later and were included in the present study. RESULTS: Our results show that women with a persistent HPV16 infection had higher HPV viral load in their primary screening test than women with transient infections (p = 5.33e-03). This was also true for sum of viral load for all hrHPV types in the primary screening test (p = 3.88e-07). 48% of women with persistent HPV16 infection and CIN2+ had an increase in HPV16 titer in the follow-up test, as compared to only 20% of women with persistent infection but without CIN2+ lesions. For the sum of all hrHPV types, 41% of women with persistent infection and CIN2+ had an increase in titer as compared to 26% of women without CIN2 + . CONCLUSIONS: The results show that hrHPV viral load in the primary screening HPV test is associated with the presence of CIN2+ and could be used in triaging hrHPV positive women for different follow-up strategies or recall times. Serial testing of hrHPV viral load has the potential to distinguish women with CIN2+ lesions from women with persistent infection but without CIN2+ lesions.

Clinical validation of the HPVIR high-risk HPV test on cervical samples according to the international guidelines for human papillomavirus DNA test requirements for cervical cancer screening.

BACKGROUND: The indicating FTA card is a dry medium used for collection of cervical samples. HPVIR is a multiplex real-time PCR test that detects 12 high-risk human papillomavirus types (hrHPV) and provides single genotype information for HPV16, - 31, - 35, - 39, - 51, - 56, and - 59 and pooled type information for HPV18/45 and HPV33/52/58. The aim of this study was to evaluate whether a strategy with cervical samples collected on the FTA card and subsequently analysed with the HPVIR test complies with the criteria of the international guidelines for a clinically validated method for cervical screening. METHODS: We performed a non-inferiority test comparing the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a clinically validated reference test (Cobas® HPV test) based on liquid-based cytology (LBC) samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. For evaluation of the specificity we used 799 women without ≥ CIN2, and for clinical sensitivity we used 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples. RESULTS: The clinical sensitivity and specificity for samples collected on the FTA card and analysed using the HPVIR test were non-inferior to samples analysed with the Cobas® HPV test based on LBC samples (non-inferiority test score, p = 1.0 × 10- 2 and p = 1.89 × 10- 9, respectively). Adequate agreement of > 87% was seen in both the intra- and inter-laboratory comparisons. CONCLUSIONS: Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.

Diagnostic excision of the cervix in women over 40 years with human papilloma virus persistency and normal cytology.

OBJECTIVE: Persistent infection with human papillomavirus (HPV) is recognized as the main risk factor of cervical cancer. Investigation via cytology and colposcopy have lower sensitivity than HPV testing in the diagnosis of high-grade cervical intraepithelial neoplasia (CIN2+). Despite normal cytology and colposcopy findings women with persistent HPV infection have an increased risk of CIN2+. The aim of the study was to evaluate the proportion of histologically confirmed CIN2+ in women with persistent HPV infection and normal Pap smears. STUDY DESIGN: From April 2013 until March 2016 we prospectively recruited 91 women over 40 years with persistent HPV infection without any abnormalities in cytology. Of these, 40 women attended a gynecological examination including an HPV test, Pap smear, endocervical cytology, colposcopy with biopsies and diagnostic loop electrosurgical excision procedure (LEEP). Biopsy and LEEP samples were subjected to histological examination. RESULTS: CIN2+ was verified by histological examination of the LEEP sample in 6/40 (15%) of the women. All the cytological samples were normal and none of the biopsies confirmed CIN2+. Only 19/40 women still had a persistent HPV infection at the study visit. None of the 21/40 women who had cleared their HPV infection at the study visit had CIN2+ in histology of the LEEP sample. CONCLUSIONS: A persistent HPV infection needs to be monitored despite normal Pap smears, since 6/40 (15%) women older than 40 years, was revealed to have an undiagnosed CIN2+ when LEEP was performed. Counseling women regarding the risk of cervical cancer and the expected effect of an eventual LEEP can help them to make an optimal informed choice.

Randomised study of HPV prevalence and detection of CIN2+ in vaginal self-sampling compared to cervical specimens collected by medical personnel.

