Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries.

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Encouraging Patients to Ask Questions: Development and Pilot Testing of a Question Prompt List for Patients Undergoing a Biopsy for Suspected Prostate Cancer.

This study assessed the acceptability and feasibility of a question prompt list (QPL) to facilitate informed treatment decision-making in men with suspected localised prostate cancer, which involves values-based choices between options with similar efficacy but different side effects. The QPL was developed through iterative consultation with consumers, clinicians and researchers. Acceptability was assessed using study-specific questions regarding QPL satisfaction and usefulness and qualitative interviews. Feasibility was determined via the proportion of men given the QPL according to medical records and the completion of standardised measures of decisional outcomes. Quantitative data were analysed using descriptive and univariate statistics. Qualitative data were thematically analysed. Fifty-two men consented; 34 provided data for analysis. The QPL recipients reported moderate-high content satisfaction (70.6%) and perceived usefulness in guiding appointments when receiving biopsy results (64.7%). Two main qualitative themes also indicated the QPL acceptability: (1) the freedom to ask-acceptable timing, flexible usage and usefulness of the QPL, and (2) satisfaction with the QPL content. However, only 18.4% of eligible men received the QPL, indicating limited feasibility. The QPL is safe and acceptable, but further research is needed regarding how to facilitate the uptake of the question prompt list in clinical practice.

Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce.

Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID50 assay and for future experiments. Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.

Low conflict and high satisfaction: Decisional outcomes after attending a combined clinic to choose between robotic prostatectomy and radiotherapy for prostate cancer.

BACKGROUND: Decisional conflict and post-treatment decisional regret have been documented in men with localised prostate cancer (LPC). However, there is limited evidence regarding decisional outcomes associated with the choice between robotic-assisted radical prostatectomy (RARP) and radiotherapy, when both treatment options are available in the public health system. There is increasing support for multidisciplinary approaches to guide men with LPC in their decision-making process. This study assessed decisional outcomes in men deciding between RARP or radiotherapy treatment before and after attending a LPC combined clinic (CC). METHODS: Quantitative longitudinal data were collected from 52 men who attended a LPC CC, where they saw both a urologist and radiation oncologist. Patients completed questionnaires assessing involvement in decision-making, decisional conflict, satisfaction and regret before and after the CC, three months, six months and 12 months post-treatment. Urologists and radiation oncologists also reported their perceptions regarding patients' suitability for, openness to, perceived preferences and appropriateness for each treatment. Data was analysed using paired/independent samples t-tests and McNemar's tests. RESULTS: Most participants (n = 37, 71%) opted for RARP over radiotherapy (n = 14, 27%); one participant deferred treatment (2%). Urologists and radiation oncologists reported low agreement (κ = 0.26) regarding the most appropriate treatment for each patient. Participants reported a desire for high levels of control over their decision-making process (77.5% patient-led, 22.5% shared) and high levels of decisional satisfaction (M = 4.4, SD = 0.47) after the CC. Decisional conflict levels were significantly reduced (baseline: M = 29.3, SD = 16.9, post-CC: M = 16.3, SD = 11.5; t = 5.37, P < 0.001) after the CC. Mean decisional regret scores were 'mild' at three-months (M = 16.0, SD = 17.5), six-months (M = 18.8, SD = 18.7) and 12-months (M = 18.2, SD = 15.1) post-treatment completion. CONCLUSION: This is the first Australian study to assess decisional outcomes when patients are offered the choice between RARP and radiotherapy in the public health system. A CC seems to support decision-making in men with LPC and positively impact some decisional outcomes. However, larger-scale controlled studies are needed to confirm these findings.

Variation in Health Care Access and Quality Among US States and High-Income Countries With Universal Health Insurance Coverage.

