Mapping the expression of key functional genes associated with murine corneal endothelial development.

Master of Science in Biology. University of KwaZulu-Natal, Durban 2015.The cornea is a complex, layered, transparent structure that is responsible for transmitting and refracting light to ensure vision. Of all the layers, the corneal endothelium is physiologically the most important layer. Its principle function is to regulate deturgescence, a requirement for corneal transparency. Corneal deturgescence is regulated by key functional genes that include a network of tight junctions, water channels and ion pumps. In this study, we created an in vitro model of murine corneal endothelial development by immortalising endothelial cells at significant stages in corneal endothelial morphogenesis. Primary cultures of presumptive corneal endothelial cells were immortalised from wild-type mouse embryos at embryonic day (E) 14.5 and E15.5, and mature corneal endothelial cells were immortalised from wild-type pups at post-natal day (P) 13. These cell lines, together with already established cell lines at earlier stages in corneal endothelial development (E12.5 and E13.5), were used to map the expression patterns of three functional genes associated with corneal endothelial function, Zo1, Aqp1 and Slc4a4. Zo1 is a tight junction protein that facilitates the formation of the resilient monolayer that is characteristic of the corneal endothelium. Aqp1 is a water channel protein that mediates transcellular fluid transport while Slc4a4 is an ion pump that transports cellular bicarbonate ions across plasma membranes. Aqp1 and Slc4a4 are disease target genes which is indicative of their functional significance in corneal endothelial physiology. Quantitative real-time PCR (qPCR) and western blot analysis showed that Zo1 expression at both mRNA and protein level was progressively down-regulated from E12.5 to E15.5, after which it was up-regulated at P13 (p˂0.05 relative to E15.5 at mRNA level). Aqp1 gene expression was up-regulated at E13.5, with subsequent down-regulation at E14.5. Thereafter, Aqp1 gene expression was up-regulated at E15.5. Interestingly, Aqp1 gene expression was significantly down-regulated at P13 when the endothelium is completely matured (p˂0.05 relative to all other cell lines). Western blot analysis demonstrated Aqp1 expression throughout development in this model. Densitometric analysis showed that glycosylated Aqp1 protein expression levels decreased gradually from E12.5 to P13. Unglycosylated Aqp1 protein expression was only present at E12.5 and E13.5, and was up-regulated at E13.5. Slc4a4 was expressed at low levels during corneal endothelial development. However, it was highly expressed at P13 (p˂0.05 relative to all other cell lines). Protein expression for Slc4a4 could not be determined due to the lack of a commercially available antibody specific for murine Slc4a4. Confocal microscopy analysis demonstrated the protein localisation patterns for Zo1 and Aqp1 in monolayer and hanging drop culture, mimicking the 3D environment in which corneal endothelial development occurs. The results of this study provide valuable insight into the expression patterns of key functional genes associated with corneal endothelial development

Concert recording 2022-10-02

[Track 1]. Grand duo concertant, op. 48. I. Allegro con fuoco ; II. Andante con moto ; III. Ronda, Allegro / Carl Maria von Weber -- [Track 2]. Impromptu: duo for clarinet and marimba / William A.R. May -- [Track 3]. Sonata in E-flat major, op. 120, no. 2. I. Allegro amabile ; II. Allegro appassionato ; III. Andante con moto, Allegro / Johannes Brahms -- [Track 4]. Adoration / Florence Price ; arr. Larkin Sanders -- [Track 5]. Blue skies / Irving Berlin ; arr. R. Percival

Concert recording 2022-10-02

[Track 1]. Grand duo concertant, op. 48. I. Allegro con fuoco ; II. Andante con moto ; III. Ronda, Allegro / Carl Maria von Weber -- [Track 2]. Impromptu: duo for clarinet and marimba / William A.R. May -- [Track 3]. Sonata in E-flat major, op. 120, no. 2. I. Allegro amabile ; II. Allegro appassionato ; III. Andante con moto, Allegro / Johannes Brahms -- [Track 4]. Adoration / Florence Price ; arr. Larkin Sanders -- [Track 5]. Blue skies / Irving Berlin ; arr. R. Percival

Developing inclusive digital health diagnostic for schistosomiasis: a need for guidance via target product profiles

IntroductionThe INSPIRED project aims to develop inclusive Digital Optical Diagnostic Devices (DODDs) for schistosomiasis, to support disease management by enabling rapid diagnostic results, to improve efficient data management to guide decision-making and to provide healthcare workers with critical health information to facilitate follow-up action. Due to the non-availability of Target Product Profiles (TPPs) for guiding the development of digital diagnostics for schistosomiasis, we explored existing diagnostic TPPs.MethodsUsing a curated open access database (Notion database), we studied a selection of TPPs for diagnosing infectious diseases, focusing on specifications related to digital health products for Neglected Tropical Diseases (NTDs).ResultsEighteen TPPs originating from 12 documents, covering 13 specific diseases, were selected and their characteristics were labeled and entered into the database. Further exploration of the database revealed several gaps, including a lack of stakeholder input, sustainability, and TPP availability. Other significant gaps related to digital health platform interconnectivity and data stewardship specifically in relation to digital diagnostics, including DODDs.DiscussionThese findings reflect two possible scenarios: (1) there is currently no need for digital diagnostic devices for schistosomiasis and, by extension for other NTDs; or (2) those needs are not yet covered by TPPs. Therefore, we recommend that digital health diagnostics are included in the use cases for schistosomiasis control and elimination, at least in the ideal/desirable scenario, as this will guide research and incentivize investment in digital health diagnostics for schistosomiasis

Two-stage automated diagnosis framework for urogenital schistosomiasis in microscopy images from low-resource settings

Purpose Automated diagnosis of urogenital schistosomiasis using digital microscopy images of urine slides is an essential step toward the elimination of schistosomiasis as a disease of public health concern in Sub-Saharan African countries. We create a robust image dataset of urine samples obtained from field settings and develop a two-stage diagnosis framework for urogenital schistosomiasis. Approach Urine samples obtained from field settings were captured using the Schistoscope device, and S. haematobium eggs present in the images were manually annotated by experts to create the SH dataset. Next, we develop a two-stage diagnosis framework, which consists of semantic segmentation of S. haematobium eggs using the DeepLabv3-MobileNetV3 deep convolutional neural network and a refined segmentation step using ellipse fitting approach to approximate the eggs with an automatically determined number of ellipses. We defined two linear inequality constraints as a function of the overlap coefficient and area of a fitted ellipses. False positive diagnosis resulting from over-segmentation was further minimized using these constraints. We evaluated the performance of our framework on 7605 images from 65 independent urine samples collected from field settings in Nigeria, by deploying our algorithm on an Edge AI system consisting of Raspberry Pi + Coral USB accelerator. Result The SH dataset contains 12,051 images from 103 independent urine samples and the developed urogenital schistosomiasis diagnosis framework achieved clinical sensitivity, specificity, and precision of 93.8%, 93.9%, and 93.8%, respectively, using results from an experienced microscopist as reference. Conclusion Our detection framework is a promising tool for the diagnosis of urogenital schistosomiasis as our results meet the World Health Organization target product profile requirements for monitoring and evaluation of schistosomiasis control programs

An adaptive phase II/III safety and efficacy randomized controlled trial of single day or three-day fixed-dose albendazole-ivermectin co-formulation versus albendazole for the treatment of Trichuris trichiura and other STH infections. ALIVE trial protocol

Background: Soil-transmitted helminths (STH) are targeted for control through mass drug-administration campaigns to prevent morbidity affecting at-risk groups in endemic regions. Although broadly successful, the use of albendazole and mebendazole achieved variable progress, with deficiencies against Trichuris trichiura and a predictable low efficacy against Strongyloides stercoralis. Novel drug combinations offer a potential solution, providing they can be delivered safely and maintain efficacy against all STH species. Here we present the protocol of a clinical trial to evaluate a fixed-dose combination (FDC) tablet containing albendazole and ivermectin that will be compared against albendazole against STH. Methods: An adaptive phase II/III randomized controlled trial will be undertaken in STH endemic sites in Ethiopia, Kenya and Mozambique to evaluate an oral FDC of 400 mg albendazole and either 9- or 18 mg ivermectin. FDC will be administered as a single dose or single doses over three-consecutive days and assessed against a single dose of 400 mg albendazole. In the phase II trial, 126 T. trichiura-infected children weighting 15 to 45 kg will be treated in a dose-escalation manner to determine safety objectives. In the phase III trial, 1097 participants aged 5 to 18 years old infected with T. trichiura, hookworm and S. stercoralis will be recruited to determine safety and efficacy. The trial will be open-label with blinded outcome assessors. Cure rate measured 21-days after-treatment in duplicate Kato-Katz is the primary efficacy outcome. Secondary objectives include efficacy evaluation by quantitative polymerase chain reaction (PCR) as an outcome measurement, description of pharmacokinetic parameters, palatability and acceptability evaluations, and monitoring of anthelmintic resistance. Conclusions: This trial with registrational goals seeks to evaluate an innovative fixed-dose combination of albendazole and ivermectin co-formulated tablets, with the goal of providing an anthelmintic regimen with improved efficacy and spectrum of coverage against STH. ClinicalTrials.gov registration: NCT05124691 (18/11/2021)

Performance evaluation of the Schistoscope 5.0 for (semi-)automated digital detection and quantification of schistosoma haematobium eggs in Urine: A field-based study in Nigeria