We conducted a randomised study to compare vaginal self-sampling with assisted sampling by medical personnel on the cervix for HPV testing in primary screening. The first aim was to determine if the HPV prevalence is independent of sampling location (vagina versus cervix) and the person performing the sampling. The second aim was to evaluate if the two sampling strategies differed in the detection rate of CIN2+. In total, 19,523 women were randomised into two groups, with 9926 invited to perform self-sampling (SS arm) using the Rover VIBA-brush and 9597 offered assisted sampling using the cytobrush (AS arm). All samples were applied to the indicating FTA elute card and analysed for high-risk HPV using the hpVIR real-time PCR assay. The outcome for the first aim was HPV prevalence and for the second aim the number of CIN2+ based on histology. In the SS arm, 52.7% of invited women participated in the study, as compared to 34.2% in the AS arm. All samples contained sufficient amount of nuclear DNA for a valid HPV result, with vaginal samples having a higher DNA amount than cervical samples (p < 4.62 × 10-11 ). HPV prevalence was 4.6% in the SS arm and 4.1% in the AS arm (p = 5.5 × 10-2 ), and the distribution of HPV types similar between arms. There was no difference in the prevalence of CIN2+ per 1000 women screened between arms (p = 0.86). The results show that vaginal self-sampling is an equivalent alternative to sampling by medical personnel for HPV typing and identification of CIN2+.

Randomised study shows that repeated self-sampling and HPV test has more than two-fold higher detection rate of women with CIN2+ histology than Pap smear cytology.

This corrects the article DOI: 10.1038/bjc.2017.85.

Daily self-sampling for high-risk human papillomavirus (HR-HPV) testing.

BACKGROUND: Self-sampling for HPV as part of primary screening is a well-tolerated method for women not attending organized Pap smear screening and could increase coverage of cervical cancer screening. OBJECTIVE: To investigate if the prevalence of HR-HPV varies from day to day in infected women and if one single sample is reliable for detecting an ongoing infection. STUDY DESIGN: This is a prospective cohort study on 12 premenopausal and 13 postmenopausal women performing daily self-sampling for HR-HPV testing. They were all HR-HPV-positive 1-3 months ago. Postmenopausal women were sampled for 28 days and premenopausal women sampled during bleeding-free days in one menstrual cycle. A possible difference in viral load between the estrogen-dominated proliferative phase and the progesterone-dominated secretory phase was analyzed. RESULTS AND CONCLUSIONS: Consistent results throughout the sampling period were observed for 19 women, with either a daily presence of HPV (14 women) or no HPV at all during the sampling period (5 women). Of 607 samples from 25 women, 596 were consistently positive or negative for HPV during the sampling period and 11 were inconsistent (2%). There was no difference in HPV copy number between the estrogen dominated proliferative or progesterone dominated secretory menstrual cycle phases. The major finding was a high degree of consistency concerning HR-HPV positivity and negativity of HR-HPV in vaginal fluid during a sustained period of daily self-sampling. It does not appear to matter whether the sample is collected in the proliferative or secretory phase.

Type-specific detection of high-risk human papillomavirus (HPV) in self-sampled cervicovaginal cells applied to FTA elute cartridge.

BACKGROUND: Most procedures for self-sampling of cervical cells are based on liquid-based media for transportation and storage. An alternative is to use a solid support, such as dry filter paper media. OBJECTIVES: To evaluate if self-sampling of cervicovaginal fluid using a cytobrush (Viba-brush; Rovers Medical Devices B.V., Oss, The Netherlands) and a solid support such as the Whatman Indicating FTA Elute cartridge (GE Healthcare, United Kingdom) can be used for reliable typing of human papillomavirus (HPV), as compared to cervical samples obtained by a physician using a cytobrush and the indicating FTA Elute Micro card and biopsy analysis. STUDY DESIGN: A total of 50 women with a previous high-risk (HR) HPV positive test were invited to perform self-sampling using the Viba-brush and the FTA cartridge and thereafter a physician obtained a cervical sample using the cytobrush and a FTA card, together with a cervical biopsy for histology and HPV typing. Detection of HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 was performed using three multiplex real-time polymerase chain reaction (PCR) assays. RESULT: All samples contained sufficient amounts of genomic DNA and the self-samples yielded on average 3.5 times more DNA than those obtained by the physician. All women that were positive for HR-HPV in the biopsy sample also typed positive both by self-sampling and physician-obtained sampling. For women with a histological diagnosis of cervical intraepithelial neoplasia grades 2-3 (CIN 2-3) all three HPV samples showed 100% concordance. A higher number of women were HPV positive by self-sampling than by physician-obtained sampling or by biopsy analysis. CONCLUSION: The Viba-brush and the FTA cartridge are suitable for self-sampling of vaginal cells and subsequent HR-HPV typing.

