Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

The contrasting phenotypes of neutrophils during asymptomatic versus symptomatic Leishmania braziliensis infection.

BACKGROUND: The mechanisms that mediate immune protection in individuals with subclinical (SC) or asymptomatic infection with L. braziliensis are largely unknown. Neutrophils (PMNs) have been implicated in progressive symptomatic cutaneous leishmaniasis (CL), but their potential participation in maintenance of subclinical infection is unexplored. The aim of this study was to compare the phenotypic and functional profiles of PMNs in individuals with SC infection versus patients with symptomatic CL due to L. braziliensis. METHODS: Subjects were recruited in the endemic region of Corte de Pedra, Bahia, Brazil. Surface markers to define activation status were characterized by flow cytometry. Functional responses of PMNs including phagocytic capacity, production of oxidative species, and oxidative killing of intracellular parasites were studied in vitro. RESULTS: PMNs from individuals with SC infection displayed a more activated phenotype and greater ability to control the infection than PMNs from patients with CL. In contrast, PMNs from patients with CL exhibited higher expression of HLA-DR and higher production of oxidative species than PMNs from subjects with SC infection. CONCLUSION: PMNs from individuals with SC infection can control the infection more efficiently than PMNs from patients with CL, despite the lower production of oxidants. Our observations suggest that L. braziliensis may evade microbicidal mechanisms of PMNs from patients with CL, contributing to parasite dissemination and the establishment of disease.

Evolution of Spatial Risk of Malaria Infection After a Pragmatic Chemoprevention Program in Response to Severe Flooding in Rural Western Uganda.

BACKGROUND: Malaria epidemics result from extreme precipitation and flooding, which are increasing with global climate change. Local adaptation and mitigation strategies will be essential to prevent excess morbidity and mortality. METHODS: We investigated the spatial risk of malaria infection at multiple timepoints after severe flooding in rural western Uganda employing longitudinal household surveys measuring parasite prevalence and leveraging remotely sensed information to inform spatial models of malaria risk in the 3 months after flooding. RESULTS: We identified clusters of malaria risk emerging in areas (1) that showed the greatest changes in Normalized Difference Vegetation Index from pre- to postflood and (2) where residents were displaced for longer periods of time and had lower access to long-lasting insecticidal nets, both of which were associated with a positive malaria rapid diagnostic test result. The disproportionate risk persisted despite a concurrent chemoprevention program that achieved high coverage. CONCLUSIONS: The findings enhance our understanding not only of the spatial evolution of malaria risk after flooding, but also in the context of an effective intervention. The results provide a "proof of concept" for programs aiming to prevent malaria outbreaks after flooding using a combination of interventions. Further study of mitigation strategies-and particularly studies of implementation-is urgently needed.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Downregulation of collagen XI during late postnatal corneal development is followed by upregulation after injury.

Collagen XI plays a role in nucleating collagen fibrils and in controlling fibril diameter. The aim of this research was to elucidate the role that collagen XI plays in corneal fibrillogenesis during development and following injury. The temporal and spatial expression of collagen XI was evaluated in C57BL/6 wild-type mice. For wound-healing studies in adult mice, stromal injuries were created using techniques that avoid caustic chemicals. The temporal expression and spatial localization of collagen XI was studied following injury in a Col11a1 inducible knockout mouse model. We found that collagen XI expression occurs during early maturation and is upregulated after stromal injury in areas of regeneration and remodeling. Abnormal fibrillogenesis with new fibrils of heterogeneous size and shape occurs after injury in a decreased collagen XI matrix. In conclusion, collagen XI is expressed in the stroma during development and following injury in adults, and is a regulator of collagen fibrillogenesis in regenerating corneal tissue.

A direct comparison in diagnostic performance of CDUS, FDG-PET/CT and MRI in patients suspected of giant cell arteritis.

