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Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of endosymbiotic viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis ofLeishmania braziliensisparasites and their endosymbioticLeishmaniaRNA virus. We show that parasite populations circulate in isolated pockets of suitable habitat and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites were geographically and ecologically dispersed, and commonly infected from a pool of genetically diverse viruses. Our results suggest that parasite hybridization, likely due to increased human migration and ecological perturbations, increased the frequency of endosymbiotic interactions known to play a key role in disease severity.

Objetivo: Evaluar la toxicidad de tres chalconas sintéticas administradas por vía intraperitoneal en ratones BALB/c. Materiales y métodos: La dosis letal media (DL50) se estimó por el método Up-and-Down de Dixon. La toxicidad subcrónica de las chalconas se evaluó a 20 y 40 mg/kg por 21 días. Se evaluó el efecto tóxico a nivel de comportamiento, fisiológico, bioquímico e histológico. Resultados: La chalcona 43 generó moco en las heces, daño visceral (hígado) y alteración en el coeficiente de órganos (riñón, p = 0,037 y cerebro, p = 0,008) en comparación con el grupo control. Además, en el análisis histológico se observó que esta chalcona produjo edema, inflamación y necrosis en los órganos evaluados, aunque no hubo diferencia significativa con el control. Todos los parámetros bioquímicos no difirieron significativamente entre los grupos de tratamiento a dosis de 40 mg/kg y el control. Conclusiones: La DL50 para las tres chalconas fue superior a 550 mg/kg de peso corporal. Las chalconas 40 y 42 son relativamente no tóxicas. Ambas pueden considerarse seguras para la aplicación vía intraperitoneal en ratones BALB/c y, en consecuencia, son posibles candidatas para ser usadas en el tratamiento contra las leishmaniosis.

Land-atmosphere interactions at different temporal and spatial scales are important for our understanding of the Earth system and its modeling. The Landscape Evolution Observatory (LEO) at Biosphere 2, managed by the University of Arizona, hosts three nearly identical artificial bare-soil hillslopes with dimensions of 11 × 30 m2 (1 m depth) in a controlled and highly monitored environment within three large greenhouses. These facilities provide a unique opportunity to explore these interactions. The dataset presented here is a subset of the measurements in each LEO’s hillslopes, from 1 July 2015 to 30 June 2019 every 15 minutes, consisting of temperature, water content and heat flux of the soil (at 5 cm depth) for 12 co-located points; temperature, relative humidity and wind speed above ground at 5 locations and 5 different heights ranging from 0.25 m to 9–10 m; 3D wind at 1 location; the four components of radiation at 2 locations; spatially aggregated precipitation rates, total subsurface discharge, and relative water storage; and the measurements from a weather station outside the greenhouses., Measurement(s) temperature of soil • wetness of soil • heat conductivity • atmospheric wind speed • Humidity • temperature of air • longwave radiation • shortwave radiation • atmospheric wind direction • hydrological precipitation process Technology Type(s) thermocouple • dielectrometry • thermopile • anemometer • weather station • pyrgeometer • pyranometer • Flowmeter Device Factor Type(s) replicate hillslope • location • height Sample Characteristic - Environment soil • atmosphere Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12881810

