De novo designed proteins neutralize lethal snake venom toxins.

Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more 1,2 . Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage 3 and inhibition of nicotinic acetylcholine receptors (nAChRs) resulting in life-threatening neurotoxicity 4 . Currently, the only available treatments for snakebite consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs 5,6,7 . Here, we use deep learning methods to de novo design proteins to bind short- and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtain protein designs with remarkable thermal stability, high binding affinity, and near-atomic level agreement with the computational models. The designed proteins effectively neutralize all three 3FTx sub-families in vitro and protect mice from a lethal neurotoxin challenge. Such potent, stable, and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective, and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our computational design methodology should help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for development of therapies to neglected tropical diseases.

Design of amyloidogenic peptide traps.

Segments of proteins with high ß-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in ß-strand and ß-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid ß1-42 (Aß42). The Aß binders block the assembly of Aß fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aß42 species.

Blueprinting extendable nanomaterials with standardized protein blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

Progress towards elimination of viral hepatitis: a Lancet Gastroenterology & Hepatology Commission update.

The top 20 highest burdened countries (in disability-adjusted life years) account for more than 75% of the global burden of viral hepatitis. An effective response in these 20 countries is crucial if global elimination targets are to be achieved. In this update of the Lancet Gastroenterology & Hepatology Commission on accelerating the elimination of viral hepatitis, we convene national experts from each of the top 20 highest burdened countries to provide an update on progress. Although the global burden of diseases is falling, progress towards elimination varies greatly by country. By use of a hepatitis elimination policy index conceived as part of the 2019 Commission, we measure countries' progress towards elimination. Progress in elimination policy has been made in 14 of 20 countries with the highest burden since 2018, with the most substantial gains observed in Bangladesh, India, Indonesia, Japan, and Russia. Most improvements are attributable to the publication of formalised national action plans for the elimination of viral hepatitis, provision of publicly funded screening programmes, and government subsidisation of antiviral treatments. Key themes that emerged from discussion between national commissioners from the highest burdened countries build on the original recommendations to accelerate the global elimination of viral hepatitis. These themes include the need for simplified models of care, improved access to appropriate diagnostics, financing initiatives, and rapid implementation of lessons from the COVID-19 pandemic.

Small-molecule binding and sensing with a designed protein family.

Despite transformative advances in protein design with deep learning, the design of small-molecule-binding proteins and sensors for arbitrary ligands remains a grand challenge. Here we combine deep learning and physics-based methods to generate a family of proteins with diverse and designable pocket geometries, which we employ to computationally design binders for six chemically and structurally distinct small-molecule targets. Biophysical characterization of the designed binders revealed nanomolar to low micromolar binding affinities and atomic-level design accuracy. The bound ligands are exposed at one edge of the binding pocket, enabling the de novo design of chemically induced dimerization (CID) systems; we take advantage of this to create a biosensor with nanomolar sensitivity for cortisol. Our approach provides a general method to design proteins that bind and sense small molecules for a wide range of analytical, environmental, and biomedical applications.

The global fatty liver disease Sustainable Development Goal country score for 195 countries and territories.

BACKGROUND AND AIMS: Fatty liver disease is highly prevalent, resulting in overarching wellbeing and economic costs. Addressing it requires comprehensive and coordinated multisectoral action. We developed a fatty liver disease Sustainable Development Goal (SDG) country score to provide insights into country-level preparedness to address fatty liver disease through a whole-of-society lens. APPROACH AND RESULTS: We developed 2 fatty liver disease-SDG score sets. The first included 6 indicators (child wasting, child overweight, noncommunicable disease mortality, a universal health coverage service coverage index, health worker density, and education attainment), covering 195 countries and territories between 1990 and 2017. The second included the aforementioned indicators plus an urban green space indicator, covering 60 countries and territories for which 2017 data were available. To develop the fatty liver disease-SDG score, indicators were categorized as "positive" or "negative" and scaled from 0 to 100. Higher scores indicate better preparedness levels. Fatty liver disease-SDG scores varied between countries and territories (n = 195), from 14.6 (95% uncertainty interval: 8.9 to 19.4) in Niger to 93.5 (91.6 to 95.3) in Japan; 18 countries and territories scored > 85. Regionally, the high-income super-region had the highest score at 88.8 (87.3 to 90.1) in 2017, whereas south Asia had the lowest score at 44.1 (42.4 to 45.8). Between 1990 and 2017, the fatty liver disease-SDG score increased in all super-regions, with the greatest increase in south Asia, but decreased in 8 countries and territories. CONCLUSIONS: The fatty liver disease-SDG score provides a strategic advocacy tool at the national and global levels for the liver health field and noncommunicable disease advocates, highlighting the multisectoral collaborations needed to address fatty liver disease, and noncommunicable diseases overall.

Blueprinting expandable nanomaterials with standardized protein building blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures 1 . Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

Demand creation for HIV testing services: A systematic review and meta-analysis.

BACKGROUND: HIV testing services (HTS) are the first steps in reaching the UNAIDS 95-95-95 goals to achieve and maintain low HIV incidence. Evaluating the effectiveness of different demand creation interventions to increase uptake of efficient and effective HTS is useful to prioritize limited programmatic resources. This review was undertaken to inform World Health Organization (WHO) 2019 HIV testing guidelines and assessed the research question, "Which demand creation strategies are effective for enhancing uptake of HTS?" focused on populations globally. METHODS AND FINDINGS: The following electronic databases were searched through September 28, 2021: PubMed, PsycInfo, Cochrane CENTRAL, CINAHL Complete, Web of Science Core Collection, EMBASE, and Global Health Database; we searched IAS and AIDS conferences. We systematically searched for randomized controlled trials (RCTs) that compared any demand creation intervention (incentives, mobilization, counseling, tailoring, and digital interventions) to either a control or other demand creation intervention and reported HTS uptake. We pooled trials to evaluate categories of demand creation interventions using random-effects models for meta-analysis and assessed study quality with Cochrane's risk of bias 1 tool. This study was funded by the WHO and registered in Prospero with ID CRD42022296947. We screened 10,583 records and 507 conference abstracts, reviewed 952 full texts, and included 124 RCTs for data extraction. The majority of studies were from the African (N = 53) and Americas (N = 54) regions. We found that mobilization (relative risk [RR]: 2.01, 95% confidence interval [CI]: [1.30, 3.09], p < 0.05; risk difference [RD]: 0.29, 95% CI [0.16, 0.43], p < 0.05, N = 4 RCTs), couple-oriented counseling (RR: 1.98, 95% CI [1.02, 3.86], p < 0.05; RD: 0.12, 95% CI [0.03, 0.21], p < 0.05, N = 4 RCTs), peer-led interventions (RR: 1.57, 95% CI [1.15, 2.15], p < 0.05; RD: 0.18, 95% CI [0.06, 0.31], p < 0.05, N = 10 RCTs), motivation-oriented counseling (RR: 1.53, 95% CI [1.07, 2.20], p < 0.05; RD: 0.17, 95% CI [0.00, 0.34], p < 0.05, N = 4 RCTs), short message service (SMS) (RR: 1.53, 95% CI [1.09, 2.16], p < 0.05; RD: 0.11, 95% CI [0.03, 0.19], p < 0.05, N = 5 RCTs), and conditional fixed value incentives (RR: 1.52, 95% CI [1.21, 1.91], p < 0.05; RD: 0.15, 95% CI [0.07, 0.22], p < 0.05, N = 11 RCTs) all significantly and importantly (≥50% relative increase) increased HTS uptake and had medium risk of bias. Lottery-based incentives and audio-based interventions less importantly (25% to 49% increase) but not significantly increased HTS uptake (medium risk of bias). Personal invitation letters and personalized message content significantly but not importantly (<25% increase) increased HTS uptake (medium risk of bias). Reduced duration counseling had comparable performance to standard duration counseling (low risk of bias) and video-based interventions were comparable or better than in-person counseling (medium risk of bias). Heterogeneity of effect among pooled studies was high. This study was limited in that we restricted to randomized trials, which may be systematically less readily available for key populations; additionally, we compare only pooled estimates for interventions with multiple studies rather than single study estimates, and there was evidence of publication bias for several interventions. CONCLUSIONS: Mobilization, couple- and motivation-oriented counseling, peer-led interventions, conditional fixed value incentives, and SMS are high-impact demand creation interventions and should be prioritized for programmatic consideration. Reduced duration counseling and video-based interventions are an efficient and effective alternative to address staffing shortages. Investment in demand creation activities should prioritize those with undiagnosed HIV or ongoing HIV exposure. Selection of demand creation interventions must consider risks and benefits, context-specific factors, feasibility and sustainability, country ownership, and universal health coverage across disease areas.

