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Background: Cryptococcal antigen (CrAg) screening in individuals with advanced HIV reduces cryptococcal meningitis (CM) cases and deaths. The World Health Organization recently recommended increasing screening thresholds from CD4 ≤100 cells/µL to ≤200 cells/µL. CrAg screening at CD4 ≤100 cells/µL is cost-effective; however, the cost-effectiveness of screening patients with CD4 101–200 cells/µL requires evaluation. Methods: Using a decision analytic model with Botswana-specific cost and clinical estimates, we evaluated CrAg screening and treatment among individuals with CD4 counts of 101–200 cells/µL. We estimated the number of CM cases and deaths nationally and treatment costs without screening. For screening we modeled the number of CrAg tests performed, number of CrAg-positive patients identified, proportion started on pre-emptive fluconazole, CM cases and deaths. Screening and treatment costs were estimated and cost per death averted or disability-adjusted life year (DALY) saved compared with no screening. Results: Without screening, we estimated 142 CM cases and 85 deaths annually among individuals with CD4 101–200 cells/µL, with treatment costs of $368,982. With CrAg screening, an estimated 33,036 CrAg tests are performed, and 48 deaths avoided (1,017 DALYs saved). While CrAg screening costs an additional $155,601, overall treatment costs fall by $39,600 (preemptive and hospital-based CM treatment), yielding a net increase of $116,001. Compared to no screening, high coverage of CrAg screening and pre-emptive treatment for CrAg-positive individuals in this population avoids one death for $2440 and $114 per DALY saved. In sensitivity analyses assuming a higher proportion of antiretroviral therapy (ART)-naïve patients (75% versus 15%), cost per death averted was $1472; $69 per DALY saved. Conclusions: CrAg screening for individuals with CD4 101–200 cells/µL was estimated to have a modest impact, involve additional costs, and be less cost-effective than screening populations with CD4 counts ≤100 cells/µL. Additional CrAg screening costs must be considered against other health system priorities.

It is challenging to associate features such as human health outcomes, diet, environmental conditions, or other metadata to microbial community measurements, due in part to their quantitative properties. Microbiome multi-omics are typically noisy, sparse (zero-inflated), high-dimensional, extremely non-normal, and often in the form of count or compositional measurements. Here we introduce an optimized combination of novel and established methodology to assess multivariable association of microbial community features with complex metadata in population-scale observational studies. Our approach, MaAsLin 2 (Microbiome Multivariable Associations with Linear Models), uses generalized linear and mixed models to accommodate a wide variety of modern epidemiological studies, including cross-sectional and longitudinal designs, as well as a variety of data types (e.g., counts and relative abundances) with or without covariates and repeated measurements. To construct this method, we conducted a large-scale evaluation of a broad range of scenarios under which straightforward identification of meta-omics associations can be challenging. These simulation studies reveal that MaAsLin 2's linear model preserves statistical power in the presence of repeated measures and multiple covariates, while accounting for the nuances of meta-omics features and controlling false discovery. We also applied MaAsLin 2 to a microbial multi-omics dataset from the Integrative Human Microbiome (HMP2) project which, in addition to reproducing established results, revealed a unique, integrated landscape of inflammatory bowel diseases (IBD) across multiple time points and omics profiles.

Background: As the relentless coronavirus disease-2019 (COVID-19) pandemic continues to spread across Africa, Botswana could face challenges maintaining the pathway towards control of its HIV epidemic. Objective: Utilising the Spectrum GOALS module (GOALS-2021), the 5-year outcomes from the implementation of the Treat All strategy were analysed and compared with the original 2016 Investment Case (2016-IC) projections. Future impact of adopting the new Joint United Nations Programme on HIV/AIDS (UNAIDS) Global AIDS Strategy (2021–2026) targets and macroeconomic analysis estimating how the financial constraints from the COVID-19 pandemic could impact the available resources for Botswana’s National HIV Response through 2030 were also considered. Method: Programmatic costs, population demographics, prevention and treatment outputs were determined. Previous 2016-IC data were uploaded for comparison, and inputs for the GOALS, AIM, DemProj, Resource Needs and Family Planning modules were derived from published reports, strategic plans, programmatic data and expert opinion. The economic projections were recalibrated with consideration of the impact of the COVID-19 pandemic. Results: Decreases in HIV infections, incidence and mortality rates were achieved. Increases in laboratory costs were offset by estimated decreases in the population of people living with HIV (PLWH). Moving forward, young women and others at high risk must be targeted in HIV prevention efforts, as Botswana transitions from a generalised to a more concentrated epidemic. Conclusion: The Treat All strategy contributed positively to decreases in new HIV infections, mortality and costs. If significant improvements in differentiated service delivery, increases in human resources and HIV prevention can be realised, Botswana could become one of the first countries with a previously high-burdened generalised HIV epidemic to gain epidemic control, despite the demands of the COVID-19 pandemic.

Introduction The functional consequences of the bacterial gut microbiome for child health are not well understood. Characteristics of the early child gut microbiome may influence the course of enteric infections, and enteric infections may change the composition of the gut microbiome, all of which may have long-term implications for child growth and development.Methods and analysis We are conducting a community-based birth cohort study to examine interactions between gut microbiome conditions and enteric infections, and how environmental conditions affect the development of the gut microbiome. We will follow 360 newborns from 3 sites along a rural–urban gradient in northern coastal Ecuador, characterising enteric infections and gut microbial communities in the children every 3 to 6 months over their first 2 years of life. We will use longitudinal regression models to assess the correlation between environmental conditions and gut microbiome diversity and presence of specific taxa, controlling for factors that are known to be associated with the gut microbiome, such as diet. From 6 to 12 months of age, we will collect weekly stool samples to compare microbiome conditions in diarrhoea stools versus stools from healthy children prior to, during and after acute enteric infections, using principal-coordinate analysis and other multivariate statistical methods.Ethics and dissemination Ethics approvals have been obtained from Emory University and the Universidad San Francisco de Quito institutional review boards. The findings will be disseminated through conference presentations and peer-reviewed journals.

