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Selective manipulation of micrometric objects in a standard microscopy environment is possible with miniaturized acoustical tweezers., Acoustical tweezers based on focalized acoustical vortices hold the promise of precise contactless manipulation of millimeter down to submicrometer particles, microorganisms, and cells with unprecedented combined selectivity and trapping force. Yet, the widespread dissemination of this technology has been hindered by severe limitations of current systems in terms of performance and/or miniaturization and integrability. Here, we unleash the potential of focalized acoustical vortices by developing the first flat, compact, paired single electrode focalized acoustical tweezers. These tweezers rely on spiraling transducers obtained by folding a spherical acoustical vortex on a flat piezoelectric substrate. We demonstrate the ability of these tweezers to grab and displace micrometric objects in a standard microfluidic environment with unique selectivity. The simplicity of this system and its scalability to higher frequencies open tremendous perspectives in microbiology, microrobotics, and microscopy.

Background Patients with Heart Failure (HF) show impaired functional capacities which have been related to their prognosis. Moreover, physical functional performance in functional tests has also been related to the prognosis in patients with HF. Thus, it would be useful to investigate how physical functional performance in functional tests could determine the prognosis in patients with HF, because HF is the leading cause of hospital admissions for people older than 65 years old. This systematic review and meta-analysis aims to summarise and synthesise the evidence published about the relationship between physical functional performance and prognosis in patients with HF, as well as assess the risk of bias of included studies and the level of evidence per outcome. Methods Major electronic databases, such as PubMed, AMED, CINAHL, EMBASE, PEDro, Web of Science, were searched from inception to March 2020 for observational longitudinal cohort studies (prospective or retrospective) examining the relationship between physical functional performance and prognosis in patients with HF. Results 44 observational longitudinal cohort studies with a total of 22,598 patients with HF were included. 26 included studies reported a low risk of bias, and 17 included studies showed a moderate risk of bias. Patients with poor physical functional performance in the Six Minute Walking Test (6MWT), in the Short Physical Performance Battery (SPPB) and in the Gait Speed Test showed worse prognosis in terms of larger risk of hospitalisation or mortality than patients with good physical functional performance. However, there was a lack of homogeneity regarding which cut-off points should be used to stratify patients with poor physical functional performance from patients with good physical functional performance. Conclusion The review includes a large number of studies which show a strong relationship between physical functional performance and prognosis in patients with HF. Most of the included studies reported a low risk of bias, and GRADE criteria showed a low and a moderate level of evidence per outcome.

BackgroundAlthough anti-inflammatory agents could theoretically have anticancer properties, results from cohort studies on nonsteroidal anti-inflammatory drugs (NSAIDs) and breast cancer (BC) risk are inconsistent.MethodsWe investigated the association between NSAID use and BC incidence in the French E3N prospective cohort, which includes 98,995 women born between 1925 and 1950 and insured by a health insurance plan that covers mostly teachers. Self-reported information on lifestyle and medical history has been collected biennially by questionnaires and matched with data from a drug reimbursement database covering the period 2004–2014. Women who self-reported current NSAID use in the 2000 or 2002 questionnaires or with at least two reimbursements in any previous 3-month period were defined as exposed to NSAIDs. Multivariable Cox regression models were used to estimate hazard ratios (HRs) for the association of NSAID use with BC risk.ResultsIn the current analysis, 62,512 postmenopausal women were followed between 2004 and 2014 (9 years on average, starting at a mean age of 63 years; 2864 incident BC). In multivariable models, there was no statistically significant association between NSAID use and BC risk [HR = 1.00 (0.92–1.08), compared with non-exposed women]. The NSAID-BC associations did not differ by NSAID types, BC subtypes, risk factors, and comorbidities, nor by duration and dose of use. However, a statistically significant interaction was observed by proton pump inhibitor (PPI) drug use (Pinteraction = 0.01) whereby a decreased risk of BC with NSAID use was only observed among women who also used PPI before.ConclusionOnly women who used NSAIDs after having used PPI had a lower risk of BC. This result is novel and requires replication in other studies.

