Longitudinal assessment of female carriers of choroideremia using multimodal retinal imaging.

BACKGROUND/AIMS: Female choroideremia carriers present with a spectrum of disease severity. Unlike in men, the rate of disease progression has not been well characterised in carriers. This longitudinal study aimed to determine the rate of retinal degeneration in choroideremia carriers, using multimodal imaging and microperimetry. METHODS: Choroideremia carriers previously seen at Oxford Eye Hospital (United Kingdom) between 2012 and 2017 returned for testing between 2015 and 2023, providing up to 11 years' follow-up data. Participants had optical coherence tomography, fundus-tracked microperimetry and fundus autofluorescence (FAF) imaging performed. RESULTS: Thirty-four eyes of 17 choroideremia carriers were examined using multimodal imaging. Median age was 44 (range: 15-73) years at baseline and median follow-up duration was 7 (range: 1-11) years. At baseline, phenotype was classified as fine (n=5 eyes), coarse (n=13 eyes), geographic (n=12 eyes) or male pattern (n=4 eyes). Thirteen patients showed no change in phenotype classification, four showed slight changes associated with choroideremia-related retinal degeneration. Despite this, carriers with severe retinal phenotypes had a statistically significant decline in average retinal sensitivity (-0.7 dB and -0.8 dB per year, respectively, p<0.001), area of geographic loss defined by FAF (+2.5 mm2 and +3.7 mm2 per year, respectively, p<0.001) and thinning of the photoreceptor complex (up to -2.8 microns and -10.3 microns per year, p<0.001). CONCLUSION: Choroideremia carriers, particularly those with severe retinal phenotypes, exhibit progressive retinal degeneration, as evident by multimodal imaging biomarkers and functional testing. Clinicians should not rely on retinal severity classification alone to assess disease progression.

What do Brazilian paediatric dentists know about bruxism in children? Cross-sectional study.

PURPOSE: Little is known about the knowledge of paediatric dentists on bruxism in children. The aim of this cross-sectional study was to assess the knowledge of paediatric dentists on the concept, associated factors of bruxism and management of sleep bruxism (SB) in children'. METHODS: An electronic questionnaire was sent to paediatric dentists in the state of Goiás, Brazil. Information was collected on (1) characteristics of the participants; (2) the concept of bruxism; (3) diagnosis; (4) associated factors; (5) strategies for the management of SB; and (6) updated knowledge on bruxism in children. The data were analysed descriptively. RESULTS: Fifty-seven paediatric dentists participated (10.7% of the total number of professionals). A high level of agreement was found with statements on the concepts of SB (94.7%) and awake bruxism (96.5%). The main strategy for the diagnosis was the combination of a parental report and a clinical examination (79.0%). Most participants indicated that bruxism is associated with anxiety/stress (96.5%), screen use (93%), airway obstruction (91.2%), and sleep apnoea (91.2%). In suspected cases of bruxism, the dentists would send the child for assessment by other health care providers (87.7%). The management options frequently indicated were the use of an occlusal splint, aromatherapy, and homeopathy. More than 70% of them considered themselves to be updated on the issue and sought information through scientific articles and discussions with colleagues. CONCLUSION: Paediatric dentists have knowledge on the concept of bruxism and associated factors. However, further information is needed on the management of this condition in children.

Retinal Characteristics of Female Choroideremia Carriers: Multimodal Imaging, Microperimetry, and Genetics.