Food Values, Food Purchasing, and Eating-Related Outcomes Among a Sample of Quebec Adults During the COVID-19 Pandemic.

Purpose: This investigation evaluated food values, food purchasing, and other food and eating-related outcomes during the COVID-19 pandemic in Quebec, Canada. The role of stress in eating outcomes was also examined.Methods: An online household survey was conducted among Quebec adults aged ≥18 years (n = 658). Changes in outcomes during, as compared to before, the pandemic were evaluated using descriptive statistics and thematic analysis of free text responses. Eating outcomes by daily stress level (low, some, high) were assessed using Cochran-Armitage test for trend.Results: Most respondents reported increased importance and purchasing of local food products (77% and 68%, respectively) and 60% reported increased grocery spending (mean ± standard deviation: 28% ± 23%). Respondents with a higher daily stress level had a higher frequency of reporting eating more than usual compared to before the pandemic (low stress 21%, some stress 34%, high stress 39%, p-trend <0.0001). Free text responses described more time spent at home as a reason for eating more than usual.Conclusions: To support healthy eating during and post-pandemic, dietitians should consider patients' mental/emotional well-being and time spent at home. Moreover, support of local food products may provide opportunities to promote healthy eating, sustainability, and post-pandemic resiliency of food systems.

Burden of disease due to liver cirrhosis in Mexico.

BACKGROUND: Liver cirrhosis is a major cause of morbidity and mortality worldwide. In Mexico, it is one of the six leading causes of death. OBJECTIVE: To analyze epidemiological data derived from the Global Burden of Disease study and their relationship with risk factors associated with the development of chronic liver diseases in Mexico. MATERIAL AND METHODS: An analysis of data from the Institute for Health Metrics and Evaluation and the National Institute of Geography and Statistics was carried out. RESULTS: Liver cirrhosis has a high prevalence in Mexico, with significant burden of disease translating into lost years of healthy life, premature death and disability. Mortality due to cirrhosis ranked sixth (3.6%) in 2021 and was the eighth cause of years of healthy life lost (2.8%). From 1990 to 2021, the mortality rate increased from 26.7 to 34.2 per 100,000 population. CONCLUSIONS: The burden of disease due to liver cirrhosis continues to be caused by alcohol consumption and hepatitis C; cirrhosis caused by steatotic liver disease has increased in terms of prevalence over the past decade. There are epidemiological changes in the frequency and burden of chronic liver disease that show territorial variations in Mexico.

ANTECEDENTES: La cirrosis hepática es una causa importante de morbilidad y mortalidad en el mundo. En México, constituye una las primeras seis causas de muerte. OBJETIVO: Analizar los datos epidemiológicos derivados del estudio de Global Burden of Disease y su relación con los factores de riesgo asociados al desarrollo de hepatopatías crónicas en México. MATERIAL Y MÉTODOS: Se realizó el análisis de datos provenientes del Instituto para la Medición y Evaluación de la Salud y del Instituto Nacional de Estadística y Geografía. RESULTADOS: La cirrosis hepática tiene una prevalencia alta en México, con una carga de enfermedad importante traducida en años perdidos de vida saludable, por muerte prematura y por discapacidad. La mortalidad por cirrosis ocupó el sexto lugar (3.6 %) en 2021 y fue la octava causa de años de vida saludable perdidos (2.8 %). De 1990 a 2021, la tasa de mortalidad se incrementó de 26.7 a 34.2 por 100 000 habitantes. CONCLUSIONES: La carga de enfermedad por cirrosis hepática se continúa derivando del consumo de alcohol y de la hepatitis C; la prevalencia de la cirrosis causada por enfermedad hepática esteatósica se ha incrementado en la última década. Existen cambios epidemiológicos en la frecuencia y carga de la hepatopatía crónica que muestra variaciones territoriales en México.

