Comparison of a multiplex real-time PCR assay with a multiplex Luminex assay for influenza virus detection.

We describe the development of a multiplex reverse transcription-PCR (RT-PCR) with Luminex microarray hybridization for detection of influenza virus subtypes (FLULUM). Performance of FLULUM was evaluated by comparing it to our real-time RT-PCR influenza virus assay on samples collected during two influenza seasons. Both assays targeted the matrix genes of influenza virus A (FluA M) and influenza virus B (FluB M) and the hemagglutinin genes of seasonal H3N2 (H3) and H1N1 (H1) and 2009 pandemic H1N1 (2009 H1). We evaluated FLULUM on both the Luminex LX200 and the Luminex MagPix instruments. Compared to real-time PCR, FLULUM tested on 259 specimens submitted in the 2010-2011 season showed sensitivities of 97.3% for FluA M, 90.5% for 2009 H1, 96.9% for H3, and 88.9% for FluB M. No specimens were positive for seasonal H1. FLULUM tested on 806 specimens submitted in the 2011-2012 season showed a sensitivity of 100% for FluA M, 89.9% for 2009 H1, 96.4% for H3, and 95.6% for FluB M. No cross-reactivity was observed for other respiratory viruses. Analytical sensitivity was assessed by testing dilutions of specimens with high viral loads. The limits of detection of FLULUM were comparable to those of the real-time PCR assay for FluA M, FluB M, and H3. The limits of detection for seasonal H1 and 2009 H1 were 10-fold higher for the FLULUM assay compared to real-time PCR. The FLULUM is an economic assay with high clinical sensitivity and specificity. It is particularly suited to high-volume detection of influenza viruses.

Increasing Clinical Trial Participation of Black Women Diagnosed with Breast Cancer.

Despite racial disparities in breast cancer mortality, Black women remain underrepresented in clinical trials. In this mixed methods research, 48 Black women were engaged via focus group discussions and in-depth interviews to better understand the lived experience of women with breast cancer. The results of this qualitative study informed the development of a subsequent online survey to identify barriers, motivators, and other factors that influence decision-making by Black women diagnosed with breast cancer when considering clinical trial participation. Among the 257 Black survey participants, most (95%) were aware of clinical trials; of those, most viewed them as lifesaving (81%) and/or benefiting others (90%). Negative perceptions such as serious side effects (58%), not receiving real treatment (52%), or risk of potential harm (62%) were indicated. Barriers included financial expenses (49%), concerns that their condition could be made worse (29%), that they would receive a placebo (28%), or that treatment was unapproved (28%). Participants were more likely than their health care providers (HCPs) to initiate discussions of clinical trials (53% versus 33%), and 29% of participants indicated a need for more information about risks and benefits, even after having those conversations. The most trustworthy sources of information on clinical trials were HCPs (66%) and breast cancer support groups (64%). These results suggest that trusted communities are key for providing education on clinical trials. However, there is also a need for HCPs to proactively discuss clinical trials with patients to ensure that they are adequately informed about all aspects of participation.

Comparison of Germline Genetic Testing Before and After a Medical Policy Covering Universal Testing Among Patients With Colorectal Cancer.

Importance: In 2020, some health insurance plans updated their medical policy to cover germline genetic testing for all patients diagnosed with colorectal cancer (CRC). Guidelines for universal tumor screening via microsatellite instability and/or immunohistochemistry (MSI/IHC) for mismatch repair protein expression for patients with CRC have been in place since 2009. Objectives: To examine whether uptake of MSI/IHC screening and germline genetic testing in patients with CRC has improved under these policies and to identify actionable findings and management implications for patients referred for germline genetic testing. Design, Setting, and Participants: The multicenter, retrospective cohort study comprised 2 analyses of patients 18 years or older who were diagnosed with CRC between January 1, 2017, and December 31, 2020. The first analysis used an insurance claims data set to examine use of MSI/IHC screening and germline genetic testing for patients diagnosed with CRC between 2017 and 2020 and treated with systemic therapy. The second comprised patients with CRC who had germline genetic testing performed in 2020 that was billed under a universal testing policy. Main Outcomes and Measures: Patient demographic characteristics, clinical information, and use of MSI/IHC screening and germline genetic testing were analyzed. Results: For 9066 patients with newly diagnosed CRC (mean [SD] age, 64.2 [12.7] years; 4964 [54.8%] male), administrative claims data indicated that MSI/IHC was performed in 6645 eligible patients (73.3%) during the study period, with 2288 (25.2%) not receiving MSI/IHC despite being eligible for coverage. Analysis of a second cohort of 55 595 patients with CRC diagnosed in 2020 and covered by insurance found that only 1675 (3.0%) received germline genetic testing. In a subset of patients for whom germline genetic testing results were available, 1 in 6 patients had pathogenic or likely pathogenic variants, with most of these patients having variants with established clinical actionability. Conclusions and Relevance: This nationwide cohort study found suboptimal rates of MSI/IHC screening and germline genetic testing uptake, resulting in clinically actionable genetic data being unavailable to patients diagnosed with CRC, despite universal eligibility. Effective strategies are required to address barriers to implementation of evidence-based universal testing policies that support precision treatment and optimal care management for patients with CRC.

