Current Testing Strategies Research Articles

Minor changes to circulating steroid hormones in female rats after perinatal exposure to diethylstilbestrol or ketoconazole

Current chemical test strategies lack sensitive markers for detecting female reproductive toxicity caused by endocrine disrupting chemicals (EDCs). In search of a potentially sensitive readout, the steroidogenic disrupting effects of the well-known EDCs ketoconazole (KTZ) and diethylstilbestrol (DES) were investigated in vitro and on circulating steroid hormones in perinatally exposed female Sprague-Dawley rats. Twenty-one steroid hormones were analysed using LC-MS/MS in plasma from female rat offspring at postnatal day (PD) 6, 14, 22, 42 and 90. Most circulating steroid hormone levels increased with age except for estrone (E1), estradiol (E2) and backdoor pathway androsterone (ANDROST), which decreased after PD 22. Perinatal exposure to DES did not affect circulating steroid hormone levels at any dose or age compared to controls. KTZ exposure resulted in dose-dependent increase of corticosterone (CORTICO) at PD 6 and PD 14, with statistical significance only at PD 14. In the in vitro gold standard H295R steroidogenesis assay, twenty-one steroid hormones were measured instead of only T and E2. DES had subtle effects on steroidogenesis, whereas KTZ decreased most steroid hormones, but increased CORTICO, progesterone (P4), estriol (E3) initially (around 0.1–1 µM) before decreasing. Our data suggests that circulating steroidomic profiling may not be a sensitive readout for EDC-induced female reproductive toxicity. Further studies are needed to associate H295R assay steroidomic profiles with in vivo profiles, especially in target tissues such as adrenals or gonads. Expanding the H295R steroidogenic assay to include a comprehensive steroidomic profile may enhance its regulatory applicability.

A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview.

The 21st-century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita-Orange Cell Viability Assay Kit (Sigma-Aldrich), CellTiter-Glo Cell Viability Assay Kit (Promega), CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma-Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma-Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure-activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.

Cost-Effectiveness of Population-Based Multigene Testing for Breast and Ovarian Cancer Prevention

The current method of BRCA testing for breast and ovarian cancer prevention, which is based on family history, often fails to identify many carriers of pathogenic variants. Population-based genetic testing offers a transformative approach in cancer prevention by allowing for proactive identification of any high-risk individuals and enabling early interventions. To assess the lifetime incremental effectiveness, costs, and cost-effectiveness of population-based multigene testing vs family history-based testing. This economic evaluation used a microsimulation model to assess the cost-effectiveness of multigene testing (BRCA1, BRCA2, and PALB2) for all women aged 30 to 35 years compared with the current standard of care that is family history based. Carriers of pathogenic variants were offered interventions, such as magnetic resonance imaging with or without mammography, chemoprevention, or risk-reducing mastectomy and salpingo-oophorectomy, to reduce cancer risk. A total of 2000 simulations were run on 1 000 000 women, using a lifetime time horizon and payer perspective, and costs were adjusted to 2022 US dollars. This study was conducted from September 1, 2020, to December 15, 2023. The main outcome measure was the incremental cost-effectiveness ratio (ICER), quantified as cost per quality-adjusted life-year (QALY) gained. Secondary outcomes included incremental cost, additional breast and ovarian cancer cases prevented, and excess deaths due to coronary heart disease (CHD). The study assessed 1 000 000 simulated women aged 30 to 35 years in the US. In the base case, population-based multigene testing was more cost-effective compared with family history-based testing, with an ICER of $55 548 per QALY (95% CI, $47 288-$65 850 per QALY). Population-based multigene testing would be able to prevent an additional 1338 cases of breast cancer and 663 cases of ovarian cancer, but it would also result in 69 cases of excess CHD and 10 excess CHD deaths per million women. The probabilistic sensitivity analyses show that the probability that population-based multigene testing is cost-effective was 100%. When the cost of the multigene test exceeded $825, population-based testing was no longer cost-effective (ICER, $100 005 per QALY; 95% CI, $87 601-$11 6323). In this economic analysis of population-based multigene testing, population-based testing was a more cost-effective strategy for the prevention of breast cancer and ovarian cancer when compared with the current family history-based testing strategy at the $100 000 per QALY willingness-to-pay threshold. These findings support the need for more comprehensive genetic testing strategies to identify pathogenic variant carriers and enable informed decision-making for personalized risk management.

