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BackgroundTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naïve tsetse with T. brucei DNA via faeces when co-housed.Methodology/principle findingsInsectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]).Conclusions/significanceOur results show that infected tsetse can contaminate naïve flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.

BackgroundTsetse flies (Glossina) transmit Trypanosoma brucei gambiense which causes Gambian human African trypanosomiasis (gHAT) in Central and West Africa. Several countries use Tiny Targets, comprising insecticide-treated panels of material which attract and kill tsetse, as part of their national programmes to eliminate gHAT. We studied how the scale and arrangement of target deployment affected the efficacy of control.Methodology and principal findingsBetween 2012 and 2016, Tiny Targets were deployed biannually along the larger rivers of Arua, Maracha, Koboko and Yumbe districts in North West Uganda with the aim of reducing the abundance of tsetse to interrupt transmission. The extent of these deployments increased from ~250 km2 in 2012 to ~1600 km2 in 2015. The impact of Tiny Targets on tsetse populations was assessed by analysing catches of tsetse from a network of monitoring traps; sub-samples of captured tsetse were dissected to estimate their age and infection status. In addition, the condition of 780 targets (~195/district) was assessed for up to six months after deployment. In each district, mean daily catches of tsetse (G. fuscipes fuscipes) from monitoring traps declined significantly by >80% following the deployment of targets. The reduction was apparent for several kilometres on adjacent lengths of the same river but not in other rivers a kilometre or so away. Expansion of the operational area did not always produce higher levels of suppression or detectable change in the age structure or infection rates of the population, perhaps due to the failure to treat the smaller streams and/or invasion from adjacent untreated areas. The median effective life of a Tiny Target was 61 (41.8-80.2, 95% CI) days.ConclusionsScaling-up of tsetse control reduced the population of tsetse by >80% across the intervention area. Even better control might be achievable by tackling invasion of flies from infested areas within and outside the current intervention area. This might involve deploying more targets, especially along smaller rivers, and extending the effective life of Tiny Targets.

BackgroundIn 2005, Bangladesh, India and Nepal agreed to eliminate visceral leishmaniasis (VL) as a public health problem. The approach to this was through improved case detection and treatment, and controlling transmission by the sand fly vector Phlebotomus argentipes, with indoor residual spraying (IRS) of insecticide. Initially, India applied DDT with stirrup pumps for IRS, however, this did not reduce transmission. After 2015 onwards, the pyrethroid alpha-cypermethrin was applied with compression pumps, and entomological surveillance was initiated in 2016.MethodsEight sentinel sites were established in the Indian states of Bihar, Jharkhand and West Bengal. IRS coverage was monitored by household survey, quality of insecticide application was measured by HPLC, presence and abundance of the VL vector was monitored by CDC light traps, insecticide resistance was measured with WHO diagnostic assays and case incidence was determined from the VL case register KAMIS.ResultsComplete treatment of houses with IRS increased across all sites from 57% in 2016 to 70% of houses in 2019, rising to >80% if partial house IRS coverage is included (except West Bengal). The quality of insecticide application has improved compared to previous studies, average doses of insecticide on filters papers ranged from 1.52 times the target dose of 25mg/m2 alpha-cypermethrin in 2019 to 1.67 times in 2018. Resistance to DDT has continued to increase, but the vector was not resistant to carbamates, organophosphates or pyrethroids. The annual and seasonal abundance of P. argentipes declined between 2016 to 2019 with an overall infection rate of 0.03%. This was associated with a decline in VL incidence for the blocks represented by the sentinel sites from 1.16 per 10,000 population in 2016 to 0.51 per 10,000 in 2019.ConclusionThrough effective case detection and management reducing the infection reservoirs for P. argentipes in the human population combined with IRS keeping P. argentipes abundance and infectivity low has reduced VL transmission. This combination of effective case management and vector control has now brought India within reach of the VL elimination targets.

