Story to Change Culture on Early Childhood in Australia.

The goal of the article is to support the early childhood sector's efforts to increase the salience of early childhood as a social issue and change policy and practice to better support young children and their families. Cultural models shape how people think about social issues and support solutions. Changing how issues are framed-how they are presented, positioned and focused-can help shift these models and facilitate culture change. Using mixed methods research, we identified cultural models that members of the Australian public use to think about early childhood and compared these mindsets to concepts that the sector seeks to advance. This revealed a set of gaps in understanding that make it difficult for the sector to advance its agenda. We then designed and tested framing strategies to address these challenges and improve the salience of early childhood as a social issue, increase understanding of key concepts and build support for policies, programs and interventions. Findings point to strategies that advocates, service providers and funders can use to communicate more effectively about the importance of the early years.

The Impact of Social Media Use and Gaming on Initiation of Smokeless Tobacco Use Among Rural Male Youth.

PURPOSE: Smokeless tobacco (SLT) use is more common among White male youth in rural locations than among other youth. Previous literature documents risk factors for SLT use (e.g., perceived harm). However, no research has examined whether SLT initiation is associated with the use of social media and video games, despite the high prevalence of those behaviors among adolescent males. METHODS: As part of the evaluation of "The Real Cost" smokeless prevention campaign, we conducted a baseline survey of a cohort of US males aged 11-16 in 2016 and four follow-up surveys conducted approximately every 8 months. We used discrete-time survival analysis to examine factors associated with SLT initiation. Logistic regression estimated the odds of initiation as a function of lagged values of key predictors (social media use in tertiles and frequency of gaming) and other baseline and lagged correlates. The model controlled for campaign exposure. RESULTS: Relative to the lowest tertile of social media use, moderate (odds ratio [OR] = 1.80) or high use (OR = 2.77) was associated with increased risk of SLT initiation at the subsequent survey wave. Relative to playing every few weeks or less, playing video games once a day (OR = 0.50) or several times a day (OR = 0.33) was associated with decreased risk of SLT initiation. DISCUSSION: SLT initiation was positively associated with social media use and negatively associated with gaming. Future research should explore reasons for the protective nature of gaming (e.g., social support, improved mood) and whether parental supervision of social media use could mitigate its negative impact.

Pudendal nerve block prior to inflatable penile prosthesis implantation: decreased intra-operative narcotic requirements.

The opioid epidemic has proven to be a public health crisis over the past two decades and efforts to decrease opioid exposure are sorely needed. Our objective was to determine whether pudendal nerve block utilization in the immediate pre-operative setting decreases intra-operative opioid analgesic requirements in veteran patients undergoing a primary inflatable penile prosthesis implantation. We performed a retrospective cohort analysis of all patients undergoing penile prosthesis implantation between January 2017 and July 2020 at the Charlie Norwood Veterans Affairs Medical Center in Augusta, GA. Univariable and multivariable gamma regression analyses were performed to evaluate the association between pudendal nerve block utilization and intra-operative opioid analgesic requirements. The study cohort consisted of 110 patients, 35 (31.8%) of whom underwent a pudendal nerve block. Median intra-operative opioid analgesic requirements were significantly lower in the pudendal nerve block group (16.3 versus 25.8 morphine milliequivalents, p = 0.037). Receipt of the nerve block was associated with significantly lower intra-operative opioid analgesic requirements on multivariable (coefficient 0.84, p = 0.038) regression analysis. There was no significant difference in post-operative opioid analgesic requirements (p = 0.18). In conclusion, pre-operative pudendal nerve blocks decrease intra-operative opioid analgesic requirements in veteran patients undergoing a primary inflatable penile prosthesis implantation.

Changes in E-cigarette Use Among Youth and Young Adults During the COVID-19 Pandemic: Insights Into Risk Perceptions and Reasons for Changing Use Behavior.