Dengue is recognized as a major health issue in large urban tropical cities but is also observed in rural areas. In these environments, physical characteristics of the landscape and sociodemographic factors may influence vector populations at small geographic scales, while prior immunity to the four dengue virus serotypes affects incidence. In 2019, a rural northwestern Ecuadorian community, only accessible by river, experienced a dengue outbreak. The village is 2-3 hours by boat away from the nearest population center and comprises both Afro-Ecuadorian and Indigenous Chachi households. We used multiple data streams to examine spatial risk factors associated with this outbreak, combining maps collected with an unmanned aerial vehicle (UAV), an entomological survey, a community census, and active surveillance of febrile cases. We mapped visible water containers seen in UAV images and calculated both the green-red vegetation index (GRVI) and household proximity to public spaces like schools and meeting areas. To identify risk factors for symptomatic dengue infection, we used mixed-effect logistic regression models to account for the clustering of symptomatic cases within households. We identified 55 dengue cases (9.5% of the population) from 37 households. Cases peaked in June and continued through October. Rural spatial organization helped to explain disease risk. Afro-Ecuadorian (versus Indigenous) households experience more symptomatic dengue (OR = 3.0, 95%CI: 1.3, 6.9). This association was explained by differences in vegetation (measured by GRVI) near the household (OR: 11.3 95% 0.38, 38.0) and proximity to the football field (OR: 13.9, 95% 4.0, 48.4). The integration of UAV mapping with other data streams adds to our understanding of these dynamics.

Ray-finned fishes (Actinopterygii) are the largest and most diverse group of vertebrates, comprising over half of all living vertebrate species. Phylogenetic relationships between ray-finned fishes have historically pivoted on the study of morphology, which has notoriously failed to resolve higher order relationships, such as within the percomorphs. More recently, comprehensive genomic analyses have provided further resolution of actinopterygian phylogeny, including higher order relationships. Such analyses are rightfully regarded as the ‘gold standard’ for phylogenetics. However, DNA retrieval requires modern or well-preserved tissue and is less likely to be preserved in archaeological or fossil specimens. By contrast, some proteins, such as collagen, are phylogenetically informative and can survive into deep time. Here, we test the utility of collagen type I amino acid sequences for phylogenetic estimation of ray-finned fishes. We estimate topology using Bayesian approaches and compare the congruence of our estimated trees with published genomic phylogenies. Furthermore, we apply a Bayesian molecular clock approach and compare estimated divergence dates with previously published genomic clock analyses. Our collagen-derived trees exhibit 77% of node positions as congruent with recent genomic-derived trees, with the majority of discrepancies occurring in higher order node positions, almost exclusively within the Percomorpha. Our molecular clock trees present divergence times that are fairly comparable with genomic-based phylogenetic analyses. We estimate the mean node age of Actinopteri at ∼293 million years (Ma), the base of Teleostei at ∼211 Ma and the radiation of percomorphs beginning at ∼141 Ma (∼350 Ma, ∼250–283 Ma and ∼120–133 Ma in genomic trees, respectively). Finally, we show that the average rate of collagen (I) sequence evolution is 0.9 amino acid substitutions for every million years of divergence, with the α3 (I) sequence evolving the fastest, followed by the α2 (I) chain. This is the quickest rate known for any vertebrate group. We demonstrate that phylogenetic analyses using collagen type I amino acid sequences generate tangible signals for actinopterygians that are highly congruent with recent genomic-level studies. However, there is limited congruence within percomorphs, perhaps due to clade-specific functional constraints acting upon collagen sequences. Our results provide important insights for future phylogenetic analyses incorporating extinct actinopterygian species via collagen (I) sequencing.

The World Health Organization (WHO) has published a guideline for the management of individuals with advanced HIV disease (AHD) to reduce HIV-related deaths. The guideline consists of a package of recommendations including interventions to prevent, diagnose and treat common opportunistic infections, including tuberculosis (TB), cryptococcosis and severe bacterial infections, along with rapid initiation of antiretroviral treatment and enhanced adherence support. Currently no clear targets exist for these key interventions. Emerging programmatic data from Uganda, Tanzania and Nigeria suggest that an estimated 80% of eligible people continue to miss the recommended cryptococcal or TB testing, highlighting the remaining challenges to the effective implementation of WHO-recommended AHD packages of care in real-world resource-limited settings. The absence of mortality indicators for the leading causes of HIV-related deaths, because of the lack of mechanisms to ascertain cause of death, has had a negative impact on establishing interventions to reduce mortality. We suggest that setting 95-95-95 targets for CD4 testing, cryptococcal antigen and TB testing, and treatment that are aligned to the WHO AHD package of care would be a step in the right direction to achieving the greater goal of the WHO End TB strategy and the proposed new strategy to end cryptococcal meningitis deaths. However, these targets will only be achieved if there is healthcare worker training, expanded access to bedside point-of-care diagnostics for hospitalised patients and those in outpatient care who meet the criteria for AHD, and health systems strengthening to minimise delays in initiating the WHO-recommended therapies for TB and cryptococcal disease.

Abstract Livestock ownership may mitigate anaemia among young children by providing access to animal‐source foods (ASFs) yet exacerbate anaemia by exposing children to animal‐source pathogens. This study aimed to assess the association between household livestock ownership and child anaemia and examine whether this relationship is mediated by child ASF consumption or by child morbidity and inflammation. We conducted a cross‐sectional study of 470 children aged 6–59 months in Greater Accra, Ghana. Child blood samples were analysed for haemoglobin concentration, iron status biomarkers and inflammatory biomarkers. Caregivers were asked about the child's frequency of ASF consumption in the past 3 months. Livestock ownership was categorized into five typologies to distinguish households by the number and combinations of species owned. In adjusted logistic regression, children from households in Type 5, owning cattle, small livestock (goats, sheep or pigs) and poultry, had lower odds of anaemia compared with those in Type 1, owning no livestock (OR [95% CI]: 0.32 [0.14, 0.71]). Although children from households that owned poultry were more likely to consume chicken meat, and children from households with cattle were more likely to drink cow's milk, consumption of these ASFs did not mediate the observed association between livestock ownership and child anaemia. There were no associations between livestock ownership and children's symptoms of illness or inflammation. Further research is needed to understand how ownership of certain livestock species, or a greater diversity of livestock species, may be associated with the risk of child anaemia, including the role of dietary and income‐based pathways.

