FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Evaluation of the applicability of internal controls on self-collected samples for high-risk human papillomavirus is needed.

BACKGROUND: Self-collection of cervical samples to detect high-risk human papillomavirus (hr-HPV) is a trending topic in primary cervical cancer screening. This study evaluates the applicability of a self-sampling device to routine molecular procedures for hr-HPV detection. METHODS: In a primary health care facility in Kinshasa, Congo, 187 self-collected samples (Evalyn Brush) were gathered and sent to Ghent University Hospital (UZ Ghent) and Algemeen Medisch Labo (AML) in Belgium where routine tests for hr-HPV were applied (Abbott RealTime hr-HPV and qPCR (E6/E7), respectively). Sample type effect was evaluated by comparing the internal control (IC) between the self-collected samples and routine, clinician-taken samples randomly selected from the UZ Ghent archive. RESULTS: In UZ Ghent an error was encountered in 9.1% (17/187) of self-collected samples due to a lack of IC signal. The hr-HPV prevalence in the remaining 170 samples was 18,8%. Comparing IC results between the self-collected and clinician-collected groups, a significant difference (p < 0,001) was found, with higher IC signals in the clinician-collected group. In AML, an error was encountered in 17.6% (33/187) of samples, including 16/17 of the UZ Ghent. The remaining sample with IC error gave a negative result in AML. Among the 154 samples without IC error at AML, a correlation of 90% was seen between both laboratories with a 77% negativity rate. CONCLUSION: Testing the self-collected specimens by 2 routine hr-HPV tests gave a high IC error rate (9.1-17.6%). A possible solution would be to differentiate cut-offs for IC values depending on sample type, as currently used cut-offs are set for clinician-taken samples.

Comparison between the Roche Cobas 4800 Human Papillomavirus (HPV), Abbott RealTime High-Risk HPV, Seegene Anyplex II HPV28, and Novel Seegene Allplex HPV28 Assays for High-Risk HPV Detection and Genotyping in Mocked Self-Samples.

Infection with high-risk human papillomavirus (hrHPV) is well recognized as the main cause of cervical cancer. The recently developed Seegene Allplex HPV28 assay is a novel quantitative PCR (qPCR) assay designed to separately detect and quantify 28 distinct HPV genotypes in a fully automated and user-friendly manner. This study evaluated and compared the performance of this new assay with the performance of the Roche Cobas 4800, the Abbott RealTime high-risk HPV, and the Seegene Anyplex II HPV28 assays. A total of 114 mocked self-samples, i.e., semicervical samples collected by gynecologists using the Viba-Brush, were analyzed with all four HPV assays. Agreement in terms of detecting and genotyping HPV was assessed by the mean of the Cohen's kappa (κ) coefficient. Results of all four HPV assays agreed in 85.9% of the cases when using the Abbott RealTime manufacturer's recommended quantification cycle (Cq) cutoff for positivity (<32.00) and 91.2% when using an adapted range (32.00 to 36.00). An intercomparison of the included assays demonstrated an overall agreement ranging from 85.9 to 100.0% (κ = 0.42 to 1.00) when using the manufacturer's guidelines and 92.9 to 100.0% (κ = 0.60 to 1.00) with the adapted range. For all assays, highly significant, strongly positive Pearson correlations were shown between the Cq values of positive test results. This study thereby shows high concordance between results of the included HPV assays on mocked self-samples. Based on these findings, we imply that the novel Allplex HPV28 assay demonstrates a comparable performance to those of available qPCR HPV assays, potentially providing opportunities for the simplification and standardization of future large-scale testing. IMPORTANCE This study proves that the novel Allplex HPV28 assay has a good diagnostic performance in comparison with the well-known, validated, and frequently used Roche Cobas 4800, Abbott RealTime, and Anyplex II HPV28 assays. According to our experience, the novel Allplex HPV28 assay had a user-friendly and automated workflow with short hands-on time, had an open platform which facilitates the use of add-on assays, and provided quick and easy-to-interpret results. Together with its ability to detect and quantify 28 HPV genotypes, the Allplex HPV28 assay could therefore potentially provide opportunities for the simplification and standardization of future diagnostic testing programs.

Offering HPV self-sampling kits: an updated meta-analysis of the effectiveness of strategies to increase participation in cervical cancer screening.