Anorectal Neisseria gonorrhoeae infections in women with and without reported anal sex and sex workers in sexual health centres in the Netherlands: a retrospective cohort study

Control of Neisseria gonorrhoeae infection (gonorrhoea) depends on effective testing strategies. Anorectal testing in women is often done on indication of anal sex; however, anorectal infections are seen with and without anal exposure, possibly caused by autoinoculation. This study aims to enhance understanding of anorectal infections in women, by identifying risk factors for anorectal diagnosis. In this retrospective cohort study we used national surveillance data from Dutch sexual health centres from Jan 1, 2016, to Dec 31, 2021. We included cisgender women having sex with men who were tested urogenitally and anorectally for gonorrhoea. Due to different testing policies, we identified three groups: women who had not reported recent anal sex (in the past 6 months), women who had reported recent anal sex, and sex workers. Extracted data for analyses included demographics, sexual behaviour, and diagnosis of a sexually transmitted infection (STI). Per group, multivariable models using Firth's penalised maximum likelihood logistic regression were constructed, identifying determinants of anorectal gonorrhoea among all women and among gonorrhoea-positive women only. Variables included in model construction were age, education level, migration background, number of partners, condom use, partner notification, STI symptoms, having a partner who has sex with men (MSM) or a migrant partner, previous STI test, anal sex, and chlamydia and gonorrhoea diagnoses per anatomical location. In total, 117 693 women were included: 43 757 women without reported recent anal sex, 51 728 women with reported recent anal sex, and 22 208 sex workers. In all three groups, around 2% of women were gonorrhoea positive, and 70% or more of women had an anorectal infection. The strongest determinant of anorectal gonorrhoea was a concurrent urogenital gonorrhoea diagnosis (adjusted odds ratios [aOR] 782 [95% CI 605-1018]) among women without reported recent anal sex (612 [490-768] among women with reported recent anal sex, and 464 [335-652] among sex workers). Among gonorrhoea-positive women, determinants of anorectal gonorrhoea were urogenital and anorectal chlamydia co-infection (aOR 2·03 [95% CI 1·38-3·02], for women without reported anal sex) and migration background (1·44 [1·02-2·06], for women with reported anal sex). Determinants among sex workers were condomless sex (2·43 [1·55-3·82]), anal sex (1·71 [1·10-2·66]), MSM or migrant partner (1·78 [1·13-2·79]), and urogenital and anorectal chlamydia co-infection (2·28 [1·11-5·14]). These findings support the possibility of an autoinoculation process from the urogenital to the anorectal location due to the very strong correlation between urogenital and anorectal gonorrhoea, and due to the similarity of results across all three groups. Current testing strategies could miss anorectal infections, which should be considered when developing gonorrhoea prevention and control guidelines. None.

A cost-consequence analysis of the Xpert Xpress CoV-2/Flu/RSV plus test strategy for the diagnosis of influenza-like illnesses