OBJECTIVES: This study directly compares diagnostic performance of Colour Duplex Ultrasound (CDUS), Fluor-18-deoxyglucose Positron Emission Tomography Computed Tomography (FDG-PET/CT) and Magnetic Resonance Imaging (MRI) in patients suspected of giant cell arteritis (GCA). METHODS: Patients with suspected GCA were included in a nested-case control pilot study. CDUS, whole body FDG-PET/CT and cranial MRI were performed within 5 working days after initial clinical evaluation. Clinical diagnosis after six months follow-up by experienced rheumatologists in the field of GCA, blinded for imaging, was used as reference standard. Diagnostic performance of the imaging modalities was determined. Stratification for GCA subtype was performed and imaging results were evaluated in different risk stratification groups. RESULTS: In total, 23 patients with GCA and 19 patients suspected of but not diagnosed with GCA were included. Sensitivity was 69.6% (95%CI 50.4%-88.8%) for CDUS, 52.2% (95%CI 31.4%-73.0%) for FDG-PET/CT and 56.5% (95%CI 35.8%-77.2%) for MRI. Specificity was 100% for CDUS, FDG-PET/CT and MRI. FDG-PET/CT was negative for GCA in all isolated cranial GCA patients (n = 8), while MRI was negative in all isolated extracranial GCA patients (n = 4). In 4 GCA patients with false-negative (n = 2; intermediate and high risk) or inconclusive (n = 2; low and intermediate risk) CDUS results, further imaging confirmed diagnosis. CONCLUSIONS: Sensitivity of CDUS was highest, while specificity was excellent in all imaging modalities. Nevertheless, confidence intervals of all imaging modalities were overlapping. Following EULAR recommendations, CDUS can be used as a first test to diagnose GCA. With insufficient evidence for GCA, further testing considering GCA subtype is warranted.

Interplay Between Ni and Brønsted and Lewis Acid Sites in the Hydrodesulfurization of Dibenzothiophene.

Ni-MoS2/γ-Al2O3 catalysts are commonly used in hydrotreating to enhance fossil fuel quality. The extensive research on these catalysts reveals a gap in understanding the role of Ni, often underestimated as an inactive sulfide phase or just a MoS2 promoter. In this work, we focused on analyzing whether well-dispersed supported nickel nanoparticles can be active in the hydrodesulfurization of dibenzothiophene. We dispersed Ni by Strong Electrostatic Adsorption (SEA) method across four supports with different types of acidity: silica (~ neutral acidity), γ-Al2O3 (Lewis acidity), H+-Y zeolite, and microporous-mesoporous H+-Y zeolite (both with Brønsted-Lewis acidity). Our findings reveal that Ni is indeed active in dibenzothiophene hydrodesulfurization, even with alumina and silica as supports, although their catalytic activity declines abruptly in the first hours. Contrastingly, the acid nature of zeolites imparts sustained stability and performance, attributed to robust metal-support interactions. The efficacy of the SEA method and the added mesoporosity in zeolites further amplify catalytic efficiency. Overall, we demonstrate that Ni nanoparticles may perform as a hydrogenating metal in the same manner as noble metals such as Pt and Pd perform in hydrodesulfurization. We discuss some of the probable reasons for such performance and remark on the role of Ni in hydrotreatment.

Characterizing Protonation-Coupled Conformational Ensembles in RNA via pH-Differential Mutational Profiling with DMS Probing.

RNA molecules undergo conformational transitions in response to cellular and environmental stimuli. Site-specific protonation, a fundamental chemical property, can alter the conformational landscape of RNA to regulate their functions. However, characterizing protonation-coupled RNA conformational ensembles on a large scale remains challenging. Here, we present pH-differential mutational profiling (PD-MaP) with dimethyl sulfate probing for high-throughput detection of protonation-coupled conformational ensembles in RNA. We demonstrated this approach on microRNA-21 precursor (pre-miR-21) and recapitulated a previously discovered A+-G-coupled conformational ensemble. Additionally, we identified a secondary protonation event involving an A+-C mismatch. We validated the occurrence of both protonation-coupled ensembles in pre-miR-21 using NMR relaxation dispersion spectroscopy. Furthermore, the application of PD-MaP on a library of well-annotated human primary microRNAs uncovered widespread protonation-coupled conformational ensembles, suggesting their potentially broad functions in biology.

Collagen XII Regulates Corneal Stromal Structure by Modulating Transforming Growth Factor-ß Activity.

