Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03–mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9–mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches

Molecular epidemiology and population genetics of Schistosoma mansoni infecting school-aged children situated along the southern shoreline of Lake Malawi, Malawi

Background In areas of low disease endemicity, highly sensitive diagnostic tools to identify, diagnose, and monitor intestinal schistosomiasis transmission are needed to reliably measure the burden and risk of infection. Here, we used highly sensitive molecular diagnostic methods to investigate Schistosoma mansoni prevalence and transmission along the southern shoreline of Lake Malawi, five years post-disease outbreak. Methodology and principal findings Faecal and urine samples were provided by school-aged children situated along the southern shoreline of Lake Malawi. Kato-Katz faecal-egg microscopy and point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic tests were then performed to diagnose infection with S. mansoni. Urine-egg microscopy was also used to diagnose infection with Schistosoma haematobium. In addition, Schistosoma miracidia were isolated from faecal material using a standard miracidium hatching technique. A two-step real-time PCR approach was then used to diagnose infection with S. mansoni using DNA isolated from faecal samples. Furthermore, isolated miracidia were genotyped to species level through PCR and Sanger sequencing. Phylogenetic analyses were then carried out to identify which previously defined S. mansoni cox1 lineage group S. mansoni miracidia were most closely related to. The measured prevalence of S. mansoni infection varied considerably depending on which diagnostic assay was used. When compared to real-time PCR, faecal-egg microscopy had a sensitivity of 9% and a specificity of 100%. When POC-CCA ‘trace’ results were considered positive, POC-CCA had a sensitivity of 73% and a specificity of 81% when compared to real-time PCR. However, when considered negative, POC-CCA sensitivity was reduced to 56%, whereas specificity was increased to 90%. In addition, a high degree of S. haematobium DNA was detected in DNA isolated from faecal samples and motile S. haematobium miracidia were recovered from faecal samples. Schistosoma mansoni miracidia were closely related to two independent cox1 lineage groups, suggesting multiple recent introduction and colonisation events originating from surrounding east African countries. Conclusions and significance Intestinal schistosomiasis is now highly prevalent along the southern shoreline of Lake Malawi just five years post-disease outbreak. In addition, a high prevalence of urogenital schistosomiasis persists. The revision of ongoing schistosomiasis control programmes in this area is therefore recommended. Our study also highlights the need for reliable diagnostic assays capable of distinguishing between Schistosoma species in multispecies co-endemic areas

Sepsis prevalence and outcome on the general wards and emergency departments in Wales: Results of a multi-centre, observational, point prevalence study

Data on sepsis prevalence on the general wards is lacking on the UK and in the developed world. We conducted a multicentre, prospective, observational study of the prevalence of patients with sepsis or severe sepsis on the general wards and Emergency Departments (ED) in Wales. During the 24-hour study period all patients with NEWS≥3 were screened for presence of 2 or more SIRS criteria. To be eligible for inclusion, patients had to have a high clinical suspicion of an infection, together with a systemic inflammatory response (sepsis) and evidence of acute organ dysfunction and/or shock (severe sepsis). There were 5317 in-patients in the 24-hour study period. Data were returned on 1198 digital data collection forms on patients with NEWS≥3 of which 87 were removed, leaving 1111 for analysis. 146 patients had sepsis and 144 patients had severe sepsis. Combined prevalence of sepsis and severe sepsis was 5.5% amongst all in-patients. Patients with sepsis had significantly higher NEWS scores (3 IQR 3–4 for non-sepsis and 4 IQR 3–6 for sepsis patients, respectively). Common organ dysfunctions in severe sepsis were hypoxia (47%), hypoperfusion (40%) and acute kidney injury (25%). Mortality at 90 days was 31% with a median (IQR) hospital free stay of 78 (36–85) days. Screening for sepsis, referral to Critical Care and completion of Sepsis 6 bundle was low: 26%, 16% and 12% in the sepsis group. Multivariable logistic regression analysis identified higher National Early Warning Score, diabetes, COPD, heart failure, malignancy and current or previous smoking habits as independent variables suggesting the diagnosis of sepsis. We observed that sepsis is more prevalent in the general ward and ED than previously suggested before and that screening and effective treatment for sepsis and severe sepsis is far from being operationalized in this environment, leading to high 90 days mortality

Detection of male genital schistosomiasis (MGS) associated with human, zoonotic and hybrid schistosomes in Southern Malawi

Background Male Genital Schistosomiasis (MGS) remains an often-overlooked chronic sequela of urogenital schistosomiasis in endemic areas of sub-Saharan Africa. As part of a 2-year longitudinal study on Hybridization of UroGenital Schistosomiasis (HUGS) in Malawi, a MGS sub-study was conducted to assess whether hybrid schistosomes were incriminated. Methods During recruitment, demographic, health and socio-economic data were collected through individual questionnaire interviews in Mthawira community from Nsanje District along Shire River and Samama community from Mangochi District along Lake Malawi shoreline. Urine and semen samples were collected and analysed to determine the identity of schistosome infection. Urine filtration and microscopy, direct microscopy of semen and its sediments (after centrifugation) were performed. Thereafter, the sediments were examined by molecular DNA analysis with a novel two-tube real-time PCR assay. The participants were also screened for Human papilloma virus (HPV) and other sexually transmitted infections (STIs). Results Twenty-two men were recruited for the sub-study, 8 in Nsanje District and 14 in Mangochi District, with a median age of 22.0 years. By microscopy, ten (45.7%) participants had Schistosoma ova in their urine, 11 (50.0%) in semen while 16 (72.7%) were positive by real-time PCR. One participant had both S. haematobium and S. mattheei ova in his semen, three showed symptoms, and one had a mixed infection of S. mansoni and possible S. haematobium-S. mattheei hybrid. Twelve men had detectable high-risk HPV serotypes 16, 18 and others while six had Trichomonas vaginalis and other STIs. Conclusion Zoonotic and hybrid schistosomes can cause MGS similar to human schistosomes, which can be co-infected with HPV and STIs, thereby posing a new challenge in diagnosis, management and control measures in resource poor settings. Increased awareness of these infections among local communities and primary healthcare workers and improvement of disease management are needed and advocated

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Establishing a statement of principles for community engagement with civil engineering

Community engagement with civil engineering is essential to deliver the United Nations sustainable development goals and to address ‘wicked’ problems such as climate change. This paper explains how the Institution of Civil Engineers developed its statement of Principles for Community Engagement with Engineering to underpin best practice across the infrastructure project lifecycle. The principles are intended to be adaptable to suit a range of contexts, sectors and scales of project, and to support civil engineers at different stages of their career and levels of influence. They provide a foundation for further development of best-practice case studies and guidance, to be shared through civil engineering education and professional development

Developing a digital data collection platform to measure the prevalence of sepsis in Wales

Objective To develop a secure, efficient, and easy-to-use data collection platform to measure the prevalence of sepsis in Wales over 24 hours. Materials and Methods Open Data Kit was used on Android devices with Google App Engine and a digital data collection form. Results A total of 184 students participated in the study using 59 devices across 16 hospitals, 1198 datasets were submitted, and 97% of participants found the Open Data Kit form easy to use. Discussion We successfully demonstrated that by combining a reliable Android device, a free open-source data collection framework, a scalable cloud-based server, and a team of 184 medical students, we can deliver a low-cost, highly reliable platform that requires little training or maintenance, providing results immediately on completion of data collection. Conclusion Our platform allowed us to measure, for the first time, the prevalence of sepsis in Wales over 24 hours

Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03–mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9–mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches