Clinical Performance of Triage Strategies for Hr-HPV-Positive Women; A Longitudinal Evaluation of Cytology, p16/K-67 Dual Stain Cytology, and HPV16/18 Genotyping.

BACKGROUND: We evaluated the longitudinal performance of three options: HPV16/18 genotyping (HPV16/18), cytology (LBC), and p16/Ki-67 dual stain cytology (DS) for the triage of high-risk Human Papillomavirus-positive (Hr-HPV+) women within the cervical screening program in Scotland. METHODS: Data were derived from a cohort of Hr-HPV+ women (n = 385) who participated in PaVDaG (Papillomavirus Dumfries and Galloway) study. Performance of triage strategies for detecting high-grade disease was assessed at 3 (in women <50 years) or 5 years (in women >50 years). Sensitivity, specificity, PPV, and cNPV of each triage test were calculated for CIN2+ and CIN3+ when used singly or sequentially. RESULTS: The sensitivity of LBC (≥ borderline), DS, and HPV 16/18 genotyping for the detection of CIN2+ was 62.7% (50.7-73.3), 77.7% (63.1-83.7), and 62.7% (50.7-73.3) with corresponding cNPVs of 10.9%, 8.4%, and 11.9%. The option with the highest sensitivity and lowest cNPV was HPV 16/18 genotyping followed by LBC of Hr-HPV other+ and then DS of the LBC negatives. This yielded sensitivity of 94.7% (86.2-98.3) and cNPV 2.7% for CIN2+. Triage performance was similar if women had tested Hr-HPV+ positive by vaginal self-sampling. CONCLUSIONS: Two-step triage with HPV 16/18 genotyping before LBC (or DS) for Hr-HPV other+ women was associated with a lower risk of significant disease at follow-up compared with single triage approaches. IMPACT: This study provides longitudinal performance data on triage strategies in Hr-HPV+ women and will be informative for the evolution of cervical screening programs that increasingly rely on molecular technologies.

Self-sampling as the principal modality for population based cervical screening: Five-year follow-up of the PaVDaG study.

Self-sampling provides a powerful means to engage women in cervical screening. In the original Papillomavirus Dumfries and Galloway study (PaVDaG), we demonstrated cross-sectional similarity of high-risk human papillomavirus (Hr-HPV) testing on self-taken vaginal vs clinician-taken samples for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+). Few data exist on the longitudinal performance of self-sampling; we present longitudinal outcomes of PaVDaG. Routinely screened women provided a self-taken and a clinician-collected sample. Ninety-one percent of 5136 women from the original cohort completed a further screening round. Sensitivity, specificity, positive predictive value and complement of the negative predictive value of the Hr-HPV test on self-samples for detection of CIN2+ and CIN3+ up-to 5 years after testing were determined. Additionally, clinical accuracy of Hr-HPV testing on vaginal and clinician-collected samples was assessed. A total of 183 CIN2+ and 102 CIN3+ lesions were diagnosed during follow-up. Risk of CIN2+ and CIN3+ following an Hr-HPV negative self-sample was 0.6% and 0.2%, respectively, for up to 5 years after testing. The relative sensitivity for CIN3+ and specificity for ≤CIN1 of Hr-HPV testing on self-taken specimens was slightly lower vs clinician-collected samples: 0.95 (95% CI: 0.90-0.99; PMcN = .0625) and 0.98 (95% CI: 0.95-1.00; PMcN = <.0000), respectively. The low risk of CIN2+ in women with Hr-HPV-self-sample(s) suggests, that the 3 to 5-year recall interval implemented in several cervical screening settings, based on clinician-taken samples, may be safe for self-samples. Future assessment will show if "universal" 5-year screening is appropriate for programs based on self-sampling.

The challenges of defining sample adequacy in an era of HPV based cervical screening.