Aims Influenza-like illnesses (ILI) affect millions each year in the United States (US). Determining definitively the cause of symptoms is important for patient management. Xpert Xpress CoV-2/Flu/RSV plus (Xpert Xpress) is a rapid, point-of-care (POC), multiplex real-time polymerase chain reaction (RT-PCR) test intended for the simultaneous qualitative detection and differentiation of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV). The objective of our analysis was to develop a cost-consequence model (CCM) demonstrating the clinico-economic impacts of implementing PCR testing with Xpert Xpress compared to current testing strategies. Methods A decision tree model, with a 1-year time horizon, was used to compare testing with Xpert Xpress alone to antigen POC testing and send-out PCR strategies in the US outpatient setting from a payer perspective. A hypothetical cohort of 1,000,000 members was modeled, a portion of whom develop symptomatic ILIs and present to an outpatient care facility. Our main outcome measure is cost per correct treatment course. Results The total cost per correct treatment course was $1,131 for the Xpert Xpress strategy compared with a range of $3,560 to $5,449 in comparators. POC antigen testing strategies cost more, on average, than PCR strategies. Limitations Simplifying model assumptions were used to allow for modeling ease. In clinical practice, treatment options, costs, and diagnostic test sensitivity and specificity may differ from what is included in the model. Additionally, the most recent incidence and prevalence data was used within the model, which is not reflective of historical averages due to the SARS-CoV-2 pandemic. Conclusion The Xpert Xpress CoV-2/Flu/RSV plus test allows for rapid and accurate diagnostic results, leading to reductions in testing costs and downstream healthcare resource utilization compared to other testing strategies. Compared to POC antigen testing strategies, PCR strategies were more efficient due to improved diagnostic accuracy and reduced use of confirmatory testing.

Known genes, new genes and new phenotypes in mitochondrial eye diseases

Primary mitochondrial diseases together represent a major cause of inherited neurological disorders that, due to dysfunction of the mitochondrial respiratory chain, can affect many parts of body, especially high energy demand organs, such as liver, heart, kidney, muscle and the eye. As the eye is particularly vulnerable to mitochondrial dysfunction, approximately half of all the patients with confirmed mitochondrial disease will develop significant ocular involvement and the complication that patients are most afraid of is visual loss. This can arise either from a primary optic neuropathy (the most common ocular involvement), from outer retinal degeneration, or in some cases from retrochiasmal visual pathway involvement resulting in varying patterns of visual field defects. To date, inherited optic neuropathies have been linked with pathogenic variants in over 40 genes across both the mitochondrial (mtDNA) and nuclear (nDNA) genomes with maternal (mtDNA) inheritance, autosomal dominant, recessive and x‐linked inheritance observed. Inherited optic neuropathies are a significant cause of blindness in both the paediatric and adult population with an estimated prevalence of 1 in 10 000 in the UK. The prevalence is estimated low because of the difficulty in making a confirmed molecular diagnosis due to multiple factors: mutations have been identified in both genomes, a limited number of genes are included in next generation sequencing (NGS) panels, phenocopies etc. Current genetic testing strategies for inherited eye diseases fail to identify pathogenic variants in a significant proportion of cases suggesting that previously unidentified genes harbouring pathogenic variants remain to be identified. However, whole exome sequencing (WES) and particularly whole genome sequencing (WGS), and unbiased interrogation of genomic variants offers an unparalleled opportunity to detect variants in both uncharacterised genes and non‐coding regions of the genome leading to previously unknown gene‐disease associations. This presentation highlights the power of unbiased WGS to elucidate the missing heritability in mitochondrial eye diseases and reports novel gene‐disease associations and novel phenotypes associated with known genes defects.

Evaluation of drug-induced liver toxicity of trovafloxacin and levofloxacin in a human microphysiological liver model