Collagen XII is a regulator of corneal stroma structure and function. The current study examined the role of collagen XII in regulating corneal stromal transforming growth factor (TGF)-ß activation and latency. Specifically, with the use of conventional collagen XII null mouse model, the role of collagen XII in the regulation of TGF-ß latency and activity in vivo was investigated. Functional quantification of latent TGF-ß in stromal matrix was performed by using transformed mink lung reporter cells that produce luciferase as a function of active TGF-ß. Col12a1 knockdown with shRNA was used to test the role of collagen XII in TGF-ß activation. Col12a1-/- hypertrophic stromata were observed with keratocyte hyperplasia. Increased collagen fibril forward signal was found by second harmonic generation microscopy in the absence of collagen XII. Collagen XII regulated mRNA synthesis of Serpine1, Col1a1, and Col5a1 and deposition of collagens in the extracellular matrix. A functional plasminogen activator inhibitor luciferase assay showed that collagen XII is necessary for latent TGF-ß storage in the extracellular matrix and that collagen XII down-regulates active TGF-ß. Collagen XII dictates stromal structure and function by regulating TGF-ß activity. A hypertrophic phenotype in Col12a1-/- corneal tissue can be explained by abnormal up-regulation of TGF-ß activation and decreased latent storage.

Collagen XII regulates stromal wound closure.

The role of collagen XII in regulating injury repair and reestablishment of corneal function is unknown. This manuscript aims to investigate the role(s) of collagen XII in the repair of incisional and debridement injuries in an adult mouse model. Two different types of injury in wild type and Col12a1-/- corneas were created to investigate the effects of collagen XII -in wound repair and scar formation-by using clinical photographs, immunohistology, second harmonic generation imaging and electron microscopy. Results showed that collagen XII is a regulator of wound closure after incisional injuries. Absence of collagen XII retarded wound closure and the wound healing process. These findings show that collagen XII regulates fibrillogenesis, CD68 cell lineage infiltration, and myofibroblast survival following injury. In vitro studies suggest that collagen XII regulates deposition of an early and provisional matrix by interacting with two proteins regulating early matrix deposition: fibronectin and LTBP1(latent transforming growth factor ß binding protein 1). In conclusion, collagen XII regulates tissue repair in corneal incisional wounds. Understanding the function of collagen XII during wound healing has significant translational value.

Evolution of erectile dysfunction in individuals infected with human T-lymphotropic virus 1: a prospective cohort study.

BACKGROUND: Virtually all patients with human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) have some degree of erectile dysfunction (ED), but ED is also found in a large percentage of HTLV-1 carriers. AIM: To evaluate the evolution of ED in individuals infected with HTLV-1 who were followed for up to 15 years. METHODS: This prospective cohort study included men infected with HTLV-1 who had ED, were aged 18 to 70 years, and were followed from January 2004 to December 2019. We used the International Index of Erectile Function-5 (IIEF-5), the Expanded Disability Status Scale and Osame Motor Disability Scale, and the Overactive Bladder Symptom Score (OABSS) to define and stratify ED, neurologic disability, and bladder dysfunction, respectively. OUTCOMES: Time to development of severe ED was the main outcome. RESULTS: We studied 90 men with ED (mean ± SD age, 52.8 ± 9.78 years). At baseline, 42 were carriers, 16 had probable HAM/TSP, and 32 had definite HAM/TSP. IIEF-5 was highest among carriers and lowest in patients with definite HAM/TSP, whereas OABSS was lowest in carriers and highest in patients with definite HAM/TSP. Median (IQR) follow-up was 8.50 years (3.00-12.00). IIEF-5 fell significantly from baseline to last follow-up among carriers and patients with probable and definite HAM/TSP. There was an inverse correlation between the IIEF-5 and the OABSS at last follow-up (r = -0.62, P < .001). In survival analysis, the time to development of severe ED was significantly shorter in patients with definite HAM/TSP when compared with carriers (P = .001) and those with probable HAM/TSP (P = .014). The presence of definite HAM/TSP at baseline was independently associated with the development of severe ED, after adjustment for baseline age and proviral load (hazard ratio, 6.74; P = .008). CLINICAL IMPLICATIONS: Formal assessment of erectile function should be part of the routine clinical assessment of individuals infected with HTLV-1; worsening erectile function should alert clinicians to the possibility of neurologic deterioration. STRENGTHS AND LIMITATIONS: This is the first prospective cohort study to describe the course of ED in men infected with HTLV-1. The small sample size and absence of seronegative controls are limitations. CONCLUSION: ED is a slowly progressive clinical manifestation of HTLV-1 infection, and the degree of neurologic compromise at baseline is the main predictor of time to progression to severe ED.

Comparing field-collected versus remotely-sensed variables to model malaria risk in the highlands of western Uganda.