Importance: Based on mortality estimates for 32 causes of death that are amenable to health care, the US health care system did not perform as well as other high-income countries, scoring 88.7 out of 100 on the 2016 age-standardized Healthcare Access and Quality (HAQ) index. Objective: To compare US age-specific HAQ scores with those of high-income countries with universal health insurance coverage and compare scores among US states with varying insurance coverage. Design, Setting, and Participants: This cross-sectional study used 2016 Global Burden of Diseases, Injuries, and Risk Factor study results for cause-specific mortality with adjustments for behavioral and environmental risks to estimate the age-specific HAQ indices. The US national age-specific HAQ scores were compared with high-income peers (Canada, western Europe, high-income Asia Pacific countries, and Australasia) in 1990, 2000, 2010, and 2016, and the 2016 scores among US states were also analyzed. The Public Use Microdata Sample of the American Community Survey was used to estimate insurance coverage and the median income per person by age and state. Age-specific HAQ scores for each state in 2010 and 2016 were regressed based on models with age fixed effects and age interaction with insurance coverage, median income, and year. Data were analyzed from April to July 2018 and July to September 2020. Main Outcomes and Measures: The age-specific HAQ indices were the outcome measures. Results: In 1990, US age-specific HAQ scores were similar to peers but increased less from 1990 to 2016 than peer locations for ages 15 years or older. For example, for ages 50 to 54 years, US scores increased from 77.1 to 82.1 while high-income Asia Pacific scores increased from 71.6 to 88.2. In 2016, several states had scores comparable with peers, with large differences in performance across states. For ages 15 years or older, the age-specific HAQ scores were 85 or greater for all ages in 3 states (Connecticut, Massachusetts, and Minnesota) and 75 or less for at least 1 age category in 6 states. In regression analysis estimates with state-fixed effects, insurance coverage coefficients for ages 20 to 24 years were 0.059 (99% CI, 0.006-0.111); 45 to 49 years, 0.088 (99% CI, 0.009-0.167); and 50 to 54 years, 0.101 (99% CI, 0.013-0.189). A 10% increase in insurance coverage was associated with point increases in HAQ scores among the ages of 20 to 24 years (0.59 [99% CI, 0.06-1.11]), 45 to 49 years (0.88 [99% CI, 0.09-1.67]), and 50 to 54 years (1.01 [99% CI, 0.13-1.89]). Conclusions and Relevance: In this cross-sectional study, the US age-specific HAQ scores for ages 15 to 64 years were low relative to high-income peer locations with universal health insurance coverage. Among US states, insurance coverage was associated with higher HAQ scores for some ages. Further research with causal models and additional explanations is warranted.

Interim Prostate-Specific Antigen: Predicting for Biochemical Failure During Salvage Radiation Therapy After Prostatectomy.

PURPOSE: A subset of patients treated with postprostatectomy radiation therapy for biochemical recurrence after surgery fail to respond because of microscopic disease beyond the irradiated prostate bed. This work aims to determine whether a rising interim prostate-specific antigen (PSA) during radiation therapy can predict the likelihood of subsequent biochemical recurrence. METHODS AND MATERIALS: Between 2010 and 2016, 185 patients had salvage radiation therapy to a dose of 68 Gy without androgen deprivation therapy for a rising PSA level after radical prostatectomy. Patients had their PSA recorded on the first day of radiation therapy and again after completing the 25th fraction (of 34 total fractions). Biochemical failure after radiation therapy was defined as a PSA value ≥0.2 ng/mL within 2 years after radiation therapy. Both univariate and multivariate Cox regression models were used for statistical analysis. Factors with a P value of <.2 in univariate analysis were then used in a multivariate analysis. RESULTS: The 2-year freedom from biochemical failure was 60% (95% confidence interval, 53%-67%). When assessing the interim PSA, 143 patients (77%) had a drop in interim PSA; of these patients, 71% had 2-year biochemical control. Forty-two patients (23%) had a stable or rising interim PSA, and only 24% of these patients had 2-year biochemical control. On multivariate analysis, a drop in PSA during radiation therapy (P < .0001) and a positive surgical margin (P < .0001) were significant factors for freedom from subsequent biochemical failure, and seminal vesicle invasion was associated with biochemical failure at 2 years (P = .019). All patients with a rising interim PSA, negative surgical margin, and seminal vesicle invasion ultimately had biochemical failure at 2 years. CONCLUSIONS: A PSA rise during salvage radiation therapy is prognostic of biochemical failure at 2 years. Factors such as seminal vesicle invasion and a negative surgical margin also predict for poor responders to salvage radiation therapy.

Reduced motion and improved rectal dosimetry through endorectal immobilization for prostate stereotactic body radiotherapy.