International audience; Objectives: Large studies are needed to update risk factors of bronchiolitis hospitalization. We performed a nationwide analysis of hospitalization rates for bronchiolitis over four consecutive bronchiolitis seasons to identify underlying medical disorders at risk of bronchiolitis hospitalization and assess their frequency.Methods: Data were retrieved from the French National Hospital Discharge database. Of all infants discharged alive from maternity wards from January 2008 to December 2013 in France (N = 3,884,791), we identified four consecutive cohorts at risk of bronchiolitis during the seasons of 2009–2010 to 2012–2013. The main outcome was bronchiolitis hospitalization during a season. Individual risk factors were collected.Results: Among infants, 6.0% were preterm and 2.0% had ≥1 chronic condition including 0.2% bronchopulmonary dysplasia (BPD) and 0.2% hemodynamically significant congenital heart disease (HS-CHD). Bronchiolitis hospitalization rates varied between seasons (min: 1.26% in 2010–2011; max: 1.48% in 2012–2013; p

Yellow fever is a vector-borne disease endemic in tropical regions of Africa, where 90% of the global burden occurs, and Latin America. It is notoriously under-reported with uncertainty arising from a complex transmission cycle including a sylvatic reservoir and non-specific symptom set. Resulting estimates of burden, particularly in Africa, are highly uncertain. We examine two established models of yellow fever transmission within a Bayesian model averaging framework in order to assess the relative evidence for each model’s assumptions and to highlight possible data gaps. Our models assume contrasting scenarios of the yellow fever transmission cycle in Africa. The first takes the force of infection in each province to be static across the observation period; this is synonymous with a constant infection pressure from the sylvatic reservoir. The second model assumes the majority of transmission results from the urban cycle; in this case, the force of infection is dynamic and defined through a fixed value of R0 in each province. Both models are coupled to a generalised linear model of yellow fever occurrence which uses environmental covariates to allow us to estimate transmission intensity in areas where data is sparse. We compare these contrasting descriptions of transmission through a Bayesian framework and trans-dimensional Markov chain Monte Carlo sampling in order to assess each model’s evidence given the range of uncertainty in parameter values. The resulting estimates allow us to produce Bayesian model averaged predictions of yellow fever burden across the African endemic region. We find strong support for the static force of infection model which suggests a higher proportion of yellow fever transmission occurs as a result of infection from an external source such as the sylvatic reservoir. However, the model comparison highlights key data gaps in serological surveys across the African endemic region. As such, conclusions concerning the most prevalent transmission routes for yellow fever will be limited by the sparsity of data which is particularly evident in the areas with highest predicted transmission intensity. Our model and estimation approach provides a robust framework for model comparison and predicting yellow fever burden in Africa. However, key data gaps increase uncertainty surrounding estimates of model parameters and evidence. As more mathematical models are developed to address new research questions, it is increasingly important to compare them with established modelling approaches to highlight uncertainty in structures and data., Author summary Yellow fever (YF) is notoriously under reported due to non-specific symptom spectrum and the true burden is highly uncertain as a result of a complex transmission cycle. As such, estimates surrounding YF burden are highly uncertain and the mechanisms behind transmission are often unclear. We assess these mechanisms and the resulting uncertainty by estimating two existing models of YF transmission within a product space framework. This allows us to produce updated estimates of transmission intensity and to compare the relative support for each model given the data. We find strong support for a model assuming a static force of infection, approximating the constant infection pressure from the sylvatic reservoir of YF. We also highlight areas where data is sparse, often the same areas estimated to have especially high transmission intensity. This is the first robust multi-model approach to applied YF modelling and provides a framework that could be extended to other disease models.