PURPOSE: To describe visual function and retinal features of female carriers of choroideremia (CHM), using multimodal imaging and microperimetry. DESIGN: Cross-sectional cohort study. PARTICIPANTS AND CONTROLS: Choroideremia carriers seen in Australia (Melbourne or Perth) or the United Kingdom (Oxford or Cambridge) between 2012 and 2023. Healthy age-matched controls seen in Melbourne, Australia, between 2022 and 2023. METHODS: Participants had visual acuity, fundus-tracked microperimetry, OCT, and fundus autofluorescence imaging performed. Choroideremia carriers were either genetically or clinically confirmed (i.e., obligate carriers). Choroideremia carriers were grouped according to their retinal phenotype and compared with healthy controls. Statistical analyses were performed on StataBE (v18.0). MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), low-luminance visual acuity (LLVA), average retinal sensitivity, volume of macular hill of vision (HoV), inner retinal thickness, and photoreceptor complex (PRC) thickness. RESULTS: Eighty-six eyes of 43 CHM carriers and 60 eyes of 30 healthy controls were examined using multimodal imaging and microperimetry. Median age was 54 and 48.5 years for CHM carriers and controls, respectively (P = 0.18). Most CHM carriers (86%) were genetically confirmed. Choroideremia carriers and controls had strong intereye correlation between eyes for BCVA and average retinal sensitivity (P < 0.001). Low-luminance visual acuity and macular HoV tests were sensitive tests to detect changes in CHM carriers with mild phenotypes (i.e., fine and coarse). Choroideremia carriers with geographic or male-pattern phenotypes had reduced BCVA, LLVA, retinal sensitivity, and retinal thinning, compared with healthy controls. Retinal thickening of the inner retina was observed in the central 1°, despite generalized thinning of the PRC in the central 7°, indicating retinal remodeling in CHM carriers, compared with controls. There were no genotype-phenotype correlations observed. CONCLUSIONS: Female carriers of CHM with severe retinal phenotypes (i.e., geographic or male pattern) have significantly decreased visual function and retinal structural changes when compared with age-matched controls and those carriers with milder phenotypes. Low-luminance visual acuity and volumetric measures of the macular HoV were found to be the most sensitive functional tests to detect milder retinal disease (fine and coarse phenotypes) in CHM carriers. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Rusa deer microbiota: the importance of preliminary data analysis for meaningful diversity comparisons.

The microbiome is an important consideration for the conservation of endangered species. Studies provided evidence of the effect of behavior and habitat change on the microbiota of wild animals and reported various inferences. It indicates the complexity of factors influencing microbiota diversity, including incomplete sampling procedures. Data abnormality may arise due to the procedures warranting preliminary analysis, such as rarefaction, before downstream analysis. This present study demonstrated the effect of data rarefaction and aggregation on the comparison of wild rusa deer's gut microbial diversity. Eighty-five feces samples were collected from 11 deer populations inhabiting three national parks in Java and Bali islands. Using the Illumina Nova-Seq platform, fragments of 16s rRNA gene were sequenced, and raw data of 51,389 reads corresponding to 2 domains, 22 phyla, 45 classes, 83 orders, 182 families, and 460 genera of bacteria were obtained. Data rarefaction was applied at two different library sizes (minimum and fixed) and aggregation (11 populations into 3 research sites) to investigate its effect on the microbial diversity comparison. There are significant differences in alpha diversity between populations, but not research sites, at all library sizes of rarefaction. A similar finding is also found in beta diversity. Moreover, data rarefaction and aggregation result in different values of the diversity metrics. This present study shows that statistical analysis remains a substantial concern in microbiome studies applied to conservation biology. It suggests reporting a more detailed data normalization in microbiome studies as an inherent control of suboptimal sampling, particularly when involving feces.

Perspectives of carriers of X-linked retinal diseases on genetic testing and gene therapy: A global survey.

Female carriers of X-linked inherited retinal diseases (IRDs) are burdened with potentially passing their disease-causing variant to future generations, as well as exhibiting signs of retinal disease themselves. This study aimed to investigate carriers' experiences of genetic testing, emotions relating to having affected children, and their knowledge regarding genetic testing and gene therapy. An online survey was advertised to self-identified carriers worldwide. Two hundred and twenty-eight carriers completed the survey with mean age of 51 years (SD ± 15.0). A majority of respondents resided in the United States of America (51%), Australia (19%), and the United Kingdom (14%). Most carriers identified with feelings of guilt (70%), concern (91%), and anxiety (88%) for their child. Female carriers who had given birth to children had significantly greater gene therapy knowledge compared to carriers who had not (p < 0.05). Respondents agreed that their eyecare provider and general practitioner helped them understand their condition (63%), however, few carriers reported receiving psychological counselling (9%) or family planning advice (5%). Most respondents (78%) agreed that gene therapy should be available to carriers. This study emphasises the importance of providing appropriate counselling to female carriers and illustrates the motivation of many to participate in emerging treatment options, such as gene therapy.

Female carriers of X-linked inherited retinal diseases - Genetics, diagnosis, and potential therapies.

