Nanopore and Illumina sequencing reveal different viral populations from human gut samples.

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

Evaluation of respondent-driven sampling in seven studies of people who use drugs from rural populations: findings from the Rural Opioid Initiative.

BACKGROUND: Accurate prevalence estimates of drug use and its harms are important to characterize burden and develop interventions to reduce negative health outcomes and disparities. Lack of a sampling frame for marginalized/stigmatized populations, including persons who use drugs (PWUD) in rural settings, makes this challenging. Respondent-driven sampling (RDS) is frequently used to recruit PWUD. However, the validity of RDS-generated population-level prevalence estimates relies on assumptions that should be evaluated. METHODS: RDS was used to recruit PWUD across seven Rural Opioid Initiative studies between 2018-2020. To evaluate RDS assumptions, we computed recruitment homophily and design effects, generated convergence and bottleneck plots, and tested for recruitment and degree differences. We compared sample proportions with three RDS-adjusted estimators (two variations of RDS-I and RDS-II) for five variables of interest (past 30-day use of heroin, fentanyl, and methamphetamine; past 6-month homelessness; and being positive for hepatitis C virus (HCV) antibody) using linear regression with robust confidence intervals. We compared regression estimates for the associations between HCV positive antibody status and (a) heroin use, (b) fentanyl use, and (c) age using RDS-1 and RDS-II probability weights and no weights using logistic and modified Poisson regression and random-effects meta-analyses. RESULTS: Among 2,842 PWUD, median age was 34 years and 43% were female. Most participants (54%) reported opioids as their drug of choice, however regional differences were present (e.g., methamphetamine range: 4-52%). Many recruitment chains were not long enough to achieve sample equilibrium. Recruitment homophily was present for some variables. Differences with respect to recruitment and degree varied across studies. Prevalence estimates varied only slightly with different RDS weighting approaches, most confidence intervals overlapped. Variations in measures of association varied little based on weighting approach. CONCLUSIONS: RDS was a useful recruitment tool for PWUD in rural settings. However, several violations of key RDS assumptions were observed which slightly impacts estimation of proportion although not associations.

The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies.

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes.

Structure- and machine learning-guided engineering demonstrate that a non-canonical disulfide in an anti-PD-1 rabbit antibody does not impede antibody developability.

Rabbits produce robust antibody responses and have unique features in their antibody repertoire that make them an attractive alternative to rodents for in vivo discovery. However, the frequent occurrence of a non-canonical disulfide bond between complementarity-determining region (CDR) H1 (C35a) and CDRH2 (C50) is often seen as a liability for therapeutic antibody development, despite limited reports of its effect on antibody binding, function, and stability. Here, we describe the discovery and humanization of a human-mouse cross-reactive anti-programmed cell death 1 (PD-1) monoclonal rabbit antibody, termed h1340.CC, which possesses this non-canonical disulfide bond. Initial removal of the non-canonical disulfide resulted in a loss of PD-1 affinity and cross-reactivity, which led us to explore protein engineering approaches to recover these. First, guided by the sequence of a related clone and the crystal structure of h1340.CC in complex with PD-1, we generated variant h1340.SA.LV with a potency and cross-reactivity similar to h1340.CC, but only partially recovered affinity. Side-by-side developability assessment of both h1340.CC and h1340.SA.LV indicate that they possess similar, favorable properties. Next, and prompted by recent developments in machine learning (ML)-guided protein engineering, we used an unbiased ML- and structure-guided approach to rapidly and efficiently generate a different variant with recovered affinity. Our case study thus indicates that, while the non-canonical inter-CDR disulfide bond found in rabbit antibodies does not necessarily constitute an obstacle to therapeutic antibody development, combining structure- and ML-guided approaches can provide a fast and efficient way to improve antibody properties and remove potential liabilities.

Adolescent Residential Addiction Treatment In The US: Uneven Access, Waitlists, And High Costs.