Drug-induced liver injury induced by already approved substances is a major threat to human patients, potentially resulting in drug withdrawal and substantial loss of financial resources in the pharmaceutical industry. Trovafloxacin, a broad-spectrum fluoroquinolone, was found to have unexpected side effects of severe hepatotoxicity, which was not detected by preclinical testing. To address the limitations of current drug testing strategies mainly involving 2D cell cultures and animal testing, a three-dimensional microphysiological model of the human liver containing expandable human liver sinusoidal endothelial cells, monocyte-derived macrophages and differentiated HepaRG cells was utilized to investigate the toxicity of trovafloxacin and compared it to the structurally-related non-toxic drug levofloxacin. In the model, trovafloxacin elicited vascular and hepatocellular toxicity associated with pro-inflammatory cytokine release already at clinically relevant concentrations, whereas levofloxacin did not provoke tissue injury. Similar to in vivo, cytokine secretion was dependent on a multicellular immune response, highlighting the potential of the complex microphysiological liver model for reliably detecting drug-related cytotoxicity in preclinical testing. Moreover, hepatic glutathione depletion and mitochondrial ROS formation were elucidated as intrinsic toxicity mechanisms contributing to trovafloxacin toxicity.

Barriers and enablers to testing for hepatitis C virus infection in people who inject drugs – a scoping review of the qualitative evidence

BackgroundInjection drug use is the primary mode of transmission of hepatitis C virus (HCV) infection in the developed world and guidelines recommend screening individuals with current or history of injection drug use for HCV; however, the majority of those living with HCV in Canada are not aware of their positive status. This low level of HCV status awareness suggests that screening is not effective with current testing strategies. The aim of this review is to determine what barriers and enablers people who inject drugs (PWID) experience surrounding testing for HCV to help inform the development of an engaging testing strategy.MethodsComprehensive literature searches were conducted using Medline, Embase and CINAHL in February 2021. Included studies investigated the barriers and enablers to testing for HCV in PWID and the experiences of PWID in testing for HCV. Studies were included if they were qualitative or mixed-methods design, involved people with current injection drug use or those with a history of injecting drugs, and were written in the English language. Studies were compared and common themes were coded and analyzed.ResultsThe literature search resulted in 1554 citations and ultimately nine studies were included. Common barriers included self-perception of low risk for HCV, fear of diagnosis, stigma associated with IV drug use and HCV, antipathy in relation to mainstream health care services, limited knowledge about HCV, lack of rapport with provider, lack of motivation or competing priority of drug use, and limited awareness of new treatment options. Common enablers to testing included increasing awareness of HCV testing and treatment and providing positive narratives around HCV care, positive rapport with provider, accessible testing options and individualized care.ConclusionWhile there has been some qualitative research on barriers and enablers to testing for HCV in PWID more research is needed to focus on this research question as a primary objective in order to provide more understanding from the participant’s perspective.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Comparing the incidence of SARS-CoV-2 across age groups considering sampling biases - use of testing data of autumn 2021 in Belgium

BackgroundTo design efficient mitigation measures against COVID-19, understanding the transmission dynamics between different age groups was crucial. The role of children in the pandemic has been intensely debated and involves both scientific and ethical questions. To design efficient age-targeted non-pharmaceutical interventions (NPI), a good view of the incidence of the different age groups was needed. However, using Belgian testing data to infer real incidence (RI) from observed incidence (OI) or positivity ratio (PR) was not trivial.MethodsBased on Belgian testing data collected during the Delta wave of Autumn 2021, we compared the use of different estimators of RI and analyzed their effect on comparisons between age groups.ResultsWe found that the RI estimator’s choice strongly influences the comparison between age groups.ConclusionThe widespread implementation of testing campaigns using representative population samples could help to avoid pitfalls related to the current testing strategy in Belgium and worldwide. This approach would also allow a better comparison of the data from different countries while reducing biases arising from the specificities of each surveillance system.

Chemical safety and the exposome

Air pollution and rapid chemical intensification are major threats to the environment and human health. Today, we have produced over 350,000 chemicals, and current testing strategies do not meet the demands. Therefore, it is important to develop new approach methodologies (NAMs) that can help fill current information gaps. Toxicology needs to evolve from hazard and risk assessments based on morphological endpoints in animal tests towards a mechanism-driven integrated approach that better includes computational modelling as well as molecular, human, and in vitro data. The application of new science and technology such as different types of imaging and omics methods can allow faster collection of high-quality toxicological data for hazard identification and better prediction of toxicological potential using advanced in silico approaches including machine learning. A shift toward active prevention of pollution through a safe and sustainable-by-design approach based on cutting-edge science could significantly help safeguard the population and planetary health. Moreover, it is necessary to improve the understanding of how interactions among chemical mixtures, climate change, infectious agents, and other stressors that constitute the exposome, may affect biota and human health. Individual responses to current exposures and susceptibility to disease are influenced by factors such as genetics, epigenetics, physiology, and health status, which involve changes in biological pathways caused by own previous exposures or even ancestral exposures. It is therefore important to better consider individual exposomes and susceptibility in future risk assessments and precision medicine. This review describes the central role of mechanistic toxicology in chemical safety and in the cross-disciplinary efforts needed to characterize the exposome and its complex interactions in detail.

DNA methylation changes from primary cultures through senescence-bypass in Syrian hamster fetal cells initially exposed to benzo[a]pyrene

Current chemical testing strategies are limited in their ability to detect non-genotoxic carcinogens (NGTxC). Epigenetic anomalies develop during carcinogenesis regardless of whether the molecular initiating event is associated with genotoxic (GTxC) or NGTxC events; therefore, epigenetic markers may be harnessed to develop new approach methodologies that improve the detection of both types of carcinogens. This study used Syrian hamster fetal cells to establish the chronology of carcinogen-induced DNA methylation changes from primary cells until senescence-bypass as an essential carcinogenic step. Cells exposed to solvent control for 7 days were compared to naïve primary cultures, to cells exposed for 7 days to benzo[a]pyrene, and to cells at the subsequent transformation stages: normal colonies, morphologically transformed colonies, senescence, senescence-bypass, and sustained proliferation in vitro. DNA methylation changes identified by reduced representation bisulphite sequencing were minimal at day-7. Profound DNA methylation changes arose during cellular senescence and some of these early differentially methylated regions (DMRs) were preserved through the final sustained proliferation stage. A set of these DMRs (e.g., Pou4f1, Aifm3, B3galnt2, Bhlhe22, Gja8, Klf17, and L1l) were validated by pyrosequencing and their reproducibility was confirmed across multiple clones obtained from a different laboratory. These DNA methylation changes could serve as biomarkers to enhance objectivity and mechanistic understanding of cell transformation and could be used to predict senescence-bypass and chemical carcinogenicity.

New genes and new phenotypes

AbstractPrimary mitochondrial diseases together represent a major cause of inherited neurological disorders that, due to dysfunction of the mitochondrial respiratory chain, can affect many parts of body, especially high energy demand organs, such as liver, heart, kidney, muscle and the eye. As the eye is particularly vulnerable to mitochondrial dysfunction, approximately half of all the patients with confirmed mitochondrial disease will develop significant ocular involvement and the complication that patients are most afraid of is visual loss. This can arise either from a primary optic neuropathy (the most common ocular involvement), from outer retinal degeneration, or in some cases from retrochiasmal visual pathway involvement resulting in varying patterns of visual field defects. To date, inherited optic neuropathies have been linked with pathogenic variants in over 40 genes across both the mitochondrial (mtDNA) and nuclear (nDNA) genomes with maternal (mtDNA) inheritance, autosomal dominant, recessive and x‐linked inheritance observed. Inherited optic neuropathies are a significant cause of blindness in both the paediatric and adult population with an estimated prevalence of 1 in 10 000 in the UK. The prevalence is estimated low because of the difficulty in making a confirmed molecular diagnosis due to multiple factors: mutations have been identified in both genomes, a limited number of genes are included in next generation sequencing (NGS) panels, phenocopies etc. Current genetic testing strategies for inherited eye diseases fail to identify pathogenic variants in a significant proportion of cases suggesting that previously unidentified genes harbouring pathogenic variants remain to be identified. However, whole exome sequencing (WES) and particularly whole genome sequencing (WGS), and unbiased interrogation of genomic variants offers an unparalleled opportunity to detect variants in both uncharacterised genes and non‐coding regions of the genome leading to previously unknown gene‐disease associations. This presentation highlights the power of unbiased WGS to elucidate the missing heritability in mitochondrial eye diseases and reports novel gene‐disease or gene‐phenotype associations.