Inherited retinal diseases (IRDs) are a group of heterogeneous conditions that cause progressive vision loss, typically due to monogenic mutations. Female carriers of X-linked IRDs have a single copy of the disease-causing gene, and therefore, may exhibit variable clinical signs that vary from near normal retina to severe disease and vision loss. The relationships between individual genetic mutations and disease severity in X-linked carriers requires further study. This review summarises the current literature surrounding the spectrum of disease seen in female carriers of choroideremia and X-linked retinitis pigmentosa. Various classification systems are contrasted to accurately grade retinal disease. Furthermore, genetic mechanisms at the early embryonic stage are explored to potentially explain the variability of disease seen in female carriers. Future research in this area will provide insight into the association between genotype and retinal phenotypes of female carriers, which will guide in the management of these patients. This review acknowledges the importance of identifying which patients may be at high risk of developing severe symptoms, and therefore should be considered for emerging treatments, such as retinal gene therapy.

The Diagnostic Yield of Next Generation Sequencing in Inherited Retinal Diseases: A Systematic Review and Meta-analysis.

PURPOSE: Accurate genotyping of individuals with inherited retinal diseases (IRD) is essential for patient management and identifying suitable candidates for gene therapies. This study evaluated the diagnostic yield of next generation sequencing (NGS) in IRDs. DESIGN: Systematic review and meta-analysis. METHODS: This systematic review was prospectively registered (CRD42021293619). Ovid MEDLINE and Ovid Embase were searched on 6 June 2022. Clinical studies evaluating the diagnostic yield of NGS in individuals with IRDs were eligible for inclusion. Risk of bias assessment was performed. Studies were pooled using a random...effects inverse variance model. Sources of heterogeneity were explored using stratified analysis, meta-regression, and sensitivity analysis. RESULTS: This study included 105 publications from 28 countries. Most studies (90 studies) used targeted gene panels. The diagnostic yield of NGS was 61.3% (95% confidence interval: 57.8-64.7%; 51 studies) in mixed IRD phenotypes, 58.2% (51.6-64.6%; 41 studies) in rod-cone dystrophies, 57.7% (46.8-68.3%; eight studies) in macular and cone/cone-rod dystrophies, and 47.6% (95% CI: 41.0-54.3%; four studies) in familial exudative vitreoretinopathy. For mixed IRD phenotypes, a higher diagnostic yield was achieved pooling studies published between 2018-2022 (64.2% [59.5-68.7%]), studies using exome sequencing (73.5% [58.9-86.1%]), and studies using the American College of Medical Genetics variant interpretation standards (65.6% [60.8-70.4%]). CONCLUSION: The current diagnostic yield of NGS in IRDs is between 52-74%. The certainty of the evidence was judged as low or very low. A key limitation of the current evidence is the significant heterogeneity between studies. Adoption of standardized reporting guidelines could improve confidence in future meta-analyses.

Primary eyecare provision for people living with dementia: what do we need to know?

Dementia comprises a group of brain disorders characterised by loss of cognitive function. Sensory loss, predominantly vision (the focus of this review) and hearing, is a significant problem for people living with dementia. Eyecare practitioners such as optometrists therefore play an important role in identifying and addressing vision-related care needs. To support provision of high quality "dementia-friendly" eyecare, this scoping review summarises recent primary research findings and available clinical practice guidelines, to identify research gaps relating to vision and dementia, and make recommendations for future research and clinical practice. The review set a priori guidelines for the population, concept and context based on the review questions. Primary research papers (2016-2021) were included via 3-step search strategy: preliminary search to index terms, full search, search reference lists of included articles for further inclusions. Additionally, websites of eyecare professional bodies in English-speaking countries were searched to identify current clinical eyecare practice guidelines relating to dementia. Study characteristics (e.g. country, study design) were reported descriptively. Patterns within findings/recommendations from included sources were identified using thematic analysis and reported as themes. 1651 titles/abstracts and 161 full-text articles were screened for eligibility. Three clinical practice guidelines were also identified. The final review included 21 sources: 18 primary research papers and 3 clinical practice guidelines. The thematic analysis reported five key themes: Diagnosis/Screening, dementia progression, findings on clinical visual testing, tailored approach to care, improving care. This scoping review demonstrated limited information about current practices of optometrists working with people living with dementia. Recent evidence reinforces the continuing need for improved eyecare for people living with dementia, taking into account their specific needs with an individualised approach. Up-to-date practical recommendations are synthesised for eyecare providers before, during and after a consultation with a person living with dementia, to better support their care.