BACKGROUND: Malaria risk is not uniform across relatively small geographic areas, such as within a village. This heterogeneity in risk is associated with factors including demographic characteristics, individual behaviours, home construction, and environmental conditions, the importance of which varies by setting, making prediction difficult. This study attempted to compare the ability of statistical models to predict malaria risk at the household level using either (i) free easily-obtained remotely-sensed data or (ii) results from a resource-intensive household survey. METHODS: The results of a household malaria survey conducted in 3 villages in western Uganda were combined with remotely-sensed environmental data to develop predictive models of two outcomes of interest (1) a positive ultrasensitive rapid diagnostic test (uRDT) and (2) inpatient admission for malaria within the last year. Generalized additive models were fit to each result using factors from the remotely-sensed data, the household survey, or a combination of both. Using a cross-validation approach, each model's ability to predict malaria risk for out-of-sample households (OOS) and villages (OOV) was evaluated. RESULTS: Models fit using only environmental variables provided a better fit and higher OOS predictive power for uRDT result (AIC = 362, AUC = 0.736) and inpatient admission (AIC = 623, AUC = 0.672) compared to models using household variables (uRDT AIC = 376, Admission AIC = 644, uRDT AUC = 0.667, Admission AUC = 0.653). Combining the datasets did not result in a better fit or higher OOS predictive power for uRDT results (AIC = 367, AUC = 0.671), but did for inpatient admission (AIC = 615, AUC = 0.683). Household factors performed best when predicting OOV uRDT results (AUC = 0.596) and inpatient admission (AUC = 0.553), but not much better than a random classifier. CONCLUSIONS: These results suggest that residual malaria risk is driven more by the external environment than home construction within the study area, possibly due to transmission regularly occurring outside of the home. Additionally, they suggest that when predicting malaria risk the benefit may not outweigh the high costs of attaining detailed information on household predictors. Instead, using remotely-sensed data provides an equally effective, cost-efficient alternative.

Adiposity and feeding practices in the first two years of life among toddlers in Guadalajara, Mexico.

BACKGROUND: Feeding practices in the first two years of life have a direct impact on nutritional status and adiposity. The purpose of this study was to identify the differences in feeding practices during the first two years of life by sex and type of feeding in the first semester of postnatal life and their relationships with adiposity in toddlers. METHODS: An analytical cross-sectional study that included 150 toddlers aged 12 to 24 months who were healthy, full-term, and had adequate weight for their gestational ages, was conducted at the New Civil Hospital and at a private practice in Guadalajara. Body compositions were obtained by bioelectrical impedance (BIA) measurements, and a modified questionnaire was used. Then, the parents completed two 24-h dietary recalls. In addition to the descriptive statistics, ANOVA, Kruskal-Wallis and Mann-Whitney U tests were used in the contrast analysis of the quantitative variables. To analyze the qualitative variables, we used X2 tests. Afterward, linear regression tests were conducted to identify the relationships between adiposity and feeding practices during the first two years. RESULTS: There were direct relationships between adiposity and duration of full breastfeeding (r = 0.610, p = 0.021), age of introduction of ultra-processed products (r = 0.311, p = 0.011), sugar (r = 0.186; p = 0.024) and age at which eggs were introduced (r = -0.202; p = 0.016). CONCLUSIONS: Adiposity was related to feeding practices in the first two years of life in toddlers.

Gurka vs Slaughter equations to estimate the fat percentage in children with cerebral palsy from all subtypes and levels of the Gross Motor Function Classification System.

BACKGROUND: Body composition assessment in children with cerebral palsy (CP) is a challenge, specially the fat percentage. There are different methods that can be used to estimate the fat percentage in this population, such as anthropometric equations, but there is still a need to determine which is the best and most accurate. The purpose of the study was to determine the method that best estimates the fat percentage in children from all CP subtypes and levels of the Gross Motor Function Classification System (GMFCS). METHODS: Analytical cross-sectional study in which 108 children with CP diagnosed by a pediatric neurologist were included with any type of dysfunction and from all levels of the GFMCS. Slaughter equation, Gurka equation and Bioelectrical impedance analysis (BIA) as reference method, were used. Groups were stratified by sex, CP subtypes, GMFCS level and Tanner stage. Median differences, Kruskal-Wallis, Mann-Whitney U test, Spearman's correlation coefficients and simple regressions were used, also multivariate models were performed. RESULTS: The Slaughter equation differed from the other methods in the total population and when it was compared by sex, CP subtypes, gross motor function and Tanner stage. The Gurka equation showed significant differences by sex and gross motor function. Gurka equation correlated positively and significantly with BIA to estimate the fat percentage in all the CP subtypes and levels of the GMFCS. Tricipital skinfold (TSF), arm fat area (AFA) and weight for age index (W/A) showed the highest variability with respect to fat percentage. CONCLUSION: Gurka equation is more appropriate and accurate than Slaughter equation to estimate the fat percentage in children with CP from all subtypes and levels of the GMFCS.

