Antifungal Resistance in Pulmonary Aspergillosis.

Aspergilli may cause various pulmonary diseases in humans, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and acute invasive pulmonary aspergillosis (IPA). In addition, chronic colonization may occur in cystic fibrosis (CF). Aspergillus fumigatus represents the main pathogen, which may employ different morphotypes, for example, conidia, hyphal growth, and asexual sporulation, in the various Aspergillus diseases. These morphotypes determine the ease by which A. fumigatus can adapt to stress by antifungal drug exposure, usually resulting in one or more resistance mutations. Key factors that enable the emergence of resistance include genetic variation and selection. The ability to create genetic variation depends on the reproduction mode, including, sexual, parasexual, and asexual, and the population size. These reproduction cycles may take place in the host and/or in the environment, usually when specific conditions are present. Environmental resistance is commonly characterized by tandem repeat (TR)-mediated mutations, while in-host resistance selection results in single-resistance mutations. Reported cases from the literature indicate that environmental resistance mutations are almost exclusively present in patients with IA indicating that the risk for in-host resistance selection is very low. In aspergilloma, single-point mutations are the dominant resistance genotype, while in other chronic Aspergillus diseases, for example, ABPA, CPA, and CF, both TR-mediated and single-resistance mutations are reported. Insights into the pathogenesis of resistance selection in various Aspergillus diseases may help to improve diagnostic and therapeutic strategies.

Longitudinal analysis on the ecological dynamics of the cervicovaginal microbiome in hrHPV infection.

The cervicovaginal microbiome (CVM) is a dynamic continuous microenvironment that can be clustered in microbial community state types (CSTs) and is associated with women's cervical health. Lactobacillus-depleted communities particularly associate with an increased susceptibility for persistence of high-risk human papillomavirus (hrHPV) infections and progression of disease, but the long-term ecological dynamics of CSTs after hrHPV infection diagnosis remain poorly understood. To determine such dynamics, we examined the CVM of our longitudinal cohort of 141 women diagnosed with hrHPV infection at baseline with collected cervical smears at two timepoints six-months apart. Here we describe that the long-term microbiome dissimilarity has a positive correlation with microbial diversity at both visits and that women with high abundance and dominance for Lactobacillus iners at baseline exhibit more similar microbiome composition at second visit than women with Lactobacillus-depleted communities at baseline. We further show that the species Lactobacillus acidophilus and Megasphaera genomosp type 1 associate with CST changes between both visits. Lastly, we also observe that Gardnerella vaginalis is associated with the stability of Lactobacillus-depleted communities while L. iners is associated with the instability of Megasphaera genomosp type 1-dominated communities. Our data suggest dynamic patterns of cervicovaginal CSTs during hrHPV infection, which could be potentially used to develop microbiome-based therapies against infection progression towards disease.

Acceptability of risk-based triage in cervical cancer screening: A focus group study.

BACKGROUND: Compared to the previous cytology-based program, the introduction of primary high-risk human papillomavirus (hrHPV) based screening in 2017 has led to an increased number of referrals. To counter this, triage of hrHPV-positive women in cervical cancer screening can potentially be optimized by taking sociodemographic and lifestyle risk factors for cervical abnormalities into account. Therefore, it is essential to gain knowledge of the views of women (30-60 years) eligible for cervical cancer screening. OBJECTIVE: The main goal of this qualitative study was to gain insight in the aspects that influence acceptability of risk-based triage in cervical cancer screening. DESIGN: A focus group study in which participants were recruited via four general medical practices, and purposive sampling was used to maximize heterogeneity with regards to age, education level, and cervical cancer screening experiences. APPROACH: The focus group discussions were transcribed verbatim and analyzed using reflexive thematic analysis. PARTICIPANTS: A total of 28 women (average age: 45.2 years) eligible for cervical cancer screening in The Netherlands participated in seven online focus group discussions. Half of the participants was higher educated, and the participants differed in previous cervical cancer screening participation and screening result. KEY RESULTS: In total, 5 main themes and 17 subthemes were identified that determine the acceptability of risk-stratified triage. The main themes are: 1) adequacy of the screening program: an evidence-based program that is able to minimize cancer incidence and reduce unnecessary referrals; 2) personal information (e.g., sensitive topics and stigma); 3) emotional impact: fear and reassurance; 4) communication (e.g., transparency); and 5) autonomy (e.g., prevention). CONCLUSION: The current study highlights several challenges regarding the development and implementation of risk-based triage that need attention in order to be accepted by the target group. These challenges include dealing with sensitive topics and a transparent communication strategy.