BackgroundThe term Nodding Syndrome (NS) refers to an atypical and severe form of childhood epilepsy characterized by a repetitive head nodding (HN). The disease has been for a long time limited to East Africa, and the cause is still unknown. The objective of this study was to confirm the existence of NS cases in Central African Republic (CAR).Methodology/principal findingsThis was a cross-sectional descriptive study in the general population. The identification of NS cases was conducted through a door-to-door survey in a village near Bangui along the Ubangui River. Based on Winkler's 2008 and the World Health Organization (WHO)'s 2012 classifications, the confirmation of cases was done by a neurologist who also performed the electroencephalograms. No laboratory tests were done during this investigation. Treatment was offered to all patients. A total of 6,175 individuals was surveyed in 799 households. After reviewing the cases, we identified 5 NS cases in girls aged between 8 and 16. The age of onset of the seizures was between 5 and 12 years of age. Two cases were classified as "HN plus" according to Winkler's 2008 classification. Four NS cases were classified as probable and one as confirmed according to the WHO's 2012 classification. Three of them presented with developmental delay and cognitive decline, and one had an abnormally low height-for-age z-score. Electroencephalographic abnormalities were found in four patients.Conclusions/significanceNodding Syndrome cases were described in CAR for the first time. Despite certain peculiarities, these cases are similar to those described elsewhere. Given that only a small part of the affected area was investigated, the study area along the Ubangui River needs to be expanded in order to investigate the association between Onchocerca volvulus and NS and also evaluate the real burden of NS in CAR.

Perioperative coagulopathy impacts patient outcome by influencing intraoperative hemodynamics and blood loss. We present two cases which despite having normal preoperative coagulation profiles and normotension during the surgery had intraoperative coagulopathy and unusual bleeding. These cases required a multidisciplinary approach to manage them successfully. A thorough knowledge of the coagulation cascade along with a high-degree of suspicion and early recognition of coagulation is required. Abnormalities are critical for a timely intervention. Point-of-care tests like thromboelastography (TEG) as well as platelet function studies helped us arrive at an early diagnosis and initiate prompt treatment.

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Abstract Background Longitudinal investigations are needed to improve understanding of the contributions of cerebral small vessel disease to the clinical manifestation of Alzheimer’s disease, particularly in the early disease stages. This study leveraged the National Alzheimer’s Coordinating Center Uniform Data Set to longitudinally examine the association between white matter hyperintensities and neuropsychological, neuropsychiatric, and functional decline among participants with normal cognition. Methods The sample included 465 participants from the National Alzheimer’s Coordinating Center Uniform Data Set who had quantitated volume of white matter hyperintensities from fluid-attenuated inversion recovery MRI, had normal cognition at the time of their MRI, and were administered the National Alzheimer’s Coordinating Center Uniform Data Set neuropsychological test battery within 1 year of study evaluation and had at least two post-MRI time points of clinical data. Neuropsychiatric status was assessed by the Geriatric Depression Scale-15 and Neuropsychiatric Inventory-Questionnaire. Clinical Dementia Rating Sum of Boxes defined functional status. For participants subsequently diagnosed with mild cognitive impairment (MCI) or dementia, their impairment must have been attributed to Alzheimer’s disease (AD) to evaluate the relationships between WMH and the clinical presentation of AD. Results Of the 465 participants, 56 converted to MCI or AD dementia (average follow-up = 5 years). Among the 465 participants, generalized estimating equations controlling for age, sex, race, education, APOE ε4, and total brain and hippocampal volume showed that higher baseline log-white matter hyperintensities predicted accelerated decline on the following neuropsychological tests in rank order of effect size: Trails B (p

Pseudoaneurysms and patch infections are known complications of carotid endarterectomy with patch angioplasty. Although they are rare occurrences, they carry high morbidity and almost uniformly require surgical intervention. Infectious pathogens are often gram-positive bacteria, most commonly Staphylococcus species, whereas gram-negative infections are less frequently observed. We present a case of recurrent pseudoaneurysm in a patient who had a carotid endarterectomy with bovine pericardial patch angioplasty complicated by Pasteurella multocida infection. This case demonstrates the need for recognition and consideration of a broad differential of pathogens in evaluating and treating vascular infections. Keywords: Carotid endarterectomy, Infection, Bovine patch, Angioplasty, Pseudoaneurysm, Complication

Enzyme substrates and products often diffuse too rapidly to assess the catalytic implications of these movements. Here, the authors characterise the structural basis of product and substrate diffusion for an exo-hydrolase and discover a substrate-product assisted processive catalytic mechanism.

Abstract In a study from the onchocerciasis-endemic area of Mahenge in southern Tanzania, Mmbando et al. [Inf Dis Poverty. 2018;7:64] demonstrate that in four selected villages the overall epilepsy prevalence was high, and significantly more elevated in the two villages of higher onchocerciasis endemicity compared to those of lower endemicity. This is replicating earlier findings from many other areas of tropical Africa. The authors are also providing data indicating that in the Mahenge focus, the prevalence of nodding syndrome may be related to that of onchocerciasis in the same way as epilepsy in general. The application of a clinical case definition for onchocerciasis-associated epilepsy (OAE) as used in the study of Mmbando et al. [Inf Dis Poverty. 2018;7:64] faces some difficulties; indeed, its precision in discerning cases of OAE from epilepsy due to other etiologies is not known, and it does not allow for a specific diagnosis in the individual patient. Because an operational surveillance tool for assessing the number of patients in the population could mean substantial advance for better estimating the burden of OAE, the proposed definition should be tried in different settings and its performance reviewed in the process.

Abstract Introduction Keloids are challenging to treat due to their inadequate response to treatment and high recurrence rate. Intralesional triamcinolone acetonide (TAC) injection with or without 5-fluorouracil (5FU) is considered the first-line treatment for keloids. Three significant disadvantages of intralesional injections are the pain associated with the procedure, the uneven topography, and epidermal atrophy. Fractionated ablative carbon dioxide (CO2) laser-assisted drug delivery (LADD) of the topical solution can help facilitate transdermal drug delivery and shows promise in scar remodeling. This study examined the use of a thermomechanical device (Tixel, Novoxel) to facilitate the transdermal delivery of TAC and 5-FU in the treatment of keloid scars. Methods Seven patients each received eight topical thermal ablations, with one ablation performed every 2–3 weeks. TAC and 5FU were applied after each ablation. Outcomes were evaluated using the Vancouver Scar Scale (VSS), and pain was assessed using the Visual Analog Scale (VAS). Results Mean keloid VSS reduced from 8.6 ± 1.2 to 5 ± 2.7 after the eight treatments. Mean treatment pain VAS score was 2.4 ± 0.7. Patients rated their satisfaction level as moderate–high. No severe adverse reactions were noted. Conclusion Thermomechanical drug delivery of TAC and 5-FU is safe and effective. This is a promising option for the treatment of keloid scars, particularly in the pediatric population.