HER2 amplification by next-generation sequencing to identify HER2-positive invasive breast cancer with negative HER2 immunohistochemistry

BackgroundHuman epidermal growth factor receptor 2 (HER2) positive breast carcinomas due to HER2 amplification are associated with aggressive behavior and a poor prognosis. Anti-HER2-targeted therapies are widely used to treat HER2-positive breast carcinomas with excellent outcomes. Accurate identification of HER2 amplification status in breast carcinomas is of important diagnostic and treatment value. Currently, HER2 amplification status is routinely determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) testing. This study will review our past HER2 data to determine and characterize discordant results between HER2 IHC and FISH. It will also determine a potential impact of HER2 amplification status by next-generation sequencing (NGS) on these patients.MethodsWe reviewed a total of 4884 breast carcinomas with coexisting HER2 IHC and HER2 FISH performed at our institution from 2010 to 2022. 57 cases also had a Next-Generation-Sequencing-based (NGS) gene panel performed. Given the advances in biostatic analysis pipelines, NGS methods were utilized to provide results on HER2 amplification status along with somatic mutations.ResultsWhile the majority (ranging from 98.5% with IHC score of 0 and 93.1% with IHC score of 1 +) of 4884 breast carcinomas had concordant results from HER2 IHC and HER2 FISH testing, a small percentage of patients (ranging from 1.5% in those with IHC score of 0, to 6.9% with IHC score of 1 +) had discordant results, with negative HER2 IHC and positive HER2 FISH results. These patients could be reported as HER2-negative breast carcinomas if only HER2 IHC testing has been performed according to a current cost-effective HER2 test strategy. 57 patients had HER2 amplification status determined by NGS, and all patients had concordant results between HER2 NGS and FISH tests. A HER2-amplified breast carcinoma by NGS had a negative IHC and a positive HER2 FISH result. This case was classified as a HER2-positive breast carcinoma, had anti-HER2-targeted therapy, and achieved a complete clinical response.ConclusionsA small percentage of HER2-positive breast carcinomas are unidentified because of a negative HER2 IHC based on our current cost-effective HER2 test strategy. It is not feasible and affordable in routine clinical practice to perform HER2 FISH for the cases with negative HER2 IHC (IHC score 0 and 1 +). Therefore, NGS assays capable of simultaneously detecting both somatic mutations and HER2 amplification could provide a more comprehensive genetic profiling for breast carcinomas in a clinical setting. Identification of HER2 amplification by NGS in HER2-positive breast carcinomas with negative HER2 IHC results is important since these cases are concealed by our current cost-effective HER2 test strategy with IHC first (for all cases) and FISH reflex (only for cases with IHC score of 2 +), and would offer the opportunity for potentially beneficial anti-HER2-targeted therapies for these patients.

A pilot surveillance report of SARS-CoV-2 rapid antigen test results among volunteers in Germany, 1st week of July 2022

PurposeWe hypothesized that SARS-CoV-2 infection numbers reported by governmental institutions are underestimated due to high dark figures as only results from polymerase chain reaction (PCR) tests are incorporated in governmental statistics and testing capacities were further restricted as of July, 2022.MethodsA point prevalence investigation was piloted by rapid antigen testing (RAT) among participants of the VACCELERATE volunteer registry. 2400 volunteers were contacted, of which 500 received a RAT including instructions for self-testing in the first week of July, 2022. Results were self-reported via e-mail.Results419 valid RAT results were collected until July 7th, 2022. Between July-1 and July-7, 2022, 7/419 (1.67%) tests were positive. Compared to reports of the German Federal Government, our results suggest a more than twofold higher prevalence. Three out of seven positive individuals did not have a PCR test and are therefore likely not to be displayed in governmental statistics.ConclusionOur findings imply that the actual prevalence of SARS-CoV-2 may be higher than detected by current surveillance systems, so that current pandemic surveillance and testing strategies may be adapted.

Residual risk estimates of transfusion transmissible hepatitis B, hepatitis C and human immunodeficiency virus using nucleic acid testing yield/window period model in an Indian setting.

Estimation of residual risk (RR) is necessary to understand status of blood safety with current testing strategies and need to improve it further. These estimates from India are lacking till date. Present study was aimed to estimate RR and incident rate (IR) of transfusion transmitted infectious disease (TTIDs) in Indian setting. Blood donor demographic data, serological testing results and nucleic acid testing (NAT) screening results were collected retrospectively from January 2015 to June 2017. Mathematical modelling using NAT yield/window period model was done to estimate IR and RR of TTIDs. Data was compared between different groups based on age-group, gender, donation repeatability and type of donor. A total 106 119 blood donors donated 109 441 units with overall TTID reactivity of 2.22%. HBV was most prevalent TTID (1.26%) with RR of 16.1 per million followed by HCV and HIV with RR of 4.4 and 3.1 per million donations, respectively. NAT testing variably reduced RR ranging from 69.4% to 96.1% depending on TTID. Younger, repeat and voluntary donors had significantly lower prevalence of TTIDs compared to older, first time and replacement donors. Gaps in the blood safety could be bridged significantly by implementing NAT testing and using quality serological assays. Comparatively high RR despite using quality serological assays and ID-NAT testing highlights need to develop long term strategies to improve blood safety by focusing on improving donor pool by recruiting regular voluntary donors among youth and imparting knowledge of healthy practices.

Missed pertussis diagnosis during co-infection with Bordetella holmesii.

The purpose of this study is to identify predictive factors associated with missed diagnosis of B. pertussis-B. holmesii co-infection by assessing the analytical performance of a commercially available multiplexed PCR assay and by building a prediction model based on clinical signs and symptoms for detecting co-infections. This is a retrospective study on the electronic health records of all clinical samples that tested positive to either B. pertussis or B. holmesii from January 2015 to January 2018 at Geneva University Hospitals. Multivariate logistic regression was used to build a model for co-infection prediction based on the electronic health record chart review. Limit of detection was determined for all targets of the commercial multiplexed PCR assay used on respiratory samples. A regression model, developed from clinical symptoms and signs, predicted B. pertussis and B. holmesii co-infection with an accuracy of 82.9% (95% CI 67.9-92.8%, p value = .012), for respiratory samples positive with any of the two tested Bordetella species. We found that the LOD of the PCR reaction targeting ptxS1 is higher than that reported by the manufacturer by a factor 10. The current testing strategy misses B. pertussis and B. holmesii co-infections by reporting only B. holmesii infections. Thus, we advocate to perform serological testing for detecting a response against pertussis toxin whenever a sample is found positive for B. holmesii. These findings are important, both from a clinical and epidemiological point of view, as the former impacts the choice of antimicrobial drugs and the latter biases surveillance data, by underestimating B. pertussis infections during co-infections.

MRD in ALL: Optimization and Innovations.

Measurable residual disease (MRD) is an important monitoring parameter that can help predict survival outcomes in acute lymphoblastic leukemia (ALL). Identifying patients with MRD has the potential to decrease the risk of relapse with the initiation of early salvage therapy and to help guide decision making regarding allogeneic hematopoietic cell transplantation. In this review, we discuss MRD in ALL, focusing on advantages and limitations between MRD testing techniques and how to monitor MRD in specific patient populations. MRD has traditionally been measured through bone marrow samples, but more data for evaluation of MRD via peripheral blood is emerging. Current and developmental testing strategies for MRD include multiparametric flow cytometry (MFC), next-generation sequencing (NGS), quantitative polymerase chain reaction (qPCR), and ClonoSeq. Novel therapies are incorporating MRD as an outcome measure to demonstrate efficacy, including blinatumomab, inotuzumab ozogamicin, and chimeric antigen receptor T (CAR-T) cell therapy. Understanding how to incorporate MRD testing into the management of ALL could improve patient outcomes and predict efficacy of new therapy options.

Genomic evaluation of hybridization in historic and modern North American Bison (Bison bison)

During the late nineteenth century North American bison underwent a significant population bottleneck resulting in a reduction in population size of over 99% and a species-level near-extinction event. Factors responsible for this destruction included indiscriminate killing, loss of access to suitable habitat, and diseases. At the nadir of this population crash, very few wild plains bison survived and were restricted to Yellowstone National Park, USA and a small number of wild wood bison remained in Wood Buffalo National Park, Canada. However, most surviving bison in the late 1800’s were maintained by cattle ranchers in private herds where hybridization between bison with various breeds of domestic cattle was often encouraged. Over the last 20 years, the legacy of this introgression has been identified using mitochondrial DNA and limited nuclear microsatellite analyses. However, no genome-wide assessment has been performed, and some herds were believed to be free of introgression based on current genetic testing strategies. Herein, we report detailed analyses using whole genome sequencing from nineteen modern and six historical bison, chosen to represent the major lineages of bison, to identify and quantitate signatures of nuclear introgression in their recent (within 200 years) history. Both low and high coverage genomes provided evidence for recent introgression, including animals from Yellowstone, Wind Cave, and Elk Island National Parks which were previously thought to be free from hybridization with domestic cattle. We employed multiple approaches, including one developed for this work, to identify putative cattle haplotypes in each bison genome. These regions vary greatly in size and frequency by sample and herd, though we detected domestic cattle introgression in all bison genomes tested. Since our sampling strategy spanned across the diversity of modern bison populations, these finding are best explained by multiple historical hybridization events between these two species with significant genetic recombination over the last 200 years. Our results demonstrate that whole genome sequencing approaches are required to accurately quantitate cattle introgression in bison.

Projections of the transmission of the Omicron variant for Toronto, Ontario, and Canada using surveillance data following recent changes in testing policies.

At the end of 2021, with the rapid escalation of COVID19 cases due to the Omicron variant, testing centers in Canada were overwhelmed. To alleviate the pressure on the PCR testing capacity, many provinces implemented new strategies that promote self testing and adjust the eligibility for PCR tests, making the count of new cases underreported. We designed a novel compartmental model which captures the new testing guidelines, social behaviours, booster vaccines campaign and features of the newest variant Omicron. To better describe the testing eligibility, we considered the population divided into high risk and non-high-risk settings. The model is calibrated using data from January 1 to February 9, 2022, on cases and severe outcomes in Canada, the province of Ontario and City of Toronto. We conduct analyses on the impact of PCR testing capacity, self testing, different levels of reopening and vaccination coverage on cases and severe outcomes. Our results show that the total number of cases in Canada, Ontario and Toronto are 2.34 (95%CI: 1.22–3.38), 2.20 (95%CI: 1.15–3.72), and 1.97(95%CI: 1.13–3.41), times larger than reported cases, respectively. The current testing strategy is efficient if partial restrictions, such as limited capacity in public spaces, are implemented. Allowing more people to have access to PCR reduces the daily cases and severe outcomes; however, if PCR test capacity is insufficient, then it is important to promote self testing. Also, we found that reopening to a pre-pandemic level will lead to a resurgence of the infections, peaking in late March or April 2022. Vaccination and adherence to isolation protocols are important supports to the testing policies to mitigate any possible spread of the virus.