The Siderophore Ferricrocin Mediates Iron Acquisition in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus utilizes two high-affinity iron uptake mechanisms, termed reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). The latter has been shown to be crucial for virulence of this fungus and is a target for development of novel strategies for diagnosis and treatment of fungal infections. So far, research on SIA in this mold focused mainly on the hyphal stage, revealing the importance of extracellular fusarinine-type siderophores in iron acquisition as well as of the siderophore ferricrocin in intracellular iron handling. The current study aimed to characterize iron acquisition during germination. High expression of genes involved in biosynthesis and uptake of ferricrocin in conidia and during germination, independent of iron availability, suggested a role of ferricrocin in iron acquisition during germination. In agreement, (i) bioassays indicated secretion of ferricrocin during growth on solid media during both iron sufficiency and limitation, (ii) ferricrocin was identified in the supernatant of conidia germinating in liquid media during both iron sufficiency and limitation, (iii) in contrast to mutants lacking all siderophores, mutants synthesizing ferricrocin but lacking fusarinine-type siderophores were able to grow under iron limitation in the absence of RIA, and (iv) genetic inactivation of the ferricrocin transporter Sit1 decreased germination in the absence of RIA. Taken together, this study revealed that ferricrocin has not only an intracellular role but also functions as an extracellular siderophore to support iron acquisition. The iron availability-independent ferricrocin secretion and uptake during early germination indicate developmental, rather than iron regulation. IMPORTANCE Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold. Previous studies demonstrated the crucial role of secreted fusarinine-type siderophores, such as triacetylfusarinine C, in iron acquisition, as well as of the ferrichrome-type siderophore ferricrocin in intracellular iron storage and transport. Here, we demonstrate that ferricrocin is also secreted to mediate iron acquisition during germination together with reductive iron assimilation. During early germination, ferricrocin secretion and uptake were not repressed by iron availability, indicating developmental regulation of this iron acquisition system in this growth phase.

Retrospective Multicenter Evaluation of the VirClia Galactomannan Antigen Assay for the Diagnosis of Pulmonary Aspergillosis with Bronchoalveolar Lavage Fluid Samples from Patients with Hematological Disease.

Galactomannan (GM) testing of bronchoalveolar lavage (BAL) fluid samples has become an essential tool to diagnose invasive pulmonary aspergillosis (IPA) and is part of diagnostic guidelines. Enzyme-linked immunosorbent assays (ELISAs) (enzyme immunoassays [EIAs]) are commonly used, but they have a long turnaround time. In this study, we evaluated the performance of an automated chemiluminescence immunoassay (CLIA) with BAL fluid samples. This was a multicenter retrospective study in the Netherlands and Belgium. BAL fluid samples were collected from patients with underlying hematological diseases with a suspected invasive fungal infection. Diagnosis of IPA was based on the 2020 European Organisation for Research and Treatment of Cancer (EORTC)/Mycoses Study Group Education and Research Consortium (MSGERC) consensus definitions. GM results were reported as optical density index (ODI) values. ODI cutoff values for positive results that were evaluated were 0.5, 0.8, and 1.0 for the EIA and 0.16, 0.18, and 0.20 for the CLIA. Probable IPA cases were compared with two control groups, one with no evidence of IPA and another with no IPA or possible IPA. Qualitative agreement was analyzed using Cohen's κ, and quantitative agreement was analyzed by Spearman's correlation. We analyzed 141 BAL fluid samples from 141 patients; 66 patients (47%) had probable IPA, and 56 cases remained probable IPA when the EIA GM result was excluded as a criterion, because they also had positive culture and/or duplicate positive PCR results. Sixty-three patients (45%) had possible IPA and 12 (8%) had no IPA. The sensitivity and specificity of the two tests were quite comparable, and the overall qualitative agreement between EIA and CLIA results was 81 to 89%. The correlation of the actual CLIA and EIA values was strong at 0.72 (95% confidence interval, 0.63 to 0.80). CLIA has similar performance, compared to the gold-standard EIA, with the benefits of faster turnaround because batching is not required. Therefore, CLIA can be used as an alternative GM assay for BAL fluid samples.