INTRODUCTION: This study assessed changes in e-cigarette use since the COVID-19 pandemic began and reasons for these changes among US youth and young adults. AIMS AND METHODS: We combined data from two cross-sectional samples of youth and young adult (15-24 years) participants of a monthly surveillance study (data collected in April and June 2021). Analyses were restricted to past-year e-cigarette users who reported using e-cigarettes before the pandemic (n = 1762). Participants reported changes in e-cigarette use since the pandemic began, reasons for changing their behavior, and their perceptions around COVID-19 risk related to e-cigarette use. Multinomial logistic regression models assessed associations between demographics and COVID vaping risk perceptions and changes in e-cigarette use. RESULTS: Over a third of the sample (37.9%) reported increasing e-cigarette use, while 28.9% reported decreasing use, and 33.1% reported no change. Analyses revealed associations between change in e-cigarette use and age, race and ethnicity, e-cigarette use status, and COVID vaping risk perceptions. The most common reason for increasing use was managing stress (36%); the most common reasons for decreasing use included fewer social interactions (22%) and concerns over general health (18%). Results also indicated differences in reasons for decreasing use by race and ethnicity, and differences in reasons for both increasing and decreasing use by age. CONCLUSIONS: This study provides recent data on how the ongoing and evolving COVID-19 pandemic has impacted youth and young adults' e-cigarette use. Findings identify subpopulations that may benefit from e-cigarette prevention interventions, as well as insights that may inform the content and delivery of such interventions. IMPLICATIONS: This study provides recent insights into if and why the ongoing COVID-19 pandemic has impacted e-cigarette use behavior among youth and young adults. Findings contribute to the understanding of the role e-cigarettes play in young people's lives-insights that can inform youth and young adult prevention efforts.

Predictive validity of the original and expanded susceptibility scales for smokeless tobacco.

BACKGROUND: This study seeks to validate the original and expanded susceptibility scales for smokeless tobacco (SLT) use among a longitudinal sample of rural male youth. We also compare the predictive validity of both scales for SLT. METHODS: Data are from a five-wave longitudinal sample of rural males in the Unites States aged 11-16 at baseline. Data were collected from January 2016 to December 2018. We used a series of logistic regressions to identify whether individual demographic variables, the original (use SLT soon, in next year, if friend offers), or the expanded (original plus curiosity) susceptibility scales predicted experimentation with SLT by 2018. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of both scales were also calculated to determine reliability. All analyses were limited to 2016 never SLT users. RESULTS: Both susceptibility scales were significant predictors of SLT initiation by follow-up. Susceptible youth had three times the odds of SLT initiation by follow-up compared to youth who were non-susceptible. Both scales have high specificity, identifying a large proportion of never users as non-susceptible. Sensitivity for the original scale was 37.0% and increased to 44.2% for the expanded scale. The PPV was approximately 20% for both the original and expanded scales. CONCLUSIONS: The SLT susceptibility scales are valid for predicting future SLT initiation; however, there remains room for improvement as the sensitivity of both scales is relatively low compared to the smoking scale. Future research should examine additional methods to identify youth at risk of SLT initiation.

Reframing Adolescent Development: Identifying Communications Challenges and Opportunities.

Understanding Americans' deeply held and widely shared assumptions about adolescents and their development can reveal key opportunities and challenges for developmental science communicators. Twenty-nine in-depth interviews were conducted with adolescents and adults about adolescence. We analyzed the cultural models the public use to make meaning about what adolescence is, what development involves, what adolescents need, and how adolescents can be supported. The analysis revealed several cultural models that may impede public engagement around youth issues. These dominant ways of thinking include a strong focus on the vulnerability of adolescence and a narrow understanding of how environments affect adolescent development. The findings have important implications for communicators seeking to expand and deepen public thinking about adolescence.

"The Real Cost" Smokeless campaign: changes in beliefs about smokeless tobacco among rural boys, a longitudinal randomized controlled field trial.

BACKGROUND: The prevalence of current smokeless tobacco (SLT) use in 2019 among high school students was 4.8%, and the overall rate of SLT use was higher among high school boys (7.5%) than girls (1.8%). The U.S. Food and Drug Administration (FDA) launched "The Real Cost" Smokeless media campaign in April 2016 to educate rural youth about the dangers of SLT use. In this study, we evaluate the effectiveness of "The Real Cost" Smokeless campaign. METHODS: We use a 3-year (Jan 2016 - Dec 2018) randomized controlled longitudinal field trial that consists of a baseline survey of boys and a parent/guardian and four follow-up surveys of the boys. The cohort includes 2200 boys who were 11 to 16 years old at baseline and lived in the rural segments of 30 media markets (15 treatment markets and 15 control). "The Real Cost" Smokeless campaign targets boys who are 12 to 17 years old in 35 media markets. It focuses primarily on graphic depictions of cosmetic and long-term health consequences of SLT use. The key outcome measures include beliefs and attitudes toward SLT that are targeted (explicitly or implicitly) by campaign messages. RESULTS: Using multivariate difference-in-difference analysis (conducted in 2019 and 2020), we found that agreement with 4 of the 11 explicit campaign-targeted belief and attitude measures increased significantly from baseline to post-campaign launch among boys 14 to 16 years old in treatment vs. control markets. Agreement did not increase for boys 11-13 years old in treatment vs. control markets and only increased for one targeted message for the overall sample. CONCLUSIONS: These findings suggest that "The Real Cost" Smokeless campaign influenced beliefs and attitudes among older boys in campaign markets and that a campaign focused on health consequences of tobacco use can be targeted to rural boys, influence beliefs about SLT use, and potentially prevent SLT use.