Genetic Testing of Inherited Retinal Disease in Australian Private Tertiary Ophthalmology Practice.

Background: To assess the prevalence of genetic testing for inherited retinal diseases (IRDs) in a tertiary practice setting. Methods: Single-centre retrospective analysis of patients with diagnosed or suspected IRD. Results: Four hundred and sixty-four patient records were analysed. Patients had received care for different IRDs grouped as follows: panretinal pigmentary retinopathies (283, 61%), macular dystrophies (136, 29.3%), stationary diseases (23, 5%), hereditary vitreoretinopathies (14, 3%), and other IRDs (8, 1.7%). The suspected pattern of inheritance of patients' IRD was predominantly autosomal recessive (205, 44.2%). Genetic testing was performed with the corresponding results available for 44 patients (9.5%). Diagnostic yield was 65.9% for the results received. Genetic test results were available mostly for younger patients (13.1% for <45 years vs 6.2% ≥45 years of age, p = 0.01) and those who received greater than 12 months of care (16% for ≥12 months vs 4% for <12 months, p < 0.01). For patients without genetic testing results, reasons include awaiting a geneticist consultation (17.9%), awaiting test results (4.5%), or patient refusal (8.4%). Most clinical records (69.2%) did not document genetic testing status. Conclusion: Genetic testing is increasingly being utilised in the work-up for patients with IRD worldwide. This large Australian private practice IRD cohort shows a low uptake of testing (around 10%), reflecting historical management patterns and accessibility of genetic counselling and testing. The results show that younger patients and those with a longer duration of care were more likely to have received genetic testing. As the importance of IRD genetic testing continues to increase, we expect to see a change in patient management within the Australian private ophthalmology system and testing rates to increase. Further research is required to identify and address clinician and patient barriers to improving genetic testing rates for IRD.

Point-of-care tools to support optometric care provision to people with age-related macular degeneration: A randomised, placebo-controlled trial.

PURPOSE: Age-related macular degeneration (AMD) is a leading cause of vision impairment. This randomised placebo-controlled trial investigated whether point-of-care tools can improve optometrists' AMD knowledge and/or care provision. METHODS: Australian optometrists (n = 31) completed a demographics survey and theoretical AMD case study multiple-choice questions (MCQs) to assess their confidence in AMD care provision and AMD knowledge. Participants were then randomly assigned to one of three point-of-care tools (online 'Classification of Age-related macular degeneration and Risk Assessment Tool' (CARAT), paper CARAT, or 'placebo') to use when providing care to their subsequent 5-10 AMD patients. Participants self-audited the compliance of their AMD care to best practice for these patients, and a similar number of consecutive patients seen prior to enrolment. Post-intervention, participants retook the AMD knowledge MCQs and confidence survey. RESULTS: A total of 29 participants completed the study. At the study endpoint, clinical confidence relative to baseline improved with the paper CARAT, relative to placebo, for knowledge of AMD risk factors, asking patients about these factors and referring for medical retinal sub-specialist care. There were no between-group differences for the change in AMD knowledge scores. Considering record documentation for patients with any AMD severity, there were no significant between-group differences for documenting patient risk factors, AMD severity, clinical examination techniques or management. In a sub-analysis, the change from baseline in compliance for documenting discussions about patient smoking behaviours for early AMD patients was higher with use of the online CARAT relative to placebo (p = 0.04). For patients with intermediate AMD, the change from baseline in documenting the risk of progression to late AMD was greater among practitioners who used the paper CARAT, relative to placebo (p = 0.04). CONCLUSIONS: This study demonstrates that point-of-care clinical tools can improve practitioner confidence and aspects of the documentation of AMD clinical care by optometrists as assessed by self-audit.

The safety and efficacy of gene therapy treatment for monogenic retinal and optic nerve diseases: A systematic review.

PURPOSE: This study aimed to systematically review and summarize gene therapy treatment for monogenic retinal and optic nerve diseases. METHODS: This review was prospectively registered (CRD42021229812). A comprehensive literature search was performed in Ovid MEDLINE, Ovid Embase, Cochrane Central, and clinical trial registries (February 2021). Clinical studies describing DNA-based gene therapy treatments for monogenic posterior ocular diseases were eligible for inclusion. Risk of bias evaluation was performed. Data synthesis was undertaken applying Synthesis Without Meta-analysis guidelines. RESULTS: This study identified 47 full-text publications, 50 conference abstracts, and 54 clinical trial registry entries describing DNA-based ocular gene therapy treatments for 16 different genetic variants. Study summaries and visual representations of safety and efficacy outcomes are presented for 20 unique full-text publications in RPE65-mediated retinal dystrophies, choroideremia, Leber hereditary optic neuropathy, rod-cone dystrophy, achromatopsia, and X-linked retinoschisis. The most common adverse events were related to lid/ocular surface/cornea abnormalities in subretinal gene therapy trials and anterior uveitis in intravitreal gene therapy trials. CONCLUSION: There is a high degree of variability in ocular monogenic gene therapy trials with respect to study design, statistical methodology, and reporting of safety and efficacy outcomes. This review improves the accessibility and transparency in interpreting gene therapy trials to date.

Clinical audit as an educative tool for optometrists: an intervention study in age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is a major cause of vision loss. This study investigated whether performing clinical audit and receiving analytical performance feedback altered documentation of the AMD care provided by optometrists. METHODS: Australian optometrists were recruited and completed a survey about their demographics and confidence in AMD care, and a three-month audit of their practice records using an AMD audit tool (termed the pre-audit evaluation). After receiving analytical feedback, participants identified areas for improvement and re-audited their practices after three months to analyse changes in performance (termed the post-audit evaluation). Paired t-tests and Wilcoxon signed-rank tests, as appropriate, were used to compare pre- and post-audit data. RESULTS: Twenty optometrists, most practising in Victoria, Australia, completed the study. Participants primarily worked in corporate practice and/or rural settings and had a range of optometric experience (2-40 years). At baseline, participants felt confident in their: knowledge of AMD risk factors (65%), advice to patients about these factors (55%) and management of earlier stages of AMD (55%). Each clinician completed (median [IQR]): 15 [IQR: 10-19] and 12 [IQR: 8-16] audits of unique patient records, pre- and post-audit, respectively. Post-audit, average record documentation (per optometrist) improved for asking about: AMD family history (94% to 100%, p = 0.03), smoking status (21% to 58%, p < 0.01), diet (11% to 29%, p < 0.01) and nutritional supplementation (20% to 51%, p < 0.01). For clinical examination, compliance with documenting pinhole visual acuity, performing an in-office Amsler grid (upon indication) and using optical coherence tomography improved post-audit (p < 0.05). Accuracy of severity documentation improved for earlier stages of AMD (p < 0.05). For earlier stages of AMD, documentation of counselling about modifiable risk factors significantly improved post-audit (p < 0.05). Aspects well-performed pre-audit that did not change included documenting: medical histories (100% at both time points, p = 0.06) and retinal imaging (77% at both time points, p = 0.97). CONCLUSIONS: Self-audit with analytical feedback improved clinical record documentation of: AMD risk factors, clinical examination, AMD severity classification and management advice. These findings support a role for audit to improve optometric clinical care of AMD, as evidenced by improved documentation of the AMD care delivered.

Plasmodium falciparum Kelch Propeller Polymorphisms in Clinical Isolates from Ghana from 2007 to 2016.

The continuous surveillance of polymorphisms in the kelch propeller domain of Plasmodium falciparum from Africa is important for the discovery of the actual markers of artemisinin resistance in the region. The information on the markers is crucial for control strategies involving chemotherapy and chemoprophylaxis for residents and nonimmune travelers to the country. Polymorphisms in the kelch propeller domain of Ghanaian malaria parasites from three different ecological zones at several time periods were assessed. A total of 854 archived samples (2007 to 2016) collected from uncomplicated malaria patients aged ≤9 years old from 10 sentinel sites were used. Eighty-four percent had wild-type sequences (PF3D7_1343700), while many of the mutants had mostly nonsynonymous mutations clustered around codons 404 to 650. Variants with different amino acid changes of the codons associated with artemisinin (ART) resistance validated markers were observed in Ghanaian isolates: frequencies for I543I, I543S, I543V, R561P, R561R, and C580V were 0.12% each and 0.6% for R539I. Mutations reported from African parasites, A578S (0.23%) and Q613L (0.23%), were also observed. Three persisting nonsynonymous (NS) mutations, N599Y (0.005%), K607E (0.004%), and V637G (0.004%), were observed in 3 of the 5 time periods nationally. The presence of variants of the validated markers of artemisinin resistance as well as persisting polymorphisms after 14 years of artemisinin-based combination therapy use argues for continuous surveillance of the markers. The molecular markers of artemisinin resistance and the observed variants will be monitored subsequently as part of ongoing surveillance of antimalarial drug efficacy/resistance studies in the country.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine combinations for uncomplicated malaria in 10 sentinel sites across Ghana: 2015-2017.

BACKGROUND: Routine surveillance on the therapeutic efficacy of artemisinin-based combination therapy (ACT) has been ongoing in Ghana since 2005. The sixth round of surveillance was conducted between 2015 and 2017 to determine the therapeutic efficacy of artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) in 10 sentinel sites across the country. METHODS: The study was a one-arm, prospective, evaluation of the clinical, parasitological, and haematological responses to directly observed treatment with AS-AQ and AL among children 6 months to 9 years old with uncomplicated falciparum malaria. The WHO 2009 protocol on surveillance of anti-malaria drug efficacy was used for the study with primary outcomes as prevalence of day 3 parasitaemia and clinical and parasitological cure rates on day 28. Secondary outcomes assessed included patterns of fever and parasite clearance as well as changes in haemoglobin concentration. RESULTS: Day 3 parasitaemia was absent in all sites following treatment with AS-AQ whilst only one person (0.2%) was parasitaemic on day 3 following treatment with AL. Day 28 PCR-corrected cure rates following treatment with AS-AQ ranged between 96.7% (95% CI 88.5-99.6) and 100%, yielding a national rate of 99.2% (95% CI 97.7-99.7). Day 28 PCR-corrected cure rates following treatment with AL ranged between 91.3% (95% CI 79.2-97.6) and 100%, yielding a national rate of 96% (95% CI 93.5-97.6). Prevalence of fever declined by 88.4 and 80.4% after first day of treatment with AS-AQ and AL, respectively, whilst prevalence of parasitaemia on day 2 was 2.1% for AS-AQ and 1.5% for AL. Gametocytaemia was maintained at low levels (< 5%) during the 3 days of treatment. Post-treatment mean haemoglobin concentration was significantly higher than pre-treatment concentration following treatment with either AS-AQ or AL. CONCLUSIONS: The therapeutic efficacy of AS-AQ and AL is over 90% in sentinel sites across Ghana. The two anti-malarial drugs therefore remain efficacious in the treatment of uncomplicated malaria in the country and continue to achieve rapid fever and parasite clearance as well as low gametocyte carriage rates and improved post-treatment mean haemoglobin concentration.

Amplification of GTP-cyclohydrolase 1 gene in Plasmodium falciparum isolates with the quadruple mutant of dihydrofolate reductase and dihydropteroate synthase genes in Ghana.

Sulfadoxine-pyrimethamine (SP) is used as malaria chemoprophylaxis for pregnant women and children in Ghana. Plasmodium falciparum resistance to SP is linked to mutations in the dihydropteroate synthase gene (pfdhps), dihydrofolate reductase gene (pfdhfr) and amplification of GTP cyclohydrolase 1 (pfgch1) gene. The pfgch1 duplication is associated with pfdhfr L164, a crucial mutant for high level pyrimethamine resistance which is rare in Ghana. The presence of amplified pfgch1 in Ghanaian isolates could be an indicator of the evolution of the L164 mutant. This study therefore determined the pfgch1 copy number variations and SP resistance mutations in clinical isolates from Ghana. One hundred and ninety-two (192) blood samples collected from children aged ≤14 years with uncomplicated malaria in 2013-14 and 2015-16 were used. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the pfgch1 copy number and nested PCR-Sanger sequencing used to detect mutations in pfdhps and pfdhfr genes. Twelve parasites (6.3%) harbored double copies of the pfgch1 gene out of the 192 samples. Of the 12, 75% had the pfdhfr I51-R59-N108, 92% had the pfdhps G437 mutant, 8% had the pfdhps E540 and 67% had the SP resistance haplotype IRNG. No L164 was detected in samples with amplified pfgch1. The rare T108 mutant associated with cycloguanil resistance showed predominance (60%) over N108 in the 2015-16 isolates. The observation of parasites with increased copy number of pfgch1 gene is indicative of the future evolution of the rare quadruple pfdhfr mutant, I51-R59-N108-L164, in Ghanaian parasites. Mutant pfdhps isolates also had increased gch1 copy number suggestive that it may also facilitate sulphadoxine resistance. The selection of parasites with pfgch1 gene amplification will enhance the sustenance and persistence of parasites with SP resistance in the country. Policy makers need to begin the search for a replacement chemoprophylaxis drug for malaria vulnerable groups in Ghana.