BACKGROUND: The implementation of Human Papillomavirus based cervical screening continues apace on a global scale. Understanding the basis and burden of inadequate or invalid samples is important to ensure confidence in high quality laboratory results and inform the development of new technologies. Here we present population based data from Scotland and Denmark which detail the extent of invalid samples for HPV detection in both clinician-taken and self-taken samples. As a comparator we report on the rate of inadequate cytology preparations in both countries. METHODS: The proportion of samples with an invalid HPV test result was calculated by retrospective analysis of routine laboratory data associated with cervical screening programmes in the two countries. Two assays were in use for the programmes at the time (the Abbott RealTime High Risk HPV assay and the BD Onclarity); both have internal endogenous controls for human genes. In addition, acellular cytology samples were reported through a prospective audit (Scotland) and National quality reporting (Denmark). RESULTS: In total, 89,418 clinician samples and 14,677 self-taken samples were assessed. We observed low rates of invalid HPV tests in clinician taken samples (0.05-0.10 %), irrespective of sample collection media (ThinPrep or SurePath), HPV test system/endogenous control type or clinical indication for testing (primary screening, triage or test of cure). For self-taken samples, the number of invalid samples was 0.18 %. Complete absence of sample material (acellular) in clinician taken samples were observed at a level of 1 in approximately 16.5 thousand. CONCLUSIONS: Clinician and self-taken samples appear robust specimens for HPV testing and acellular samples are very rare. Efforts to develop endogenous controls for HPV assays that provide greater insight into true sample adequacy for cervical disease detection, beyond measuring the presence of human cells, will be welcome.

Methylation markers FAM19A4 and miR124-2 as triage strategy for primary human papillomavirus screen positive women: A large European multicenter study.

In human papillomavirus (HPV) cervical cancer screening, cytology is used as triage to counter the low specificity of HPV testing. VALID-SCREEN is a EU-multicenter, retrospective study conducted to evaluate the clinical performance of the FAM19A4/miR124-2 methylation-based molecular triage test as a substitute or addition to cytology as reflex testing of HPV screen positive women. FAM19A4/miR124-2 methylation test (QIAsure Methylation Test) was evaluated in 2384 HPV-positive cervical screening samples, from women 29-76 years of age, derived from four EU countries. Specimens were collected in ThinPrep or SurePath media, HPV-status, concurrent cytology, and histology diagnosis were provided by the parent institutes. The control population consisted of women with no evidence of disease within 2 years of follow-up. A total of 899 histologies were retrieved; 527 showed no disease, 124 CIN2 (5.2%), 228 CIN3 (9.6%) and 20 cervical cancers (0.8%); 19 of 20 screen-detected cervical cancers were found methylation-positive (sensitivity 95%). Overall specificity of FAM19A4/miR124-2 methylation test was 78.3% (n = 2013; 95%CI: 76-80). The negative predictive value of hrHPV positive, methylation-negative outcomes were 99.9% for cervical cancer (N = 1694; 95%CI: 99.6-99.99), 96.9% for ≥CIN3 (95%CI: 96-98), and 93.0% for ≥CIN2 (95%CI: 92-94). Overall sensitivity for CIN3 using FAM19A4/miR124-2 methylation test was 77% (n = 228; 95%CI: 71-82). CIN3 sensitivity was uniform between centers independent of sample collection medias, DNA extraction methods and HPV screening tests. Being objectively reported compared to the subjectivity of cytology, equally performing across settings and screening methods, the FAM19A4/miR124-2 methylation constitute an alternative/supplement to cytology as triage method to be investigated in real-life pilot implementation.

Defining Optimal Triage Strategies for hrHPV Screen-Positive Women-An Evaluation of HPV 16/18 Genotyping, Cytology, and p16/Ki-67 Cytoimmunochemistry.

Background: Several options for the triage of high-risk HPV screen-positive (hrHPV+) women were assessed.Methods: This study incorporated CIN2+ cases and controls, all of whom tested hrHPV+ and whose results of liquid-based cytology (LBC), HPV16/18 genotyping, and p16/Ki67 cytoimmunochemistry were available. Sensitivity and specificity for the CIN2+ of these triage tests were evaluated.Results: Absolute sensitivities of HPV 16/18 typing, LBC, and p16/Ki-67 cytoimmunochemistry for CIN2+ detection were 61.7%, 68.3%, and 85.0% for women with hrHPV+ clinician-taken samples. Respective specificities were 70.5%, 89.1%, and 76.7%. The absolute accuracy of the triage tests was similar for women with a hrHPV+ self-sample. P16/Ki-67 cyto-immunochemistry was significantly more sensitive than LBC although significantly less specific.Conclusions: All three single-test triage options, if positive, exceed the threshold of 20% risk at which colposcopy would be indicated. However, none of them conferred a post-test probability of CIN2+ <2%; which would permit routine recall. P16/Ki-67 cytoimmunochemistry on HPV16/18 negative women had a post-test probability of CIN2+ of 1.7% and 0.6% if also LBC negative.Impact: This is one of the few studies to directly compare the performance of triage strategies of hrHPV+ women, in isolation and combinations. It is the only study assessing triage strategies in women who test hrHPV+ in self-taken vaginal samples. A combined triage option that incorporated HPV 16/18 typing prior to p16/ki-67 cytoimmunochemistry in HPV 16/18-negative women yielded a post-test probability of CIN2+ of >20%, whereas women who tested negative had a probability of CIN2+ of <2%. Cancer Epidemiol Biomarkers Prev; 26(11); 1629-35. ©2017 AACR.

Clinical validation of hrHPV testing on vaginal and urine self-samples in primary cervical screening (cross-sectional results from the Papillomavirus Dumfries and Galloway-PaVDaG study).

OBJECTIVES: Papillomavirus Dumfries and Galloway (PaVDaG) assessed the performance of a high-risk human papillomavirus (hrHPV) PCR-based assay to detect high-grade cervical intraepithelial neoplasia (CIN2+) in self-collected vaginal and urine samples. SETTING: Women attending routine cervical screening in primary care. PARTICIPANTS: 5318 women aged 20-60 years provided self-collected random urine and vaginal samples for hrHPV testing and a clinician-collected liquid-based cytology (LBC) sample for cytology and hrHPV testing. INTERVENTIONS: HrHPV testing. All samples were tested for hrHPV using the PCR-based cobas 4800 assay. Colposcopy was offered to women with high-grade or repeated borderline/low-grade cytological abnormalities; also to those who were LBC negative but hrHPV 16/18 positive. PRIMARY AND SECONDARY OUTCOME MEASURES: The self-tests' absolute sensitivity and specificity for CIN2+ were assessed on all biospecimens; also, their relative sensitivity and specificity compared with clinician-taken samples. Interlaboratory and intralaboratory performance of the hrHPV assay in self-collected samples was also established. RESULTS: HrHPV prevalence was 14.7%, 16.6% and 11.6% in cervical, vaginal and urine samples, respectively. Sensitivity for detecting CIN2+ was 97.7% (95% to 100%), 94.6% (90.7% to 98.5%) and 63.1% (54.6% to 71.7%) for cervical, vaginal and urine hrHPV detection, respectively. The corresponding specificities were 87.3% (86.4% to 88.2%), 85.4% (84.4% to 86.3%) and 89.8% (89.0% to 90.7%). There was a 38% (24% to 57%) higher HPV detection rate in vaginal self-samples from women over 50 years compared with those ≤29 years. Relative sensitivity and specificity of hrHPV positivity for the detection of CIN2+ in vaginal versus cervical samples were 0.97 (0.94 to 1.00) and 0.98 (0.97 to 0.99); urine versus cervical comparisons were 0.53 (0.42 to 0.67) and 1.03 (1.02 to 1.04). The intralaboratory and interlaboratory agreement for hrHPV positivity in self-samples was high (κ values 0.98 (0.96 to 0.99) and 0.94 (0.92 to 0.97) for vaginal samples and 0.95 (0.93 to 0.98) and 0.90 (0.87 to 0.94) for urine samples). CONCLUSIONS: The sensitivity of self-collected vaginal samples for the detection of CIN2+ was similar to that of cervical samples and justifies consideration of this sample for primary screening.

Cobas 4800 HPV detection in the cervical, vaginal and urine samples of women with high-grade CIN before and after treatment.

AIMS: To assess the performance of a clinically validated human papillomavirus (HPV) test (the Cobas 4800 HPV test) in urine and self-taken vaginal specimens within a colposcopy population and to assess HPV prevalence before and after treatment across the different biospecimens. METHODS: A total of 100 women attending a colposcopy clinic provided three biospecimens (a clinician-taken liquid-based cytology sample (LBC), a self-taken vaginal sample and a urine sample) for HPV testing. HPV prevalence and concordance was compared across the biospecimens and clinical performance relative to the detection of cervical intraepithelial neoplasia (CIN)2+ and CIN3+ was assessed. A total of 39 women retuned at 6 months for a post-treatment follow-up appointment, and HPV concordance in all biospecimens was measured relative to their original HPV status. RESULTS: 65 cases of CIN2+ were detected in the baseline population; sensitivity for CIN2+ was 92% (82 to 97) for the vaginal and the LBC sample and 80.0 (68% to 88%) for the urine sample. In the follow-up (post treatment) population, women were twice as likely to be HPV positive in their urine or vaginal sample compared with the equivalent LBC sample. CONCLUSIONS: Vaginal and LBC samples showed very similar performance for the detection of CIN2+ in this population using the Cobas HPV test; further validation of these findings in screening contexts will be of value. Self-taken samples may have less utility in a 'test of cure' setting-given the higher prevalence of HPV relative to LBC.

The genetic predisposition to produce high levels of TGF-beta1 impacts on the severity of eclampsia/pre-eclampsia.

OBJECTIVES: To investigate the hypothesis that women who are genetically programmed to produce higher levels of transforming growth factor-beta 1 are more likely to develop severe eclampsia/pre-eclampsia. DESIGN: Case-control study. METHODS: Blood samples from women whose pregnancy was complicated by eclampsia (n=37) or pre-eclampsia (n=49) and healthy controls (n=86) were analyzed for the presence of polymorphisms at codons 10 and 25 of the transforming growth factor-beta 1 gene. The polymorphisms are thought to determine whether an individual produces low, medium, or high levels of the cytokine. The analysis was carried out using the ARMS-PCR technique. RESULTS: Women who developed eclampsia/pre-eclampsia with severe renal and neurological complications or had neonatal deaths/still births were more likely to have the high-producer allele T in codon 10 of the transforming growth factor-beta 1 gene than healthy controls. By contrast, the transforming growth factor-beta 1 producer genotype and allele frequency as determined by gene polymorphisms at codon 25 were comparable in cases and controls. The cytokine producer status per se appears to had no bearing on whether a patient developed eclampsia/pre-eclampsia. CONCLUSIONS: Our findings suggest that women who experience eclampsia/pre-eclampsia with severe maternal and/or fetal complications are more likely to have a genetic predisposition to produce high levels of transforming growth factor-beta 1 as defined by polymorphisms at codon 10. While it is recognized that eclampsia/pre-eclampsia has heterogenous pathomechanisms, we have demonstrated a strong relationship between poor maternal and pregnancy outcomes and codon 10 polymorphisms. The characterization of the immunogenetic make-up of the women may be an additional tool in the differentiation of component pathologies and/or prediction of severity of the syndrome.

HIV/AIDS in Central Africa: pathogenesis, immunological and medical issues.

The estimated worldwide prevalence of human immunodeficiency virus (HIV) infections topped 52.5 million in June 2003, a mere 20 years after the aetiological agent was shown to be a sexually transmissible virus with a predilection for CD4+ T lymphocytes. More than 22 million people have died of the acquired immunodeficiency syndrome (AIDS) and the condition has in one generation become the most devastating and persistent epidemics in recorded history. More than two thirds of the world total of HIV-infected people live in Sub-Saharan Africa. In Central and Southern Africa at least 20% of the adult population is infected. As these adults die, they leave increasing numbers of orphans. Life expectancy at birth declined by 10 years per decade since the late 1980s to 50 years in the late 1990s, and in Botswana it is estimated to be as low as 33 years by 2010. The epidemic is increasing unabated and prospects for a curative or protective vaccine remain remote. The impact on HIV in Africa has been so profound that it influences political, economic, agriculture/food security, social, education, defence, science and health considerations. The medical and in particular immunology communities in Central Africa have the invidious challenge of on the one hand diagnosing the condition, monitoring its impact and contributing to treatment and management efforts. The science and clinical practice of immunology is challenged to find answers to the epidemic, perhaps including a vaccine. In this review we address the peculiarities of the HIV epidemic in Africa, its epidemiology and immunopathogenesis. We address the effect of the epidemic on individual patients, in their homes, workplaces and the knock-on effects on families and friends of the infected. Respective specialists discuss special groups (women, children) that are predominantly seen in Africa. We also discuss the impact of the epidemic on the clinical practice of medicine in general and challenges faced in the introduction of antiretroviral medicines. We also discuss options available for the diagnosis, treatment and monitoring of HIV-infected patients in this region.

Typing of human papillomavirus in Zimbabwean patients with invasive cancer of the uterine cervix.

BACKGROUND: Cervical cancer affects 1 in 2000 Zimbabwean women. We investigated the type-specific distribution of human papillomavirus (HPV) infection in Zimbabwean women with invasive cervical cancer. METHODS: We conducted a descriptive study on 98 women with invasive cervical cancer. The methods used were a nested polymerase chain reaction (PCR) for amplification of HPV-DNA and restriction fragment length polymorphism (RFLP) to characterize the HPV types. RESULTS: HPV-DNA was identified in 97% of the cases. HPV types 16, 33, 18 and 31 were identified in 61%, 39%, 18% and 4% of the patients, respectively. We typed one case each of HPV types 35 and 58. Multiple HPV infections were present in 24%. All patients (n = 3) with adenocarcinoma of the cervix were infected with the HPV. Patients infected with HPV-16 alone presented at a median age of 46 years while those infected with HPV-33 alone presented at 43 years. However, patients coinfected with both HPV-16 and HPV-33 were between 10 and 13 years older (median age of 56 years) than patients with either HPV-16 or HPV-33 as single infections. These differences were marginally significant (p = 0.08) or significant (p = 0.02), respectively. CONCLUSION: We present the first prevalence data on HPV types in patients with cervical cancer in Zimbabwe and show that, provided appropriate techniques are employed, HPV infection can be identified in a majority of the patients. The distribution of HPV types should be taken into consideration in tailoring locally relevant vaccines against HPV.

Detection of human papillomavirus in urine and cervical swabs from patients with invasive cervical cancer.

Despite the high prevalence of both human papillomavirus (HPV) infections and cervical cancer among Zimbabwean women, the ability to test for HPV infection of the uterine cervix is limited by a lack of an easy sample collection method that does not require gynecological examination. The presence of HPVs in urine and cervical swab samples collected from 43 women who presented with invasive cervical cancer was investigated. HPV detection was done by means of degenerate primers in a nested polymerase chain reaction (PCR). Typing of HPVs was done using restriction fragment length polymorphism (RFLP) analysis. HPV was identified and typed in 98% (42/43) of cervical swabs and 72% (31/43) of paired urine samples. HPV type 16 was the most common (25/42, 59%), followed by types: 33 (13/42, 31%), 18 (6/42, 14%), and 31 (1/42, 2%). Type-specific concordance between cervical and urine samples was high (22/28, 79%). Therefore, the HPV types identified in urine samples in most cases represent the same HPV type infecting the cervical epithelium. The results suggest that urine may be a practical sample for testing of HPV urogenital infection. Further research is required before the detection of HPV in urine can be applied in the routine cervical screening programs.

Ethnic differences in allelic distribution of IFN-g in South African women but no link with cervical cancer.

BACKGROUND: The failure of specific types of human papillomaviruses (HPV) to raise effective immune responses may be important in the pathogenesis of cervical cancer, the second most common cancer in South African women. Polymorphisms of a number of cytokine genes have been implicated in inducing susceptibility or resistance to cancers caused by infectious agents owing to their role in determining host immune response. Polymorphisms of IL-10 and IFN-gamma genes are believed to influence the expression and/or secretion levels of their respective cytokines. METHODS AND RESULTS: In this study, women with histologically proven cancer of the cervix (n = 458) and hospital-based controls (n = 587) were investigated for bi-allelic -1082 (A/G) polymorphisms of IL-10 and the bi-allelic +874(A/T) polymorphisms of IFN-gamma. In addition, the distributions of the allelic frequencies were stratified in both the African and mixed race population groups of South Africa. We found striking differences in the allele distribution of IFN-gamma (X2 = 0.02) among the two ethnic groups. A significant increase in the allele distribution of the IFN-gamma AA genotype was found in the African group compared to the mixed population group (OR, 0.5; 95% CI, 0.2-1.0). For IL-10 there were no significant allelic differences between the two South African ethnic groups. Furthermore, when the ethnic groups were combined the IL-10 allelic frequencies in the combined South African data were similar to those observed in an Oriental population from Southern China and in an Italian population. However, the allele frequencies of the IFN-gamma genotype among the two South African ethnic groups were different when compared to an Italian Caucasoid group. While crude analysis of these data showed both statistically significantly increased and diminished risks of cervical cancer among high producers of INF-gamma and low producers of IL-10 respectively, these associations were no longer significant when the data were adjusted for confounding factors. CONCLUSION: These findings demonstrate a clear correlation between ethnicity and IFN-gamma polymorphism across different population groups. However, these differences in ethnicity and gene polymorphisms in the aforementioned cytokines are suggested not to influence the development of invasive cervical cancer but may represent an important susceptibility biomarker for other diseases and should be explored further.