Drug overdose deaths among adolescents are increasing in the United States. Residential treatment facilities are one treatment option for adolescents with substance use disorders, yet little is known about their accessibility or cost. Using the Substance Abuse and Mental Health Services Administration's treatment locator and search engine advertising data, we identified 160 residential addiction treatment facilities that treated adolescents with opioid use disorder as of December 2022. We called facilities while role-playing as the aunt or uncle of a sixteen-year-old child with a recent nonfatal overdose, to inquire about policies and costs. Eighty-seven facilities (54.4 percent) had a bed immediately available. Among sites with a waitlist, the mean wait time for a bed was 28.4 days. Of facilities providing cost information, the mean cost of treatment per day was $878. Daily costs among for-profit facilities were triple those of nonprofit facilities. Half of facilities required up-front payment by self-pay patients. The mean up-front cost was $28,731. We were unable to identify any facilities for adolescents in ten states or Washington, D.C. Access to adolescent residential addiction treatment centers in the United States is limited and costly.

Dissemination and implementation of an evidence-based voluntary medical male circumcision program: The Spear & Shield program.

Despite compelling evidence linking voluntary medical male circumcision (VMMC) with 60-70% HIV risk reduction in sub-Saharan Africa, Zambian men have been especially reluctant to undergo VMMC. The Government of Zambia set targets for VMMC uptake and promoted community-level interventions. Spear & Shield (S&S) is an innovative, evidence-based, service program promoting VMMC uptake while ensuring both VMMC supply and demand. This study assessed the large-scale provincial rollout of the program (S&S2) utilizing the RE-AIM model for translating interventions into the community. The S&S2 study was conducted between November 2015 and December 2020, and sequentially rolled out over four Zambian provinces in 96 clinics; 24 observation clinics received VMMC training only. Local clinic healthcare workers were trained to conduct the VMMC procedure and HIV counselors were trained to lead S&S group sessions. Using the RE-AIM model, primary outcomes were: Reach, the number, proportion, and representativeness of S&S attendees; Effectiveness, the impact of S&S2 on VMMC uptake; Adoption, the number, proportion, and representativeness of clinics implementing S&S2; Implementation, fidelity to the S&S intervention manual; and Maintenance, the extent to which S&S2 became an element of standard care within community clinics. Initially, n = 109 clinics were recruited; 96 were sustained and randomized for activation (Adoption). A total of 45,630 clinic patients (n = 23,236 men and n = 22,394 women) volunteered to attend the S&S sessions (Reach). The S&S2 program ran over 2,866 clinic-months (Implementation). Although the study did not target individual-level VMMCs, ~58,301 additional VMMCs were conducted at the clinic level (Effectiveness). Fidelity to the S&S intervention by group leaders ranged from 42%-95%. Sustainability of the program was operationalized as the number of CHCs initially activated that sustained the program. Intervention delivery ended, however, when study funding ceased (Maintenance). The S&S2 program successfully utilized the RE-AIM model to achieve study goals for implementation and dissemination in four Zambian provinces. Innovative VMMC programs such as S&S2 can improve the uptake of VMMC, one of the most effective strategies in the HIV prevention arsenal.

Associations between stimulant use and return to illicit opioid use following initiation onto medication for opioid use disorder.