Resumen Antecedentes La histoplasmosis diseminada (HD) es una infeccion fungica oportunista en pacientes con infeccion por VIH gravemente inmunocomprometidos. El sindrome hemofagocitico (SHF), que puede presentarse en estos pacientes coinfectados cuando la respuesta inmunitaria esta significativamente alterada, suele estar asociado a una elevada mortalidad. Objetivos Describir las caracteristicas epidemiologicas, clinicas, analiticas y microbiologicas, asi como evaluar la presencia de SHF, en pacientes con HD-VIH. Metodos Estudio retrospectivo de serie de casos, consistente en la revision de registros clinicos de pacientes con diagnostico de HD e infeccion VIH, durante los anos 2014 y 2015. Resultados El 1,3% (8/597) de los pacientes VIH presentaron HD. El 100% se hallaban en estadio C3 y el 75% (6/8) no se encontraban en terapia antirretroviral combinada (TARVc). Los dos pacientes restantes habian comenzado recientemente el tratamiento con TARVc (posible sindrome de reconstitucion inmunologica). El 62,5% (5/8) cumplieron con criterios diagnosticos de SHF. Las manifestaciones clinicas mas frecuentes fueron el sindrome linfoproliferativo y consuntivo, el compromiso respiratorio y la citopenia. En el 75% (6/8) de los pacientes se aislo Histoplasma en ganglios, en el 25% (2/8) en muestras hematicas y en uno adicionalmente en tejido intestinal. La terapia antifungica fue anfotericina B desoxicolato; no se emplearon adyuvantes. La mortalidad global fue del 50%. Conclusiones En nuestra serie la coinfeccion HD-VIH progreso en la mayoria de los casos a SHF con elevada mortalidad. El cuadro clinico puede asemejarse al de otras enfermedades sistemicas, como la tuberculosis, o presentarse simultaneamente a ellas. Con el fin de obtener un diagnostico precoz y poder prescribir la terapia especifica oportuna es importante poseer un adecuado indice de sospecha en pacientes con sindrome linfoproliferativo y consuntivo asociado a citopenia grave.

This study discusses the synoptic situations associated with cases of high pollution in terms of black carbon (BC) concentrations observed at the research site during the four months of the VAMPIRE campaign that is described fully in the companion study by Marin et al. (2017). Cases in which the concentration of BC exceeded the 85th percentile were selected to evaluate composite synoptic situations, resulting in a total of fifteen cases analyzed. These fifteen cases occurred during one of the three prevalent synoptic situations identified and labeled as: i) Coastal low, characterized by weak easterly winds and low planetary boundary layer (PBL) height due to an enhancement of the temperature inversion before the arrival of the coastal low; ii) Pre-frontal, also characterized by low PBL height, with a cold front approaching from the South and the coastal low retreating northwards, also associated with pollution episodes in Santiago (Rutllant and Garreaud, 1995); and iii) SEP anticyclone characterized by large stability, very low gradient in the mean sea level pressure and weak westerly-southwesterly wind due to the dominance of the southeast Pacific anticyclone. Coastal low cases were observed not only during winter but also in all four months of the VAMPIRE field campaign, while Pre-frontal cases were observed only at the end of July and August 2014. All three synoptic situations are consistent with a reduced synoptic forcing, in which the mesoscale sea-land breeze predominates and advects large pollutant concentrations from local/regional emission sources to the research site. Detailed analysis is presented here for two case studies: one Coastal low and one Pre-frontal, combining the observations of pollutants as well as mesoscale modeling with the WRF model to estimate back-trajectories and evaluate the relative role of the synoptic and mesoscale forcing on the pollution episodes in Valparaiso. These synoptic conditions and their interplay with meso-scale circulations identified during VAMPIRE provide evidence and insight that will aid in future air quality forecasts for Valparaiso.

Droughts are a worldwide concern, thus assessment efforts are conducted by many centers around the world, mainly through simple drought indices which usually neglect important hydrometeorological processes or require variables available only from complex Land Surface Models (LSMs). The U.S. Climate Prediction Center (CPC) uses the Leaky Bucket (LB) water-balance model to post-process temperature and precipitation, providing soil moisture (SM) anomalies to assess drought conditions. However, despite its crucial role in the water cycle, snowpack has been neglected by LB and most drought indices.Taking advantage of the high-quality snow water equivalent (SWE) data from the University of Arizona (UA), a single-layer snow scheme, forced by daily temperature and precipitation only, is developed for LB implementation and tested with two independent forcing datasets. Compared against the UA and SNOTEL SWE data over CONUS, LB outperforms a sophisticated LSM (Noah/NLDAS-2), with the median LB vs SNOTEL correlation (RMSE) about 40% (26%) higher (lower) than that from Noah/NLDAS-2, with only slight differences due to different forcing datasets.The changes in the temporal variability of SM due to the snowpack treatment lead to improved temporal and spatial distribution of drought conditions in the LB simulations compared to the reference U.S. Drought Monitor maps, highlighting the importance of snowpack inclusion in drought assessment. The simplicity but reasonable reliability of the LB with snowpack treatment makes it suitable for drought monitoring and forecasting in both snow-covered and snow-free areas, while only requiring precipitation and temperature data (markedly less than other water-balance-based indices).