High prevalence of oncogenic human papilloma virus in women not attending organized cytological screening.

Since the introduction of organized cytological screening in Sweden, most women currently presenting with cervical cancer are those who have not attended the programme and who have no cytological screening history. The aims of this study were: (i) to measure the response rate among women not attending organized cytological screening who were offered a device for self-sampling a vaginal smear at home; (ii) to examine the prevalence of high-risk human papilloma virus (HPV) among women performing self-sampling. Women aged 35-50 years, who had not participated in organized cytological screening for more than 6 years, were offered the opportunity to collect vaginal samples at home using a self-sampling device (Qvintip). The material collected was analysed for high-risk HPV using the Hybrid Capture 2 method. Of 369 women included in the study, 179 (49%) ordered the self-sampling device and 117 (32%) performed self-sampling at home and sent the sample to our laboratory for analysis. The mean prevalence of high-risk HPV was 26% (30/117), 31% (25/80) in women aged 35-42 years and 14% (5/37) in women aged 43-50 years. There was no significant difference in the participation rate with regard to age. The prevalence of high-risk HPV in women not covered by organized screening was considerably higher than in the general population; therefore they may represent a category at high risk of cervical cancer. The study shows that the use of a disposable self-sampling device for HPV testing is a relevant method to increase the participation rate in countries with organized cytological screening.

Attitudes to self-sampling of vaginal smear for human papilloma virus analysis among women not attending organized cytological screening.

BACKGROUND: The major problem with the cytological screening is the non-optimal participation rate among women invited for cervical smear collection. The aim of the present investigation was to examine the attitudes of the non-responding women to perform self-sampling of vaginal smear at home as a method to increase the coverage of the screening and to examine the prevalence of high-risk human papilloma virus (HPV) among the responding women. METHODS: From the database of the Department of Cytology, University Hospital of Uppsala 198 women, aged 35-55 years, who had not attended the organized gynaecological screening for over 6 years were identified. They were sent a letter of information about the study and one week later a self-sampling device aimed to collect vaginal smear. The vaginal smear of the women responding to the offer of self-sampling was analysed for high-risk HPV using Hybrid Capture 2 method or polymerase chain reaction amplification of HPV DNA. All women in the study also received a questionnaire in order to investigate their attitudes towards self-sampling as an alternative in the organized screening. RESULTS: Of the 198 women 15 women had to be excluded. Fifty-eight per cent of the women responded and collected vaginal smear at home and among them 7% were positive for high-risk HPV. The questionnaire revealed no significant difference of age, country of birth and occupation or marital status, on using self-sampling of vaginal smear at home. The attitudes among responding and non-responding women differed. The responding women who contributed by sampling vaginal smear were more positive to self-sampling of vaginal smear (p<0.01). CONCLUSIONS: Offering self-sampling of vaginal smear in women not attending the organized cytological screening increases the coverage and identifies an additional group of women with an increased risk to develop cervical cancer. The attitude towards self-sampling was mainly positive.

Accuracy of HPV testing of vaginal smear obtained with a novel self-sampling device.