The study of seabird behaviour has largely relied on animal-borne tags to gather information, requiring interpretation to estimate at-sea behaviours. Details of shallow-diving birds' foraging are less known than deep-diving species due to difficulty in identifying shallow dives from biologging devices. Development of smaller video loggers allow a direct view of these birds' behaviours, at the cost of short battery capacity. However, recordings from video loggers combined with relatively low power usage accelerometers give a means to develop a reliable foraging detection method. Combined video and acceleration loggers were attached to streaked shearwaters in Funakoshi-Ohshima Island (39°24'N,141°59'E) during the breeding season in 2018. Video recordings were classified into behavioural categories (rest, transit, and foraging) and a detection method was generated from the acceleration signals. Two foraging behaviours, surface seizing and foraging dives, are reported with video recordings. Surface seizing was comprised of successive take-offs and landings (mean duration 0.6 and 1.5s, respectively), while foraging dives were shallow subsurface dives (3.2s mean duration) from the air and water surface. Birds were observed foraging close to marine predators, including dolphins and large fish. Results of the behaviour detection method were validated against video recordings, with mean true and false positive rates of 90% and 0%, 79% and 5%, and 66% and

AimTo capture pandemic experiences of people with opioid use disorder (OUD) to better inform the programs that serve them.DesignWe designed, conducted, and analyzed semi-structured qualitative interviews using grounded theory. We conducted interviews until theme saturation was reached and we iteratively developed a codebook of emerging themes. Individuals with lived experience of substance use provided feedback at all steps of the study.SettingWe conducted phone or in-person interviews in compliance with physical distancing and public health regulations in outdoor Vancouver parks or well-ventilated indoor spaces between June to September 2020.ParticipantsUsing purposive sampling, we recruited participants (n = 19) who were individuals with OUD enrolled in an intensive community outreach program, had visited one of two emergency departments, were over 18, lived within catchment, and were not already receiving opioid agonist therapy.MeasurementsWe audio-recorded interviews, which were later transcribed verbatim and checked for accuracy while removing all identifiers. Interviews explored participants' knowledge of COVID-19 and related safety measures, changes to drug use and healthcare services, and community impacts of COVID-19.ResultsOne third of participants were women, approximately two thirds had stable housing, and ages ranged between 23 and 59 years old. Participants were knowledgeable on COVID-19 public health measures. Some participants noted that fear decreased social connection and reluctance to help reverse overdoses; others expressed pride in community cohesion during crisis. Several participants mentioned decreased access to housing, harm reduction, and medical care services. Several participants reported using drugs alone more frequently, consuming different or fewer drugs because of supply shortages, or using more drugs to replace lost activities.ConclusionCOVID-19 had profound effects on the social lives, access to services, and risk-taking behaviour of people with opioid use disorder. Pandemic public health measures must include risk mitigation strategies to maintain access to critical opioid-related services.

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

Bats are primary consumers of nocturnal insects, disperse nutrients across landscapes, and are excellent bioindicators of an ecosystem's health, however four of the seventeen Great British species are listed as declining. In this study we aim to investigate the link between bat guano morphology and diet, specifically looking at the ability to predict 1) species, 2) dietary guild, and 3) bat size, using guano morphology alone. Guano from 16 bat species sampled from across Great Britain were analysed to determine various morphological metrics. These data were coupled with diet data obtained by an extensive literature review. It was found that guano morphology overlapped too much to make predictions on the species of bat which deposited the guano, however, in some cases, it could be used to indicate the dietary guild to which the bat belonged. In general, guano morphology seems more correlated to diet than species. This enables the identification of the most important prey taxa within a local environment; a crucial step for informing conservation strategies.