Although complex hydrological models with detailed physics are becoming more common, lumped and semi-distributed models are still used for many applications and offer some advantages, such as reduced computational cost. Most of these semi-distributed models use the concept of the hydrological response unit or HRU. In the original conception, HRUs are defined as homogeneous structured elements with similar climate, land use, soil and/or pedotransfer properties, and hence a homogeneous hydrological response under equivalent meteorological forcing. This work presents a quantitative methodology, called hereafter the principal component analysis and hierarchical cluster analysis or PCA/HCPC method, to construct HRUs using gridded meteorological data and hydrological parameters. The PCA/HCPC method is tested using the water evaluation and planning system (WEAP) model for the Alicahue River Basin, a small and semi-arid catchment of the Andes, in Central Chile. The results show that with four HRUs, it is possible to reduce the relative within variance of the catchment up to about 10%, an indicator of the homogeneity of the HRUs. The evaluation of the simulations shows a good agreement with streamflow observations in the outlet of the catchment with an Nash&ndash, Sutcliffe efficiency (NSE) value of 0.79 and also shows the presence of small hydrological extreme areas that generally are neglected due to their relative size.

Wildfire is a major concern worldwide and particularly in Australia. The 2019&ndash, 2020 wildfires in Australia became historically significant as they were widespread and extremely severe. Linking climate and vegetation settings to wildfires can provide insightful information for wildfire prediction, and help better understand wildfires behavior in the future. The goal of this research was to examine the relationship between the recent wildfires, various hydroclimatological variables, and satellite-retrieved vegetation indices. The analyses performed here show the uniqueness of the 2019&ndash, 2020 wildfires. The near-surface air temperature from December 2019 to February 2020 was about 1 &deg, C higher than the 20-year mean, which increased the evaporative demand. The lack of precipitation before the wildfires, due to an enhanced high-pressure system over southeast Australia, prevented the soil from having enough moisture to supply the demand, and set the stage for a large amount of dry fuel that highly favored the spread of the fires.

Significance Parasites are interesting models for studying speciation processes because they have a high potential for specialization, thanks to the intimate ecological association with their hosts and vectors. Yet little is known about the circumstances under which new parasite lineages emerge. Here we studied the genome diversity of parasites of the Leishmania braziliensis species complex that inhabit both Amazonian and Andean biotas in Peru. We identify three major parasite lineages that occupy particular ecological niches and show that these emerged during forestation changes over the past 150,000 y. We furthermore discovered that meiotic recombination between Amazonian and Andean lineages resulted in full-genome hybrids presenting mixed mitochondrial genomes, providing insights into the genetic consequences of hybridization in parasitic protozoa., The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host–parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles—but not maxicircles—show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.

For 60 years, Universidad Peruana Cayetano Heredia (UPCH) in Lima, Peru, has contributed to world-class knowledge in tropical medicine, high-altitude medicine and physiology, public health, and basic sciences with the highest research standards and ethics. We therefore read with interest the World Report by Lucien Chauvin,1 in which we found some inaccuracies that we would like to clarify...