Cerebral aneurysms can be complex and variable in size, position, and morphology, resulting in difficult surgical exposure and secure clip placement. They have a high mortality rate when ruptured, and though endovascular techniques have emerged to tackle this, surgical clip ligation remains the preferred modality for some aneurysms. Various techniques that help the surgeon dissect the aneurysmal dome and apply the clip are temporary clip ligation of proximal vessels, deep hypothermic circulatory arrest, and balloon suction decompression. All of them require significant logistics and result in increased morbidity and mortality. Adenosine is a suitable alternative for inducing a temporary flow arrest, causing a short period of controlled hypotension. Its rapid onset and offset property along with low incidence of adverse events makes it an ideal agent in this regard. We present here a review on its use, contraindications, safety profile, and future applications.

Background and Aims: General anesthesia using agents like Desflurane or Sevoflurane are beneficial for early recovery especially for ambulatory procedures. The aim of this randomised controlled double-blind study was to compare the early recovery profiles of sevoflurane and desflurane in patients undergoing laparoscopic cholecystectomy. Material and Methods: ASA I, II patients, undergoing laparoscopic cholecystectomy were randomly assigned to receive desflurane (n = 30) or sevoflurane (n = 30), using Bispectral Index System (BIS) to determine the depth of anaesthesia. An independent adjudicator, who was blinded to the agent used, recorded the events during the recovery phase. The time required for extubation, eye opening, verbal response and achievement of a modified Aldrete score of 9 were recorded. Results: The time required for extubation and for eye opening was significantly shorter in the Desflurane group as compared to the Sevoflurane group [9.1 min ± 5.0 versus 12.5 min ± 7.1, P = 0.049 and 10.1 min ± 5.2 versus 6.3 min ± 4.0, P = 0.008]. Verbal Response also occurred significantly faster in the Desflurane group [12.7 min ± 5.4 versus 8.7 min ± 4.7, P = 0.002]. A significantly higher mean modified Aldrete score was seen at extubation [7.1 ± 0.6 vs 6.0 ± 0.8, P < 0.001] in the Desflurane group, which also achieved a modified Aldrete score of ≥9 significantly sooner [11.1 min ± 4.6 versus 17.8 min ± 6.9, P < 0.001] than the Sevoflurane group. The frequency of adverse effects was not significantly different in either of the groups. Conclusion: The time required for early recovery from anaesthesia, was significantly shorter in the Desflurane group compared to the Sevoflurane group.

Abstract Type II endoleak is a common complication following endovascular aortic aneurysm repair and can lead to an increased risk of aneurysmal expansion and rupture. The most frequently employed strategies to treat Type II endoleak involves catheterization of the branch vessels responsible for the endoleak or accessing the aneurysm sac through a percutaneous approach. An endovascular transcaval approach for embolization of the aneurysmal sac provides an alternate strategy with comparable success rates. This technique is advantageous when the endoleak is predominantly on the right side of the aneurysm sac and/or when a direct access to the aneurysm sac through a percutaneous approach is not feasible.

Cumulative sums (Cusums) are a simple, efficient statistical method developed for process control and increasingly used to determine underlying features of time series. Here, two useful applications of Cusums to environmental time series are presented: Cusums in the time domain and plotting Cusum-transformed variables against non-transformed variables to extract meaning in the context of driver-response relationships. These statistical analyses are simple to conduct and provide valuable information about trends, patterns and thresholds of time-series over time and in relation to potential driver variables. In addition, this work investigates the robustness of the Cusum transform to various characteristics of environmental time series that challenge conventional statistical methods. In summary, this work presents: • Cusum methods to derive meaning from complex environmental time series. • Effects of common time series issues on the Cusums method. • Application to real-world datasets. Method name: Applying cumulative sums to environmental data, Keywords: Cumulative sums, Driver-response, Time series, Everglades restoration, Nutrients, Water management

Abstract Background High epilepsy prevalence and incidence have been reported in areas with high onchocerciasis transmission. Recent findings suggest that proper community-directed treatment with ivermectin (CDTI) is potentially able to prevent onchocerciasis-associated epilepsy (OAE). We assessed the epilepsy prevalence and onchocerciasis transmission in two Nigerian villages following more than 20 years of CDTI. Methods A cross-sectional door-to-door survey was performed in two villages in the Imo River Basin reported to be mesoendomic for onchocerciasis (Umuoparaodu and Umuezeala). Individuals were screened for epilepsy using a validated 5-item questionnaire. Persons suspected to have epilepsy were examined by a neurologist or a physician with training in epilepsy for confirmation. Onchocerciasis was investigated via skin snip microscopy and rapid diagnostic tests for Ov16 antibodies. Results were compared with previous findings from the Imo river basin. Results A total of 843 individuals from 257 households in the two villages were encountered. We detected four persons with epilepsy (PWE) giving a crude epilepsy prevalence of 0.5%. This finding differs from observations reported 14 years ago which showed an epilepsy prevalence of 2.8% in the neighbouring village of Umulolo (P = 0.0001), and 1.2% from 13 villages in the Imo river basin (P = 0.07). The seroprevalence of Ov16 antibodies was found to be 0%. Only 4.6% of skin snips were positive compared to 26.8% in previous surveys (P

Boron arsenate, BAsO4, is a β-cristobalite-like crystal which has been reported to exhibit the rather unusual property of negative linear compressibility behaviour at elevated pressures, that is expanding rather than shrinking in a linear dimension when subjected to pressure. This work proposes a ‘geometry—deformation mechanism’-based mathematical model to aid the discernment of the manner how this anomalous pressure behaviour is achieved. The model makes use of data obtained from DFT simulations over an extended range of pressures, including extreme pressure conditions, and rigorously explains the macroscopic properties of this material in terms of the nanoscale deformations. More specifically, through this model, it was possible to decipher the different contributions to the deformation mechanism and compressibility properties of BAsO4. Moreover, for the first time, it was shown that a rule related to the sum of angles of tetrahedrally coordinated atoms is so robust that it applies at the extreme pressures studied here.

