Intra- and interlaboratory reproducibility evaluation toward international validation status of the AmpFire assay.

To meet the screening goal of WHO's 90-70-90 strategy aimed at eliminating cervical cancer (CC) by 2030, clinical validation of human papillomavirus (HPV) assays is essential to provide accurate and valid results through fulfilling three criteria of the international validation guidelines (IVGs). Previously, the clinical accuracy of the AmpFire® HPV Screening 16/18/HR assay (AmpFire assay) was reported but reproducibility data are lacking. Here, we aim to evaluate the intra- and inter-laboratory reproducibility of the AmpFire assay. The reproducibility of the isothermal AmpFire assay was assessed using 556 cervical cell samples collected from women attending CC screening and biobanked in a Belgian HPV national reference center. This assay detects HPV16, HPV18, and 12 other high-risk HPV (hrHPV) types (31/33/35/39/45/51/52/56/58/59/66/68) in aggregate. Lower 95% confidence interval bound around the assay's reproducibility should exceed 87%, with κ ≥ 0.50. Additionally, a literature review of the assay's clinical performance was performed. The AmpFire assay showed an excellent intralaboratory (96.4%, 95% CI:94.5-97.8%, κ = 0.920) and interlaboratory (95.3%, 95% CI:93.2-96.9%, κ = 0.897) reproducibility. One study demonstrated noninferior sensitivity of a prototype AmpFire assay targeting 15 hrHPV types (including HPV53) to detect CIN2+. However, clinical specificity became similar to the comparator after removing HPV53 from analyses. The low-cost and easy-to-use AmpFire assay presents excellent reproducibility and-after removing HPV53 from the targeted types-fulfills also clinical accuracy requirements. Inclusion of HPV53, which is not recognized as carcinogenic, comprises clinical specificity of screening assays.

Genital Tract Infections in an Isolated Community: 100 Women of the Príncipe Island.

Objective: To characterize the vaginal microbiome and the rate of sexually transmitted infections (STIs) in the women of Príncipe (São Tomé and Príncipe). Methods: Cross-sectional study of 100 consecutive women, invited for a free appointment and cervical cancer screening. A vaginal slide (wet mount microscopy) and a cervical sample (ThinPrep®) (Pap test, high risk human papillomavirus [HR-HPV], N. gonorrhea [NG], T. vaginalis [TV], and C. trachomatis [CT]) were obtained. Results: TV, NG, CT, and HIV were found in 8.0%, 2.0%, 3.0%, and 2.0%, respectively, and were more prevalent in younger women. HR-HPV was positive in 36.7%; 2 were positive for HPV18, but none for HPV16. Coinfection of HPV with other STIs was 8.3%. Prevalence of abnormal vaginal flora (AVF) was 82.5%, mostly bacterial vaginosis (BV) 54.6%, and moderate/severe aerobic vaginitis (msAV) 25.8%. HR-HPV was not related to BV (p = 0.67). The association of abnormal Pap test with msAV was not significant (p = 0.08). Conclusion: The prevalence of NG, CT, TV, and HR-HPV was according to expected, while that of HR-AVF was higher. The surprisingly low prevalence of HPV16 and HPV18 must be considered in the design of programs for prevention and vaccination; this setting can be useful as a model for postvaccination scenarios.

Development, Validation, and Implementation of an Augmented Multiwell, Multitarget Quantitative PCR-Human Papillomavirus Genotyping Analysis through Software Automation, Data Science, and Artificial Intelligence.

The value of human papillomavirus (HPV) testing for cervical cancer screening is well established, where its use as a primary screening option or as a reflex test after atypical cytology results has recently gained wide acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management. In this study, a new analysis method for the RIATOL quantitative PCR assay was developed, validated, and implemented in the laboratory of clinical molecular pathology at AML, under national accreditation and following the International Organization for Standardization guidelines. It presents the successful validation of a high-throughput, multitarget HPV analysis method, with enhanced accuracy on both qualitative and quantitative end results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence. Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources. Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL real-time quantitative PCR assay proved to be a remarkable advancement in high-throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.

Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis.

BACKGROUND: Bacterial vaginosis (BV), an alteration of vaginal flora involving a decrease in Lactobacilli and predominance of anaerobic bacteria, is among the most common cause of vaginal complaints for women of childbearing age. It is well known that BV has an influence in acquisition of certain genital infections. However, association between BV and cervical human papillomavirus (HPV) infection has been inconsistent among studies. The objective of this meta-analysis of published studies is to clarify and summarize published literature on the extent to which BV is associated with cervical HPV infection. METHODS: Medline and Web of Science were systematically searched for eligible publications until December 2009. Articles were selected based on inclusion and exclusion criteria. After testing heterogeneity of studies, meta-analysis was performed using random effect model. RESULTS: Twelve eligible studies were selected to review the association between BV and HPV, including a total of 6,372 women. The pooled prevalence of BV was 32%. The overall estimated odds ratio (OR) showed a positive association between BV and cervical HPV infection (OR, 1.43; 95% confidence interval, 1.11-1.84). CONCLUSION: This meta-analysis of available literature resulted in a positive association between BV and uterine cervical HPV infection.

Mothers of adolescent girls and Human Papilloma Virus (HPV) vaccination in Western Kenya.

INTRODUCTION: human papilloma virus (HPV) which is preventable is the main cause of cervical cancer and it targets mostly young adolescents. The study was to determine the practice desire, attitude and knowledge of mothers of adolescent girls on HPV vaccination in Western Kenya. METHODS: this was a descriptive cross-sectional study design. Data was obtained using semi-structured questionnaires and analyzed using both descriptive and inferential statistics at 95% confidence level using the SPSS software version 22. A p-value ≤ 0.05 was considered statistically significant. RESULTS: ninety five percent of the mothers had intentions to vaccinate their daughters and also had a positive attitude and their response to HPV vaccination was significantly lower than those without intentions p=0.02, 95% CI, OR=0.48 (0.90-0.89). Vaccination against HPV was low at 9.4% with a mean age of 34 years. Our results found a high level of cervical cancer awareness (85.0%), HPV and vaccine awareness respectively (62.0%, and 64.0%). "Vaccination of my daughters will prompt early sexual activity and the cost of HPV vaccination being a barrier to vaccination" had a statistically significant influence on the practice of vaccination. Negative attitude to daughters´ early onset of sexual activity significantly reduced up take while positive attitude to cost of HPV vaccine significantly increased up take of HPV vaccination with p value of 0.007 and 0.04 respectively. CONCLUSION: awareness of HPV and HPV vaccine prevention is low among mothers of adolescent girls in Western Kenya. There was a positive attitude and high desire towards the use of HPV vaccination therefore a need for awareness, policy and unify efforts to reduce cervical cancer burden.

Distinct role of clathrin-mediated endocytosis in the functional uptake of cholera toxin.

The involvement of the clathrin-mediated endocytic internalization route in the uptake of cholera toxin (CT) was investigated using different cell lines, including the human intestinal Caco-2 and T84 cell lines, green monkey Vero cells, SH-SY5Y neuroblastoma cells and Madin-Darby canine kidney cells. Suppression of the clathrin-mediated endocytic pathway by classical biochemical procedures, like intracellular acidification and potassium depletion, inhibited cholera toxin uptake by up to about 50% as well as its ability to raise intracellular levels of cAMP. Also prior exposure of these cell types to the cationic amphiphilic drug chlorpromazine reduced the functional uptake of cholera toxin, even to a greater extent. These effects were dose- and cell type-dependent, suggesting an involvement of clathrin-mediated endocytosis in the functional uptake of cholera toxin. For a more straightforward approach to study the role of the clathrin-mediated uptake in the internalization of cholera toxin, a Caco-2(eps-) cell line was exploited. These Caco-2(eps-) cells constitutively suppress the expression of epsin, an essential accessory protein of clathrin-mediated endocytosis, thereby selectively blocking this internalization route. CT uptake was found to be reduced by over 60% in Caco-2(eps-) paralleled by a diminished ability of CT to raise the level of cAMP. The data presented suggest that the clathrin-mediated uptake route fulfils an important role in the functional internalization of cholera toxin in several cell types.