The city of Valparaiso is home to one of the largest commercial ports on the west coast of South America. Port activities, that continue year-round, 24 h a day and seven days a week, produce emissions of pollutants, particularly aerosol particles composed of black and brown carbon (BC and BrC) that have serious impact on human population and the local environment. A measurement program was launched to document the magnitude of selected pollutants, to identify their sources and to evaluate the meteorological processes that enhance and transport these pollutants locally and regionally. In this study, we report the measurements made during four months: 25July - 25August 2014 (referred to as August 2014 throughout the manuscript), December 2014, January 2015 and March 2015. The daily mass concentrations of equivalent black carbon (eBC), derived from measurements of the light absorption coefficient, regularly exceed 5 μg m −3 , a magnitude similar to values found in megacities such as Mexico City. The daily maximum number concentration of condensation nuclei (CN) is usually larger than 30000 cm −3 . The Angstrom absorption exponent (AAE), derived from the absorption coefficients at 550 nm and 870 nm, is used to identify the primary sources of BC and BrC. In colder weather, emissions from the diesel fueled buses and trucks and the consumption of kerosene and wood for residential heating are the main sources of BC. In December, local wildfires contributed to the particle mass loading, but the day-to-day variability in boundary layer height and the presence of clouds and fog in occasions inhibited high concentrations. In March, the port activities reach a yearly peak during the seasonal export of agricultural products that translates into much more ship and truck traffic leading to very high eBC concentrations, comparable to values observed during August. The variability in weather patterns underscores the complexity of meteorological processes that drive the evolution and transport of pollution in Valparaiso.

Cutaneous Leishmaniasis is a skin infection which is commonly present in underdeveloped countries. The incidence is particularly high in amazonic countries of Latin-America like Brazil, Colombia and Perú and is usually reported as endemic. In Perú, more than one million people are at risk of infection and approximately 6,000 new cases are detected each year. The present work proposes to integrate a set of control, monitoring and disease quantification procedures in: (1) an automated tool to expedite the analysis in laboratories studying parasiticidal agents and (2) a non-invasive treatment monitoring protocol. The first, consists in the adaptation of an optical microscope KRUSS MBL3100 to perform a fast image capture and automated promastigotes identification. This may be of value to evaluate the effectiveness of various parasiticidal agents. The counting process is performed by an automatic segmentation in the RGB green color space discriminating elements by their area. The second, proposes a protocol for monitoring the evolution of the disease treatment divided into three stages: supra-skin modeling and reconstruction, subcutaneous exploration by textural characteristics and volumetric segmentation. This protocol is performed using a Next Engine HD 3D scanner and a Vevo Visualsonix 2100 ultrasonic scanner. The results show improvements in sample processing time, accuracy and inter- and intra-operational variability. The sensitivity and accuracy of the microscopic identification system was of 97% and 92% respectively. The exactitude and precision error was up to 2% and the sensitivity and specificity went as high as 71% in the 3D reconstruction and texture analysis respectively.

Under stressful conditions some microorganisms adopt a reversible non or slow proliferative quiescent stage that allows their survival. Although quiescence has been described broadly in bacteria, this phenotype has been only recently discovered inLeishmania. In the present work we developed a biosensor of quiescence that allows to monitor the physiological stage of the parasite at population and single cell levels. We inserted a GFP gene into the ribosomal DNA locus and followed the expression of this reporter gene, driven by the ribosomal promotor (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during thein vitrotransition from extracellular promastigotes to intracellular amastigotes ofL. mexicanaanL. braziliensisand that the decrease in rGFP expression was coupledin vitrowith a decrease in replication as measured by BrdU incorporation. Quiescence was not only observed in reference laboratory strains, but also among clinical isolates. We found that quiescence was reversible as the parasites could rapidly resume their metabolically active and proliferative stage when they were put back in an optimal environment for growth. We demonstrated for the first time in live cells that amastigotes are a heterogeneous population in which shallow and deep quiescent stages may coexist. Finally, we showed that rGFP expression could be monitoredin vivoand that quiescent amastigotes could reside in tissues of animals with latent infections ofL. braziliensisorL. mexicana. We propose rGFP expression as a simple parameter to define quiescent cells and further characterize them.IMPORTANCEQuiescence is a physiological diversification that allows pathogens to overcome chemotherapy without the development of drug resistance and to be invisible to the immune system of their host. Quiescent pathogens can cause latent infections and (re-) emerge in an unpredictable time during the lifetime of the individual. The phenomenon was recently described inLeishmaniain which it could explain several clinical and sub-clinical features, like therapeutic failure, reactivation of the disease and asymptomatic infections. However, a simple biosensor of quiescence forLeishmaniais not yet available. We show for the first time that the integration of GFP within the rDNA locus and the subsequent quantification of its expression can be used as a biosensor to distinguish quiescent subpopulations among live amastigotes. Moreover, we show quiescence is quickly reversible bothin vivoandin vitro. We offer a tool that will allow the further molecular characterization of quiescent parasites.

The presentation of syphilis as a manifestation of immune reconstitution inflammatory syndrome in patients with human immunodeficiency virus (HIV) infection is rare and can be associated with the varied clinical expression of unusual syphilitic manifestations. We report a case of immune reconstitution syndrome with dermatologic, ophthalmologic and neurologic compromise of secondary syphilis in a patient with HIV infection.

Access to nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) first-line antiretroviral therapy (ART) for HIV has been increasing in Peru since a national ART program was initiated in 2004. Between 2007 and 2009, we found a 1% prevalence of pre-ART HIV drug resistance (PDR) among antiretroviral (ARV)-naive Peruvians. Given that PDR has been associated with virologic failure (VF) of ART, in 2014–2015 we enrolled a follow-up cohort at the same institution to determine whether the rate of transmitted resistance had increased and compared virologic outcomes of those with and without PDR. Blood specimens from ARV-naive individuals were assessed for PDR to NNRTI-based ART by an oligonucleotide ligation assay (OLA) sensitive to 2% mutant within an individual's HIV quasispecies at reverse transcriptase codons M41L, K65R, K103N, Y181C, M184V, and G190A, and by Sanger consensus sequencing (CS). Rates of VF (plasma HIV RNA >200 copies/mL) were compared between those with and without PDR. Among 122 ARV-naive adults, PDR was detected by OLA in 17 (13.9%) adults. Compared with the 2007–2009 cohort, the proportion with PDR at OLA codons was significantly increased (p

The management and disposal of concentrate is often considered to be the most significant obstacle to the implementation of membrane technology. Faced with the need to manage and dispose of up to 1.0 million gallons per day (mgd) of concentrate generated from a new 4.0-mgd low pressure reverse osmosis (LPRO) expansion of their water treatment plant (WTP), the City of Ormond Beach embarked on a mission to implement an innovative and sustainable method of concentrate management which consisted of blending the concentrate with treated wastewater at the City’s wastewater treatment plant (WWTP). Implementation of this concentrate management strategy allowed the City to reclaim and reuse a traditionally unutilized resource as well as augment its reclaimed water supplies without consuming treatment capacity of the WWTP. The success of this innovative and sustainable concentrate management strategy has been demonstrated throughout the past four years of operation. Analysis and examination of data preceding and succeeding the LPRO expansion revealed the following: • The City’s reclaimed water supplies increased by an average of 0.34 mgd following the commissioning of the LPRO expansion.

Mucosal leishmaniasis (ML) is a disfiguring manifestation of Leishmania (Viannia) infection. We evaluated parasite load (PL) over time as a potential biomarker of treatment outcome in ML. PL was assessed with kinetoplast DNA quantitative real-time polymerase chain reaction (kDNA-qPCR) at enrollment, days 14 and 21-28 of therapy and 3, 6, 12-18, and 18-24 months after treatment of ML and correlated to demographic, clinical, and parasitologic factors. Forty-four patients were enrolled: 30 men and 14 women. Enrollment PL differed significantly by causative species (P < 0.001), and was higher in patients with severe ML (nasal and laryngeal involvement) compared with those with only isolated nasal involvement (median = 1,285 versus 51.5 parasites/mug tissue DNA; P = 0.005). Two patterns of PL emerged: pattern 1 (N = 23) was characterized by a sequential decline in PL during and after therapy until kDNA was undetectable. Pattern 2 (N = 18) was characterized by clearance of detectable kDNA during treatment, followed by an increased PL thereafter. All patients who failed treatment (N = 4) demonstrated pattern 1. Leishmania (Viannia) braziliensis was overrepresented among those with pattern 2 (P = 0.019). PL can be quantified by cytology brush qPCR during and after treatment in ML. We demonstrate that treatment failure was associated with undetectable PL, and L. (V.) braziliensis infection was overrepresented in those with rebounding PL.

Urban pollution can often impact surrounding, non-urban regions, through advection and dispersal of pollutants by the prevailing winds. Urban regions located upstream of high mountains, such as the Andes, can potentially impact the cryosphere by deposition of particles onto the surface of the snowpack and glaciers. Santiago, the capital of Chile, has more than 6 million inhabitants and regularly experiences episodes of severe pollution, particularly during the austral winter. Some studies have hypothesized that particle pollution from Santiago can reach the cryosphere downwind of the city, but the scarcity of measurements made high in the mountains prevents the validation of mesoscale models so the proof of actual impact remains elusive. A research project was designed to provide some insight into this question. The Pollution Impact on Snow in the Cordillera - Experiments and Simulations (PISCES) project was carried out in 2014 and includes both observational and modeling components. A five-week field campaign was conducted at the end of winter, at an elevated site in a mountain valley, 65 km to the southeast of the center of Santiago, to characterize some aspects of particulate pollution. During synoptic conditions that result in clear days at the site, the mesoscale mountain-valley circulation is effective in transporting pollutants upwards during the day, leading to diluted particle concentrations beyond the summits of the highest peaks. Cloudy days with reduced up-valley circulation do not show increased concentrations associated with transport. Back trajectories indicate that airmasses reaching the site during the field campaign are seldom influenced by pollution from Santiago.

OBJETIVO: Evaluar las características del trabajo de parto, parto y resultados perinatales de las pacientes que fueron inducidas con misoprostol intravaginal. MATERIALES Y MÉTODOS: Estudio descriptivo y retrospectivo desarrollado en el Departamento de Ginecología y Obstetricia del Hospital Nacional Cayetano Heredia durante el período julio 2003 a junio 2004. Se evaluó las historias clínicas de 139 pacientes con indicación de terminar gestación y a las que se había administrado una dosis de 25 microgramos de misoprostol intravaginal. RESULTADOS: Se obtuvo 113 partos vaginales, de los cuales 85 (61,2%) ocurrieron antes de las 24 horas, observándose un menor tiempo en las multíparas (17,4 ± 10,4 horas) versus las nulíparas (21,1 ± 13,3 horas); 58 (41,7%) pacientes sólo requirieron 1 dosis de misoprostol para tener un parto vaginal. La tasa de ceráreas fue 18,7% (26 casos), siendo la causa principal patrón no tranquilizador en 8 pacientes (30,8%). hubo 2 casos de taquisistolia (1,4%) y 1 de síndrome de hiperestimulación (0,7%). El 20% de partos presentó líquido amniótico meconial. hubo 1 (0,7%) caso de Ápgar

Graphical abstract, Highlights • Functional cloning identifies a Leishmania-specific 58 kD antimony resistance marker, ARM58. • ARM58 overexpression confers antimony resistance. • A neighbouring, structurally related gene lacks antimony resistance function. • ARM58 comprises 4 related domains of unknown function, classified as DUF1935. • Cosmid-based functional cloning is feasible in L. braziliensis in spite of RNAi., Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts.

In order to understand the epidemiological dynamics of antimonial (SbV) resistance in zoonotic tegumentary leishmaniasis and its link with treatment outcome, we analyzed the population structure of 24 Peruvian Leishmania braziliensis clinical isolates with known in vitro antimony susceptibility and clinical phenotype by multilocus microsatellite typing (14 microsatellite loci). The genetic variability in the Peruvian isolates was high and the multilocus genotypes were strongly differentiated from each other. No correlation was found between the genotypes and in vitro drug susceptibility or clinical treatment outcome. The finding of a polyphyletic pattern among the SbV-resistant L. braziliensis might be explained by (i) independent events of drug resistance emergence, (ii) sexual recombination and/or (iii) other phenomena mimicking recombination signals. Interestingly, the polyphyletic pattern observed here is very similar to the one we observed in the anthroponotic Leishmania donovani (Laurent et al., 2007), hereby questioning the role of transmission and/or chemotherapeutic drug pressure in the observed population structure.