Cost Effectiveness of the Tips From Former Smokers® Campaign-U.S., 2012-2018.

INTRODUCTION: Since 2012, the Centers for Disease Control and Prevention has conducted the national Tips From Former Smokers® public education campaign, which motivates smokers to quit by featuring people living with the real-life health consequences of smoking. Cost effectiveness, from the healthcare sector perspective, of the Tips From Former Smokers® campaign was compared over 2012-2018 with that of no campaign. METHODS: A combination of survey data from a nationally representative sample of U.S. adults that includes cigarette smokers and literature-based lifetime relapse rates were used to calculate the cumulative number of Tips From Former Smokers® campaign‒associated lifetime quits during 2012-2018. Then, lifetime health benefits (premature deaths averted, life years saved, and quality-adjusted life years gained) and healthcare sector cost savings associated with these quits were assessed. All the costs were adjusted for inflation in 2018 U.S. dollars. The Tips From Former Smokers® campaign was conducted and the survey data were collected during 2012-2018. Analyses were conducted in 2019. RESULTS: During 2012-2018, the Tips From Former Smokers® campaign was associated with an estimated 129,100 premature deaths avoided, 803,800 life years gained, 1.38 million quality-adjusted life years gained, and $7.3 billion in healthcare sector cost savings on the basis of an estimated 642,200 campaign-associated lifetime quits. The Tips From Former Smokers® campaign was associated with cost savings per lifetime quit of $11,400, per life year gained of $9,100, per premature deaths avoided of $56,800, and per quality-adjusted life year gained of $5,300. CONCLUSIONS: Mass-reach health education campaigns, such as Tips From Former Smokers®, can help smokers quit, improve health outcomes, and potentially reduce healthcare sector costs.

Effect of menthol cigarette and other menthol tobacco product bans on tobacco purchases in the RTI iShoppe virtual convenience store.

OBJECTIVE: To test how a potential US ban of menthol products or replacement with 'green' products and ads could influence tobacco purchases. METHODS: US adult menthol smokers (N=1197) were recruited via an online panel and randomly assigned to complete a shopping task in one of four versions (experimental conditions) of the RTI iShoppe virtual store: (1) no ban, (2) replacement of menthol cigarettes and ads with green replacement versions, (3) menthol cigarette ban and (4) all menthol tobacco product ban. Logistic regressions assessed the effect of condition on tobacco purchases. RESULTS: Participants in the menthol cigarette ban (OR=0.67, 95% CI 0.48 to 0.92) and all menthol product ban conditions (OR=0.60, 95% CI 0.43 to 0.83) were less likely to purchase cigarettes of any type than participants in the no ban condition. Participants in the green replacement (OR=1.74, 95% CI 1.13 to 2.70), menthol cigarette ban (OR=3.40, 95% CI 2.14 to 5.41) and all menthol product ban conditions (OR=3.14, 95% CI 1.97 to 5.01) were more likely to purchase a cigarette brand different from their usual brand than participants in the no ban condition. CONCLUSIONS: Our findings suggest that menthol bans could have great public health impact by reducing cigarette purchases. However, tobacco marketing strategies, such as creating green (or other replacement) versions of menthol cigarettes, may undermine public health benefits of a menthol ban by prompting purchases of non-menthol cigarettes. Our findings highlight the importance of taking tobacco marketing tactics into consideration in tobacco product regulation.

Comparison of Transcription-Mediated Amplification and Real-Time PCR Assays for Hepatitis B Virus DNA Quantitation in Serum.