AIM: The aim of this study was to estimate how ongoing stimulant use affects return to illicit opioid use after initiation onto medication for opioid use disorder (MOUD). DESIGN: This was a secondary analysis of pooled data from two clinical trials comparing buprenorphine (BUP-NX) and extended-release naltrexone (XR-NTX). SETTING: Thirteen opioid treatment programs and HIV clinics across 10 states in the United States from 2014 to 2019 took part in this study. PARTICIPANTS: A total of 528 participants who initiated MOUD as part of trial participation were included. Nearly half (49%) were between 30 and 49 years of age, 69% were male and 66% were non-Hispanic White. MEASUREMENTS: The primary outcome was first self-reported day of non-prescribed opioid use following MOUD initiation, and the exposure of interest was daily stimulant use (methamphetamine, amphetamines or cocaine). Both were defined using time-line follow-back. Among participants reporting at least 1 day of illicit opioid use, we also examined relapse to ongoing use, defined as (1) 7 days of continuous opioid use or (2) 4 consecutive weeks with self-reported opioid use, one or more positive urine drug screens (UDS) for opioids or one or more missing UDS. FINDINGS: Forty-seven per cent of participants reported stimulant use following MOUD initiation, 58% returned to illicit opioid use and 66% of those relapsed to ongoing use. Stimulant use was strongly associated with increased risk of misusing opioids after MOUD initiation when measured daily [adjusted hazard ratio (aHR) = 9.23, 95% confidence interval (CI) = 6.80-12.50, P < 0.001] and over a 7-day period (aHR = 1.27 for each additional day, CI = 1.18-1.37, P < 0.001). Using stimulants weekly or more often was associated with increased likelihood of relapse to ongoing opioid use compared with less than weekly or no stimulant use (adjusted odds ratio = 2.30, CI = 1.05-5.39, P = 0.044). CONCLUSIONS: People initiated on medication for opioid use disorder who subsequently use stimulants appear to be more likely to return to and continue using non-prescribed opioids compared with those without stimulant use. The association appears to be stronger among patients who initiate buprenorphine compared with those who initiate extended-release naltrexone.

Recent Incarceration, Substance Use, Overdose, and Service Use Among People Who Use Drugs in Rural Communities.

Importance: Drug use and incarceration have a substantial impact on rural communities, but factors associated with the incarceration of rural people who use drugs (PWUD) have not been thoroughly investigated. Objective: To characterize associations between recent incarceration, overdose, and substance use disorder (SUD) treatment access among rural PWUD. Design, Setting, and Participants: For this cross-sectional study, the Rural Opioid Initiative research consortium conducted a survey in geographically diverse rural counties with high rates of overdose across 10 US states (Illinois, Wisconsin, North Carolina, Oregon, Kentucky, West Virginia, Ohio, Massachusetts, New Hampshire, and Vermont) between January 25, 2018, and March 17, 2020, asking PWUD about their substance use, substance use treatment, and interactions with the criminal legal system. Participants were recruited through respondent-driven sampling in 8 rural US regions. Respondents who were willing to recruit additional respondents from their personal networks were enrolled at syringe service programs, community support organizations, and through direct community outreach; these so-called seed respondents then recruited others. Of 3044 respondents, 2935 included participants who resided in rural communities and reported past-30-day injection of any drug or use of opioids nonmedically via any route. Data were analyzed from February 8, 2022, to September 15, 2023. Exposure: Recent incarceration was the exposure of interest, defined as a report of incarceration in jail or prison for at least 1 day in the past 6 months. Main Outcomes and Measures: The associations between PWUD who were recently incarcerated and main outcomes of treatment use and overdose were examined using logistic regression. Results: Of 2935 participants, 1662 (56.6%) were male, 2496 (85.0%) were White; the mean (SD) age was 36 (10) years; and in the past 30 days, 2507 (85.4%) reported opioid use and 1663 (56.7%) reported injecting drugs daily. A total of 1224 participants (41.7%) reported recent incarceration, with a median (IQR) incarceration of 15 (3-60) days in the past 6 months. Recent incarceration was associated with past-6-month overdose (adjusted odds ratio [AOR], 1.38; 95% CI, 1.12-1.70) and recent SUD treatment (AOR, 1.62; 95% CI, 1.36-1.93) but not recent medication for opioid use disorder (MOUD; AOR, 1.03; 95% CI, 0.82-1.28) or currently carrying naloxone (AOR, 1.02; 95% CI, 0.86-1.21). Conclusions and Relevance: In this cross-sectional study of PWUD in rural areas, participants commonly experienced recent incarceration, which was not associated with MOUD, an effective and lifesaving treatment. The criminal legal system should implement effective SUD treatment in rural areas, including MOUD and provision of naloxone, to fully align with evidence-based SUD health care policies.