The Amyloid Precursor Protein Is a Conserved Receptor for Slit to Mediate Axon Guidance.

The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and ß-amyloid production play a central role in Alzheimer's disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation.

Genetic diversity of Plasmodium falciparum isolates from uncomplicated malaria cases in Ghana over a decade.

BACKGROUND: Genotyping malaria parasites to assess their diversity in different geographic settings have become necessary for the selection of antigenic epitopes for vaccine development and for antimalarial drug efficacy or resistance investigations. This study describes the genetic diversity of Plasmodium falciparum isolates from uncomplicated malaria cases over a ten year period (2003-2013) in Ghana using the polymorphic antigenic marker, merozoite surface protein 2 (msp2). METHODS: Archived filter paper blood blots from children aged nine years and below with uncomplicated malaria collected from nine sites in Ghana were typed for the presence of the markers. A total of 880 samples were genotyped for msp2 for the two major allelic families, FC27 and 3D7, using nested polymerase chain reaction (PCR). The allele frequencies and the multiplicity of infection were determined for the nine sites for five time points over a period of ten years, 2003-2004, 2005-2006, 2007-2008, 2010 and 2012-2013 malaria transmission seasons. RESULTS: The number of different alleles detected for the msp2 gene by resolving PCR products on agarose gels was 14. Both of the major allelic families, 3D7 and FC27 were common in all population samples. The highest multiplicity of infection (MOI) was observed in isolates from Begoro (forest zone, rural site): 3.31 for the time point 2007-2008. A significant variation was observed among the sites in the MOIs detected per infection (Fisher's exact test, P < 0.001) for the 2007 isolates and also at each of the three sites with data for three different years, Hohoe, P = 0.03; Navrongo, P < 0.001; Cape Coast, P < 0.001. Overall, there was no significant difference between the MOIs of the three ecological zones over the years (P = 0.37) and between the time points when data from all sites were pooled (P = 0.40). CONCLUSIONS: The diversity and variation between isolates detected using the msp2 gene in Ghanaian isolates were observed to be profound; however, there was homogeneity throughout the three ecological zones studied. This is indicative of gene flow between the parasite populations across the country probably due to human population movements (HPM).

The effect of leptin concentrations and other maternal characteristics on gestational weight gain is different according to pre-gestational BMI: results from a prospective cohort.

OBJECTIVE: To evaluate the effect of leptin and other selected variables on gestational weight gain (GWG) according to pre-gestational body mass index (BMI). DESIGN: Prospective cohort. SETTING: Public Health Center, Rio de Janeiro, Brazil. SAMPLE: Two hundred and twenty-eight pregnant women. METHODS: Women were followed at the 5-13, 20-26 and 30-36th gestational weeks. The effects of independent variables on GWG in normal weight (BMI 18.5-24.9 kg/m(2) ), overweight (BMI 25.0-29.9 kg/m(2) ) and obese (BMI ≥ 30.0 kg/m(2) ) women were assessed using longitudinal linear mixed-effects models. MAIN OUTCOME MEASURE: Maternal body weight (kg) throughout pregnancy. RESULTS: Leptin concentrations were associated with GWG in normal weight (ß = 0.048, P < 0.001) and overweight (ß = 0.023, P = 0.048) women, but not in obese ones (ß = 0.011, P = 0.308). Additionally, the number of hours slept per night decreased the effect of leptin on GWG in OW women (ß = -0.013, P = 0.002). The effect of other maternal characteristics on GWG was different depending on the BMI category. CONCLUSIONS: Leptin concentrations were positively associated with GWG in normal weight and overweight women, but not in obese ones. Maternal height was associated with GWG in all BMI categories, but other variables such as sleep duration, QUICKI values, HDL-c, smoking habit and marital status presented differential effects according to BMI. We encourage further studies to investigate the association between leptin and gestational weight gain, taking into account the pre-pregnancy weight and sleep duration, in order to compare and confirm our results. TWEETABLE ABSTRACT: Leptin is associated with weight gain in normal weight and overweight pregnant women, but not in obese ones.