BACKGROUND: The quantification of hepatitis B (HBV) DNA in serum is critical to identify patients requiring antiviral therapy and to monitor the response to treatment. METHOD: This study describes the evaluation of the Aptima HBV Quant Dx assay (Aptima HBV) performed on the automated Panther system. RESULTS: Aptima HBV was linear from 1.70 to 7.70 log10 IU/mL with a commercial reference panel, as well as clinical specimens representing genotypes B and C, and total imprecision, as measured by the percentage coefficient of variation (%CV) at 2.0 log10 IU/mL was <10%. The specificity of Aptima HBV was 94.7% (126/133) and 96.6% (84/87) for serum specimens from individuals without HBV exposure and individuals with resolved HBV infection, respectively. The qualitative agreement and quantitative accuracy of Aptima HBV was compared to the COBAS AmpliPrep/COBAS TaqMan HBV Test v2.0 (CAP/CTM). Overall agreement was 90.8% (187/206) with a κ statistic of 0.708 (standard error, 0.063; 95% CI, 0.585-0.831). Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% CI of the slope, 0.883-1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (Aptima - CAP/CTM) showed a slight negative bias (-0.054 log10 IU/mL, and 95% limits of agreement of -1.093 to 0.984). CONCLUSIONS: The Aptima HBV test affords a suitable alternative to CAP/CTM for serum virus load testing and provides a key component of the diagnostic algorithm for the global eradication of viral hepatitis.

Antismoking Advertisements and Price Promotions and Their Association With the Urge to Smoke and Purchases in a Virtual Convenience Store: Randomized Experiment.

BACKGROUND: Point of sale (POS) advertising is associated with smoking initiation, current smoking, and relapse among former smokers. Price promotion bans and antismoking advertisements (ads) are 2 possible interventions for combating POS advertising. OBJECTIVE: The purpose of this analysis was to determine the influence of antismoking ads and promotions on urges to smoke and tobacco purchases. METHODS: This analysis examined exposure to graphic (graphic images depicting physical consequences of tobacco use) and supportive (pictures of and supportive messages from former smokers) antismoking ads and promotions in a virtual convenience store as predictors of urge to smoke and buying tobacco products among 1200 current cigarette smokers and 800 recent quitters recruited via a Web-based panel (analytical n=1970). We constructed linear regression models for urge to smoke and logistic regression models for the odds of purchasing tobacco products, stratified by smoking status. RESULTS: The only significant finding was a significant negative relationship between exposure to supportive antismoking ads and urge to smoke among current smokers (beta coefficient=-5.04, 95% CI -9.85 to -0.22; P=.04). There was no significant relationship between graphic antismoking ads and urge to smoke among current smokers (coefficient=-3.77, 95% CI -8.56 to 1.02; P=.12). Neither relationship was significant for recent quitters (graphic: coefficient=-3.42, 95% CI -8.65 to 1.81; P=.15 or supportive: coefficient=-3.82, 95% CI -8.99 to 1.36; P=.20). There were no significant differences in urge to smoke by exposure to promotions for current smokers (coefficient=-1.06, 95% CI -4.53 to 2.41; P=.55) or recent quitters (coefficient=1.76, 95% CI -2.07 to 5.59; P=.37). There were also no differences in tobacco purchases by exposure to graphic (current smokers: coefficient=0.93, 95% CI 0.67 to 1.29; P=.66 and recent quitters: coefficient=0.73, 95% CI 0.44 to 1.19; P=.20) or supportive (current smokers: coefficient=1.05, 95% CI 0.75 to 1.46; P=.78 and recent quitters: coefficient=0.73, 95% CI 0.45 to 1.18; P=.20) antismoking ads or price promotions (current smokers: coefficient=1.09, 95% CI 0.86 to 1.38; P=.49 and recent quitters: coefficient=0.90, 95% CI 0.62 to 1.31; P=.60). CONCLUSIONS: The results of this analysis support future research on the ability of supportive antismoking ads to reduce urges to smoke among current cigarette smokers. Research on urges to smoke has important tobacco control implications, given the relationship between urge to smoke and smoking cigarettes, time to next smoke, and amount smoked.

Warning Labels on Sugar-sweetened Beverages: An Eye Tracking Approach.

Objectives: In this study, we examined visual attention of a warning label on a sugar-sweetened beverage (SSB) and its effects on visual attention to SSB product descriptors and perceptions of SSB using eye tracking technology. Methods: We had 180 young adults view an image of a generic soda can with or without a text warning on a computer monitor. Results: Participants spent less time looking at marketing elements on the can in the "Warning" condition compared to the "No warning" (control) condition. Compared to the control, participants in the "Warning" condition viewed the sugar-sweetened beverage as less healthy (1.78 warning vs 2.21 control, p < .01) and believed that drinking SSBs contributed to diabetes (5.70 warning vs 5.27 control, p < .01). Visual attention to warning label was associated with correct recall of the warning and opting out of purchasing the can. Conclusions: Textual warning on SSB reduced visual attention to marketing elements on the can. Although there were few statistically significant differences between the conditions on most measures of product appeal or risk perception, warnings increased some perceived risks of SSBs indicating that warning labels on SSBs might be a promising strategy in informing consumers, particularly young adults, about risks of added sugars.