Viral metagenomics reveals diverse virus-host interactions throughout the soil depth profile.

IMPORTANCE: Soil viruses can moderate the roles that their host microbes play in global carbon cycling. However, given that most studies investigate the surface layer (i.e., top 20 cm) of soil, the extent to which this occurs in subsurface soil (i.e., below 20 cm) is unknown. Here, we leveraged public sequencing data to investigate the interactions between viruses and their hosts at soil depth intervals, down to 115 cm. While most viruses were detected throughout the soil depth profile, their adaptation to host microbes varied. Nonetheless, we uncovered evidence for the potential of soil viruses to encourage their hosts to recycle plant-derived carbon in both surface and subsurface soils. This work reasons that our understanding of soil viral functions requires us to continue to dig deeper and compare viruses existing throughout soil ecosystems.

Treatment Initiation, Substance Use Trajectories, and the Social Determinants of Health in Persons Living With HIV Seeking Medication for Opioid Use Disorder.

BACKGROUND: People living with HIV and opioid use disorder (OUD) are disproportionally affected by adverse socio-structural exposures negatively affecting health, which have shown inconsistent associations with uptake of medications for OUD (MOUD). This study aimed to determine whether social determinants of health (SDOH) were associated with MOUD uptake and trajectories of substance use in a clinical trial of people seeking treatment. METHODS: Data are from a 2018 to 2019 randomized trial comparing the effectiveness of different MOUD to achieve viral suppression among people living with HIV and OUD. SDOH were defined by variables mapping to Healthy People 2030 domains: education (Education Access and Quality), income (Economic Stability), homelessness (Neighborhood and Built Environment), criminal justice involvement (Social and Community Context), and recent SUD care (Health Care Access and Quality). Associations between SDOH and MOUD initiation were assessed with Cox proportional hazards models, and SDOH and substance use over time with generalized estimating equation models. RESULTS: Participants (N = 114) averaged 47 years old, 63% were male, 56% were Black, and 12% Hispanic. Participants reported an average of 2.3 out of 5 positive SDOH indicators (SD = 1.2). Stable housing was the most commonly reported SDOH (61%), followed by no recent criminal justice involvement (59%), having a high-school level education or greater (56%), income stability (45%), and recent SUD care (13%). Each additional favorable SDOH was associated with a 25% increase in the likelihood of MOUD initiation during the study period [adjusted HR = 1.25, 95% CI = (1.01, 1.55), P = .044]. Positive SDOH were also associated with a decrease in the odds of baseline opioid use and a greater reduction in opioid use during subsequent weeks of the study (P < .001 for a joint test of baseline and slope differences). CONCLUSIONS: Positive social determinants of health, in aggregate, may increase the likelihood of MOUD treatment initiation among people living with HIV and OUD.

Increasing early infant male circumcision uptake in Zambia: Like father like son.

Voluntary Medical Male Circumcision (VMMC) is an effective strategy for HIV prevention in areas with high prevalence of, and risk for, HIV. More than 361,000 male neonates are born each year in Zambia, many of whom could be eligible for Early-Infant Medical Circumcision (EIMC). Building on successful implementation strategies utilized in our Spear & Shield program, this pilot study, "Like Father, Like Son" (LFLS), evaluated the feasibility and acceptability of offering combined EIMC and VMMC services and couple-level behavioral interventions. A total of N = 702 pregnant women and their male partners (n = 351 couples) were recruited and enrolled. Couples were assessed twice pre-birth, 2 weeks post birth, and 6 months post birth. Expectant mothers were an average of 15.05 weeks pregnant (SD = 8.83). Thirty-nine pregnancies did not result in a live birth (11%), 14 couples withdrew from the study or were lost to follow-up prior to delivery (4%), and 148 babies were born female (42%), leaving 150 couples with a male infant in the analytic sample (43%). The LFLS study achieved significantly higher EIMC rates (35%) in comparison with previously observed EIMC study rates in Zambia (11%), and significantly higher than hypothetical comparison rates up to 30%. Relative to baseline rates, odds of VMMC among couples' older sons increased by 31% at post-intervention and by 90% at two-weeks following birth. Overall, this pilot study found the LFLS intervention to be feasible, acceptable, and effective in doubling the rate of EIMC in comparison with a previous longitudinal study in Zambia. Future research should consider a family-centric approach to promotion of male circumcision for infants and adolescents. LFLS may be effective in promoting father-son "bonding" by MC status; a bond that may be a bridge to increase both EIMC and VMMC uptake in newborns and couples' older sons and is a novel leverage point for promotion of this HIV prevention strategy.