Evaluation of DNA methylation biomarkers ASCL1 and LHX8 on HPV-positive self-collected samples from primary HPV-based screening.

BACKGROUND: Host-cell DNA methylation analysis can be used to triage women with high-risk human papillomavirus (HPV)-positive self-collected cervicovaginal samples, but current data are restricted to under-/never-screened women and referral populations. This study evaluated triage performance in women who were offered primary HPV self-sampling for cervical cancer screening. METHODS: Self-collected samples from 593 HPV-positive women who participated in a primary HPV self-sampling trial (IMPROVE study; NTR5078), were tested for the DNA methylation markers ASCL1 and LHX8 using quantitative multiplex methylation-specific PCR (qMSP). The diagnostic performance for CIN3 and cervical cancer (CIN3 + ) was evaluated and compared with that of paired HPV-positive clinician-collected cervical samples. RESULTS: Significantly higher methylation levels were found in HPV-positive self-collected samples of women with CIN3 + than control women with no evidence of disease (P values <0.0001). The marker panel ASCL1/LHX8 yielded a sensitivity for CIN3 + detection of 73.3% (63/86; 95% CI 63.9-82.6%), with a corresponding specificity of 61.1% (310/507; 95% CI 56.9-65.4%). The relative sensitivity for detecting CIN3+ was 0.95 (95% CI 0.82-1.10) for self-collection versus clinician-collection, and the relative specificity was 0.82 (95% CI 0.75-0.90). CONCLUSIONS: The ASCL1/LHX8 methylation marker panel constitutes a feasible direct triage method for the detection of CIN3 + in HPV-positive women participating in routine screening by self-sampling.

Value of routine follow-up in oropharyngeal squamous cell cancer patients treated with curative intent.

BACKGROUND: The major goal of routine follow-up in oropharyngeal squamous cell carcinoma (OPSCC) patients is the asymptomatic detection of new disease in order to improve survival. This study evaluated the effect of routine follow-up on overall survival (OS). METHODS: A retrospective cohort of 307 consecutive OPSCC patients treated with curative intent between 2006 and 2012 was analyzed. The effectiveness of routine follow-up was studied by comparing treatment-intent and OS in patients with asymptomatically versus symptomatically detected new disease. RESULTS: Three- and five-year risks of new disease were 29% (95% CI: 24-34) and 33% (95% CI: 27-39). Of the 81 patients with locoregional recurrence or second primary head and neck cancer, 8 (10%) were detected asymptomatically with no difference in OS with those detected with symptoms. CONCLUSIONS: Asymptomatic detection of new disease during routine visits was not associated with improved OS. The focus of follow-up should be on providing psychosocial care and rehabilitation.

The added value of digital imaging to reflex cytology for triage of high-risk human papillomavirus positive self-sampled material in cervical cancer screening: A prospective cohort study.