OBJECTIVE: PROMETHEUS (ACTRN12615000223538) is a multicentre clinical trial investigating the feasibility of 19 Gy in 2 fractions of stereotactic body radiotherapy (SBRT) as a boost technique for prostate cancer. The objective of this substudy was to evaluate intrafraction motion using cine MRI and assess the dosimetric impact of using a rectal displacement device (RDD). METHODS: The initial 10 patients recruited underwent planning CT and MRI, with and without a RDD. Cine MRI images were captured using an interleaved T2 HASTE sequence in sagittal and axial planes with a temporal resolution of 5.2 s acquired over 4.3 min. Points of interest (POIs) were defined and a validated tracking algorithm measured displacement of these points over the 4.3 min in the anteroposterior, superior-inferior and left-right directions. Plans were generated with and without a RDD to examine the impact on dosimetry. RESULTS: There was an overall trend for increasing displacement in all directions as time progressed when no RDD was in situ . points of interest remained comparatively stable with the RDD. In the sagittal plane, the RDD resulted in statistically significant improvement in the range of anteroposterior displacement for the rectal wall, anterior prostate, prostate apex and base. Dosimetrically, the use of a RDD significantly reduced rectal V16, V14 and Dmax, as well as the percentage of posterior rectal wall receiving 8.5 Gy. CONCLUSION: The RDD used in stereotactic prostate radiotherapy leads to reduced intrafraction motion of the prostate and rectum, with increasing improvement with time. It also results in significant improvement in rectal wall dosimetry. ADVANCES IN KNOWLEDGE: It was found that the rectal displacement device improved prostate stabilization significantly, improved rectum stabilization and dosimetry significantly. The rectal displacement device did not improve target volume dosimetry.

Long-term outcomes in 1121 Australian prostate cancer patients treated with definitive radiotherapy.

INTRODUCTION: Optimal definitive treatment of prostate cancer is controversial, especially in high-risk patients. We report the largest prospective cohort of Australian patients treated with radiotherapy for localised prostate cancer. METHODS: One thousand, one hundred and twenty-one patients with prostate cancer were prospectively registered and treated to a dose of 70-74 Gy. Patients were classified as low, intermediate or high risk based on PSA, clinical staging and Gleason score. Intermediate-risk patients were treated with 0-6 months of hormonal therapy (ADT) and high-risk patients were offered neoadjuvant and adjuvant ADT. Overall survival (OS) and biochemical relapse-free survival (bNED) were calculated using the Kaplan-Meier method. RESULTS: Median follow-up was 92 months. Eight-year OS and bNED were 78.4% and 68.1% respectively in the entire cohort. OS for the low, intermediate and high-risk groups was 84.5%, 78.4% and 68% respectively. For these risk groups, bNED was 80.3%, 65.7% and 53.7% respectively. In the intermediate and high-risk group, OS and bNED decreased with increasing number of risk factors. CONCLUSION: Definitive radiotherapy is an effective treatment for prostate cancer, including in high-risk cases.

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing.

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

Health system changes under pay-for-performance: the effects of Rwanda's national programme on facility inputs.

Pay-for-performance (P4P) programmes have been introduced in numerous developing countries with the goal of increasing the provision and quality of health services through financial incentives. Despite the popularity of P4P, there is limited evidence on how providers achieve performance gains and how P4P affects health system quality by changing structural inputs. We explore these two questions in the context of Rwanda's 2006 national P4P programme by examining the programme's impact on structural quality measures drawn from international and national guidelines. Given the programme's previously documented success at increasing institutional delivery rates, we focus on a set of delivery-specific and more general structural inputs. Using the programme's quasi-randomized roll-out, we apply multivariate regression analysis to short-run facility data from the 2007 Service Provision Assessment. We find positive programme effects on the presence of maternity-related staff, the presence of covered waiting areas and a management indicator and a negative programme effect on delivery statistics monitoring. We find no effects on a set of other delivery-specific physical resources, delivery-specific human resources, delivery-specific operations, general physical resources and general human resources. Using mediation analysis, we find that the positive input differences explain a small and insignificant fraction of P4P's impact on institutional delivery rates. The results suggest that P4P increases provider availability and facility operations but is only weakly linked with short-run structural health system improvements overall.

Temperature and density dependent induction of a cytopathic effect following infection with non-cytopathic HAV strains.

Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (<34°C) and reduced cell density in both monkey (FRhK-4) and human (A549) cells. This phenotype is not virus-strain restricted, as it was also observed in cells infected with HAV strains, HAS-15 and LSH/S. Cytopathic effect was accompanied by rRNA cleavage, indicating activation of the RNase L pathway, viral negative strand synthesis, caspase-3 activation, and apoptosis. The results indicate that a cytopathic phenotype may be present in some HAV strains that can be induced under appropriate conditions, suggesting the potential for development of a plaque assay for this virus.

Activation of the 2-5OAS/RNase L pathway in CVB1 or HAV/18f infected FRhK-4 cells does not require induction of OAS1 or OAS2 expression.

The latent, constitutively expressed protein RNase L is activated in coxsackievirus and HAV strain 18f infected FRhK-4 cells. Endogenous oligoadenylate synthetase (OAS) from uninfected and virus infected cell extracts synthesizes active forms of the triphosphorylated 2-5A oligomer (the only known activator of RNase L) in vitro and endogenous 2-5A is detected in infected cell extracts. However, only the largest OAS isoform, OAS3, is readily detected throughout the time course of infection. While IFNbeta treatment results in an increase in the level of all three OAS isoforms in FRhK-4 cells, IFNbeta pretreatment does not affect the temporal onset or enhancement of RNase L activity nor inhibit virus replication. Our results indicate that CVB1 and HAV/18f activate the 2-5OAS/RNase L pathway in FRhK-4 cells during permissive infection through endogenous levels of OAS, but contrary to that reported for some picornaviruses, CVB1 and HAV/18f replication is insensitive to this activated antiviral pathway.

Apoptosis induced by a cytopathic hepatitis A virus is dependent on caspase activation following ribosomal RNA degradation but occurs in the absence of 2'-5' oligoadenylate synthetase.

We have presented previously evidence that the cytopathogenic 18f strain of hepatitis A virus (HAV) induced degradation of ribosomal RNA (rRNA) in infected cells [Arch. Virol. 148 (2003) 1275-1300]. In contrast, the non-cytopathogenic parent virus HM175 clone 1 had no effect on rRNA integrity. We present here data showing that rRNA degradation is followed by apoptosis accompanied by characteristic DNA laddering in the cytoplasm of 18f infected cells. The DNA laddering coincided with the detection of caspase 3 and PARP-1 cleavage and was dependent upon activation of the caspase pathway, since treatment with Z-VAD-FMK, a pan-caspase inhibitor, inhibited both events. RNase L mRNA was present in both virus-infected and uninfected cells. Messenger RNA for the interferon inducible enzyme 2'-5' oligoadenylate synthetase (2'-5' OAS), which polymerizes ATP into 2'-5' oligo adenylate (2-5A, the activator of RNase L) in the presence of double-stranded RNA, was not detected following virus infection. 2'-5' OAS mRNA was induced by treatment of the cells with interferon-beta (IFN-beta). IFN-beta mRNA was marginally induced following infection. However, phosphorylated STAT 1, a key regulator of interferon-stimulated gene transcription was not detected in virus infected cells. STAT 1 phosphorylation in response to IFN treatment was lower in virus-infected cells, compared to uninfected cells treated with interferon, suggesting that 18f virus infection interferes with interferon signaling. The results suggest that 18f infection causes the induction of a 2-5A independent RNase L like activity.

CXCR3 is induced early on the pathway of CD4+ T cell differentiation and bridges central and peripheral functions.

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs, or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4(+) T cells. Using CD4(+) T cells from cord blood, we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu, although on resting cells, expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils, CXCR3(+)CD4(+) T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4(+) T cells by comparing its pattern of expression with that of CCR5, another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5, supporting a model of sequential induction of chemokine receptors during CD4(+) T cell differentiation. Taken together, our data show that CXCR3 can be expressed at all stages of CD4(+) T cell activation and differentiation, bridging central function in lymphoid organs and effector function in peripheral tissues.

A polymerase chain reaction-based method for the detection of hepatitis A virus in produce and shellfish.

Outbreaks of gastroenteritis that are suspected to be of viral origin are on the rise. Thus, there is a need for regulatory agencies entrusted with food safety to develop adequate techniques for the detection of viruses in foods. We have established a general procedure for the detection of hepatitis A virus (HAV) in shellfish that, with minor modifications, is also applicable to fresh produce such as cilantro. Total RNA was isolated from shellfish or cilantro, followed by isolation of poly(A)-containing RNA. Because HAV genomic RNA contains a poly(A) tail, the isolation of poly(A)-containing RNA also enriches HAV genomic RNA. Reverse transcription was used to convert the RNA to cDNA, and then amplification was carried out by polymerase chain reaction (PCR). Reamplification with internal primers was used to improve the quality and the quantity of amplified DNA, allowing for post-PCR analysis such as sequence identification of the viral strain. With this procedure, multiple samples could be analyzed in four working days by a single trained individual. The nominal sensitivity of detection of the procedure was 0.15 TCID50 (50% tissue culture infective dose) per 0.62 g of tissue with a test virus. The direct RNA isolation protocol avoided pitfalls associated with whole-virus purification procedures by replacing virus precipitation steps involving polyethylene glycol and Procipitate with phenol extraction. The method is straightforward and reliable. We successfully used this procedure to detect naturally occurring HAV in clams involved in a gastroenteritis outbreak, as well as in cilantro artificially contaminated with a test virus.