Clinical Utility of Universal Germline Genetic Testing for Patients With Breast Cancer.

Importance: National Comprehensive Cancer Network guidelines currently recommend germline testing for high-risk genes in selected patients with breast cancer. The clinical utility of recommending testing all patients with breast cancer with multigene panels is currently under consideration. Objective: To examine the implications of universal testing of patients with breast cancer with respect to clinical decision-making. Design, Setting, and Participants: Patients from a previously reported cohort were assessed as in-criteria or out-of-criteria according to the 2017 guidelines and underwent testing with a multigene germline panel between 2017 to 2018. Patients were women and men aged 18 to 90 years, with a new and/or previous diagnosis of breast cancer who had not undergone either single or multigene testing. Clinicians from 20 community and academic sites documented patient clinical information and changes to clinical recommendations made according to test findings. Association between prevalence of pathogenic or likely pathogenic germline variants and previously unreported clinical features, including scores generated by the BRCAPRO statistical model, was determined. Data were analyzed from April 2020 to May 2022. Exposure: New and/or previous diagnosis of breast cancer. Main Outcomes and Measures: Disease management recommendations that were changed as a result of genetic testing results are reported. Results: Clinicians were asked to assess changes to clinical management as a result of germline genetic testing for 952 patients. Informative clinician-reported recommendations were provided for 939 (467 in-criteria and 472 out-of-criteria) of the patients with breast cancer (936 [99.7%] female; 702 [74.8%] White; mean [SD] age at initial diagnosis, 57.6 [11.5] years). One or more changes were reported for 31 of 37 (83.8%) in-criteria patients and 23 of 34 (67.6%) out-of-criteria patients with a pathogenic or likely pathogenic variant. Recommendations were changed as a result of testing results for 14 of 22 (63.6%) out-of-criteria patients who had a variant in a breast cancer predisposition gene. Clinicians considered testing beneficial for two-thirds of patients with pathogenic or likely pathogenic variants and for one-third of patients with either negative results or variants of uncertain significance. There was no difference in variant rate between patients meeting the BRCAPRO threshold (≥10%) and those who did not (P = .86, Fisher exact test). No changes to clinical recommendations were made for most patients with negative results (345 of 349 patients [98.9%]) or variants of uncertain significance (492 of 509 patients [96.7%]). Conclusions and Relevance: In this cohort study, germline genetic testing was used by clinicians to direct treatment for most out-of-criteria patients with breast cancer with pathogenic or likely pathogenic germline variants, including those with moderate-risk variants. Universal germline testing informs clinical decision-making and provides access to targeted treatments and clinical trials for all patients with breast cancer.

Identifying residual hotspots and mapping lower respiratory infection morbidity and mortality in African children from 2000 to 2017.

Lower respiratory infections (LRIs) are the leading cause of death in children under the age of 5, despite the existence of vaccines against many of their aetiologies. Furthermore, more than half of these deaths occur in Africa. Geospatial models can provide highly detailed estimates of trends subnationally, at the level where implementation of health policies has the greatest impact. We used Bayesian geostatistical modelling to estimate LRI incidence, prevalence and mortality in children under 5 subnationally in Africa for 2000-2017, using surveys covering 1.46 million children and 9,215,000 cases of LRI. Our model reveals large within-country variation in both health burden and its change over time. While reductions in childhood morbidity and mortality due to LRI were estimated for almost every country, we expose a cluster of residual high risk across seven countries, which averages 5.5 LRI deaths per 1,000 children per year. The preventable nature of the vast majority of LRI deaths mandates focused health system efforts in specific locations with the highest burden.

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection.

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

Clinical benefit and preservation of flavonols in dark chocolate manufacturing.

The consumption of high-cacao-content chocolate has been associated with positive health benefits ascribed to flavanol [corrected] antioxidants derived from the ground, fermented cocoa seeds of Theobroma cacao. However, flavanols [corrected] impart a bitter, astringent flavor to foodstuffs, frequently masked in chocolates and confections by aggressive processing and adulteration with other flavors. Recent reports have implied that not all varieties of dark chocolate are created equally, and significant caveats exist regarding its potential health benefits. It is perhaps not surprising that extensive processing, dilution, and the addition of flavor modifiers may improve the palatability of chocolate, but could have negative nutritional and clinical benefits. This article examines the chemical composition of chocolate and the clinical data associated with the consumption of flavonoid-rich cocoa. We review the steps in chocolate manufacturing that directly affect the antioxidant levels in chocolate products, and the caveats associated with claims of health benefits from the consumption of dark chocolate.