A randomized controlled trial for a peer-facilitated telemedicine hepatitis c treatment intervention for people who use drugs in rural communities: study protocol for the "peer tele-HCV" study.

BACKGROUND: Hepatitis C virus (HCV) transmission is primarily driven by injection drug use, and acute HCV infection rates are increased in rural communities with substantial barriers to care. Treatment of HCV in persons who use drugs (PWUD) is cost effective, decreases high risk behaviors and HCV transmission, and achieves high rates of treatment completion and sustained viral response. Adapting HCV care delivery to utilize peer support specialists, telemedicine technology, and streamlined testing and treatment strategies can better reach rural populations living with HCV. METHODS: This is an open label, two-arm, non-blinded, randomized controlled trial designed to test the superiority of peer-facilitated and streamlined telemedicine HCV care (peer tele-HCV) compared to enhanced usual care (EUC) among PWUD in rural Oregon. In the intervention arm, peers conduct HCV screening in the community, facilitate pretreatment evaluation and linkage to telemedicine hepatitis C treatment providers, and support participants in HCV medication adherence. For participants assigned to EUC, peers facilitate pretreatment evaluation and referral to community-based treatment providers. The primary outcome is sustained virologic response at 12 weeks post treatment (SVR12). Secondary outcomes include: (1) HCV treatment initiation, (2) HCV treatment completion, (3) engagement with harm reduction resources, (4) rates of substance use, and (5) engagement in addiction treatment resources. The primary and secondary outcomes are analyzed using intention-to-treat (ITT) comparisons between telemedicine and EUC. A qualitative analysis will assess patient, peer, and clinician experiences of peer-facilitated telemedicine hepatitis C treatment. DISCUSSION: This study uses a novel peer-based telemedicine delivery model with streamlined testing protocols to improve access to HCV treatment in rural communities with high rates of injection drug use and ongoing disease transmission. We hypothesize that the peer tele-HCV model will increase treatment initiation, treatment completion, SVR12 rates, and engagement with harm reduction services compared to EUC. Trial registration This trial has been registered with ClinicalTrials.gov (clinicaltrials.gov NCT04798521).

A gender-based secondary analysis of the ADAPT-2 combination naltrexone and bupropion treatment for methamphetamine use disorder trial.