Immune Response to LinB13, a Lutzomyia Intermedia Salivary Protein Correlates With Disease Severity in Tegumentary Leishmaniasis.

BACKGROUND: We have previously shown that seropositivity to rLinB-13, a salivary protein from Lutzomyia intermedia, predicted sand fly exposure and was associated with increased risk of developing cutaneous leishmaniasis (CL). METHODS: Here, we investigated the cellular immune response to saliva from Lu. intermedia, using rLinB-13 as a surrogate antigen in naturally exposed individuals presenting positive serology to LinB-13. We also investigated the response to rLinB-13 in leishmaniasis patients, displaying active ulcers and positive PCR for Leishmania braziliensis. RESULTS: Peripheral blood mononuclear cells (PBMCs) stimulated in vitro with rLinB-13 secreted elevated levels of interleukin-10 (IL-10), IL-4, IL-1ß, IL-1α, IL-6, and chemokines (CCL3, CCL4, CCL5, and CXCL5). CL and disseminated leishmaniasis (DL) patients displayed a significantly higher immunoglobulin G (IgG) response to rLinB-13 compared with healthy subjects, and anti-rLinB-13 IgG was positively correlated with the number of lesions in DL patients. Positive serology to rLinB-13 was also associated with chemotherapy failure. PBMCs from DL patients stimulated with rLINB-13 secreted significantly higher levels of IL-10 and IL-1ß compared with CL individuals. CONCLUSIONS: In this study, we observed an association between humoral and cellular immune response to the sand fly salivary protein rLinB-13 and disease severity in tegumentary leishmaniasis. This study brings evidence that immunity to rLinB-13 influences disease outcome in L. braziliensis infection and results indicate that positive serology to rLinB-13 IgG can be used as a marker of DL, an emerging and severe form of disease caused by L. braziliensis.

Dihydroartemisinin-Piperaquine Chemoprevention and Malaria Incidence After Severe Flooding: Evaluation of a Pragmatic Intervention in Rural Uganda.

BACKGROUND: Malaria epidemics are a well-described phenomenon after extreme precipitation and flooding. Yet, few studies have examined mitigation measures to prevent post-flood malaria epidemics. METHODS: We evaluated a malaria chemoprevention program implemented in response to severe flooding in western Uganda. Children aged ≤12 years from 1 village were eligible to receive 3 monthly rounds of dihydroartemisinin-piperaquine (DP). Two neighboring villages served as controls. Malaria cases were defined as individuals with a positive rapid diagnostic test result as recorded in health center registers. We performed a difference-in-differences analysis to estimate changes in the incidence and test positivity of malaria between intervention and control villages. RESULTS: A total of 554 children received at least 1 round of chemoprevention, with 75% participating in at least 2 rounds. Compared with control villages, we estimated a 53.4% reduction (adjusted rate ratio [aRR], 0.47; 95% confidence interval [CI]: .34-.62; P < .01) in malaria incidence and a 30% decrease in the test positivity rate (aRR, 0.70; 95% CI: .50-.97; P = .03) in the intervention village in the 6 months post-intervention. The impact was greatest among children who received the intervention, but decreased incidence was also observed in older children and adults (aRR, 0.57; 95% CI: .38-.84; P < .01). CONCLUSIONS: Three rounds of chemoprevention with DP delivered under pragmatic conditions reduced the incidence of malaria after severe flooding in western Uganda. These findings provide a proof-of-concept for the use of malaria chemoprevention to reduce excess disease burden associated with severe flooding.

RXam2, a NLR from cassava (Manihot esculenta) contributes partially to the quantitative resistance to Xanthomonas phaseoli pv. manihotis.