Migraine is a severe neurovascular disease manifested mainly as unilateral throbbing headaches. Triptans are agonists for serotonin receptors. Zolmitriptan (ZMP) is a biopharmaceutics classification system (BCS) class III medication with an absolute oral bioavailability of less than 40%. As a result, our research intended to increase ZMP bioavailability by developing transdermal nanostructured lipid carriers (NLCs). NLCs were prepared utilizing a combination of hot melt emulsification and high-speed stirring in a 32 full factorial design. The studied variables were liquid lipid type (X1) and surfactant type (X2). The developed NLCs were evaluated in terms of particle size (Y1, nm), polydispersity index (Y2, PDI), zeta potential (Y3, mV), entrapment efficacy (Y4, %) and amount released after 6 h (Q6h, Y5, %). At 1% Mygliol as liquid lipid component and 1% Span 20 as surfactant, the optimized formula (NLC9) showed a minimum particle size (138 ± 7.07 nm), minimum polydispersity index (0.39 ± 0.001), acceptable zeta potential (−22.1 ± 0.80), maximum entrapment efficiency (73 ± 0.10%) and maximum amount released after 6 h (83.22 ± 0.10%). The optimized formula was then incorporated into gel preparation (HPMC) to improve the system stability and ease of application. Then, the pharmacokinetic study was conducted on rabbits in a cross-over design. The calculated parameters showed a higher area under the curve (AUC0–24, AUC0–∞ (ng·h/mL)) of the developed ZMP-NLCs loaded gel, with a 1.76-fold increase in bioavailability in comparison to the orally administered marketed product (Zomig®). A histopathological examination revealed the safety of the developed nanoparticles. The declared results highlight the potential of utilizing the proposed NLCs for the transdermal delivery of ZMP to improve the drug bioavailability.

Cerebrospinal fluid (CSF) viral escape has been poorly described among people with HIV-associated cryptococcal meningitis. We determined the prevalence of CSF viral escape and HIV-1 viral load (VL) trajectories in individuals treated for HIV-associated cryptococcal meningitis. A retrospective longitudinal study was performed using paired CSF and plasma collected prior to and during the antifungal treatment of 83 participants recruited at the Botswana site of the phase-3 AMBITION-cm trial (2018–2021). HIV-1 RNA levels were quantified then CSF viral escape (CSF HIV-1 RNA ≥ 0.5 log10 higher than plasma) and HIV-1 VL trajectories were assessed. CSF viral escape occurred in 20/62 (32.3%; 95% confidence interval [CI]: 21.9–44.6%), 13/52 (25.0%; 95% CI: 15.2–38.2%) and 1/33 (3.0%; 95% CI: 0.16–15.3%) participants at days 1, 7 and 14 respectively. CSF viral escape was significantly lower on day 14 compared to days 1 and 7, p = 0.003 and p = 0.02, respectively. HIV-1 VL decreased significantly from day 1 to day 14 post antifungal therapy in the CSF but not in the plasma (β = −0.47; 95% CI: −0.69 to −0.25; p < 0.001). CSF viral escape is high among individuals presenting with HIV-associated cryptococcal meningitis; however, antifungal therapy may reverse this, highlighting the importance of rapid initiation of antifungal therapy in these patients.

Abstract Background A recent study in the Logo and Rethy health zones in the Ituri Province in the Democratic Republic of Congo (DRC) reported that the majority of the persons with epilepsy (PWE) had not been treated with anti-epileptic medication (AEM) or had stopped treatment. Prior to the implementation of an epilepsy treatment programme in these health zones, this study investigated the perceptions and experiences regarding epilepsy and its treatment amongst community leaders, PWE and/or their families, traditional healers and health professionals. Methods A total of 14 focus group discussions (FGD) and 39 semi-structured interviews (SSI) were conducted with PWE and/or their family members, community leaders, traditional healers, and health professionals in the Logo and Rethy health zones during February 2–19, 2017. Results In the two health zones, the clinical signs of convulsive epilepsy were recognized by community members. However, a variety of misconceptions about epilepsy were identified including the beliefs that epilepsy is a family-related condition, a contagious disease, is transmitted by insects, saliva or by touching a person of the same sex during seizures, or is caused by evil spirits and witchcraft. The role of traditional healers in spreading these beliefs was revealed. The study also reported sexual abuse on PWE, stigmatisation of PWE and loss of productivity of PWE and their families. Some PWE had been using AEM and traditional treatment but were not convinced about the efficacy of these treatment options. The lack of training of health providers about epilepsy care, financial barriers in obtaining AEM, and the shortage of AEM at primary health facilities were revealed. As remedies, the community requested access to a decentralized center for epilepsy treatment. They also proposed using churches and community health workers as communication channels for information about epilepsy. Conclusions Clinical signs of convulsive epilepsy were recognized by the community in the Logo and Rethy health zones but many misconceptions about epilepsy were identified. A comprehensive community-based epilepsy treatment programme with an affordable uninterrupted AEM supply needs to be established. Such a programme should address stigma, misconceptions, sexual abuse and foster the rehabilitation of PWE to alleviate poverty.

BackgroundIt is essential that clinical trial participants are representative of the population under investigation. Using HIV-associated cryptococcal meningitis (CM) as a case study, we conducted a systematic review of clinical trials to determine how inclusive and representative they were both in terms of the affected population and the involvement of local investigators.MethodsWe searched Medline, EMBASE, Cochrane, Africa-Wide, CINAHL Plus, and Web of Science. Data were extracted for 5 domains: study location and design, screening, participants, researchers, and funders. Data were summarised and compared over 3 time periods: pre-antiretroviral therapy (ART) (pre-2000), early ART (2000 to 2009), and established ART (post-2010) using chi-squared and chi-squared for trend. Comparisons were made with global disease burden estimates and a composite reference derived from observational studies.ResultsThirty-nine trials published between 1990 and 2019 were included. Earlier studies were predominantly conducted in high-income countries (HICs) and recent studies in low- and middle-income countries (LMICs). Most recent studies occurred in high CM incidence countries, but some highly affected countries have not hosted trials. The sex and ART status of participants matched those of the general CM population. Patients with reduced consciousness and those suffering a CM relapse were underrepresented. Authorship had poor representation of women (29% of all authors), particularly as first and final authors. Compared to trials conducted in HICs, trials conducted in LMICs were more likely to include female authors (32% versus 20% p = 0.014) but less likely to have authors resident in (75% versus 100%, p < 0.001) or nationals (61% versus 93%, p < 0.001) of the trial location.ConclusionsThere has been a marked shift in CM trials over the course of the HIV epidemic. Trials are primarily performed in locations and populations that reflect the burden of disease, but severe and relapse cases are underrepresented. Most CM trials now take place in LMICs, but the research is primarily funded and led by individuals and institutions from HICs.

Passive thermography is commonly used for composites load testing to detect damage formation as a function of the applied load. The advantages of passive thermography are real time implementation, large area coverage, and noncontact measurement. Passive thermography is able to detect the damage location and size, however, damage depth has been a challenge for quasistatic loading. Recent work has shown that damage formation during loading produces heating that is composed of two heat generation components. The first component is an instantaneous thermal response due to an irreversible thermoelastic strain release due to rapid damage formation. The second component observed is mechanical heating, at the interface of failure, due to fracture damage that produces a transient rise in surface temperature as a function of damage depth. The first component defines the thermal start time for the transient response. A one-dimensional thermal model, that is independent of delamination damage gap spacing, is presented and fitted to the data pixel by pixel, to produce imagery of the damage depth. The percent difference between thermal results, as compared to the ultrasonic measurements of damage length and width, was on average 15%. The percent difference between the thermal results, as compared to the X-ray CT measurements for damage depth was on average 7%. This same processing technique was applied for detection of damage depth during cyclic loading as well.

Summary: Inflammation during neonatal brain infections leads to significant secondary sequelae such as hydrocephalus, which often follows neonatal sepsis in the developing world. In 100 African hydrocephalic infants we identified the biological pathways that account for this response. The dominant bacterial pathogen was a Paenibacillus species, with frequent cytomegalovirus co-infection. A proteogenomic strategy was employed to confirm host immune response to Paenibacillus and to define the interplay within the host immune response network. Immune activation emphasized neuroinflammation, oxidative stress reaction, and extracellular matrix organization. The innate immune system response included neutrophil activity, signaling via IL-4, IL-12, IL-13, interferon, and Jak/STAT pathways. Platelet-activating factors and factors involved with microbe recognition such as Class I MHC antigen-presenting complex were also increased. Evidence suggests that dysregulated neuroinflammation propagates inflammatory hydrocephalus, and these pathways are potential targets for adjunctive treatments to reduce the hazards of neuroinflammation and risk of hydrocephalus following neonatal sepsis.

Introduction Individuals recruited into clinical trials for life-threatening illnesses are particularly vulnerable. This is especially true in low-income settings. The decision to enrol may be influenced by existing inequalities, poor healthcare infrastructure and fear of death. Where patients are confused or unconscious the responsibility for this decision falls to relatives. This qualitative study is nested in the ongoing AMBIsome Therapy Induction OptimisatioN (AMBITION) Trial. AMBITION is recruiting participants from five countries in sub-Saharan Africa and is trialling a novel treatment approach for HIV-associated cryptococcal meningitis, an infection known to affect brain function. We aim to learn from the experiences of participants, relatives and researchers involved in AMBITION.Methods and analysis We will collect data through in-depth interviews with trial participants and the next of kin of participants who were confused at enrolment and therefore provided surrogate consent. Data will be collected in Gaborone, Botswana; Kampala, Uganda and Harare, Zimbabwe. Interviews will follow a narrative approach including participatory drawing of participation timelines. This will be supplemented by direct observation of the research process at each of the three recruiting hospitals. Interviews will also take place with researchers from the African and European institutions that form the partnership through which the trial is administered. Interviews will be transcribed verbatim, translated (if necessary) and organised thematically for narrative analysis.Ethics and dissemination This study has been approved by the Health Research Development Committee, Gaborone (Reference: HPDME:13/18/1); Makerere School of Health Sciences Institutional Review Board, Kampala (Reference: 2019–061); University of Zimbabwe Joint Research Ethics Committee, Harare (Reference: 219/19), and the London School of Hygiene and Tropical Medicine (Reference: 17957). Study findings will be shared with research participants from the sites, key stakeholders at each research institution and ministries of health to help inform the development and implementation of future trials. The findings of this study will be published in journals and presented at academic meetings.Trial registration Registered at www.clinicaltrials.gov:NCT04296292.

Introduction: Hip fractures are a significant economic burden to our healthcare system. As there have been efforts made to create an alternative payment model for hip fracture care, it will be imperative to risk-stratify reimbursement for these medically comorbid patients. We hypothesized that patients readmitted to the hospital within 90 days would be more likely to have a recent previous hospital admission, prior to their injury. Patients with a recent prior admission could therefore be considered higher risk for readmission and increased cost. Methods: A retrospective chart review identified 598 patients who underwent surgical fixation of a hip or femur fracture. Data on readmissions within 90 days of surgical procedure and previous admissions in the year prior to injury resulting in surgical procedure were collected. Logistic regression analysis was used to determine if recent prior admission had increased risk of 90-day readmission. A subgroup analysis of geriatric hip fractures and of readmitted patients were also performed. Results: Having a prior admission within one year was significantly associated (p < 0.0001) for 90-day readmission. Specifically, logistic regression analysis revealed that a prior admission was significantly associated with 90-day readmission with an odds ratio of 7.2 (95% CI: 4.8-10.9). Discussion: This patient population has a high rate of prior hospital admissions, and these prior admissions were predictive of 90-day readmission. Alternative payment models that include penalties for readmissions or fail to apply robust risk stratification may unjustly penalize hospital systems which care for more medically complex patients. Conclusions: Hip fracture patients with a recent prior admission to the hospital are at an increased risk for 90-day readmission. This information should be considered as alternative payment models are developed for hip fracture care.

There is a prevalence of high fertility among the Mbaise of Imo State, Nigeria. This is linked to the perceptions, beliefs and practices of the people. The voluntaristic social action theory by Talcott Parsons and Proximate determinants of fertility framework by Bongaarts were utilized to highlight the links between Catholicism and fertility, as well as socio-cultural variables and fertility behaviour by showing strength of their separate and combined relationships. Data were gathered through survey questionnaire and in-depth interview. The findings from the study show that the factors affecting fertility among the Mbaise include communal and individual norms and practices, and their persistent influence signal a need to investigate their separate and combined influences on fertility behaviour. Specific findings were that: (1) Roman Catholicism is a factor responsible for the perceived high fertility among Mbaise people, (2) Roman Catholic fertility practices conform to the socio-cultural environment in the area (3) knowledge of contraceptives use is significantly low among the people of Mbaise. The negligible proportion that has used family planning methods did so for child spacing rather than for limiting fertility.