Evaluation of the Aptima HCV Quant Dx Assay Using Serum and Dried Blood Spots.

Hepatitis C virus (HCV) RNA quantitation is the primary method by which active HCV infections are identified and the response to direct-acting antiviral therapy is monitored. This study describes the evaluation of the Aptima HCV Quant Dx assay (Aptima HCV) performed on the Panther system. The clinical performance of Aptima HCV was compared to that of the Cobas AmpliPrep/Cobas TaqMan HCV test v2.0 (CAP/CTM). Overall agreement was 84.9% (186/219) with a kappa statistic of 0.755 (standard error, 0.037; 95% confidence interval [CI], 0.682 to 0.828). Passing-Bablok regression of log10 IU/ml values revealed a regression line of Y = 1.163 × X - 0.991 (95% CI of the slope, 1.103 to 1.221, and intercept, -1.341 to -0.642). The 95% lower limit of detection (LLOD) for Aptima HCV on dried blood spot (DBS) samples was calculated to be 2.43 log10 IU/ml (267 IU/ml; 95% CI, 2.31 to 2.73 log10 IU/ml [204 to 540 IU/ml]). A comparison of Aptima HCV testing on paired DBS and serum specimens collected from patients at the time of routine blood collection for CAP/CTM demonstrated an overall agreement of 90.1% (82/91) with a kappa statistic of 0.657 (standard error, 0.101; 95% CI, 0.458 to 0.855). In conclusion, Aptima HCV provides a suitable alternative for HCV RNA testing on serum and DBS samples.

Reframing Aging: Effect of a Short-Term Framing Intervention on Implicit Measures of Age Bias.

OBJECTIVES: Evidence suggests that agist biases may operate implicitly (i.e., automatically and unconsciously) to affect discriminatory attitudes and behaviors toward older adults. However, few studies have tested the malleability of implicit age bias. The present study tests the effect of a framing intervention on implicit age bias in a nationally representative sample of American adults. METHOD: Participants (N = 767) were randomly assigned to read 1 of 3 framed messages, to an unframed message about aging, or to a control message unrelated to aging. Framed messages emphasized (a) the contributions of older adults to society; (b) aging as a process of accumulating wisdom and energy; and (c) mechanisms through which prejudice against older adults operates. Participants subsequently completed an aging implicit association test (IAT) to assess implicit bias. RESULTS: Relative to the control condition, participants in the 3 framed message conditions displayed lower implicit age bias. No differences were observed between participants in the control condition and those who read the unframed message. DISCUSSION: Findings indicate that reframing messages about aging can decrease implicit bias against older adults. This study highlights ways for communicators to promote a positive understanding of the aging process, thereby mitigating sources of implicit prejudice.

Deception and Shopping Behavior Among Current Cigarette Smokers: A Web-Based, Randomized Virtual Shopping Experiment.

BACKGROUND: Virtual stores can be used to identify influences on consumer shopping behavior. Deception is one technique that may be used to attempt to increase the realism of virtual stores. OBJECTIVE: The objective of the experiment was to test whether the purchasing behavior of participants in a virtual shopping task varied based on whether they were told that they would receive the products they selected in a virtual convenience store (a form of deception) or not. METHODS: We recruited a US national sample of 402 adult current smokers by email from an online panel of survey participants. They completed a fully automated randomized virtual shopping experiment with a US $15 or US $20 budget in a Web-based virtual convenience store. We told a random half of participants that they would receive the products they chose in the virtual store or the cash equivalent (intervention condition), and the other random half simply to conduct a shopping task (control condition). We tested for differences in demographics, tobacco use behaviors, and in-store purchases (outcome variable, assessed by questionnaire) by experimental condition. RESULTS: The characteristics of the participants (398/402, 99.0% with complete data) were comparable across conditions except that the intervention group contained slightly more female participants (103/197, 52.3%) than the control group (84/201, 41.8%; P=.04). We did not find any other significant differences in any other demographic variables or tobacco use, or in virtual store shopping behaviors, including purchasing any tobacco (P=.44); purchasing cigarettes (P=.16), e-cigarettes (P=.54), cigars (P=.98), or smokeless tobacco (P=.72); amount spent overall (P=.63) or on tobacco (P=.66); percentage of budget spent overall (P=.84) or on tobacco (P=.74); number of total items (P=.64) and tobacco items purchased (P=.54); or total time spent in the store (P=.07). CONCLUSIONS: We found that telling participants that they will receive the products they select in a virtual store did not influence their purchases. This finding suggests that deception may not affect consumer behavior and, as a result, may not be necessary in virtual shopping experiments.