BACKGROUND AND AIMS: Socio-cultural (gender) and biological (sex)-based differences contribute to psychostimulant susceptibility, potentially affecting treatment responsiveness among women with methamphetamine use disorder (MUD). The aims were to measure (i) how women with MUD independently and compared with men respond to treatment versus placebo and (ii) among women, how the hormonal method of contraception (HMC) affects treatment responsiveness. DESIGN: This was a secondary analysis of ADAPT-2, a randomized, double-blind, placebo-controlled, multicenter, two-stage sequential parallel comparison design trial. SETTING: United States. PARTICIPANTS: This study comprised 126 women (403 total participants); average age = 40.1 years (standard deviation = 9.6) with moderate to severe MUD. INTERVENTIONS: Interventions were combination intramuscular naltrexone (380 mg/3 weeks) and oral bupropion (450 mg daily) versus placebo. MEASUREMENTS: Treatment response was measured using a minimum of three of four negative methamphetamine urine drug tests during the last 2 weeks of each stage; treatment effect was the difference between weighted treatment responses of each stage. FINDINGS: At baseline, women used methamphetamine intravenously fewer days than men [15.4 versus 23.1% days, P = 0.050, difference = -7.7, 95% confidence interval (CI) = -15.0 to -0.3] and more women than men had anxiety (59.5 versus 47.6%, P = 0.027, difference = 11.9%, 95% CI = 1.5 to 22.3%). Of 113 (89.7%) women capable of pregnancy, 31 (27.4%) used HMC. In Stage 1 29% and Stage 2 5.6% of women on treatment had a response compared with 3.2% and 0% on placebo, respectively. A treatment effect was found independently for females and males (P < 0.001); with no between-gender treatment effect (0.144 females versus 0.100 males; P = 0.363, difference = 0.044, 95% CI = -0.050 to 0.137). Treatment effect did not differ by HMC use (0.156 HMC versus 0.128 none; P = 0.769, difference = 0.028, 95% CI -0.157 to 0.212). CONCLUSIONS: Women with methamphetamine use disorder receiving combined intramuscular naltrexone and oral bupropion treatment achieve greater treatment response than placebo. Treatment effect does not differ by HMC.

Substance use disorder treatment and technology access among people who use drugs in rural areas of the United States: A cross-sectional survey.

PURPOSE: To evaluate how technology access affected substance use disorder (SUD) treatment prior to COVID-19 for people who use drugs in rural areas. METHODS: The Rural Opioid Initiative (January 2018-March 2020) was a cross-sectional study of people with prior 30-day injection drug or nonprescribed opioid use from rural areas of 10 states. Using multivariable mixed-effect regression models, we examined associations between participant technology access and SUD treatment. FINDINGS: Of 3,026 participants, 71% used heroin and 76% used methamphetamine. Thirty-five percent had no cell phone and 10% had no prior 30-day internet use. Having both a cell phone and the internet was associated with increased days of medication for opioid use disorder (MOUD) use (aIRR 1.29 [95% CI 1.11-1.52]) and a higher likelihood of SUD counseling in the prior 30 days (aOR 1.28 [95% CI 1.05-1.57]). Lack of cell phone was associated with decreased days of MOUD (aIRR 0.77 [95% CI 0.66-0.91]) and a lower likelihood of prior 30-day SUD counseling (aOR 0.77 [95% CI 0.62-0.94]). CONCLUSIONS: Expanding US rural SUD treatment engagement via telemedicine may require increased cell phone and mobile network access.

Trauma-Informed Supervision and Related Predictors of Burnout and Secondary Traumatic Stress Among Prelicensed Counsellors During the COVID-19 Pandemic.

In the current study, we examined individual factors, organizational factors, COVID-19 anxiety, and trauma-informed supervision as predictors of prelicensed counsellor burnout and secondary traumatic stress (STS). In a sample of 282 prelicensed counsellors, we conducted two hierarchical regression analyses for counsellor burnout and STS. The variables of interest accounted for 38% of the variance in counsellor burnout and 32% of the variance in counsellor STS. In the model of counsellor burnout, statistically significant variables included age, caseload, setting, COVID-19 anxiety, and trauma-informed supervision. For STS, statistically significant predictor variables were age, caseload, COVID-19 anxiety, and trauma-informed supervision. Implications of the findings for prelicensed counsellors, supervisors, and counselling researchers are discussed.

Contraception and Healthcare Utilization by Reproductive-Age Women Who Use Drugs in Rural Communities: a Cross-Sectional Survey.