BACKGROUND: Human papillomavirus (HPV) testing on self-samples represents a great opportunity to increase cervical cancer screening uptake among under-screened women. METHODS: A systematic review and meta-analysis on randomised controlled trials (RCTs) were performed to update the evidence on the efficacy of strategies for offering self-sampling kits for HPV testing compared to conventional invitations and to compare different self-sampling invitation scenarios. Four experimental invitational scenarios were considered. Women in the control group were invited for screening according to existing practice: collection of a cervical specimen by a healthcare professional. Random-effects models were used to pool proportions, relative participation rates and absolute participation differences. RESULTS: Thirty-three trials were included. In the intention-to-treat analysis, all self-sampling invitation scenarios were more effective in reaching under-screened women compared to controls. Pooled participation difference (PD) and 95% confidence interval (CI) for experimental vs. control was 13.2% (95% CI = 11.0-15.3%) for mail-to-all, 4.4% (95% CI = 1.2-7.6%) for opt-in, 39.1% (95% CI = 8.4-69.9%) for community mobilisation & outreach and 28.1% (23.5-32.7%) for offer at healthcare service. PD for the comparison opt-in vs. mail-to-all, assessed in nine trials, was -8.2% (95% CI = -10.8 to -5.7%). DISCUSSION: Overall, screening participation was higher among women invited for self-sampling compared to control, regardless of the invitation strategy used. Opt-in strategies were less effective than send-to-all strategies.

Barriers and facilitators to cervical cancer screening among under-screened women in Cuenca, Ecuador: the perspectives of women and health professionals.

BACKGROUND: Cervical cancer screening is a cost-effective method responsible for reducing cervical cancer-related mortality by 70% in countries that have achieved high coverage through nationwide screening strategies. However, there are disparities in access to screening. In Ecuador, although cervical cancer is the second most common cancer in women, only 58.4% of women of reproductive age have ever been screened for cervical cancer. METHODOLOGY: A qualitative study was performed to understand the current barriers to screening and to identify strategies that could increase uptake in Azuay province, Ecuador. Seven focus group discussions (FGDs) were conducted with under-screened women and health professionals (HPs). The FGDs were recorded and transcribed. Content analysis was done using the socio-ecological framework to categorize and analyse the data. RESULTS: Overall, 28 women and 27 HPs participated in the study. The two groups perceived different barriers to cervical cancer screening. The HPs considered barriers to be mainly at the policy level (lack of a structured screening plan; lack of health promotion) and the individual level (lack of risk perception; personal beliefs). The women identified barriers mainly at organizational level, such as long waiting times, lack of access to health centres, and inadequate patient-physician communication. Both groups mentioned facilitators at policy level, such as national campaigns promoting cervical cancer screening, and at community and individual level, including health literacy and women's empowerment. CONCLUSIONS: The women considered access to health services the main barrier to screening, while the HPs identified a lack of investment in screening programmes and cultural patterns at the community level as major obstacles. To take an integrated approach to cervical cancer prevention, the perspectives of both groups should be taken into account. Additionally, new strategies and technologies, such as self-administered human papillomavirus (HPV) testing and community participation, should be implemented to increase access to cervical cancer screening.

Universal cervical cancer control through a right to health lens: refocusing national policy and programmes on underserved women.

BACKGROUND: Cervical cancer claims 311,000 lives annually, and 90% of these deaths occur in low- and middle-income countries. Cervical cancer is a highly preventable and treatable disease, if detected through screening at an early stage. Governments have a responsibility to screen women for precancerous cervical lesions. Yet, national screening programmes overlook many poor women and those marginalised in society. Under-screened women (called hard-to-reach) experience a higher incidence of cervical cancer and elevated mortality rates compared to regularly-screened women. Such inequalities deprive hard-to-reach women of the full enjoyment of their right to sexual and reproductive health, as laid out in Article 12 of the International Covenant on Economic, Social and Cultural Rights and General Comment No. 22. DISCUSSION: This article argues first for tailored and innovative national cervical cancer screening programmes (NCSP) grounded in human rights law, to close the disparity between women who are afforded screening and those who are not. Second, acknowledging socioeconomic disparities requires governments to adopt and refine universal cancer control through NCSPs aligned with human rights duties, including to reach all eligible women. Commonly reported- and chronically under-addressed- screening disparities relate to the availability of sufficient health facilities and human resources (example from Kenya), the physical accessibility of health services for rural and remote populations (example from Brazil), and the accessibility of information sensitive to cultural, ethnic, and linguistic barriers (example from Ecuador). Third, governments can adopt new technologies to overcome individual and structural barriers to cervical cancer screening. National cervical cancer screening programmes should tailor screening methods to under-screened women, bearing in mind that eliminating systemic discrimination may require committing greater resources to traditionally neglected groups. CONCLUSION: Governments have human rights obligations to refocus screening policies and programmes on women who are disproportionately affected by discrimination that impairs their full enjoyment of the right to sexual and reproductive health. National cervical cancer screening programmes that keep the right to health principles (above) central will be able to expand screening among low-income, isolated and other marginalised populations, but also women in general, who, for a variety of reasons, do not visit healthcare providers for regular screenings.