Multiscale Risk Factors of Cardiovascular Disease: CLSA Analysis of Genetic and Psychosocial Factors.

Background: Cardiovascular disease (CVD) is a complex disease resulting from multiscale risk factors including genetics, age, and psychosocial factors (PSFs) such as depression and social isolation. However, previous research has lacked in operationalizing multiscale risk factors to determine individual and interactive associations over the life course. Therefore, this study aimed to evaluate individual and interactive associations of multiscale risk factors for CVD outcomes including genetics and PSFs at middle and older-aged stages of the life course. Methods: Baseline data from the Canadian Longitudinal Study on Aging (CLSA; n = 9,892 with genome-wide genotyping data) was used for this investigation. A 39 single nucleotide polymorphism polygenic risk score (PRS) for CVD was constructed. PSFs consisted of: (1) Depressive symptoms categorized into: "none" (Group 1, reference), "current" (Group 2), "clinical depression with no current symptoms" (Group 3), and "potential, recurrent depression" (Group 4); and (2) Social isolation index as a binary variable comprised of marital status, living arrangements, retirement status, contacts, and social participation. Heart-related disorders (HRD: myocardial infarction, angina and heart disease) was the primary outcome of interest and peripheral/vascular-related disorders (PVRD: stroke, peripheral vascular disease and hypertension) was the secondary outcome. Multivariable logistic regression models adjusted for socio-demographic factors were conducted stratified by age group (middle-aged: 45-69 years, older-aged: ≥70 years). Results: PRS was associated with HRD among middle- and older-aged participants [OR (95% confidence interval)] [1.06 (1.03-1.08), 1.06 (1.03-1.08), respectively]. Most depressive symptoms groups compared to the reference associated with HRD and PVRD, but only Group 4 associated with PVRD among older-aged [1.69 (1.08-2.64)]. Social isolation was associated with only PVRD among middle-aged [1.84 (1.04-3.26)]; however, socially isolated CLSA participants were underrepresented in the genotyped cohort (1.2%). No significant PRS*PSFs interactions were observed. Conclusions: Genetics and PSFs are independently associated with CVD. Varying observations across age groups underscores the need to advance research on multiscale risk factors operating both at a given point in time and over the life course. Future cohort studies may benefit from use of mobile assessment units to enable better reach to socially isolated participants for collection of biospecimens.

Characterizing a sexual health and HIV risk stratification scale for sexually active adolescent girls and young women (AGYW) in Tanzania.

