Impact of HIV-Related Immune Impairment of Yellow Fever Vaccine Immunogenicity in People Living with HIV-ANRS 12403.

The yellow fever (YF) vaccine is one of the safest and most effective vaccines currently available. Still, its administration in people living with HIV (PLWH) is limited due to safety concerns and a lack of consensus regarding decreased immunogenicity and long-lasting protection for this population. The mechanisms associated with impaired YF vaccine immunogenicity in PLWH are not fully understood, but the general immune deregulation during HIV infection may play an important role. To assess if HIV infection impacts YF vaccine immunogenicity and if markers of immune deregulation could predict lower immunogenicity, we evaluated the association of YF neutralization antibody (NAb) titers with the pre-vaccination frequency of activated and exhausted T cells, levels of pro-inflammatory cytokines, and frequency of T cells, B cells, and monocyte subsets in PLWH and HIV-negative controls. We observed impaired YF vaccine immunogenicity in PLWH with lower titers of YF-NAbs 30 days after vaccination, mainly in individuals with CD4 count <350 cells/mm3. At the baseline, those individuals were characterized by having a higher frequency of activated and exhausted T cells and tissue-like memory B cells. Elevated levels of those markers were also observed in individuals with CD4 count between 500 and 350 cells/mm3. We observed a negative correlation between the pre-vaccination level of CD8+ T cell exhaustion and CD4+ T cell activation with YF-NAb titers at D365 and the pre-vaccination level of IP-10 with YF-NAb titers at D30 and D365. Our results emphasize the impact of immune activation, exhaustion, and inflammation in YF vaccine immunogenicity in PLWH.

Sharing lessons learned from COVID-19 vaccine introductions: a global community forum for countries.

To optimize the efficient introduction and deployment of COVID-19 vaccines across the globe during the COVID-19 pandemic, in April 2021 WHO launched a new process and tools for countries to rapidly review the early phase of countries' COVID-19 vaccine introduction. This methodology is called the COVID-19 vaccination intra-action review, also known as mini COVID-19 vaccine post-introduction evaluation (mini-cPIE). As of November 2022, 46 mini-cPIEs had been conducted. In collaboration with Project ECHO, WHO convened and facilitated real-time experience sharing and peer-learning among countries following their mini-cPIEs through a virtual global real-time learning forum. This five-session clinic series was attended by 736 participants from 129 countries. Based on post-session feedback surveys, when asked about the utility of the sessions, half of the participants said that sessions led them to review national guidelines and protocols or make other changes to their health systems. The post-series survey sent following the end of the clinic series showed that at least eight countries subsequently conducted a mini-cPIE after participating in the clinics, and participants from at least nine countries indicated the experience shared by peer countries on the clinic largely benefited their COVID-19 vaccine introduction and deployment. In this article, we highlight the benefits and importance of creating a global experience-sharing forum for countries to connect and share pertinent learnings in real-time during an international public health emergency. Moving forward, it is critical to foster a culture of individual and collective learning within and between countries during public health emergencies, with WHO playing an important convening role.

Predictors of Trypanosoma cruzi PCR positivity in patients with chronic Chagas disease.

BACKGROUND: A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD). OBJECTIVES: To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD. METHODS: This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant. FINDINGS: Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 [95% confidence interval (CI) 0.30-0.93]; p = 0.03}, right bundle branch block [OR 1.78 (95% CI 1.09-2.89); p = 0.02], and history of trypanocidal treatment [OR 0.13 (95% CI 0.04-0.38); p = 0.0002]. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR. MAIN CONCLUSIONS: Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.

Biomarkers and Echocardiographic Predictors of Cardiovascular Outcome in Patients With Chronic Chagas Disease.