Background: Cassava is a major staple root crop in Ghana, which serves as a food security and an income generating crop for farming families. In spite of its importance, the crop is plagued with biotic factors such as pests and diseases, resulting in yield and income reductions. Methods: Farmers’ knowledge, attitudes and practices towards cassava pest and disease management were investigated. A mixed research questionnaire was used to collect both qualitative and quantitative data from 94 cassava farm households across two major cassava growing agro-ecologies. Results: Using descriptive statistics, parametric and non-parametric analysis, our study revealed that farmers’ knowledge on cassava pests was high but low for diseases. Whiteflies (Bemisia tabaci Gennadius), grasshoppers (Zonocerus variegatus), aphids (Aphis gossypii Glover), mealybugs (Phenacoccus manihoti), termites (Isoptera), and grasscutters (Thryonomys swinderianus) were perceived as the most common damaging pests. Farmers’ descriptions showed that disease pathogens attacked foliar tissues, stem and root tissues and caused leaf dropping and die back. Cassava mosaic disease and root rot were the most common diseases; however, disease descriptions suggested the incidence of viral, bacterial and fungal diseases. Some of the farmers observed mixed symptoms on their farms. The results also showed that only 25.5% cultivated improved varieties. Management actions applied included field sanitation practices and pesticide application. The effectiveness level of the control actions was rated moderately effective. Conclusions: The analysis showed heterogeneity in personal and farm level characteristics of respondents across the two agro-ecologies, but agro-ecologies were independent of the management practices employed. There is a need to improve farmers’ access to improved disease-free planting materials through efficient dissemination pathways and increase farmers’ knowledge on cassava pests, diseases and integrated management through publfic awareness creation and capacity building by extension agents and research institutions. Continued government investment is needed to achieve sustainable outcomes.

Summary: We resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes.

IntroductionExposure to media content can shape public opinions about tobacco. In early September 2019, the outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI) became headline news in the United States.MethodsIn August and September 2019, we conducted two cross-sectional online surveys with current and former smokers assessing attitudes and beliefs about e-cigarettes. Study one (n = 865) was collected before the EVALI outbreak was widely covered and study two (n = 344) was collected after the outbreak had become nation-wide news. We examined differences in perceptions and beliefs between time points.ResultsE-cigarette harm perceptions increased between study one (mean = 2.67) and study two (mean = 2.90, p < .05). Ever-users of e-cigarettes largely account for this change. Endorsement of the belief that e-cigarettes were risky and more likely to cause lung damage compared to cigarettes increased between studies (p < .05). Seventy eight percent of participants at study two were aware of the vaping illness story. Being aware of the story was associated with more endorsement of the belief that e-cigarettes were risky to use, but not that using e-cigarettes would make the participant more likely to get damaged lungs.DiscussionWhen the stories about the health and safety of tobacco products dominate the public information environment, it presents an opportunity to change beliefs that are frequently targeted by paid health campaigns. Changes in participant's perceptions of e-cigarettes were associated with coverage of this large news story, underscoring the importance of working to ensure that coverage is a scientifically accurate as possible.

BackgroundRefugees are at increased risk for COVID-19 infection in part due to their living conditions, which make it harder to adopt and adhere to widely accepted preventive measures. Little empirical evidence exists about what refugees know about COVID-19 and what they do to prevent infection. This study explored what refugee women and their health care workers understand about COVID-19 prevention, the extent of their compliance to public health recommendations, and what influences the adoption of these measures.MethodsIn October 2020, we conducted 25 in-depth interviews with facility and community health care staff (n = 10) and refugee women attending antenatal and postnatal care services (n = 15) in Eastleigh, Nairobi.FindingsWhile researchers found a high level of awareness about COVID-19 and related prevention and control measures among refugee women, various barriers affected compliance with such measures, due in part to poverty and in part to rampant misconceptions informed by religious beliefs and political narratives about the virus.ConclusionsThese findings indicated that Kenya's Ministry of Health needs to institute a concerted and continuous education program to bring refugee communities up to speed about COVID-19 and its prevention. In addition to disseminating information about the need to wear masks and repeatedly wash hands, supplies-masks, soap and access to water-need to be made available to poor refugee communities. Future research could explore which measures for disseminating factual information work best in refugee populations with different cultural norms and how best to target interventions to these groups.

Background. Surgical patients frequently seek information from digital sources, particularly before common operations such as laparoscopic cholecystectomy (LC). YouTube provides a large amount of free educational content; however, it lacks regulation or peer review. To inform patient education, we evaluated the quality of YouTube videos on LC. Methods. We searched YouTube with the phrase “laparoscopic cholecystectomy.” Two authors independently rated quality of the first 50 videos retrieved using the JAMA, Health on the Net (HON), and DISCERN scoring systems. Data collected for each video included total views, time since upload, video length, total comments, and percentage positivity (proportion of likes relative to total likes plus dislikes). Interobserver reliability was assessed using an intraclass correlation coefficient (ICC). Association between quality and video characteristics was tested. Results. Mean video quality scores were poor, scoring 1.9/4 for JAMA, 2.0/5.0 for DISCERN, and 4.9/8.0 for HON. There was good interobserver reliability with an ICC of 0.78, 0.81, and 0.74, respectively. Median number of views was 21,789 (IQR 3000–61,690). Videos were mostly published by private corporations. No video characteristic demonstrated significant association with video quality. Conclusion. YouTube videos for LC are of low quality and insufficient for patient education. Treating surgeons should advise of the website’s limitations and direct patients to trusted sources of information.

Background: The coronavirus disease of 2019 (COVID-19) has extensively impacted various sectors globally, including higher education. The indefinite closure of Universities has necessitated the need to introduce alternative teaching and learning methods and tools. E-learning a disruptive innovation has provided an opportunity to allow for continuity of teaching and learning in Universities during these closures. Objectives: The study adopts Teo's model as the analytical tool to investigate factors that influence University lecturers to adopt e-learning platforms in South Africa. Method: The study followed a quantitative research approach with stratified sampling as a data collection approach using a sample size of 132 respondents. Structural Equation Modelling (SEM) was adopted to deduce factors that influence the behavioural Intention (BIU) to adopt e-learning as well as to test the Model fit of Teo's model in the South African context. Results: The study revealed that the lecturer's attitude towards the use (ATU) of e-learning is the most influential construct towards lecturers behavioural Intention to use (BIU) e-learning platforms. The empirical evidence also revealed that Teo's model is a reasonable fit to the data to understand the adoption of e-learning by lecturers in South African universities. Conclusion: The empirical evidence from this study supports the viewpoint that in order for lecturers to successfully adopt e-learning platforms, their attitude towards the use of these platforms is a critical factor. In order to understand lecturer’s adoption of e-learning platforms successfully, Teo's model is a reasonably good framework to use.