Lipid changes throughout pregnancy according to pre-pregnancy BMI: results from a prospective cohort.

OBJECTIVE: To evaluate the rate of change in serum lipids and the factors associated with their variations, stratifying for pre-pregnancy body mass index (BMI) categories. DESIGN: Prospective cohort. SETTING: Public Health centre, Rio de Janeiro, Brazil. POPULATION: Two hundred and twenty-five healthy pregnant women recruited between 2009 and 2011. METHODS: Women were evaluated during the three trimesters of pregnancy (5th-13th, 20th-26th and 30th-36th gestational weeks). Pre-pregnancy BMI (kg/m²) was classified as normal weight (NW = 18.5-24.9), overweight (OW = 25.0-29.9) or obese (OB ≥ 30.0). The independent variables included maternal socioeconomic, demographic, biochemical and lifestyle factors. We performed linear mixed-effects models adjusted for gestational age and body weight, reporting coefficient (ß) and 95% confidence interval (95% CI). MAIN OUTCOME MEASURES: Longitudinal total cholesterol (TC), high-density lipoprotein (HDL-c), low-density lipoprotein (LDL-c) and triglyceride (TG) measurements. RESULTS: OW and OB women presented higher mean TG, TC and LDL-c compared with their NW counterparts (P < 0.05). The mean HDL-c concentrations were higher in NW than in OB women (P = 0.03). OW and OB women presented lower serum TC (ßOW  = -0.014; 95% CI = -0.026 to -0.002; P = 0.022; ßOB  = -0.015; 95% CI -0.015 to 0.001; P = 0.066) and LDL-c (ßOW  = -0.012; 95% CI = -0.021 to -0.002; P = 0.017; ßOB  = -0.018; 95% CI = -0.031 to -0.005; P = 0.005) rates of change (mmol/l per gestational week) compared with the NW. Pre-gestational BMI was the main factor associated with the rate of changes in TC and LDL-c concentrations. CONCLUSION: Pre-pregnancy BMI was the main factor associated with the rate of change in TC and LDL-c throughout pregnancy, and OW and OB women presented lower rates of change compared with NW controls.

An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs.

BACKGROUND: The recently introduced SYBR Green1 (SG) assay for testing parasites susceptibility to anti-malarial drugs needs further improvement. This has been necessitated by various setbacks, the major one being the low fluorescence intensity associated with it use. This shortcoming diminishes the anticipated hope that this novel method was going to replace the more traditional ones, such as the isotopic and microscopy. In order to restore confidence in its use, series of experiments to determine conditions that give the best fluorescence intensity were conducted. METHODS: Conditions that yield the maximum fluorescent signal were ascertained by measuring the fluorescence after incubation of Plasmodium falciparum culture at different parasites concentration with lysis buffer containing SYBR Green (LBS) at different time period. In order to ascertain the effect of freeze-thaw on fluorescence intensity, P. falciparum culture was frozen for 1 h, thawed, incubated with LBS and the fluorescence measured. The optimized conditions determined in this study were then used to assess the susceptibility of clinical isolates of P. falciparum to artesunate, chloroquine and mefloquine. The concentration of anti-malarial drug inhibiting parasite growth by 50 % (IC50) for each drug was estimated using the online ICEstimator. The IC50 generated using the optimized SG method determined in this study was compared with that obtained using microscopic method and the previously reported standard SG method. RESULTS: Over all, the SG method was found to be easy to perform and sensitive. Freeze-thaw of parasite culture followed by incubation with lysis buffer containing the dye for 3 h was consistently observed to give the highest fluorescence signal. The IC50 values for chloroquine, mefloquine and artesunate determined were consistent and comparable with that determined with the previously reported standard SG method and the microscopic method. CONCLUSION: The authors conclude that freezing and thawing of parasite culture, followed by incubation with LBS in the dark for 3 h provided a significant improvement in fluorescence signal. The IC50 generated using the improved SG method is comparable with that from microscopy and the standard method.