Seeing Context through Metaphor: Using Communications Research to Bring a Social Determinants Perspective to Public Thinking about Child Abuse and Neglect.

Human beings think in metaphor and reason through analogy. The metaphors through which we think influence how we understand and feel about social issues as well as the actions that we see as appropriate and important. Metaphors can be used to increase understanding of how issues work and increase the salience of a given issue, build support for programs and policies necessary to address the issue, and instigate demand for change and civic action. In this paper, we use a mixed methods research design, including brief qualitative interviews, experimental surveys, and focus groups, to test the ability of different metaphors to influence public understanding of the social determinants of child abuse and neglect in the UK. We find one metaphor in particular that improves people's understanding of the social causes of child maltreatment and increases support for structural solutions. This metaphor can be used to build support for preventative public health solutions.

Smokers' Attention to Point-of-Sale Antismoking Ads: An Eye-tracking Study.

OBJECTIVES: We used eye-tracking to examine smokers' visual attention in one of 4 antismoking ad contexts (alone, next to cigarette ad, tobacco display, or cooler). Participants viewed 4 ad types (graphic, intended emotive, and benefits of quitting-graphic ads, and benefits of quitting-informational ads), each with 3 areas of interest (AOI) (anti-ad image, anti-ad text, and other text). METHODS: Current smokers (N = 153) viewed ads for 10 seconds each. Multivariable random effect linear regressions with post-test comparisons (with sidak-adjusted p-values) were used to test for differences in fixations and dwell time by ad context and type while adjusting for covariates. Visual attention was adjusted by percentage of anti-ad area taken up by each AOI. RESULTS: Adjusting for covariates, there were no differences by ad context (p > .05). Fixations and dwell time were greatest for the image of the benefits of quitting-graphic ad, the text of the graphic ad, and the other text of the intended emotive ad (all ps < .005). Conclusions: Visual attention to antismoking ads did not vary by ad context but varied significantly by ad type.

Beyond Hat in Hand: Science Advocacy Is Foundational for Policy Decisions.

Beyond those to whom neuroscientists typically communicate exciting discoveries-that is, those who can provide more funding for researchers-there are important audiences that are positioned to use neuroscience findings to affect policy and improve societal outcomes. Showing the utility of research that policymakers, service providers, and the public can use to make decisions will enhance views of the value of scientific research. The ingredients of successful communications between neuroscientists and other stakeholders are different from those that characterize effective communications between scientists. Here, we discuss our experiences in the communication of the science of early childhood and brain development and our recommendations to help neuroscientists better communicate the benefits of their research to those who make practice and policy decisions.

Stability of Zika virus in urine: Specimen processing considerations and implications for the detection of RNA targets in urine.

BACKGROUND: Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine. METHODS: To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels. Urine samples from healthy donors were spiked with ZIKV using the Exact Diagnostics® ZIKV Verification Panel, a commercially available panel composed of heat-inactivated ZIKV, at concentrations of 5.0 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4°C, or -80°C and frozen aliquots were exposed to no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield (Zymo Research, Irvine, CA). RESULTS: ZIKV RNA levels in urine declined steadily at room temperature, though was not significant by 48h (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4°C, suggesting that storage at 4°C can slow the progression of RNA degradation. Freezing urine samples at -80°C resulted in a significant loss of detectable ZIKV RNA in the ZIKV-low group. ZIKV RNA was detected in 5/6 replicates at 3days, 1/6 replicates at 10days, and 1/3 replicates at 30days, with findings reproducible on repeat testing. Presence of either nucleic acid stabilizer in urine corrected this effect, and resulted in recovery of ZIKV RNA in all replicates. Use of a nucleic acid stabilizer in the ZIKV-high group did not add incremental benefit for the detection or quantitation of ZIKV RNA. CONCLUSIONS: ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80°C for 10days. Detection of ZIKV-positive urine samples, particularly those containing low ZIKV titers may be aided with the addition of a nucleic acid stabilizer during urine specimen processing.

Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus.

Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R2 = 0.99; secondary standard, Y = 1.04X - 0.26, R2 = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log10; copies/ml, -0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.