Background Performing well-powered randomised controlled trials (RCTs) of new treatments for rare diseases is often infeasible. However, with the increasing availability of historical data, incorporating existing information into trials with small sample sizes is appealing in order to increase the power. Bayesian approaches enable one to incorporate historical data into a trial’s analysis through a prior distribution. Methods Motivated by a RCT intended to evaluate the impact on event-free survival of mifamurtide in patients with osteosarcoma, we performed a simulation study to evaluate the impact on trial operating characteristics of incorporating historical individual control data and aggregate treatment effect estimates. We used power priors derived from historical individual control data for baseline parameters of Weibull and piecewise exponential models, while we used a mixture prior to summarise aggregate information obtained on the relative treatment effect. The impact of prior-data conflicts, both with respect to the parameters and survival models, was evaluated for a set of pre-specified weights assigned to the historical information in the prior distributions. Results The operating characteristics varied according to the weights assigned to each source of historical information, the variance of the informative and vague component of the mixture prior and the level of commensurability between the historical and new data. When historical and new controls follow different survival distributions, we did not observe any advantage of choosing a piecewise exponential model compared to a Weibull model for the new trial analysis. However, we think that it remains appealing given the uncertainty that will often surround the shape of the survival distribution of the new data. Conclusion In the setting of Sarcome-13 trial, and other similar studies in rare diseases, the gains in power and accuracy made possible by incorporating different types of historical information commensurate with the new trial data have to be balanced against the risk of biased estimates and a possible loss in power if data are not commensurate. The weights allocated to the historical data have to be carefully chosen based on this trade-off. Further simulation studies investigating methods for incorporating historical data are required to generalise the findings. Electronic supplementary material The online version of this article (10.1186/s12874-019-0714-z) contains supplementary material, which is available to authorized users.

A thorough understanding of the heritability, genetic correlations and additive and non-additive variance components of tree growth and wood properties is a requisite for effective tree breeding. This knowledge is essential to maximize genetic gain, that is, the amount of increase in trait performance achieved annually through directional selection. Understanding the genetic attributes of traits targeted by breeding is also important to sustain decade-long genetic progress, that is, the progress made by increasing the average genetic value of the offspring as compared to that of the parental generation. In this study, we report quantitative genetic parameters for fifteen growth, wood chemical and physical traits for the world-famous Eucalyptus urograndis hybrid (E. grandis × E. urophylla). These traits directly impact the optimal use of wood for cellulose pulp, paper, and energy production. A population of 1,000 trees sampled in a progeny trial was phenotyped directly or following the development and use of near-infrared spectroscopy calibration models. Trees were genotyped with 33,398 SNPs and 24,001 DArT-seq genome-wide markers and genomic realized relationship matrices (GRM) were used for parameter estimation with an individual-tree additive-dominant mixed model. Wood chemical properties and wood density showed stronger genetic control than growth, cellulose and fiber traits. Additive effects are the main drivers of genetic variation for all traits, but dominance plays an equally or more important role for growth, singularly in this hybrid. GRM´s with >10,000 markers provided stable relationships estimates and more accurate parameters than pedigrees by capturing the full genetic relationships among individuals and disentangling the non-additive from the additive genetic component. Low correlations between growth and wood properties indicate that simultaneous selection for wood traits can be applied with minor effects on genetic gain for growth. Conversely, moderate to strong correlations between wood density and chemical traits exist, likely due to their interdependency on cell wall structure such that responses to selection will be connected for these traits. Our results illustrate the advantage of using genomewide marker data to inform tree breeding in general and have important consequences for operational breeding of eucalypt urograndis hybrids. Fil: de Lima, Bruno Marco. Universidade de Sao Paulo; Brasil. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil Fil: Cappa, Eduardo Pablo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Silva Junior, Orzenil B.. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil. Universidade Católica de Brasília; Brasil Fil: Garcia, Carla. No especifíca; Fil: Mansfield, Shawn D.. University of British Columbia; Canadá Fil: Grattapaglia, Dario. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil. Universidade Católica de Brasília; Brasil

Recent years have seen an increased interest towards utilizing bio-based and biodegradable materials for barrier packaging applications. Most of the above said materials usually have certain shortcomings that discourage their adoption as a preferred material of choice. Nanocellulose falls into such category. It has excellent barrier against grease, mineral oils and oxygen, but poor tolerance against water vapor, which makes it unsuitable to be used at high humidity. In addition, nanocellulose suspensions' high viscosity and yield stress already at low solids content, and poor adhesion to substrates create additional challenges for high-speed processing. Polylactic acid (PLA) is another potential candidate that has a reasonably high tolerance against water vapor, but rather poor barrier against oxygen. The current work explores the possibility to combine both these materials into thin multilayer coatings onto paperboard. A custom-built slot-die was used to coat either microfibrillated cellulose (MFC) or cellulose nanocrystals (CNCs) onto pigment-coated baseboard in a continuous process. These were subsequently coated with PLA using a pilot scale extrusion coater. Low-density polyethylene (LDPE) was used as a reference extrusion coating. Cationic starch pre-coating and corona treatment improved the adhesion at nanocellulose/baseboard and nanocellulose/PLA interfaces, respectively. Water vapor transmission rate for nanocellulose + PLA coatings remained lower than the control PLA coating, even at a high relative humidity of 90% (38 oC). The multilayer coating had 98% lower oxygen transmission rate compared to just PLA coated baseboard and heptane vapor transmission rate reduced by 99% in comparison to baseboard. Grease barrier for nanocellulose + PLA coatings increased 5-fold compared to nanocellulose alone and 2-fold compared to PLA alone. This approach of processing nanocellulose and PLA into multiple layers utilizing slot-die and extrusion coating in tandem has the potential to produce a barrier packaging paper that is both 100% bio-based and biodegradable. publishedVersion

Objectives Our objective was to evaluate the effectiveness of peer-led self-management education in improving glycaemic control in patients with type 2 diabetes in a low-income country (Mali). Methods We conducted an open-label randomised controlled trial. A total of 151 adults (76% women, mean age 52.5) with type 2 diabetes (HbA1c≥8%), treated in the diabetes consultation units of two secondary health centres in Bamako, were allocated to peer-led structured patient education (n = 76) or conventional care alone (n = 75). The intervention group received 1 year of culturally tailored structured patient education (3 courses of 4 sessions) delivered in the community by five trained peer educators. Both groups underwent conventional diabetes monitoring and follow-up. Primary outcome was the mean absolute change in HbA1c from baseline to 12 months. Results 177 education sessions were delivered to the intervention group. Patient attrition was 8%. From baseline to 12 months, the decrease in HbA1c levels was 1.05% (SD = 2.0; CI95%: 1.54;-0.56) in the intervention group compared with 0.15% (SD = 1.7; CI95%: -0.56; 0.26) in the control group, p = 0.006. Mean BMI change was -1.65 kg/m2 (SD = 2.5; CI95%: -2.25; -1.06) in the intervention group and +0.05 kg/m2 (SD = 3.2; CI95%: -0.71; 0.81) in the control group, p = 0.0005. Mean waist circumference decreased by 3.34 cm (SD = 9.3; CI95%: -5.56;-1.13) in the intervention group and increased by 2.65 cm (SD = 10.3; CI95%: 0.20; 5.09) in the control group, p = 0.0003. Conclusions Peer-led structured patient education delivered over 1 year to patients with poorly controlled type 2 diabetes in Mali yielded substantial improvements in glycaemic control and anthropometric parameters. This is of importance for the scaling up of efficient interventions in low-resource settings in the future. Trial registration ClinicalTrials.gov NCT01485913

Background In 2012, the World Health Organization recommended blocking the transmission of Plasmodium falciparum with single low-dose primaquine (SLDPQ, target dose 0.25 mg base/kg body weight), without testing for glucose-6-phosphate dehydrogenase deficiency (G6PDd), when treating patients with uncomplicated falciparum malaria. We sought to develop an age-based SLDPQ regimen that would be suitable for sub-Saharan Africa. Methods Using data on the anti-infectivity efficacy and tolerability of primaquine (PQ), the epidemiology of anaemia, and the risks of PQ-induced acute haemolytic anaemia (AHA) and clinically significant anaemia (CSA), we prospectively defined therapeutic-dose ranges of 0.15–0.4 mg PQ base/kg for children aged 1–5 years and 0.15–0.5 mg PQ base/kg for individuals aged ≥6 years (therapeutic indices 2.7 and 3.3, respectively). We chose 1.25 mg PQ base for infants aged 6–11 months because they have the highest rate of baseline anaemia and the highest risks of AHA and CSA. We modelled an anthropometric database of 661,979 African individuals aged ≥6 months (549,127 healthy individuals, 28,466 malaria patients and 84,386 individuals with other infections/illnesses) by the Box–Cox transformation power exponential and tested PQ doses of 1–15 mg base, selecting dosing groups based on calculated mg/kg PQ doses. Results From the Box–Cox transformation power exponential model, five age categories were selected: (i) 6–11 months (n = 39,886, 6.03%), (ii) 1–5 years (n = 261,036, 45.46%), (iii) 6–9 years (n = 20,770, 3.14%), (iv) 10–14 years (n = 12,155, 1.84%) and (v) ≥15 years (n = 328,132, 49.57%) to receive 1.25, 2.5, 5, 7.5 and 15 mg PQ base for corresponding median (1st and 99th centiles) mg/kg PQ base of: (i) 0.16 (0.12–0.25), (ii) 0.21 (0.13–0.37), (iii) 0.25 (0.16–0.38), (iv) 0.26 (0.15–0.38) and (v) 0.27 (0.17–0.40). The proportions of individuals predicted to receive optimal therapeutic PQ doses were: 73.2 (29,180/39,886), 93.7 (244,537/261,036), 99.6 (20,690/20,770), 99.4 (12,086/12,155) and 99.8% (327,620/328,132), respectively. Conclusions We plan to test the safety of this age-based dosing regimen in a large randomised placebo-controlled trial (ISRCTN11594437) of uncomplicated falciparum malaria in G6PDd African children aged 0.5 − 11 years. If the regimen is safe and demonstrates adequate pharmacokinetics, it should be used to support malaria elimination. Electronic supplementary material The online version of this article (doi:10.1186/s12916-017-0990-6) contains supplementary material, which is available to authorized users.

Background Rift Valley fever (RVF) is a vector-borne disease affecting ruminants and humans. Madagascar was heavily affected by RVF in 2008–2009, with evidence of a large and heterogeneous spread of the disease. The identification of at-risk environments is essential to optimize the available resources by targeting RVF surveillance in Madagascar. Herein, the objectives of our study were: (i) to identify the environmental factors and areas favorable to RVF transmission to both cattle and human and (ii) to identify human behaviors favoring human infections in Malagasy contexts. Methodology/Principal Findings First, we characterized the environments of Malagasy communes using a Multiple Factor Analysis (MFA). Then, we analyzed cattle and human serological data collected at national level using Generalized Linear Mixed Models, with the individual serological status (cattle or human) as the response, and MFA factors, as well as other potential risk factors (cattle density, human behavior) as explanatory variables. Cattle and human seroprevalence rates were positively associated to humid environments (p, Author Summary Rift Valley fever virus (RVFV) is a pathogen that causes a vector-borne tropical disease. The disease affects ruminants and humans and severely impacts the health and economy of affected countries. Madagascar was heavily affected by Rift Valley fever (RVF) in 2008–2009, with evidence of a large and heterogeneous spread of the disease. Our study aims at identifying environmental and human-related risk factors for RVFV transmission. First, we characterized Malagasy environments according to their putative influence on RVFV mosquito density and population dynamics. Then we statistically analyzed cattle and human serological data collected at a national level with the individual serological status as response, and Malagasy environments previously characterized by climatic and landscape variables as well as other potential risk factors as explanatory variables. Our results identified humid environments of the western, north-western and eastern parts of the island as risky areas. The identification of at-risk environments is essential to focus veterinary surveillance and control of RVFV.