The bioassay-guided purification of the ethanolic extract from the leaves of Piper hispidum led to the isolation of one new amide, N -2-(3′,4′,5′-trimethoxyphenyl)ethyl-2-hydroxybenzamide ( 1 ) as well as two known chalcones 2′-hydroxy-3′,4′,6′-trimethoxychalcone ( 2 ); 2′,4′-dihydroxy-6′-methoxychalcone (cardamonin, 3 ) and one known flavanone, 5,7-dihydroxyflavanone (Pinocembrin, 4 ). Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. The isolated compounds were tested against Leishmania amazonensis axenic amastigotes. The results showed that the known chalcone 2 exhibited the most potent antileishmanial activity with an IC 50 of 0.8 μM (amphotericin B: IC 50 = 0.2 μM) but was shown to exhibit mild cytotoxicity.

We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.

Three new caffeic acid esters (1-3), four new lignans (4-7), and the known compounds (7'S)-parabenzlactone (8), dihydrocubebin (9), and justiflorinol (10) have been isolated from leaves of Piper sanguineispicum. Their structures were determined by spectroscopic methods, including 1D and 2D NMR, HRCIMS, CD experiments, and chemical methods. Compounds 1-10 were assessed for their antileishmanial potential against axenic amastigote forms of Leishmania amazonensis. Caffeic acid esters 1 and 3 exhibited the best antileishmanial activity (IC(50) 2.0 and 1.8 μM, respectively) with moderate cytotoxicity on murine macrophages.

From December 2005 to April 2007, we enrolled 60 adults starting antiretroviral therapy (ART) in Lima, Peru to receive community-based accompaniment with supervised antiretrovirals (CASA), consisting of 12 months of DOT-HAART, as well as microfinance assistance and/or psychosocial support group according to individuals’ need. We matched 60 controls from a neighboring district, and assessed final clinical and psychosocial outcomes at 24 months. CASA support was associated with higher rates of virologic suppression and lower mortality. A comprehensive, tailored adherence intervention in the form of community-based DOT-HAART and matched economic and psychosocial support is both feasible and effective for certain individuals in resource-poor settings.

The synthesis of 2-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone-derivatives (BTPs) and their in vitro evaluation against Trypanosoma cruzi trypomastigotes, Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, and six human cancer cell lines is described. The in vivo activity of the most active and least toxic compounds against T. cruzi and L. amazonensis was also studied. BTPs constitute a new family of drug leads with potential activity against infectious diseases. Due to their drug-like properties, this series of compounds can potentially serve as templates for future drug-optimization and drug-development efforts for use as therapeutic agents in developing countries.

BACKGROUND Traditional detection of Leishmania from ulcers involves collection of invasive specimens that cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared traditional diagnostic methods with a molecular noninvasive filter paper-based method for the diagnosis of cutaneous leishmaniasis. METHODS Consecutive patients presenting to the Leishmania Clinic at Hospital Nacional Cayetano Heredia were enrolled. Polymerase chain reaction (PCR) was performed on lesion scrapings, aspirates, and filter paper impressions. The reference standard was any 2 of 5 tests positive: smear, aspirate culture, invasive-specimen PCR (scrapings and aspirates), filter paper PCR, and leishmanin skin test. Outcome measures were sensitivity and specificity. Leishmania speciation was performed by PCR-restriction fragment length polymorphism (RFLP) of positive specimens. RESULTS Forty-five patients with 66 lesions were enrolled. Of 52 lesions diagnosed as cutaneous leishmaniasis, 50 were positive by PCR of invasive specimens versus 48 by PCR of filter papers (P=.930). Sensitivity and specificity of PCR on invasively obtained specimens were 94.2% (95% confidence interval [CI], 87.9%-100%) and 92.9% (95% CI, 79.4%-100%). Sensitivity and specificity of filter paper PCR were 92.3% (95% CI, 85.1%-99.5%) and 100%. Culture, smear, and leishmanin skin test all had inferior sensitivities, compared with PCR of invasive or noninvasive specimens (P

A multilocus microsatellite typing (MLMT) approach based on the analysis of 15 independent loci has been developed for the discrimination of strains belonging to different Viannia species. Thirteen microsatellite loci were isolated de novo from microsatellite-enriched libraries for both Leishmania braziliensis and L. guyanensis . Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Markers were designed to contain simple dinucleotide repeats flanked by the minimal possible number of nucleotides in order to allow variations in repeat numbers to be scored as size variations of the PCR products. The 15 markers in total were amplified for almost all of the strains of Viannia tested; one marker did not amplify from the two L. peruviana strains included in the study. When 30 strains of L. braziliensis , 21 strains of L. guyanensis , and 2 strains of L. peruviana were tested for polymorphisms, all strains except two strains of L. guyanensis had individual MLMT types. Distance-based analysis identified three main clusters. All strains except one strain of L. guyanensis grouped together. Two clusters consisted of strains of L. braziliensis according to their geographical origins. The two strains of L. peruviana grouped together with strains of L. braziliensis from Peru and the adjacent Brazilian state of Acre. MLMT has proven capable of individualizing strains even from the same areas of endemicity and of detecting genetic structures at different levels. MLMT is thus applicable for epidemiological and population genetic studies of strains within the subgenus Viannia .

Diabetic retinopathy (DR) remains the major threat to sight in the working age population. Diabetic macular edema (DME) is a manifestation of DR that produces loss of central vision. Macular edema within 1 disk diameter of the fovea is present in 9% of the diabetic population. Proliferative diabetic retinopathy (PDR) is a major cause of visual loss in diabetic patients. In PDR, the growth of new vessels from the retina or optic nerve, is thought to occur as a result of vascular endothelial growth factor (VEGF) release into the vitreous cavity as a response to ischemia. Furthermore, VEGF increases vessel permeability leading to deposition of proteins in the interstitium that facilitate the process of angiogenesis and macular edema. This review demonstrates multiple benefits of intravitreal bevacizumab on DR including DME and PDR. The results indicate that intravitreal bevacizumab injections may have a beneficial effect on macular thickness and visual acuity (VA), independent of the type of macular edema that is present. Therefore, in the future this new treatment modality could replace or complement focal/grid laser photocoagulation in DME. In addition, in PDR, this new option could be an adjuvant agent to PRP so that more selective therapy may be applied.

The mechanisms linking the immune response to cutaneous and mucosal leishmaniasis (CL and ML, respectively) lesions and the response to treatment are incompletely understood. Our aims were to prospectively assess, by quantitative reverse transcription-PCR, the levels of mRNA for gamma interferon, tumor necrosis factor alpha, interleukin-10 (IL-10), IL-4, and IL-13, as well as the presence of T cells (CD2) and macrophages (CD68), in CL and ML lesions and to follow their changes in response to treatment with pentavalent antimonials. The leishmanin skin test (LST) was performed on all CL and ML patients before treatment. The patient population included individuals living in areas of Peru where the disease is endemic, i.e., 129 with CL and 43 with ML. Compared to CL patients, the LST induration size was larger, the levels of all cytokine mRNAs but IL-10 were higher, T-cell mRNA was similar, and macrophage mRNA was lower in ML patients. The proportion of CL patients with an LST induration size of >8 mm was higher among responders to treatment. In CL, the pretreatment levels of cytokine mRNAs did not discriminate between responders and nonresponders; however, treatment was more often accompanied by a reduction in the levels of T-cell and cytokine mRNAs in responders than in nonresponders. Furthermore, the production of cytokines per T cell and macrophage decreased with treatment but IL-10 production remained high in nonresponders. Overall, these findings point to complex relationships among New World Leishmania parasites, skin and mucosal immune responses, and treatment outcome. The persistence of high levels of IL-10 in CL is characteristically associated with a poor response to treatment.