OBJECTIVE: Cytology performed directly on hrHPV-positive self-samples (reflex cytology) is feasible and for women with abnormal cytology, an additional cytology test at the general practitioner could be omitted. The aim of this study is to assess the added value of digital imaging (ThinPrep® Imaging System) on the clinical utility of reflex cytology by reducing screening error. DESIGN: A secondary analysis of a prospective cohort study. SETTING: One of five Dutch screening laboratories. POPULATION: Women tested hrHPV-positive on self-samples between December 2018 and August 2019. METHODS: Self-samples were used for reflex cytology with and without digital imaging. The follow-up data (cytological and histological results within 1 year of follow-up) were obtained through the Dutch Pathology Registry (PALGA). MAIN OUTCOME MEASURES: Test performance of the reflex cytology was determined by comparing it with physician-collected follow-up results. RESULTS: The sensitivity for detecting abnormal cells by reflex cytology on self-samples increased significantly from 26.3% (42/160; 95% confidence interval [CI] 19.6-33.8) without digital imaging to 35.4% (56/158; 95% CI 28-43.4) with digital imaging (P < 0.05) without compromising specificity. Importantly, 41.7% of women with ≥CIN2 (35/84) and 45.6% with ≥CIN3 (26/57) were detected by reflex cytology with digital imaging on hrHPV-positive self-samples. CONCLUSION: Digital imaging is of added value to reflex cytology on hrHPV-positive self-samples with a 9% increase in sensitivity. If reflex cytology on self-samples analysed with digital imaging had been implemented in the screening programme, 35.4% of the hrHPV-positive women with abnormal cytology on additional physician-collected samples could have been referred directly for colposcopy.

Sociodemographic Characteristics and Screening Outcomes of Women Preferring Self-Sampling in the Dutch Cervical Cancer Screening Programme: A Population-Based Study.

BACKGROUND: In the Netherlands, lower high-risk human papillomavirus (hrHPV) positivity but higher cervical intraepithelial neoplasia (CIN) 2+ detection were found in self-collected compared with clinician-collected samples. To investigate the possible reason for these differences, we compared sociodemographic and screening characteristics of women and related these to screening outcomes. METHODS: We extracted data from PALGA on all primary hrHPV screens and associated follow-up tests for 857,866 screened women, invited in 2017 and 2018. We linked these data with sociodemographic data from Statistics Netherlands. Logistic regression was performed for hrHPV positivity and CIN 2+/3+ detection. RESULTS: Out of the 857,866 women, 6.8% chose to use a self-sampling device. A higher proportion of self-sampling users was ages 30 to 35 years, was not previously screened, was living in a one-person household, or was the breadwinner in the household. After adjustment for these factors self-sampling had lower hrHPV positivity (aOR, 0.65; 95% CI, 0.63-0.68)) as compared with clinician-collected sampling, as well as lower odds of CIN 2+ (aOR, 0.76; 95% CI, 0.70-0.82) and CIN 3+ (aOR, 0.86; 95% CI, 0.78-0.95) detection. CONCLUSIONS: It is likely that the observed differences between the two sampling methods are not only related to sociodemographic differences, but related to differences in screening test accuracy and/or background risk. IMPACT: Self-sampling can be used for targeting underscreened women, as a more convenient screening tool. Further investigation is required to evaluate how to implement self-sampling, when it is used as a primary instrument in routine screening. See related commentary by Arbyn et al., p. 159.

Adjunctive use of p16 immunohistochemistry for optimizing management of CIN lesions in a high-risk human papillomavirus-positive population.

INTRODUCTION: Immunostaining with p16INK4a (p16), a tumor-suppressor surrogate protein biomarker for high-risk human papillomavirus (hrHPV) oncogenic activity, may complement standard hematoxylin and eosin (H&E) histology review, and provide more objective criteria to support the cervical intraepithelial neoplasia (CIN) diagnosis. With this study we assessed the impact of p16 immunohistochemistry on CIN grading in an hrHPV-based screening setting. MATERIAL AND METHODS: In this post-hoc analysis, 326 histology follow-up samples from a group of hrHPV-positive women were stained with p16 immunohistochemistry. All H&E samples were centrally revised. The pathologists reported their level of confidence in classifying the CIN lesion. RESULTS: Combining H&E and p16 staining resulted in a change of diagnosis in 27.3% (n = 89) of cases compared with the revised H&E samples, with a decrease of 34.5% (n = 18) in CIN1 and 22.7% (n = 15) in CIN2 classifications, and an increase of 18.3% (n = 19) in no CIN and 20.7% (n = 19) in CIN3 diagnoses. The level of confidence in CIN grading by the pathologist increased with adjunctive use of p16 immunohistochemistry to standard H&E. CONCLUSIONS: This study shows that adjunctive use of p16 immunohistochemistry to H&E morphology reduces the number of CIN1 and CIN2 classifications with a proportional increase in no CIN and CIN3 diagnoses, compared with standard H&E-based CIN diagnosis alone. The pathologists felt more confident in classifying the material with H&E and p16 immunohistochemistry than by using H&E alone, particularly during assessment of small biopsies. Adjunctive use of p16 immunohistochemistry to standard H&E assessment of CIN would be valuable for the diagnostic accuracy, thereby optimizing CIN management and possibly decreasing overtreatment.

In-depth insights into cervicovaginal microbial communities and hrHPV infections using high-resolution microbiome profiling.

The cervicovaginal microbiome (CVM) correlates with women's cervical health, and variations in its composition are associated with high-risk human papillomavirus (hrHPV) infection outcomes. Cervicovaginal microbes have been grouped into five community state types (CSTs) based on microbial community composition and abundance. However, studying the impact of CSTs in health and disease is challenging because the current sequencing technologies have limited confident discrimination between closely related and yet functionally different bacterial species. Circular probe-based RNA sequencing (ciRNAseq) achieves high-resolution microbiome profiling and therefore provides in-depth and unambiguous knowledge about the composition of the CVM. Based on ciRNAseq profiling of a large cohort of cervical smears (n = 541), we here define subgroups of CSTs I, III, and IV based on intra-CST differences with respect to abundances of Lactobacillus acidophilus (CSTs I-A vs. I-B and CSTs III-A vs. III-B), Lactobacillus iners (CSTs I-A vs. I-B and CSTs III-A vs. III-B), and Megasphaera genomosp type 1 (CSTs IV-A vs. IV-B). Our results further support the existence of subgroups of CST IV-C that are dominant for non-Lactobacillus species and have intermediate microbial diversity. We also show that CST V is associated with uninfected conditions, and CST IV-A associates with hrHPV-induced cervical disease. In conclusion, we characterized new subdivisions of cervicovaginal CSTs, which may further advance our understanding of women's cervical health and hrHPV-related progression to disease.

Assessing the Cervicovaginal Microbiota in the Context of hrHPV Infections: Temporal Dynamics and Therapeutic Strategies.

Cervical cancer is the third leading cause of female cancers globally, resulting in more than 300,000 deaths every year. The majority of all cervical cancers are caused by persistent infections with high-risk human papillomaviruses (hrHPV) that can progress to cancer via a series of premalignant lesions. Most women, however, clear this infection within a year, concomitant with disease regression. Both hrHPV clearance and disease regression have been associated with the composition of the cervicovaginal microenvironment, which is defined by the host immune system and the cervicovaginal microbiome (CVM). A healthy microbiome is generally characterized by a high abundance of Lactobacillus species, and a change in the composition may cause bacterial vaginosis (BV), which is associated with an increased susceptibility to persistent hrHPV infections and disease. In this review, the composition of the CVM is discussed, with emphasis on the possible causes that drive changes in the cervicovaginal microbiota in relation to hrHPV infections, disease progression, and disease regression. The literature search focused on the composition of the CVM and its correlation with hrHPV infections and neoplastic lesions as well as the current efforts to adjust the microbiome against adverse viral outcomes.

Clinical use of the mRNA urinary biomarker SelectMDx test for prostate cancer.

BACKGROUND: Molecular biomarker tests are developed as diagnostic tools for prostate cancer (PCa) diagnosis. The SelectMDx (MDxHealth, Nijmegen, The Netherlands) test is a urinary-based biomarker test intended to be used to predict presence of high-grade PCa upon biopsy in men with elevated serum prostate-specific antigen (PSA) levels. Previous validation of the SelectMDx test revealed that 53% of the unnecessary biopsies (biopsies indicating no- or GG1 PCa) could be avoided using the SelectMDx test as a decision-tool to select men for prostate biopsy. The objective of this study is to examine the use of the commercially available SelectMDx test under routine, real-life practice. METHODS: Men that underwent a SelectMDx test between May 2019 and December 2020 and that were originating from countries that perform the SelectMDx test on a regular basis were included in this study, resulting in 5157 cases from 10 European countries. Clinical parameters, urinary RNA scores, and test outcomes were compared between PSA groups, age groups, countries, and the validation cohort (described previously [4]) using the Mann-Whitney U test, Chi-Square test, Benjamini-Hochberg and Kruskal-Wallis tests. RESULTS: 40.72% of the cases received a negative SelectMDx result. The test is also used in patients outside the intended-use population (PSA < 3 and >10 ng/mL). Clinical parameters (age, PSA density, DRE outcome) varied between patient population from individual countries and the validation cohort, resulting in differences in the potential number of saved biopsies using the test. CONCLUSIONS: The potential number of reduced biopsies in clinical use was 40,72% using the SelectMDx test, assuming a negative SelectMDx test resulted in the decision not to biopsy the patient. This is higher compared to the validation cohort, which is explained by differences in patient population.

Targeted RNA next generation sequencing analysis of cervical smears can predict the presence of hrHPV-induced cervical lesions.

BACKGROUND: Because most cervical cancers are caused by high-risk human papillomaviruses (hrHPVs), cervical cancer prevention programs increasingly employ hrHPV testing as a primary test. The high sensitivity of HPV tests is accompanied by low specificity, resulting in high rates of overdiagnosis and overtreatment. Targeted circular probe-based RNA next generation sequencing (ciRNAseq) allows for the quantitative detection of RNAs of interest with high sequencing depth. Here, we examined the potential of ciRNAseq-testing on cervical scrapes to identify hrHPV-positive women at risk of having or developing high-grade cervical intraepithelial neoplasia (CIN). METHODS: We performed ciRNAseq on 610 cervical scrapes from the Dutch cervical cancer screening program to detect gene expression from 15 hrHPV genotypes and from 429 human genes. Differentially expressed hrHPV- and host genes in scrapes from women with outcome "no CIN" or "CIN2+" were identified and a model was built to distinguish these groups. RESULTS: Apart from increasing percentages of hrHPV oncogene expression from "no CIN" to high-grade cytology/histology, we identified genes involved in cell cycle regulation, tyrosine kinase signaling pathways, immune suppression, and DNA repair being expressed at significantly higher levels in scrapes with high-grade cytology and histology. Machine learning using random forest on all the expression data resulted in a model that detected 'no CIN' versus CIN2+ in an independent data set with sensitivity and specificity of respectively 85 ± 8% and 72 ± 13%. CONCLUSIONS: CiRNAseq on exfoliated cells in cervical scrapes measures hrHPV-(onco)gene expression and host gene expression in one single assay and in the process identifies HPV genotype. By combining these data and applying machine learning protocols, the risk of CIN can be calculated. Because ciRNAseq can be performed in high-throughput, making it cost-effective, it can be a promising screening technology to stratify women at risk of CIN2+. Further increasing specificity by model improvement in larger cohorts is warranted.

Performance of DNA methylation analysis of ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST for the triage of HPV-positive women: Results from a Dutch primary HPV-based screening cohort.

Methylation of host-cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high-risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host-cell DNA methylation markers for triage in an hrHPV-positive cohort derived from primary HPV-based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation-specific PCR (qMSP) in clinician-collected cervical samples (n = 715) from hrHPV-positive women (age 29-60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end-point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single-marker and bi-marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV-positive clinician-collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi-marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3-85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1-77.9%). In conclusion, our study shows that the individual host-cell DNA methylation classifiers and the bi-marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV-positive women invited for routine screening.

Clinical performance of high-risk HPV testing on self-samples versus clinician samples in routine primary HPV screening in the Netherlands: An observational study.

BACKGROUND: High-risk human papillomavirus (hrHPV) testing on self-collected samples has potential as a primary screening tool in cervical screening, but real-world evidence on its accuracy in hrHPV-based screening programmes is lacking. METHODS: In the Netherlands, women aged 30-60 years invited for cervical screening can choose between sampling at the clinician's office (Cervex Brush) or self-sampling at home (Evalyn Brush). HrHPV testing is performed using Roche Cobas 4800. We collected screening test results between January 2017 and March 2018 and histological follow-up until August 2019. The main outcome measures were mean cycle threshold (Ct) value, cervical intraepithelial neoplasia (CIN) grade 3 or cancer (CIN3+) and CIN grade 2 or worse (CIN2+). FINDINGS: 30,808 women had a self-collected and 456,207 had a clinician-collected sample. In hrHPV-positive women with adequate cytology, Ct values were higher for self-collection than clinician-collection with a mean Ct difference of 1·25 (95% CI 0·98-1·52) in women without CIN2+, 2·73 (1·75-3·72) in CIN2 and 3·59 (3·03-4·15) in CIN3+. The relative sensitivity for detecting CIN3+ was 0·94 (0·90-0·97) for self-collection versus clinician-collection and the relative specificity was 1·02 (1·02-1·02). INTERPRETATION: The clinical accuracy of hrHPV testing on a self-collected sample for detection of CIN3+ is high and supports its use as a primary screening test for all invited women. Because of the slightly lower sensitivity of hrHPV testing on a self-collected compared to a clinician-collected sample, an evaluation of the workflow procedure to optimise clinical performance seems warranted. FUNDING: National Institute for Public Health and the Environment (the Netherlands) and the European Commission.

Novel high-resolution targeted sequencing of the cervicovaginal microbiome.

BACKGROUND: The cervicovaginal microbiome (CVM) plays a significant role in women's cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM. RESULTS: We introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes' abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women. CONCLUSIONS: CiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions.

Cervical intraepithelial neoplasia and the risk of spontaneous preterm birth: A Dutch population-based cohort study with 45,259 pregnancy outcomes.

BACKGROUND: Excisional procedures of cervical intraepithelial neoplasia (CIN) may increase the risk of preterm birth. It is unknown whether this increased risk is due to the excision procedure itself, to the underlying CIN, or to secondary risk factors that are associated with both preterm birth and CIN. The aim of this study is to assess the risk of spontaneous preterm birth in women with treated and untreated CIN and examine possible associations by making a distinction between the excised volume of cervical tissue and having cervical disease. METHODS AND FINDINGS: This Dutch population-based observational cohort study identified women aged 29 to 41 years with CIN between 2005 and 2015 from the Dutch pathology registry (PALGA) and frequency matched them with a control group without any cervical abnormality based on age at and year of pathology outcome (i.e., CIN or normal cytology) and urbanization (<100,000 inhabitants or ≥100,000 inhabitants). All their 45,259 subsequent singleton pregnancies with a gestational age ≥16 weeks between 2010 and 2017 were identified from the Dutch perinatal database (Perined). Nineteen potential confounders for preterm birth were identified. Adjusted odds ratios (ORs) were calculated for preterm birth comparing the 3 different groups of women: (1) women without CIN diagnosis; (2) women with untreated CIN; and (3) women with treated CIN prior to each childbirth. In total, 29,907, 5,940, and 9,412 pregnancies were included in the control, untreated CIN, and treated CIN group, respectively. The control group showed a 4.8% (1,002/20,969) proportion of spontaneous preterm birth, which increased to 6.9% (271/3,940) in the untreated CIN group, 9.5% (600/6,315) in the treated CIN group, and 15.6% (50/321) in the group with multiple treatments. Women with untreated CIN had a 1.38 times greater odds of preterm birth compared to women without CIN (95% confidence interval (CI) 1.19 to 1.60; P < 0.001). For women with treated CIN, these odds 2.07 times increased compared to the control group (95% CI 1.85 to 2.33; P < 0.001). Treated women had a 1.51 times increased odds of preterm birth compared to women with untreated CIN (95% CI 1.29 to 1.76; P < 0.001). Independent from cervical disease, a volume excised from the cervix of 0.5 to 0.9 cc increased the odds of preterm birth 2.20 times (37/379 versus 1,002/20,969; 95% CI 1.52 to 3.20; P < 0.001). These odds further increased 3.13 times and 5.93 times for women with an excised volume of 4 to 8.9 cc (90/724 versus 1,002/20,969; 95% CI 2.44 to 4.01; P < 0.001) and ≥9 cc (30/139 versus 1,002/20,969; 95% CI 3.86 to 9.13; P < 0.001), respectively. Limitations of the study include the retrospective nature, lack of sufficient information to calculate odds of preterm birth <24 weeks, and that the excised volume could only be calculated for a select group of women. CONCLUSIONS: In this study, we observed a strong correlation between preterm birth and a volume of ≥0.5 cc excised cervical tissue, regardless of the severity of CIN. Caution should be taken when performing excisional treatment in women of reproductive age as well as prudence in case of multiple biopsies. Fertile women with a history of performing multiple biopsies or excisional treatment for CIN may benefit from close surveillance during pregnancy.