Erratum in - Correction: Temporal Patterns of Influenza A and B in Tropical and Temperate Countries: What Are the Lessons for Influenza Vaccination? PLoS One. 2016 May 2;11(5):e0155089. doi: 10.1371/journal.pone.0155089. Introduction: Determining the optimal time to vaccinate is important for influenza vaccination programmes. Here, we assessed the temporal characteristics of influenza epidemics in the Northern and Southern hemispheres and in the tropics, and discuss their implications for vaccination programmes. Methods: This was a retrospective analysis of surveillance data between 2000 and 2014 from the Global Influenza B Study database. The seasonal peak of influenza was defined as the week with the most reported cases (overall, A, and B) in the season. The duration of seasonal activity was assessed using the maximum proportion of influenza cases during three consecutive months and the minimum number of months with 80% of cases in the season. We also assessed whether co-circulation of A and B virus types affected the duration of influenza epidemics. Results: 212 influenza seasons and 571,907 cases were included from 30 countries. In tropical countries, the seasonal influenza activity lasted longer and the peaks of influenza A and B coincided less frequently than in temperate countries. Temporal characteristics of influenza epidemics were heterogeneous in the tropics, with distinct seasonal epidemics observed only in some countries. Seasons with co-circulation of influenza A and B were longer than influenza A seasons, especially in the tropics. Discussion: Our findings show that influenza seasonality is less well defined in the tropics than in temperate regions. This has important implications for vaccination programmes in these countries. High-quality influenza surveillance systems are needed in the tropics to enable decisions about when to vaccinate. The Global Influenza B Study is supported by an unrestricted research grant from Sanofi Pasteur. info:eu-repo/semantics/publishedVersion

Background: Mobile learning (m-learning) has becoming very popular in education due to the rapidly advancing technology in our society. The potentials of the mobile applications should be used to enhance the education process. Few mobile applications have been designed to complement the study of physical therapy skills for physiotherapy students. The aim of this study was to investigate whether a mobile application, as a supplement to traditional learning, is useful for physiotherapy students in the acquisition of palpation and ultrasound skills in the shoulder area. Methods: Forty-nine students participated in this single-blinded, randomized controlled study. They were randomly distributed into two groups: experimental, with free access to the mobile application; and control, with access to traditional learning materials on the topic. Objective structured clinical evaluation (OSCE) and multiple-choice questionnaire (MCQ) were used to assess the educational intervention. Then, we also assessed the time taken to get a reliable ultrasound image and to localize a specific shoulder structure by palpation. Results: There was no significant intergroup difference in the acquisition of theoretical knowledge (p = .089). Scores were significantly higher in the experimental group than in the control group for the majority items in the ultrasound assessment; positioning of patient (p < .001), positioning of ultrasound probe (p = 0.007), handling of ultrasound probe (p = .013) and global OSCE (p < .001) and skills in palpation of the shoulder; position of patient (p = .009), direction of palpation contact (p = .021) and global OSCE (p = .034). There were no significant differences in the time required to perform the examination between groups in ultrasound (p = .944) and palpation (p = .393). The results from the post-program survey assessing the global satisfaction with the mobile application were high (8.200 ± .767), on an 11 numeric point rating scale. Conclusion: These results suggest the effectiveness of an m-learning program as a complement to traditional education for developing skills in ultrasound and palpation of the shoulder region in undergraduate physiotherapy students., The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by a grant from the Educational Innovation Unit of the University of Granada, Spain (PID 13–86).

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden., Instituto de Biotecnologia y Biologia Molecular, Facultad de Ciencias Exactas, Facultad de Ciencias Médicas

Background Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis. Methods and Findings In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island. Conclusions Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures.