Aproximación a un sistema de vigilancia molecular de Leishmania infantum y sus vectores

Los protozoos del género Leishmania son transmitidos al hombre mediante las picaduras infectivas de hembras de distintas especies de flebótomo (Diptera: Psychodidae), convirtiéndose en agentes causales de la leishmaniasis, cuyas manifestaciones clínicas dependen de diferentes factores asociados al parásito y al hospedador. Estas formas clínicas varían desde lesiones cutáneas localizadas hasta formas viscerales, las últimas pueden ser mortales sin tratamiento.En Europa Leishmania infantum es el principal agente causal de la leishmaniasis cutánea y visceral, y las hembras de distintas especies del género Phlebotomus son los vectores implicados en su transmisión, la cual depende de un ciclo zoonótico en el que el perro es considerado el principal reservorio. La incidencia regional de la leishmaniasis se estima en menos del 2% de la carga global de la leishmaniasis de acuerdo con la estimación reciente de la OMS. Sin embargo,se considera que hay una importante subnotificación debido a la ausencia de un sistema de vigilancia centralizado. Durante la última década varios estudios epidemiológicos han demostrado un incremento de la prevalencia de leishmaniasis en algunos países endémicos, así como la aparición de nuevos focos relacionados con la propagación de la enfermedad hacia el norte del continente europeo..

DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.This work was supported in part by Instituto de Salud Carlos III (MPY-1248/12) grant and (PI14CIII/00016) funded by AESI. Ivonne Pamela Llanes-Acevedo was granted a student fellowship by COLCIENCIAS/COLFUTURO.S

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector.

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector.

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals

BioClimate: a Science Gateway for Climate Change and Biodiversity research in the EUBrazilCloudConnect project

[EN] Climate and biodiversity systems are closely linked across a wide range of scales. To better understand the mutual interaction between climate change and biodiversity there is a strong need for multidisciplinary skills, scientific tools, and access to a large variety of heterogeneous, often distributed, data sources. Related to that, the EUBrazilCloudConnect project provides a user-oriented research environment built on top of a federated cloud infrastructure across Europe and Brazil, to serve key needs in different scientific domains, which is validated through a set of use cases. Among them, the most data-centric one is focused on climate change and biodiversity research. As part of this use case, the BioClimate Science Gateway has been implemented to provide end-users transparent access to (i) a highly integrated user-friendly environment, (ii) a large variety of data sources, and (iii) different analytics & visualization tools to serve a large spectrum of users needs and requirements. This paper presents a complete overview of BioClimate and the related scientific environment, in particular its Science Gateway, delivered to the end-user community at the end of the project.This work was supported by the EU FP7 EUBrazilCloudConnect Project (Grant Agreement 614048), and CNPq/Brazil (Grant Agreement no 490115/2013-6).Fiore, S.; Elia, D.; Blanquer Espert, I.; Brasileiro, FV.; Nuzzo, A.; Nassisi, P.; Rufino, LAA.... (2019). BioClimate: a Science Gateway for Climate Change and Biodiversity research in the EUBrazilCloudConnect project. Future Generation Computer Systems. 94:895-909. https://doi.org/10.1016/j.future.2017.11.034S8959099

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery

Molecular typing of Leishmania infantum isolates from a leishmaniasis outbreak in Madrid, Spain, 2009 to 2012

Abstract: Leishmaniasis is endemic in south-west Europe. Recent data point to the spread and (re-)emergence of this disease in previously endemic and non-endemic European countries. A recent example is the urban community outbreak of cutaneous and visceral leishmaniasis in the south-west of Madrid autonomous community, Spain, which began on 1 July 2009. A total of 446 cases associated to this outbreak were reported up to 31 December 2012. We show molecular typing data for 73 Leishmania infantum isolates obtained from January 2008 to July 2012 from different areas of Madrid, including those affected by the outbreak. Seven different genotypes were identified by combining data from two targets: the ribosomal internal transcribed spacers (ITS)-1 and -2 and the haspb (k26) gene. The results contribute to a better understanding of the parasite population circulating in the region, and indicate that most of the outbreak-associated isolates (22/31) were infected by parasites with the same combined genotype. Additional data from 82 L. infantum isolates typed as either MON-1 or MON-24 by isoenzyme analysis indicate that far from concluding that the outbreak was caused by a ‘new’ emerging genotype, further molecular typing-based surveillance studies are required to better understand the epidemiology of leishmaniasis in the region. Resumen: La leishmaniasis es endémica en el suroeste de Europa. Datos recientes apuntan a la difusión y la (re) emergencia de esta enfermedad tanto en países europeos anteriormente endémicos como no endémicos. Un ejemplo reciente es el brote urbano de leishmaniasis cutánea y visceral en el sur- oeste de Madrid, Comunidad de Madrid, España, que comenzó el 1 de julio de 2009. Un total de 446 casos relacionados con este brote se registraron hasta el 31 de diciembre de 2012. Se muestran los datos de tipificación molecular de 73 aislados de Leishmania infantum obtenidos de enero de 2008 a julio de 2012 desde las diferentes áreas de Madrid, incluidas las afectadas por el brote. Siete diferentes genotipos fueron identificados mediante la combinación de los datos de dos marcadores moleculares: los espaciadores transcritos internos (ITS) -1 y -2 y el gen (k26) haspb . Nuestros resultados contribuyen a una mejor comprensión de la población de parásitos que circula en la región, e indican que la mayoría de los aislados asociados al brote (22/31) se debían a parásitos con el mismo genotipo combinado. Adicionalmente, caracterizamos 82 aislados de L. infantum tipados como MON -1 o MON- 24 mediante el análisis de isoenzimas, y los datos obtenidos a partir de este segundo conjunto de cepas indican que el brote no fue causado por un genotipo emergente o "nuevo", sin embargo se requieren de estudios de vigilancia en tipificación para entender mejor la epidemiología de la leishmaniasis en la región

Molecular typing of Leishmania infantum isolates from a leishmaniasis outbreak in Madrid, Spain, 2009 to 2012

Leishmaniasis is endemic in south-west Europe. Recent data point to the spread and (re-)emergence of this disease in previously endemic and non-endemic European countries. A recent example is the urban community outbreak of cutaneous and visceral leishmaniasis in the south-west of Madrid autonomous community, Spain, which began on 1 July 2009. A total of 446 cases associated to this outbreak were reported up to 31 December 2012. We show molecular typing data for 73 Leishmania infantum isolates obtained from January 2008 to July 2012 from different areas of Madrid, including those affected by the outbreak. Seven different genotypes were identified by combining data from two targets: the ribosomal internal transcribed spacers (ITS)-1 and -2 and the haspb (k26) gene. The results contribute to a better understanding of the parasite population circulating in the region, and indicate that most of the outbreak-associated isolates (22/31) were infected by parasites with the same combined genotype. Additional data from 82 L. infantum isolates typed as either MON-1 or MON-24 by isoenzyme analysis indicate that far from concluding that the outbreak was caused by a 'new' emerging genotype, further molecular typing-based surveillance studies are required to better understand the epidemiology of leishmaniasis in the region.This study was supported in part by Instituto de Salud Carlos III (Grant number MPY-1248/12). Ivonne Pamela Llanes Acevedo is granted by Fundación para el Futuro de Colombia, COLFUTURO).S

Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain

<div><p>Background</p><p>Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is endemic in Europe with Mediterranean countries reporting endemic status alongside a worrying northward spread. Serological diagnosis, including immunochromatographic test based on the recombinant antigen rK39 (rK39-ICT) and a direct agglutination test (DAT) based on the whole parasite antigen, have been validated in regions with high VL burden, such as eastern Africa and the Indian subcontinent. To date, no studies using a large set of patients have performed an assessment of both methods within Europe.</p><p>Methodology/Principal findings</p><p>We selected a range of clinical serum samples from patients with confirmed VL (including HIV co-infection), Chagas disease, malaria, other parasitic infections and negative samples (n = 743; years 2009–2015) to test the performance of rK39-ICT rapid test (Kalazar Detect Rapid Test; InBios International, Inc., USA) and DAT (ITM-DAT/VLG; Institute of Tropical Medicine Antwerp, Belgium). An in-house immunofluorescence antibody test (IFAT), was included for comparison. Estimated sensitivities for rK39-ICT and DAT in HIV-negative VL patients were 83.1% [75.1–91.2] and 84.2% [76.3–92.1], respectively. Sensitivity was reduced to 67.3% [52.7–82.0] for rK39 and increased to 91.3% [82.1–100.0] for DAT in HIV/VL co-infected patients. The in-house IFAT was more sensitive in HIV-negative VL patients, 84.2% [76.3–92.1] than in HIV/VL patients, 79.4% [73.3–96.2]. DAT gave 32 false positives in sera from HIV-negative VL suspects, compared to 0 and 2 for rK39 and IFAT, respectively, but correctly detected more HIV/VL patients (42/46) than rK39 (31/46) and IFAT (39/46).</p><p>Conclusions/Significance</p><p>Though rK39-ICT and DAT exhibited acceptable sensitivity and specificity a combination with other tests is required for highly sensitive diagnosis of VL cases in Spain. Important variation in the performance of the tests were seen in patients co-infected with HIV or with other parasitic infections. This study can help inform the choice of serological test to be used when screening or diagnosing VL in a European Mediterranean setting.</p></div