KEY MESSAGE: The overexpression of RXam2, a cassava NLR (nucleotide-binding leucine-rich repeat) gene, by stable transformation and gene expression induction mediated by dTALEs, reduce cassava bacterial blight symptoms. Cassava (Manihot esculenta) is a tropical root crop affected by different pathogens including Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of cassava bacterial blight (CBB). Previous studies have reported resistance to CBB as a quantitative and polygenic character. This study sought to validate the functional role of a NLR (nucleotide-binding leucine-rich repeat) associated with a QTL to Xpm strain CIO151 called RXam2. Transgenic cassava plants overexpressing RXam2 were generated and analyzed. Plants overexpressing RXam2 showed a reduction in bacterial growth to Xpm strains CIO151, 232 and 226. In addition, designer TALEs (dTALEs) were developed to specifically bind to the RXam2 promoter region. The Xpm strain transformed with dTALEs allowed the induction of the RXam2 gene expression after inoculation in cassava plants and was associated with a diminution in CBB symptoms. These findings suggest that RXam2 contributes to the understanding of the molecular basis of quantitative disease resistance.

Immunogenicity and safety of fractional doses of yellow fever vaccines: a randomised, double-blind, non-inferiority trial.

BACKGROUND: Stocks of yellow fever vaccine are insufficient to cover exceptional demands for outbreak response. Fractional dosing has shown efficacy, but evidence is limited to the 17DD substrain vaccine. We assessed the immunogenicity and safety of one-fifth fractional dose compared with standard dose of four WHO-prequalified yellow fever vaccines produced from three substrains. METHODS: We did this randomised, double-blind, non-inferiority trial at research centres in Mbarara, Uganda, and Kilifi, Kenya. Eligible participants were aged 18-59 years, had no contraindications for vaccination, were not pregnant or lactating, had no history of yellow fever vaccination or infection, and did not require yellow fever vaccination for travel. Eligible participants were recruited from communities and randomly assigned to one of eight groups, corresponding to the four vaccines at standard or fractional dose. The vaccine was administered subcutaneously by nurses who were not masked to treatment, but participants and other study personnel were masked to vaccine allocation. The primary outcome was proportion of participants with seroconversion 28 days after vaccination. Seroconversion was defined as post-vaccination neutralising antibody titres at least 4 times pre-vaccination measurement measured by 50% plaque reduction neutralisation test (PRNT50). We defined non-inferiority as less than 10% decrease in seroconversion in fractional compared with standard dose groups 28 days after vaccination. The primary outcome was measured in the per-protocol population, and safety analyses included all vaccinated participants. This trial is registered with ClinicalTrials.gov, NCT02991495. FINDINGS: Between Nov 6, 2017, and Feb 21, 2018, 1029 participants were assessed for inclusion. 69 people were ineligible, and 960 participants were enrolled and randomly assigned to vaccine manufacturer and dose (120 to Bio-Manguinhos-Fiocruz standard dose, 120 to Bio-Manguinhos-Fiocruz fractional dose, 120 to Chumakov Institute of Poliomyelitis and Viral Encephalitides standard dose, 120 to Chumakov Institute of Poliomyelitis and Viral Encephalitides fractional dose, 120 to Institut Pasteur Dakar standard dose, 120 to Institut Pasteur Dakar fractional dose, 120 to Sanofi Pasteur standard dose, and 120 to Sanofi Pasteur fractional dose). 49 participants had detectable PRNT50 at baseline and 11 had missing PRNT50 results at baseline or 28 days. 900 were included in the per-protocol analysis. 959 participants were included in the safety analysis. The absolute difference in seroconversion between fractional and standard doses by vaccine was 1·71% (95% CI -2·60 to 5·28) for Bio-Manguinhos-Fiocruz, -0·90% (-4·24 to 3·13) for Chumakov Institute of Poliomyelitis and Viral Encephalitides, 1·82% (-2·75 to 5·39) for Institut Pasteur Dakar, and 0·0% (-3·32 to 3·29) for Sanofi Pasteur. Fractional doses from all four vaccines met the non-inferiority criterion. The most common treatment-related adverse events were headache (22·2%), fatigue (13·7%), myalgia (13·3%) and self-reported fever (9·0%). There were no study-vaccine related serious adverse events. INTERPRETATION: Fractional doses of all WHO-prequalified yellow fever vaccines were non-inferior to the standard dose in inducing seroconversion 28 days after vaccination, with no major safety concerns. These results support the use of fractional dosage in the general adult population for outbreak response in situations of vaccine shortage. FUNDING: The study was funded by Médecins Sans Frontières Foundation, Wellcome Trust (grant no. 092654), and the UK Department for International Development. Vaccines were donated in kind.