Background Chagas disease (CD) presents an ominous prognosis. The predictive value of biomarkers and new echocardiogram parameters in adjusted models have not been well studied. Methods and Results There were 361 patients with chronic CD (57.6% men, 61±11 years of age, clinical forms: indeterminate 27.1%, cardiac 56.6%, digestive 3.6%, cardiodigestive 12.7%) included in this single-center, observational, prospective longitudinal study. Echocardiographic evaluation included strain analyses of left atrial, left ventricular (LV), and right ventricular and 3-dimensional analyses of left atrial and LV volumes. Biomarkers included cardiac troponin I, brain natriuretic peptide, transforming growth factor ß1, tumor necrosis factor, matrix metalloproteinases, and Trypanosoma cruzi polymerase chain reaction. The studied end point was a composite of CD-related mortality, heart transplant, hospital admission due to worsening heart failure, or new cardiac device insertion. Event-free survival was analyzed by multivariable regression analyses adjusted for competing risks. P values <0.05 were considered significant. The composite event occurred in 79 patients after 4.9±2.0 years follow-up. LV end-diastolic volume (hazard ratio [HR], 1.01 [95% CI, 1.00-1.02]; P=0.02), peak negative global atrial strain (HR, 1.08 [95% CI, 1.00-1.17]; P=0.04), LV global circumferential strain (HR, 1.12 [95% CI, 1.04-1.21]; P=0.003), LV torsion (HR, 0.55 [95% CI, 0.35-0.81]; P=0.003), brain natriuretic peptide (HR, 2.03 [95% CI, 1.23-3.34]; P=0.005), and positive T cruzi polymerase chain reaction (HR, 1.80 [95% CI, 1.12-2.91]; P=0.01) were end point predictors independent from age, sex, 2-dimensional echocardiographic indexes, hypertension, previous cardiac device, and CD cardiac form. Conclusions Two-dimensional strain- and 3-dimensional-derived parameters, brain natriuretic peptide, and positive T cruzi polymerase chain reaction can be useful for prediction of CD cardiovascular events.

Monitoring the Evolution of the Aroma Profile of Lager Beer in Aluminium Cans and Glass Bottles during the Natural Ageing Process by Means of HS-SPME/GC-MS and Multivariate Analysis.

Headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME/GC-MS), sensory evaluation, and multivariate analysis were applied to monitor and compare the evolution of the aromatic profile of a lager beer in different types of containers (aluminum cans and glass bottles) during the natural ageing process. Samples were aged naturally for a year in the absence of light with a controlled temperature of around 14 °C +/- 0.5 °C. The sensory evaluation applied was a blind olfactometric triangle test between canned and bottled samples at different periods of aging: fresh, 6 months, and 11 months. The sensory evaluation showed that the panelists were able to differentiate between samples, except for the fresh samples from the brewery. A total of 34 volatile compounds were identified using the HS-SPME/GC-MS technique for both packaging types in this experiment. The application of multivariate analysis to the GC-MS data showed that the samples could not be differentiated according to the type of packaging but could be differentiated by the ageing time. The results showed that the combination of sensory, HS-SPME-GC-MS, and multivariate analysis seemed to be a valuable tool for monitoring and identifying possible changes in the aroma profile of a beer during its shelf life. Furthermore, the results showed that storing beer under optimal conditions helped preserve its quality during its shelf life, regardless of the type of packaging (aluminum can and glass bottle).

Multivariate Analysis of the Influence of Microfiltration and Pasteurisation on the Quality of Beer during Its Shelf Life.

Gas chromatography-mass spectrometry (GC-MS), physicochemical and microbiological analyses, sensory descriptive evaluation, and multivariate analyses were applied to evaluate the efficiencies of microfiltration and pasteurization processes during the shelf life of beer. Samples of microfiltered and pasteurised beer were divided into fresh and aged groups. A forced ageing process, which consisted of storing fresh samples at 55° C for 6 days in an incubator and then keeping them under ambient conditions prior to analysis, was applied. Physicochemical analysis showed that both microfiltered and pasteurised samples had a slight variation in apparent extract, pH, and bitterness. The samples that underwent heat treatment had lower colour values compared with those that were microfiltered. Chromatographic peak areas of vicinal diketones increased in both fresh and aged samples. The results of the microbiological analysis revealed spoilage lactic acid bacteria (Lactobacillus) and yeasts (Saccharomyces and non-Saccharomyces) in fresh microfiltered samples. In the GC-MS analysis, furfural, considered by many authors as a heat indicator, was detected only in samples that underwent forced ageing and not in samples that were subjected to thermal pasteurisation. Finally, sensory analysis found differences in the organoleptic properties of fresh microfiltered samples compared with the rest of the samples.

Predictors of Trypanosoma cruzi PCR positivity in patients with chronic Chagas disease

BACKGROUND A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD). OBJECTIVES To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD. METHODS This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant. FINDINGS Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 [95% confidence interval (CI) 0.30-0.93]; p = 0.03}, right bundle branch block [OR 1.78 (95% CI 1.09-2.89); p = 0.02], and history of trypanocidal treatment [OR 0.13 (95% CI 0.04-0.38); p = 0.0002]. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR. MAIN CONCLUSIONS Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.

Ensino híbrido na formação em saúde: uma revisão sistemática / Hybrid education in healthcare education - A systematic review

Introdução: A partir da necessidade de reestruturação das instituições de ensino em saúde devido à pandemia da COVID-19, o ensino híbrido vem se destacando como possibilidade de reorganização das atividades educativas. O objetivo deste estudo foi escrever o desenvolvimento do ensino híbrido na formação de profissionais da área da saúde. Materiais e Métodos: Revisão sistemática da literatura, baseada nas recomendações da Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). A pesquisa foi desenvolvida em pares, entre julho a setembro de 2020, em quatro bases de dados eletrônicas. Os descritores foram os termos "Educação a Distância", "Educação Superior", "Aprendizagem", "Saúde", "Enfermagem", "Medicina", "Odontologia" e "Fisioterapia" e "Ensino híbrido". Os artigos foram classificados conforme seu Nível de Evidência. Resultados: 49 artigos foram selecionados, entre estudos quantitativos, qualitativos e de método misto. Foram encontradas experiências do desenvolvimento do ensino híbrido nos diferentes cursos de formação na área da saúde. Observou-se aplicação do ensino híbrido segundo o modelo de Rotação, modelo à la carte e o modelo Flex. Discussão: o ensino híbrido vem ganhando destaque cada vez maior no cenário da educação acadêmica em saúde. Foi visto que, a partir dele, o aluno destaca-se em sua aprendizagem, pois é o principal gerenciador deste processo, aprendendo ativamente por diversos instrumentos educativos a partir da condução do professor. Conclusões: O êxito do ensino híbrido pode estar relacionado ao seu caráter inovador, flexível, com boa relação custo-benefício e capaz de tornar os alunos protagonistas do seu processo de ensino-aprendizagem, influenciando no desempenho acadêmico dos alunos.

Introduction: Given the need to restructure healthcare education institutions due to the COVID-19 pandemic, hybrid education has been identified as an alternative to reorganize educational activities. The objective of this study is to describe hybrid education development in healthcare education. Materials and Methods: A systematic literature review was conducted based on the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guideline. A peer-reviewed search was conducted across four electronic databases between July and September 2020. Descriptors used were "distance education", "learning", "health", "nursing", "medicine", "dentistry", "physical therapy" and "hybrid education". Selected articles were classified based on their level of evidence. Results: 49 articles were selected, including quantitative, qualitative and mixed methods studies. Experiences in the development of hybrid education in multiple healthcare courses were found. Hybrid education has been implemented based on the rotation model, a la carte model and Flex model. Discussion: Hybrid education has been gaining more and more importance in the academic scenario of healthcare education. Based on this, students manage to stand out in their learning process as they become the main agent of this process by actively learning using multiple education tools provided by their teachers. Conclusions: Successful hybrid education can be related to its innovative, flexible, cost-effective nature and the ability to turn students into key players in their teaching-learning process, which ends up influencing their own academic performance.