BACKGROUND: Women who use drugs (WWUD) have low rates of contraceptive use and high rates of unintended pregnancy. Drug use is common among women in rural U.S. communities, with limited data on how they utilize reproductive, substance use disorder (SUD), and healthcare services. OBJECTIVE: We determined contraceptive use prevalence among WWUD in rural communities then compared estimates to women from similar rural areas. We investigated characteristics of those using contraceptives, and associations between contraceptive use and SUD treatment, healthcare utilization, and substance use. DESIGN: Rural Opioids Initiative (ROI) - cross-sectional survey using respondent-driven sampling (RDS) involving eight rural U.S. regions (January 2018-March 2020); National Survey on Family Growth (NSFG) - nationally-representative U.S. household reproductive health survey (2017-2019). PARTICIPANTS: Women aged 18-49 with prior 30-day non-prescribed opioid and/or non-opioid injection drug use; fecundity determined by self-reported survey responses. MAIN MEASURES: Unweighted and RDS-weighted prevalence estimates of medical/procedural contraceptive use; chi-squared tests and multi-level linear regressions to test associations. KEY RESULTS: Of 855 women in the ROI, 36.8% (95% CI 33.7-40.1, unweighted) and 38.6% (95% CI 30.7-47.2, weighted) reported contraceptive use, compared to 66% of rural women in the NSFG sample. Among the ROI women, 27% had received prior 30-day SUD treatment via outpatient counseling or inpatient program and these women had increased odds of contraceptive use (aOR 1.50 [95% CI 1.08-2.06]). There was a positive association between contraception use and recent medications for opioid use disorder (aOR 1.34 [95% CI 0.95-1.88]) and prior 6-month primary care utilization (aOR 1.32 [95% CI 0.96-1.82]) that did not meet the threshold for statistical significance. CONCLUSION: WWUD in rural areas reported low contraceptive use; those who recently received SUD treatment had greater odds of contraceptive use. Improvements are needed in expanding reproductive and preventive health within SUD treatment and primary care services in rural communities.

Microcomputed tomography void analysis after cement cleanup methods.

STATEMENT OF PROBLEM: Cement at the restorative margin after implementation of the manufacturer's recommended procedure for cement cleanup has not been extensively evaluated. PURPOSE: The purpose of this in vitro study was to use microcomputed tomography (µCT) to evaluate the number, volume, and width of voids left at the margin when dental cement is cleaned during the cementation process as per 2 manufacturer-prescribed methods for cement cleanup by evaluating a self-adhesive resin cement and a resin-modified glass ionomer cement. MATERIAL AND METHODS: Twenty molar Ivorine Typodont prepared teeth were scanned, and lithium disilicate crowns were milled and cemented to the prepared teeth as per the manufacturer-recommended cementation process. Two methods of cement cleanup were performed: the excess cement was tack-polymerized with a polymerization light, or a delay of 3 minutes was used for the excess cement to reach partial polymerization, and the excess cement was removed with a sharp sickle scaler. These 2 methods of cement cleanup were performed with 2 cements: a resin-modified glass ionomer cement and a self-adhesive resin. Each specimen was then scanned with a µCT machine. The buccal margin of each specimen, from mesiobuccal line angle to distobuccal line angle, was examined, and the number, volume, and width of any voids remaining at the margin open to the environment were measured. For statistical analysis, the specimens were first grouped by method of cement cleanup and then grouped by cement type. The Wilcoxon ranked sum test was implemented because of the lack of a normal distribution and the heterogeneity of the data (α=.05). RESULTS: No statistically significant difference was found in the median number of voids, median volume of voids, or median width per void between the 2 cleanup methods, irrespective of cement type (P>.05). Significant differences were found in the median number of voids, median volume of voids, and median width per void when comparing cement types, irrespective of cement cleanup methods (P<.05). CONCLUSIONS: The choice of cement may be more important than the method of cement cleanup when considering voids left at the margin. Microcomputed tomography is an excellent nondestructive tool for volumetric measurements of voids at the margin.

The Effect of Finishing and Polishing with Proprietary Vs Interchanged Polishing Kits on the Surface Roughness of Different Ceramic Materials.

PURPOSE: To evaluate the surface roughness of glazed and polished monolithic ceramic materials and to use this as a baseline for comparison after adjustment and polishing with both the recommended kit for the specific materials as well as interchanged polishing kits. MATERIAL AND METHODS: Flat ceramic specimens (n = 150) were fabricated from IPS ZirCAD Prime, IPS E.max, and Vitabloc Mark II. The specimens were adjusted and polished using either the proprietary polishing kit for the material or interchanged kits. The surface roughness was objectively assessed using Atomic Force Microscopy (AFM) and profilometer. Subjective assessment of surface finish was performed using a scanning electron microscope (SEM). Gravimetric weight loss of each sample was measured before and after polishing using a digital microscale. The mean surface roughness and standard deviation was calculated for each ceramic- polisher pair. A factorial ANOVA was used to compare the mean surface roughness values in nanometers between multiple groups (α = 0.05). The subjective results from the SEM were reported as descriptive statistics RESULTS: The zirconia polishing system produced surfaces with the lowest surface roughness regardless of the ceramic material. The polisher-ceramic combination was found to have a statistically significant effect on both Ra and RMS values when the AFM was used (p = 0.039 and 0.010, respectively). CONCLUSION: The zirconia polishing system resulted in the lowest surface roughness values regardless of ceramic materials tested. The zirconia polishing system also did not result in significant gravimetric weight loss regardless of the ceramic material used.

Predicting stop codon reassignment improves functional annotation of bacteriophages.

The majority of bacteriophage diversity remains uncharacterised, and new intriguing mechanisms of their biology are being continually described. Members of some phage lineages, such as the Crassvirales, repurpose stop codons to encode an amino acid by using alternate genetic codes. Here, we investigated the prevalence of stop codon reassignment in phage genomes and subsequent impacts on functional annotation. We predicted 76 genomes within INPHARED and 712 vOTUs from the Unified Human Gut Virome catalogue (UHGV) that repurpose a stop codon to encode an amino acid. We re-annotated these sequences with modified versions of Pharokka and Prokka, called Pharokka-gv and Prokka-gv, to automatically predict stop codon reassignment prior to annotation. Both tools significantly improved the quality of annotations, with Pharokka-gv performing best. For sequences predicted to repurpose TAG to glutamine (translation table 15), Pharokka-gv increased the median gene length (median of per genome medians) from 287 to 481 bp for UHGV sequences (67.8% increase) and from 318 to 550 bp for INPHARED sequences (72.9% increase). The re-annotation increased mean coding density from 66.8% to 90.0%, and from 69.0% to 89.8% for UHGV and INPHARED sequences. Furthermore, the proportion of genes that could be assigned functional annotation increased, including an increase in the number of major capsid proteins that could be identified. We propose that automatic prediction of stop codon reassignment before annotation is beneficial to downstream viral genomic and metagenomic analyses.

Antimicrobial resistance in dairy slurry tanks: A critical point for measurement and control.

Waste from dairy production is one of the largest sources of contamination from antimicrobial resistant bacteria (ARB) and genes (ARGs) in many parts of the world. However, studies to date do not provide necessary evidence to inform antimicrobial resistance (AMR) countermeasures. We undertook a detailed, interdisciplinary, longitudinal analysis of dairy slurry waste. The slurry contained a population of ARB and ARGs, with resistances to current, historical and never-used on-farm antibiotics; resistances were associated with Gram-negative and Gram-positive bacteria and mobile elements (ISEcp1, Tn916, Tn21-family transposons). Modelling and experimental work suggested that these populations are in dynamic equilibrium, with microbial death balanced by fresh input. Consequently, storing slurry without further waste input for at least 60 days was predicted to reduce ARB spread onto land, with > 99 % reduction in cephalosporin resistant Escherichia coli. The model also indicated that for farms with low antibiotic use, further reductions are unlikely to reduce AMR further. We conclude that the slurry tank is a critical point for measurement and control of AMR, and that actions to limit the spread of AMR from dairy waste should combine responsible antibiotic use, including low total quantity, avoidance of human critical antibiotics, and choosing antibiotics with shorter half-lives, coupled with appropriate slurry storage.