Conventional microscopy is the standard procedure for the diagnosis of schistosomiasis, despite its limited sensitivity, reliance on skilled personnel, and the fact that it is error prone. Here, we report the performance of the innovative (semi-)automated Schistoscope 5.0 for optical digital detection and quantification of Schistosoma haematobium eggs in urine, using conventional microscopy as the reference standard. At baseline, 487 participants in a rural setting in Nigeria were assessed, of which 166 (34.1%) tested S. haematobium positive by conventional microscopy. Captured images from the Schistoscope 5.0 were analyzed manually (semiautomation) and by an artificial intelligence (AI) algorithm (full automation). Semi- and fully automated digital microscopy showed comparable sensitivities of 80.1% (95% confidence interval [CI]: 73.2–86.0) and 87.3% (95% CI: 81.3–92.0), but a significant difference in specificity of 95.3% (95% CI: 92.4–97.4) and 48.9% (95% CI: 43.3–55.0), respectively. Overall, estimated egg counts of semi- and fully automated digital microscopy correlated significantly with the egg counts of conventional microscopy (r = 0.90 and r = 0.80, respectively, P < 0.001), although the fully automated procedure generally underestimated the higher egg counts. In 38 egg positive cases, an additional urine sample was examined 10 days after praziquantel treatment, showing a similar cure rate and egg reduction rate when comparing conventional microscopy with semiautomated digital microscopy. In this first extensive field evaluation, we found the semiautomated Schistoscope 5.0 to be a promising tool for the detection and monitoring of S. haematobium infection, although further improvement of the AI algorithm for full automation is required

Validation of artificial intelligence-based digital microscopy for automated detection of Schistosoma haematobium eggs in urine in Gabon

Introduction Schistosomiasis is a significant public health concern, especially in Sub-Saharan Africa. Conventional microscopy is the standard diagnostic method in resource-limited settings, but with limitations, such as the need for expert microscopists. An automated digital microscope with artificial intelligence (Schistoscope), offers a potential solution. This field study aimed to validate the diagnostic performance of the Schistoscope for detecting and quantifying Schistosoma haematobium eggs in urine compared to conventional microscopy and to a composite reference standard (CRS) consisting of real-time PCR and the up-converting particle (UCP) lateral flow (LF) test for the detection of schistosome circulating anodic antigen (CAA). Methods Based on a non-inferiority concept, the Schistoscope was evaluated in two parts: study A, consisting of 339 freshly collected urine samples and study B, consisting of 798 fresh urine samples that were also banked as slides for analysis with the Schistoscope. In both studies, the Schistoscope, conventional microscopy, real-time PCR and UCP-LF CAA were performed and samples with all the diagnostic test results were included in the analysis. All diagnostic procedures were performed in a laboratory located in a rural area of Gabon, endemic for S. haematobium. Results In study A and B, the Schistoscope demonstrated a sensitivity of 83.1% and 96.3% compared to conventional microscopy, and 62.9% and 78.0% compared to the CRS. The sensitivity of conventional microscopy in study A and B compared to the CRS was 61.9% and 75.2%, respectively, comparable to the Schistoscope. The specificity of the Schistoscope in study A (78.8%) was significantly lower than that of conventional microscopy (96.4%) based on the CRS but comparable in study B (90.9% and 98.0%, respectively). Conclusion Overall, the performance of the Schistoscope was non-inferior to conventional microscopy with a comparable sensitivity, although the specificity varied. The Schistoscope shows promising diagnostic accuracy, particularly for samples with moderate to higher infection intensities as well as for banked sample slides, highlighting the potential for retrospective analysis in resource-limited settings

Towards Inclusive Diagnostics for Neglected Tropical Diseases:: User Experience of a New Digital Diagnostic Device in Low-Income Settings

Designing new and inclusive diagnostic tools to detect Neglected Tropical Diseases (NTDs) to achieve rational disease control requires a co-design process where end-users’ input is important. Failure to involve all potential end-users in new diagnostics for NTDs can result in low use and adoption failure, leading to persistent infection hot spots and ineffective disease control. There are different categories of potential end-users of new diagnostic tools for NTD control, and it is unclear if there are differences between the user efficiency, effectiveness, perception, and acceptability across these end-user categories. This study evaluated the usability, user perception, contextual factors affecting the user’s experience, and acceptability of a new digital optical diagnostic device for NTDs across three types of potential end users. A total of 21 participants were tested. Laboratory scientists, technicians, and Community Health Extension Workers (CHEWs) in training achieved similar scores on the usability and user perception questionnaires with no statistically significant difference between end-user categories. All participants also have high scores for the user perception domains which strongly correlate with the acceptability of the AiDx NTDx Assist device. This study indicates that, by providing digital diagnostic tools in combination with minimal training and support, CHEWs undergoing training and, by extension, CHEWs post-training, can be involved in the diagnoses of NTDs, potentially enhancing a community’s capabilities to diagnose, treat, and control NTDs