Validation of the FAM19A4/mir124-2 DNA methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women

Objectives DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples. Methods We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined. Results Training set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4-80.6) at a specificity of 67.8% (95%CI: 62.7-73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8-80.1) at a 76.4% (95%CI: 70.2-82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4-95.6) and 46.0% (95%CI: 40.4-51.5) for lavage self-samples, and 84.7% (95%CI: 76.4-93.0) and 54.9% (95%CI: 47.7-62.2) for brush self-samples. Conclusions FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive la

Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazol

Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazol

Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; an expression profile of clinical isolates upon exposure to itraconazole

Background The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any “hard-wired” genetic mutations arise

Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazol

Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; An expression profile of clinical isolates upon exposure to itraconazole

Background: The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results: After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions: Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any "hard-wired" genetic mutations arise.</p

Adjuvant VACcination against HPV in surgical treatment of Cervical Intra-epithelial Neoplasia (VACCIN study) a study protocol for a randomised controlled trial

BACKGROUND: Cervical cancer is caused by Human Papilloma viruses (HPV) and is preceded by precursor stages: Cervical Intraepithelial Neoplasia (CIN). CIN is mostly found in women in their reproductive age and treated with a Loop Electrosurgical E

Quantitative analysis of single-nucleotide polymorphism for rapid detection of TR34/L98H- and TR46/Y121F/T289A-positive Aspergillus fumigatus isolates obtained from patients in Iran from 2010 to 2014

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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

Discrimination of aspergillosis, mucormycosis, fusariosis, and scedosporiosis in formalin-fixed paraffin-embedded tissue specimens by use of multiple real-time quantitative PCR assays

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