Introducción: Ante la necesidad de reestructurar las instituciones de educación en salud a causa de la pandemia por COVID-19, se ha señalado a la educación híbrida como una alternativa para reorganizar las actividades educativas. El objetivo de este estudio es describir el desarrollo de la educación híbrida en la formación de los profesionales en el área de la salud. Materiales y métodos: Revisión sistemática de la literatura basada en las recomendaciones de los elementos de informes preferidos para revisiones sistemáticas y metaanálisis (PRISMA). La búsqueda se realizó por pares entre julio y septiembre de 2020 en cuatro bases de datos electrónicas. Los descriptores utilizados fueron "educación a distancia", "educación superior", "aprendizaje", "salud", "enfermería", "medicina", "odontología", "fisioterapia" y "educación híbrida". A su vez, los artículos seleccionados se clasificaron según su nivel de evidencia. Resultados: Se seleccionaron 49 artículos, entre los que se incluyeron estudios cuantitativos, cualitativos y de métodos mixtos. Se identificaron experiencias en el desarrollo de la educación híbrida en diferentes cursos de formación en el área de la salud. Se observó que la educación híbrida se implementó de acuerdo con el modelo de rotación, el modelo a la carta y el modelo Flex. Discusión: La enseñanza híbrida ha venido ganando cada vez más importancia en el escenario académico de la educación en salud. Se observó que, a partir de esta, el estudiante logra destacarse en su aprendizaje, ya que se convierte en el principal gestor de este proceso al aprender de forma activa mediante diversas herramientas educativas guiadas por el docente. Conclusiones: El éxito de la educación híbrida se puede relacionar con su carácter innovador, flexible, con buena relación costo-beneficio y la habilidad de convertir a los estudiantes en protagonistas de su proceso de enseñanza-aprendizaje, lo que influye en su propio rendimiento académico.

An Overview of the Application of Multivariate Analysis to the Evaluation of Beer Sensory Quality and Shelf-Life Stability.

Achieving beer quality and stability remains the main challenge for the brewing industry. Despite all the technologies available, to obtain a high-quality product, it is important to know and control every step of the beer production process. Since the process has an impact on the quality and stability of the final product, it is important to create mechanisms that help manage and monitor the beer production and aging processes. Multivariate statistical techniques (chemometrics) can be a very useful tool for this purpose, as they facilitate the extraction and interpretation of information from brewing datasets by managing the connections between different types of data with multiple variables. In addition, chemometrics could help to better understand the process and the quality of the product during its shelf life. This review discusses the basis of beer quality and stability and focuses on how chemometrics can be used to monitor and manage the beer quality parameters during the beer production and aging processes.

Blood Feeding Sources of Nyssomyia antunesi (Diptera: Psychodidae): A Suspected Vector of Leishmania (Kinetoplastida: Trypanosomatidae) in the Brazilian Amazon.

Present work aimed to identify blood feeding sources and attempt to detect Leishmania DNA in Nyssomyia antunesi, suspected vector of Leishmania sp., from a park in the urban center of Belém, the capital of Pará State, in the Brazilian Amazon. Entire bodies and gut contents of Ny. antunesi engorged females, previously captured in the urban park with Centers for Disease Control (CDC) light traps and aspiration on tree bases, were subjected to Leishmania and vertebrate DNA detection through amplification of the Leishmania mini-exon and vertebrate cytochrome b (cyt b) gene regions, respectively. The quality of DNA extraction from entire bodies was ensured through amplification of the dipteran cyt b region. The vertebrate cyt b amplicons were sequenced and compared with those available on GenBank. A maximum likelihood phylogenetic tree was constructed to assess the clustering patterns of these sequences. Leishmania DNA was not detected. The sequences of 13 vertebrate cyt b amplicons were considered informative, exhibiting similarity and clustering with the following six vertebrate species: Dasyprocta leporina (1), Cuniculus paca (1), Tamandua tetradactyla (4), Choloepus didactylus (4), Pteroglossus aracari aracari (2), Homo sapiens (1). The samples of D. leporina and C. paca were obtained from the CDC canopy, whereas the others were by aspiration from tree bases. The present results revealed the eclectic and opportunist blood-feeding behavior of Ny. antunesi, with birds and mammals, these last ones acting as potential reservoirs for Leishmania species, distributed throughout the vertical forest strata.

Fruitful exchanges: social networks and food resources amidst change.