Benefit and burden in the Dutch cytology-based vs high-risk human papillomavirus-based cervical cancer screening program.

BACKGROUND: In 2017, the Dutch cervical cancer screening program had replaced the primary cytology-based screening with primary high-risk human papillomavirus-based screening, including the opportunity to participate through self-sampling. Evaluation and balancing benefit (detection of high-grade cervical intraepithelial neoplasia) and burden of screening (unnecessary referrals, invasive diagnostics, and overtreatment) is needed. OBJECTIVE: This study aimed to compare the referral rates, detection of high-grade cervical intraepithelial neoplasia, overdiagnosis, and overtreatment in the new high-risk human papillomavirus-based screening program, including physician-sampled and self-sampled material, with the previous cytology-based screening program in the Netherlands. STUDY DESIGN: A retrospective cohort study was conducted within the Dutch population-based cervical cancer screening program. Screenees with referrals for colposcopy between 2014 and 2015 (cytology-based screening) and 2017 and 2018 (high-risk human papillomavirus-based screening) were included. Data were retrieved from the Dutch Pathology Registry (PALGA) and compared between the 2 screening programs. The main outcome measures were referral rate, detection of high-grade cervical intraepithelial neoplasia or worse, overdiagnosis (cervical intraepithelial neoplasia grade 1 or less in the histologic specimen), and overtreatment (cervical intraepithelial neoplasia grade 1 or less in the treatment specimen). RESULTS: Of the women included in the study, 19,109 received cytology-based screening, and 26,171 received high-risk human papillomavirus-based screening. Referral rates increased from 2.5% in cytology-based screening to 4.2% in high-risk human papillomavirus-based screening (+70.2%). Detection rates increased to 46.2% for cervical intraepithelial neoplasia grade 2 or worse, 32.2% for cervical intraepithelial neoplasia grade 3 or worse, and 31.0% for cervical cancer, and overdiagnosis increased to 143.4% with high-risk human papillomavirus-based screening. Overtreatment rates were similar in both screening periods. The positive predictive value of referral for detection of cervical intraepithelial neoplasia grade 2 or worse in high-risk human papillomavirus-based screening was 34.6% compared with 40.2% in cytology-based screening. Women screened through self-sampling were at higher risk of cervical intraepithelial neoplasia grade 2 or worse detection (odds ratio, 1.38; 95% confidence interval, 1.20-1.59) and receiving treatment (odds ratio, 1.31; 95% confidence interval, 1.16-1.48) than those screened through physician-sampling. CONCLUSION: Compared with cytology-based screening, high-risk human papillomavirus-based screening increases detection of high-grade cervical intraepithelial neoplasia, with 462 more cervical intraepithelial neoplasia grade 2 or worse cases per 100,000 women but at the expense of 850 more cases per 100,000 women with invasive diagnostics indicating cervical intraepithelial neoplasia grade 1 or less.