Adolescent girls and young women (AGYW) aged 15 to 24 years face disproportionately high risks of acquiring HIV and other sexually transmitted infections (STIs). A sexual health risk stratification tool can support the development and implementation of tailored HIV and STI prevention services for sub-groups of at-risk AGYW. Data were collected among sexually active AGYW aged 15 to 24 years in Tanzania between April 2015 and March 2017. Exploratory and confirmatory factor analyses were conducted to construct and assess the latent structure of a ten-item scale for rapid assessment of sexual health risks. Items with high factor loadings and minimal cross loadings were retained in the final scale. Scale performance was appraised against condomless sex (defined as unprotected vaginal or anal intercourse) reported by AGYW for construct validity. A three-factor structure of vulnerability to HIV among AGYW was supported with subscales for socioeconomic vulnerability; lack of adult support; and sexual behavioral risks. The chi-square goodness-of-fit test, root mean square error of approximation, comparative fit index, and Tucker-Lewis index indicated a strong goodness-of-fit of the three-factor scale. Cronbach alphas (0.55 for socioeconomic vulnerability, 0.55 for lack of support, and 0.48 for sexual risk) indicated sub-optimal internal consistency for all sub-scales. The factor-item and factor-factor correlations identified in these analyses were consistent with the conceptual framework of vulnerability of HIV infection in AGYW, suggesting good construct validity. The scale also demonstrated a statistically significant association with condomless sex and could be potentially used for sexual health risk stratification (OR = 1.17, 95% CI: 1.12, 1.23). The sexual health and HIV risk stratification scale demonstrated potential in identifying sexually active AGYW at high risk for HIV and other STIs. Ultimately, all AGYW in Tanzania are not at equal risk for HIV and this scale may support directing resources towards those at highest risk of HIV.

Longitudinal Patterns of Food Procurement Over the Course of the COVID-19 Pandemic: Findings From a Canadian Online Household Survey.

Introduction: Consumer food procurement during the COVID-19 pandemic has been understudied. This investigation aimed to longitudinally evaluate food procurement patterns, concern of virus exposure in grocery retailers, and food access challenges over the pandemic among a sample of households in Quebec, Canada. Methods: Online surveys were collected at three time points of the pandemic: first wave in spring 2020 (lockdown period), summer 2020 (deconfinement period), and second wave in winter 2021 (curfew period). Respondents were the household's primary grocery shopper (n = 491). Non-parametric tests and multivariable logistic regression were conducted to compare responses over time and to evaluate characteristics of respondents who regularly used no-contact grocery methods (store pick-up or home delivery). Results: Frequency of in-store grocery shopping was lowest during the lockdown (once per week or less), and significantly increased over time to resemble pre-pandemic frequency. Concern of virus exposure in grocery retailers and disinfection/discarding of food packaging was highest during the lockdown, but significantly decreased over time. At all time points, use of public transit, walking or cycling for grocery shopping was associated with regular use of no-contact grocery methods (curfew odds ratio (OR): 3.13 (95% confidence interval 1.60, 6.14). Age (60 years+) was associated with regular use during the lockdown [OR: 2.27 (1.13, 4.59)]. Conclusion: Among our sample, frequency of in-store grocery shopping was lowest and concern of virus exposure in stores was highest during the lockdown period. No-contact grocery use was associated with transportation mode and potentially with personal risk perception (age).

Sexual behaviour change following HIV testing services: a systematic review and meta-analysis.

INTRODUCTION: Learning one's HIV status through HIV testing services (HTS) is an essential step toward accessing treatment and linking to preventive services for those at high HIV risk. HTS may impact subsequent sexual behaviour, but the degree to which this varies by population or is true in the setting of contemporary HIV prevention activities is largely unknown. As part of the 2019 World Health Organization Consolidated Guidelines on HTS, we undertook a systematic review and meta-analysis to determine the effect of HTS on sexual behaviour. METHODS: We searched nine electronic databases for studies published between July 2010 and December 2019. We included studies that reported on at least one outcome (condom use [defined as the frequency of condom use or condom-protected sex], number of sex partners, HIV incidence, STI incidence/prevalence). We included studies that prospectively assessed outcomes and that fit into one of three categories: (1) those evaluating more versus less-intensive HTS, (2) those of populations receiving HTS versus not and (3) those evaluating outcomes after versus before HTS. We conducted meta-analyses using random-effects models. RESULTS AND DISCUSSION: Of 29 980 studies screened, 76 studies were included. Thirty-eight studies were randomized controlled trials, 36 were cohort studies, one was quasi-experimental and one was a serial cross-sectional study. There was no significant difference in condom use among individuals receiving more-intensive HTS compared to less-intensive HTS (relative risk [RR]=1.03; 95% CI: 0.99 to 1.07). Condom use was significantly higher after receiving HTS compared to before HTS for individuals newly diagnosed with HIV (RR = 1.65; 95% CI: 1.36 to 1.99) and marginally significantly higher for individuals receiving an HIV-negative diagnosis (RR = 1.63; 95% CI: 1.01 to 2.62). Individuals receiving more-intensive HTS reported fewer sex partners at follow-up than those receiving less-intensive HTS, but the finding was not statistically significant (mean difference = -0.28; 95% CI: -3.66, 3.10). CONCLUSIONS: Our findings highlight the importance of using limited resources towards HTS strategies that focus on early HIV diagnosis, treatment and prevention services rather than resources dedicated to supplementing or enhancing HTS with additional counselling or other interventions.

Diet Quality and Food Prices Modify Associations between Genetic Susceptibility to Obesity and Adiposity Outcomes.

The role of the retail food environment in obesity risk is unclear, which may be due in part to the lack of consideration of individual differences in the responsivity to food cues. This cross-sectional investigation geo-temporally linked the CARTaGENE biobank (including genetic, dietary, lifestyle, and anthropometric data) with in-store retail food environment data to examine interactions between a polygenic risk score (PRS) for obesity and (1) diet quality (n = 6807) and (2) in-store retail food measures (n = 3718). The outcomes included adiposity-related measures and diet quality assessed using the 2010 Canadian-adapted Healthy Eating Index. A vegetable:soft drink ratio was constructed for each retail measure to assess the relative healthfulness of exposures. Generalized linear models adjusted for individual and neighborhood socio-demographic factors were used to evaluate main and interactive effects. Diet quality significantly modified the association between polygenic risk of obesity and body mass index, waist circumference, and body fat percent. A significant interaction was also observed between PRS and frequency of price discount of vegetables in relation to soft drinks on waist circumference. These results replicate previous reports of diet moderating polygenic risk of obesity and suggest that prices of low vs. high-energy density foods are an intervention target to address population obesity rates.

Assisted HIV partner services training in three sub-Saharan African countries: facilitators and barriers to sustainable approaches.

INTRODUCTION: Healthcare worker training is essential to successful implementation of assisted partner services (aPS), which aims to improve HIV testing and linkage-to-care outcomes for previously unidentified HIV-positive individuals. Cameroon, Kenya and Mozambique are three African countries that have implemented aPS programmes and are working to bring those programmes to scale. In this paper, we present and compare different aPS training strategies implemented by these three countries, and discuss facilitators and barriers associated with implementation of aPS training in sub-Saharan Africa. DISCUSSION: aPS training programmes in Cameroon, Kenya and Mozambique share the following components: the development of comprehensive and interactive training curricula, recruitment of qualified trainees and trainers with intimate knowledge of the community served, continuous training, and rigorous monitoring and evaluation activities. Cameroon and Kenya were able to engage various stakeholders early on, establishing multilateral coalitions that facilitated attainment of long-term buy-in from the local governments. Ministries of Health and various implementing partners are often included in strategic planning and delivery of training curricula to ensure sustainability of the training programmes. Kenya and Mozambique have integrated aPS training into the national HTS guidelines, which are being rolled out nationwide by the Ministries of Health and implementing partners. Continual revision of training curricula to reflect the country context, as well as ongoing monitoring and evaluation, have also been identified as key facilitators to sustain aPS training programmes. Some of the barriers to scale-up and sustainability of aPS training include limited funding and resources for training and scale-up and shortage of aPS providers to facilitate on-the-job mentorship. CONCLUSIONS: These three programmes demonstrate that aPS training can be implemented and scaled up in sub-Saharan Africa. As countries plan for initial implementation or national scale-up of aPS services, they will need to establish government buy-in, expand funding sources, address the shortage of staff and resources to provide aPS and on-the-job mentorship, and continuously collect data to evaluate and improve aPS training plans. Development of national standards for aPS training, empowered healthcare providers, increased government commitment, and sustained funding for aPS services and training will be crucial for successful aPS implementation.

Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay.

We have developed a generalizable "smart molecular diagnostic" capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.