BACKGROUND: The Amazon region of Brazil is known both for its significant biological and cultural diversity. It is also a region, like many parts of the country, marked by food insecurity, even amongst its rural agricultural populations. In a novel approach, this paper addresses the networks of exchanges of local food and their relationship to the agrobiodiversity of traditional riverine peoples' (ribeirinho) households in the Central Amazon. Methodologically, it involves mapping the social networks and affinities between households, inventories of known species, and, finally, statistical tests of the relationships between network and subsequent agrobiodiversity. RESULTS: The diversity per area of each land type where food cultivation or management takes place shows how home gardens, fields and orchards are areas of higher diversity and intense cultivation compared to fallow areas. Our findings, however, indicate that a household's income does appear to be strongly associated with the total agrobiodiversity across cultivation areas. In addition, a household's agrobiodiversity is significantly associated with the frequency and intensity of food exchanges between households. CONCLUSIONS: Agrobiodiversity cannot be considered separate from the breadth of activities focused on sustenance and yields from the cash economy, which riverine people engage in daily. It seems to be connected to quotidian social interactions and exchanges in both predictable and occasionally subtler ways. Those brokers who serve as prominent actors in rural communities may not always be the most productive or in possession of the largest landholdings, although in some cases they are. Their proclivity for cultivating and harvesting a wide diversity of produce may be equally important if not more so. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40066-021-00342-5.

Building global capacity for COVID-19 vaccination through interactive virtual learning.

BACKGROUND: To support the introduction of the COVID-19 vaccine, the World Health Organization and its partners developed an interactive virtual learning initiative through which vaccination stakeholders could receive the latest guidance, ask questions, and share their experiences. This initiative, implemented between 9 February 2021 and 15 June 2021, included virtual engagement between technical experts and participants during a 15-session interactive webinar series as well as web and text-messaging discussions in English and French. METHODS: This article uses a mixed-methods approach to analyze survey data collected following each webinar and a post-series survey conducted after the series had concluded. Participant data were tracked for each session, and feedback surveys were conducted after each session to gauge experience quality and content usability. Chi-square tests were used to compare results across professions (health workers, public health practitioners, and others). RESULTS: The COVID-19 Vaccination: Building Global Capacity webinar series reached participants in 179 countries or 93% of the WHO Member States; 75% of participants were from low- and middle-income countries. More than 60% of participants reported using the resources provided during the sessions, and 47% reported sharing these resources with colleagues. More than 79% of participants stated that this initiative significantly improved their confidence in preparing for and rolling out COVID-19 vaccinations; an additional 20% stated that the initiative "somewhat" improved their confidence. In the post-series survey, 70% of participants reported that they will "definitely use" the knowledge derived from this learning series in their work; an additional 20% will "probably use" and 9% would "possibly use" this knowledge in their work. CONCLUSION: The COVID-19 Vaccination: Building Global Capacity learning initiative used a digital model of dynamic, interactive learning at scale. The initiative enhanced WHO's ability to disseminate knowledge, provide normative guidance, and share best practices to COVID-19 vaccination stakeholders in real time. This approach allowed WHO to hear the information needs of stakeholders and respond by developing guidance, tools, and training to support COVID-19 vaccine introduction. WHO and its partners can learn from this capacity-building experience and apply best practices for digital interactive learning to other health programs moving forward.

Conditional Cash Transfers in the Amazon: From the Nutrition Transition to Complex Dietary Behavior Change.

Nutrition transition theory describes a progressive substitution of local staples for industrialized processed foods in local diets, a process documented diversely across world regions, and increasingly observed in rural areas of the global south. Here we examine the role of conditional cash transfer programs, in particular the emblematic Brazilian Bolsa Família (BFP), in driving nutritional transition in rural areas of the Amazon. Based on ethnographic research with both participating and nonparticipating women in the Amanã Sustainable Development Reserve (SDR), our analysis integrates Food Frequency Questionnaires (FFQ), seasonal 24-hour food intake recalls, and stable isotope ratios in fingernails to examine dietary behavioral change. Contrary to dietary changes observed elsewhere in the Amazon, participation in the BFP is not associated with a significant substitution of local staples for industrialized processed foods in Amanã. While an increase in the consumption of some industrialized foods was observed, it has been selective and it has not changed the structure of diets. Factors such as social and cultural value of local staples, resident's involvement with the SDR, their relationship with lake and upland forest and agricultural environments, and limited market access have buffered the expansion of industrialized processed foods as observed elsewhere.

Toll-like receptors 2, 4, and 9 expressions over the entire clinical and immunopathological spectrum of American cutaneous leishmaniasis due to Leishmania(V.) braziliensis and Leishmania (L.) amazonensis.

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-ß cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-ß+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL.

High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

BACKGROUND: Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. METHODS/PRINCIPAL FINDINGS: Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. CONCLUSIONS/SIGNIFICANCE: HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years.

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.