Associations Between Vaginal Infections and Potential High-risk and High-risk Human Papillomavirus Genotypes in Female Sex Workers in Western Kenya.

PURPOSE: Infection with and persistence of high-risk human papillomavirus (HR HPV) are the strongest risk factors for cervical cancer. Little is known about the prevalence and role of concurrent sexually transmitted infections (STIs) found in HPV-infected female sex workers (FSW) in Africa. This study purports to test our a priori hypotheses that STIs are associated with genotypes pertaining to the α-group species 9. The objectives were to determine the prevalence of bacterial vaginosis (BV), Trichomonas vaginalis, and Candida spp in FSW, the association between these STIs and the prevalence of any potential HR and HR HPV genotypes in FSWs. METHODS: A cross-sectional study design of 616 FSW from Western Kenya aged between 18 and 61 years during 2009-2015 using a peer recruitment sampling strategy. Inclusion criteria for the study entailed female sex and >18 years of age and having engaged in transactional sex in exchange for money, goods, services, or drugs in the last 3 months. Women were excluded if they were pregnant, <18 years of age, had a history of cervical dysplasia or cancer, had current abnormal bleeding, or had a hysterectomy. FINDINGS: Of the FSW, 33.3% had HIV and 57.7% harbored a potential HR and HR HPV genotype. The 2 most prevalent potential HR and HR genotypes were HPV 16 (16.10%) and HPV 59 (12.20%). BV was the most common infection (48.3%), followed by Trichomonas vaginalis (31.4%) and Candida spp (19.9%). A multivariate regression revealed significant associations with both α-group 9 and 6; BV and HPV 58 (adjusted odds ratio [aOR] = 2.3; 95% CI, 1.0-5.2; P = 0.05), Trichomonas vaginalis and HPV 31 and HPV 35 (aOR = 2.0; 95% CI, 1.0-3.8; P = 0.04 and aOR = 1.8; 95% CI, 1.0-3.3, P = 0.05 respectively); and between Candida spp and HPV 53 (aOR = 2.0; 95% CI, 1.1-4.0; P = 0.03) and 16 (aOR = 1.9; 95% CI, 1.1-3.3; P = 0.03). IMPLICATIONS: Snowball sampling may have inadvertently excluded FSW less likely to benefit from a social network. Significant associations between BV and HPV 58 and between Candida spp and HPV 16 and 53 suggest the need for sexually transmitted disease management within a cervical cancer prevention program. The probable synergistic effects of the vaginal microbiota should be elucidated, especially within this vulnerable population. Given the potential for FSW to transmit STIs, robust epidemiologic sampling methods are urgently required that account for the heterogeneity of the FSW population.

Surveillance of effects of HPV vaccination in Belgium.

BACKGROUND: Early effects of HPV (human papillomavirus) vaccination are reflected by changes observable in young women attending cervical cancer screening. SUBJECT AND METHODS: The SEHIB study included HPV geno-typing of ∼6000 continuous and 650 pathological cervical cell specimen as well as biopsies, collected from women in Belgium in 2010-2014. Data were linked to vaccination status. RESULTS: HPV vaccination offered protection among women aged <30years against infection with HPV16 (vaccine effectiveness [VE]=67%, 95% CI: 48-79%), HPV18 (VE=93%, 95% CI: 52-99%), and high-risk HPV (VE=16%, 95% CI: 2-29%). Vaccination protected also against cytological lesions. Vaccination protected against histologically confirmed lesions: significantly lower absolute risks of CIN1+ (risk difference [RD]=-1.6%, 95% CI: -2.6% to -0.7%) and CIN3+ associated with HPV16/18 (RD=-0.3%, 95% CI -0.6% to -0.1%). Vaccine effectiveness decreased with age. Protection against HPV16 and 18 infection was significant in all age groups, however no protection was observed against cytological lesions associated with these types in age-group 25-29. CONCLUSION: The SEHIB study demonstrates the effectiveness of HPV vaccination in Belgian young women in particular in age group 18-19. Declining effectiveness with increasing age may be explained by higher tendency of women already exposed to infection to get the vaccine.

Comparative analysis of cervical cytology and human papillomavirus genotyping by three different methods in a routine diagnostic setting.

Application of Bethesda guidelines on cervical cytology involves human papillomavirus (HPV) determinations on all ASC-US and ASC-H results. We compared HPV DNA results in view of the eventual development of a cervical intraepithelial neoplasia lesion determined either on cytology or histology. A total of 214 liquid-based cytology samples were analysed. Three different HPV DNA methods were applied: the Abbott RealTime High Risk HPV test, INNO-Lipa HPV Genotyping Extra and Full Spectrum PCR HPV Amplification and Detection/Genotyping System by Lab2Lab Diagnostic Service. A comparison of these three methods showed full concordance only for 49 samples (23%), and 27 (13%) of the samples were discordant in indicating the presence of the high-risk HPV type. Out of 214 patients, 88 were selected who presented with a cervical intraepithelial neoplasia or a VAIN lesion at follow-up cytology or histology. In this group, full concordance with HPV genotyping was present only in 19 (22%) follow-up samples. Nine (10%) follow-up samples showed discordant results for the presence of a high-risk genotype between the three genotyping methods tested either by negativity for high-risk HPV by one of the methods (n=6) or by failure to genotype HPV (n=2), or by a combination of both (n=1). Moreover, discordance for the detection of HPV16 or HPV18 was observed between the three HPV DNA genotyping methods used in 9 (10%) follow-up samples. In addition, the performance of genotyping methods on 20 external quality samples was assessed, showing discordant results for HPV16 and HPV18. Major differences were found in the genotyping results according to the HPV DNA method. Our findings highlight the importance of careful interpretation of data from studies using different HPV genotyping methods and underline the need for standardization by method validation in clinical laboratories, especially in the setting of primary HPV screening.

HIV testing and sexually transmitted infection care among sexually active youth in the Balkans.

In light of the imminent threat of a growing HIV epidemic in east and southeast Europe, optimal accessibility of primary and secondary HIV preventative interventions, including HIV testing and sexually transmitted infection (STI) care, are fast becoming public health priorities. We surveyed 2150 high school students in Bosnia and Herzegovina, FYR of Macedonia, Serbia, and Montenegro to examine the uptake of HIV testing and associated predictors. Among sexually active youth (n = 651), 5.9% had already been tested for HIV. In marginal logistic regression, country of origin, type of high school, knowing a friend or relative with HIV, poor self-assessed health status, suspicion of having had an STI, and not having used a condom at first sex were independently associated with HIV testing. Fear of the diagnosis, fear of violation of confidentiality, and not knowing where to go for HIV testing were reported as barriers to HIV testing. Of sexually active adolescents who thought they might have contracted an STI, only 42% had subsequently visited a doctor or health facility. The main reasons for not doing so were spontaneous disappearance of the complaints, fear of the diagnosis and being ashamed of discussing the problem. In conclusion, the uptake of HIV testing among this population of sexually active, urban high school students was found to be low, although a higher prevalence of HIV testing history was observed among students showing evidence of risky sexual behavior. Practical and psychological factors seem to challenge the accessibility of facilities for HIV testing and STI care.