Purpose: To define the time required to achieve the minimally clinically important difference (MCID), substantial clinical benefit (SCB) and patient acceptable symptomatic state (PASS) for isolated arthroscopic partial meniscectomy (APM), and define preoperative and intraoperative factors that predict both early and late achievement of the stated metrics. Methods: Patients who underwent isolated APM between 2014 and 2017 were retrospectively included. Patients without preoperative and 6-month patient-reported outcome measure scores, revision procedures, and significant concomitant procedures were excluded. The MCID, SCB, and PASS were calculated for knee-based patient-reported outcome measure scores using receiver operating curve analysis. Kaplan-Meier survival analysis established the time required to achieve MCID, SCB and PASS. Hazard ratios from multivariate Cox regression allowed for the isolation of demographic and intraoperative factors predictive of the delayed time required to achieve MCID, SCB and PASS. Results: A total of 126 patients (42.86% female, age: 48.9 ± 12.4 years) were included. Overall achievement rates ranged between 73.0% and 89.7% for MCID, 43.7% and 68.2% for SCB, and 50.8% and 68.3% for PASS. Median achievement time for MCID was 5.68-5.78 months, 5.73-6.05 months for SCB and 6.54-7.72 months for PASS. Multivariate Cox regression identified older age, workers’ compensation status, diabetes, and various tear types (i.e., longitudinal, transverse, bucket handle, complex) as predictors of early clinically significant outcome achievement (hazard ratio: 1.02-24.72), whereas subsequent steroid injection, higher preoperative scores and root and flap tears predicted delays in clinically significant outcome achievement (hazard ratio: 0.12-0.99). Conclusions: The majority of patients undergoing APM achieve benefit within 6 months of surgery, with diminishing proportions at later timepoints. Important factors for consideration of the the timeline of achieving clinically significant outcome include age, diabetes, workers’ compensation, preoperative score, and tear type. The timeline for achieving improvement that was established by this study may aid in setting patient expectations and designing future outcome studies involving APM. Study design: Level IV, Therapeutic Case Series.

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.

OBJECTIVES/GOALS: Wild-type transthyretin amyloid (ATTRwt) deposits have been found to deposit in the ligamentum flavum (LF) of spinal stenosis patients prior to systemic and cardiac amyloidosis, and is implicated in LF hypertrophy. Currently, no precise method of quantifying amyloid deposits exists. Here, we present our machine learning quantification method. METHODS/STUDY POPULATION: Images of ligamentum flavum specimens stained with Congo red are obtained from spinal stenosis patients undergoing laminectomies and confirmed to be positive for ATTRwt. Amyloid deposits in these specimens are classified and quantified by TWS through training the algorithm via user-directed annotations on images of LF. TWS can also be automated through exposure to a set of training images with user- directed annotations, and then application to a set of new images without additional annotations. Additional methods of color thresholding and manual segmentation are also used on these images for comparison to TWS. RESULTS/ANTICIPATED RESULTS: We develop the use of TWS in images of LF and demonstrate its potential for automated quantification. TWS is strongly correlated with manual segmentation in the training set of images with user-directed annotations (R = 0.98; p = 0.0033) as well as in the application set of images where TWS was automated (R = 0.94; p = 0.016). Color thresholding was weakly correlated with manual segmentation in the training set of images (R = 0.78; p = 0.12) and in the application set of images (R = 0.65; p = 0.23). DISCUSSION/SIGNIFICANCE: Our machine learning method correlates with the gold standard comparator of manual segmentation and outperforms color thresholding. This novel machine learning quantification method is a precise, objective, accessible, high throughput, and powerful tool that will hopefully pave the way towards future research and clinical applications.

The increasing use of reclaimed water for irrigation in areas lacking access to advanced wastewater treatment and reclaimed water distribution systems calls for an examination of irrigation-site-based treatment technologies that can improve the quality of this alternative water source. To address this need, we investigated the impact of zero-valent iron (ZVI)-sand filtration on the bacterial community structure and functional potential of conventionally treated reclaimed water utilized in downstream applications. Over a 2-month period, reclaimed water was collected from a tertiary wastewater treatment plant in the Mid-Atlantic, U.S. and trucked to our greenhouse facility. The water was stored in rain barrels and then filtered through one ZVI-sand filter every 5 days. Filtrate was then subjected to enumeration, phylotyping, shiga toxin screening, and antimicrobial susceptibility testing of Escherichia coli. Aliquots of filtrate were also DNA extracted, and purified DNA was subjected to 16S rRNA gene sequencing and metagenomic shotgun sequencing. The genera Dechloromonas, Desulfotomaculum, Leptonema, and Thermomonas, which contain denitrifying and sulfate reducing species, commonly used in bioremediation, and known to increase the inherent reactivity of ZVI, were significantly more relatively abundant in ZVI-sand filtered reclaimed water compared to reclaimed water. The concentration of E. coli in ZVI-sand filtered reclaimed water was significantly lower compared to that of reclaimed water, and cefoxitin- and tetracycline-resistant E. coli were undetectable after ZVI-sand filtration. ZVI-sand filtration reduced the occurrence of human as well as plant pathogenic genera (Aeromonas, Mycobacterium, Shewanella, Acidovorax, Agrobacterium, and Clavibacter) but increased the proportion of Azospira, a nitrogen fixing bacterial genera, in the microbial community. Our exploratory functional analysis showed a modest non-significant increase in the proportion of open reading frames for genes associated with iron uptake, oxidative stress, as well as defense and repair mechanisms after ZVI-sand filtration. These data indicate an iron rich environment in the filter causing an oxidative stress response by the bacterial community present in the reclaimed water. Our findings demonstrate that ZVI-sand filtration effectively filters conventionally treated reclaimed water. Longer-term, field-based studies are needed to evaluate the effectiveness of the filter in agricultural settings and inform the development of future agricultural water reuse regulations.