BACKGROUND: Most of women diagnosed as having cervical cancer have not participated in organized cytological screening. Aim. A study was conducted to evaluate the accuracy of human papilloma virus testing by self-collected vaginal samples in comparison to regular cytological screening. The agreement of hybrid capture 2 assay and polymerase chain reaction assay for detection of human papilloma virus DNA in self-collected vaginal samples and clinician-obtained cervical smears was investigated. METHOD: Forty-three women aged 23-58 years admitted for further examination due to previous positive cytology in the organized screening participated in self-collecting of vaginal samples with a novel self-sampling device. During the visit a clinician also collected a cervical smear using a cytobrush. The vaginal samples collected with the self-sampling device were analyzed for high-risk human papilloma virus with the hybrid capture 2 assay technique and the cervical smears were Pap-stained, examined cytologically and after that reanalyzed for human papilloma virus DNA using a polymerase chain reaction assay. RESULT: The vaginal samples were positive for high-risk human papilloma virus in 37% of the cases using hybrid capture 2 assay. Twelve of the 43 Pap smears showed positive cytology (ASCUS-CIN 3), of which 4 showed CIN 2-3. When polymerase chain reaction assay was performed, human papilloma virus DNA was detected in 40% of the glass slides. The agreement between cytology and the two human papilloma virus testing techniques was 67-74% (kappa 0.27-0.45) and the agreement between the two human papilloma virus tests was 70% (kappa 0.36). CONCLUSION: Testing for high-risk human papilloma virus can identify more women at risk of developing cervical cancer than cytology irrespective of the sampling method. Furthermore, offering a self-sampling device for collection of vaginal smear seems to be a useful screening tool for cervical cancer among women not responding to an invitation for smear sampling.

Significance of HPV tests on women with cervical smears showing ASCUS.

BACKGROUND: The relationship between the human papillomavirus (HPV) tests and the cytological findings was evaluated. METHODS: Totally, 161 women participating in the organized cytological screening in the County of Uppsala and showing atypical squamous cells of uncertain significance (ASCUS) in their cervical smears were identified during 2002 and 2003. Those women were invited for a repeated examination about 3 months later. At that occasion, a smear sample was collected and used for cytological examination and a concomitant HPV test. Oncogenic HPV was identified by polymerase chain reaction and HPV deoxyribonucleoside acid (DNA) sequence analysis in 50 cases and by Hybrid Capture II in 111 cases. RESULTS: Women with an ASCUS diagnosis showed a normal cytology and a negative HPV test in 30% of the cases, abnormal cytology and a positive HPV test in 32% of the cases; in 26% of the women, the HPV test was positive, whereas cytology was normal; and in 11% of the women, the HPV test was negative, whereas cytology was abnormal (ASCUS or more). CONCLUSION: The results show that HPV tests identify a larger group of women at risk to develop cervical cancer in comparison with cytological examination. As the HPV tests have a higher sensitivity than the cytological screening, it is suggested that an HPV test in conjunction with a diagnosis of ASCUS can be employed to more efficiently select women, to whom further follow up is recommended.

Association between human papillomavirus 16 E6 variants and human leukocyte antigen class I polymorphism in cervical cancer of Swedish women.

Persistent infection with human papillomavirus (HPV), particularly HPV16, represents the prime risk factor in cervical carcinogenesis. HPV variants (e.g., within the E6 gene) together with immunogenetic factors of the host may be responsible either for effective viral clearance, or alternatively, for viral persistence. Peripheral blood from 27 HPV16 positive Swedish women with cervical carcinoma, who had previously been tested for HPV16 E6 variants, was used for human leukocyte antigen (HLA) class I typing. Women with HLA-B*44, HLA-B*51, or HLA-B*57 who were infected with the HPV16 E6 variant L83V had an approximately four- to fivefold increased risk for cancer compared with controls (odds ratio [OR] = 3.5, 95% CI = 1.1-11.1, OR = 4.2, 95% CI = 1.19-14.69, or OR = 4.67, 95% CI = 1.2-18.6, respectively). Epitope predictive algorithm with SYFPEITHI revealed that the variant at amino acid 83 affects the binding affinity in association with HLA-B*44. Interestingly, the HLA-B*15 allele seems protective because it was absent in HPV16 positive cancer. It is concluded that specific HLA class I alleles, combined with certain HPV16 E6 variants, may be crucial for immune surveillance in cervical carcinogenesis. The evaluation of associations of HLA alleles with HPV variants may be helpful in defining prognostic markers and in designing vaccines capable of mediating immune protection against HPV infection.