BackgroundAgitation is a common, challenging symptom affecting large numbers of people with dementia and impacting on quality of life (QoL). There is an urgent need for evidence-based, cost-effective psychosocial interventions to improve these outcomes, particularly in the absence of safe, effective pharmacological therapies. This study aimed to evaluate the efficacy of a person-centred care and psychosocial intervention incorporating an antipsychotic review, WHELD, on QoL, agitation, and antipsychotic use in people with dementia living in nursing homes, and to determine its cost.Methods and findingsThis was a randomised controlled cluster trial conducted between 1 January 2013 and 30 September 2015 that compared the WHELD intervention with treatment as usual (TAU) in people with dementia living in 69 UK nursing homes, using an intention to treat analysis. All nursing homes allocated to the intervention received staff training in person-centred care and social interaction and education regarding antipsychotic medications (antipsychotic review), followed by ongoing delivery through a care staff champion model. The primary outcome measure was QoL (DEMQOL-Proxy). Secondary outcomes were agitation (Cohen-Mansfield Agitation Inventory [CMAI]), neuropsychiatric symptoms (Neuropsychiatric Inventory-Nursing Home Version [NPI-NH]), antipsychotic use, global deterioration (Clinical Dementia Rating), mood (Cornell Scale for Depression in Dementia), unmet needs (Camberwell Assessment of Need for the Elderly), mortality, quality of interactions (Quality of Interactions Scale [QUIS]), pain (Abbey Pain Scale), and cost. Costs were calculated using cost function figures compared with usual costs. In all, 847 people were randomised to WHELD or TAU, of whom 553 completed the 9-month randomised controlled trial. The intervention conferred a statistically significant improvement in QoL (DEMQOL-Proxy Z score 2.82, p = 0.0042; mean difference 2.54, SEM 0.88; 95% CI 0.81, 4.28; Cohen's D effect size 0.24). There were also statistically significant benefits in agitation (CMAI Z score 2.68, p = 0.0076; mean difference 4.27, SEM 1.59; 95% CI -7.39, -1.15; Cohen's D 0.23) and overall neuropsychiatric symptoms (NPI-NH Z score 3.52, p < 0.001; mean difference 4.55, SEM 1.28; 95% CI -7.07,-2.02; Cohen's D 0.30). Benefits were greatest in people with moderately severe dementia. There was a statistically significant benefit in positive care interactions as measured by QUIS (19.7% increase, SEM 8.94; 95% CI 2.12, 37.16, p = 0.03; Cohen's D 0.55). There were no statistically significant differences between WHELD and TAU for the other outcomes. A sensitivity analysis using a pre-specified imputation model confirmed statistically significant benefits in DEMQOL-Proxy, CMAI, and NPI-NH outcomes with the WHELD intervention. Antipsychotic drug use was at a low stable level in both treatment groups, and the intervention did not reduce use. The WHELD intervention reduced cost compared to TAU, and the benefits achieved were therefore associated with a cost saving. The main limitation was that antipsychotic review was based on augmenting processes within care homes to trigger medical review and did not in this study involve proactive primary care education. An additional limitation was the inherent challenge of assessing QoL in this patient group.ConclusionsThese findings suggest that the WHELD intervention confers benefits in terms of QoL, agitation, and neuropsychiatric symptoms, albeit with relatively small effect sizes, as well as cost saving in a model that can readily be implemented in nursing homes. Future work should consider how to facilitate sustainability of the intervention in this setting.Trial registrationISRCTN Registry ISRCTN62237498.

Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds.

Cortical oscillations are likely candidates for segmentation and coding of continuous speech. Here, we monitored continuous speech processing with magnetoencephalography (MEG) to unravel the principles of speech segmentation and coding. We demonstrate that speech entrains the phase of low-frequency (delta, theta) and the amplitude of high-frequency (gamma) oscillations in the auditory cortex. Phase entrainment is stronger in the right and amplitude entrainment is stronger in the left auditory cortex. Furthermore, edges in the speech envelope phase reset auditory cortex oscillations thereby enhancing their entrainment to speech. This mechanism adapts to the changing physical features of the speech envelope and enables efficient, stimulus-specific speech sampling. Finally, we show that within the auditory cortex, coupling between delta, theta, and gamma oscillations increases following speech edges. Importantly, all couplings (i.e., brain-speech and also within the cortex) attenuate for backward-presented speech, suggesting top-down control. We conclude that segmentation and coding of speech relies on a nested hierarchy of entrained cortical oscillations.

Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures.The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability.In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

BACKGROUND:Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion. METHODOLOGY/PRINCIPAL FINDINGS:To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation. CONCLUSIONS/SIGNIFICANCE:Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation.