Cielo M. León

Taxonomy, diversity, temporal and geographical distribution of Cutaneous Leishmaniasis in Colombia: A retrospective study

Leishmaniases are tropical zoonotic diseases, caused by kinetoplastid parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. Currently, few studies show the relative distribution of Leishmania species related to cutaneous Leishmaniasis (CL) in South America due to the lack of accurate surveillance and public health systems. Herein, we conducted a systematic estimation of the Leishmania species causing CL in Colombia from 1980 to 2001 via molecular typing and isoenzymes. A total of 327 Leishmania isolates from humans, sandflies and reservoirs were typed as L. panamensis 61.3% (201), L. braziliensis 27.1% (88), L. lainsoni 0.6% (2), L. guyanensis 0.9% (3), L. infantum chagasi 4% (12), L. equatoriensis 0.6% (2), L. mexicana 2.1% (8), L. amazonensis 2.8% (9) and L. colombiensis 0.6% (2). This is the first report of two new Leishmania species circulating in Colombia and suggests the need to convince the Colombian government about the need to deploy and standardize tools for the species identification to provide adequate management to individuals suffering this pathology.

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

BackgroundLeishmaniases are tropical zoonotic diseases, caused by parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species.FindingsHerein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL.ConclusionsThis is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.

Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia

Trypanosoma cruzi is the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruzi I (TcI) shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.

Murine models susceptibility to distinct Trypanosoma cruzi I genotypes infection.

Chagas disease is a complex zoonosis that affects around 8 million people worldwide. This pathology is caused by Trypanosoma cruzi, a kinetoplastid parasite that shows tremendous genetic diversity evinced in six distinct Discrete Typing Units (TcI-TcVI) including a recent genotype named as TcBat and associated with anthropogenic bats. TcI presents a broad geographical distribution and has been associated with chronic cardiomyopathy. Recent phylogenetic studies suggest the existence of two genotypes (Domestic (TcIDom) and sylvatic TcI) within TcI. The understanding of the course of the infection in different mouse models by these two genotypes is not yet known. Therefore, we infected 126 animals (ICR-CD1, National Institute of Health (NIH) and Balb/c) with two TcIDom strains and one sylvatic strain for a follow-up period of 60 days. We quantified the parasitaemia, immune response and histopathology observing that the maximum day of parasitaemia was achieved at day 21 post-infection. Domestic strains showed higher parasitaemia than the sylvatic strain in the three mouse models; however in the survival curves Balb/c mice were less susceptible to infection compared with NIH and ICR-CD1. Our results suggest that the genetic background plays a fundamental role in the natural history of the infection and the sympatric TcI genotypes have relevant implications in disease pathogenesis.

Molecular Epidemiology of Entamoeba: First Description of Entamoeba moshkovskii in a Rural Area from Central Colombia

Background Entamoeba histolytica, E. dispar and E. moshkovskii are the most frequent species described in human infection where E. histolytica is the only true pathogen. The epidemiology of this infection is complex due to the absence of a routine exam that allows a correct discrimination of the Entamoeba species complex. Therefore, molecular methods appear as the unique epidemiological tool to accomplish the species discrimination. Herein, we conducted a cross-sectional study to determine the frequency of Entamoeba species infections in a group of asymptomatic individuals from a rural area in central Colombia. Methodology/Principal Findings A total of 181 fecal samples from asymptomatic children under 16 years old from the hamlet La Vírgen, Cundinamarca (Colombia) that voluntarily accepted to participate in the study were collected. The fecal samples were examined by light microscopy and DNA-extracted, subsequently submitted to molecular discrimination of E. dispar/E. histolytica/E. moshkovskii infection based on a multiplex PCR assay targeting the 18S rRNA fragment. To confirm the species description, twenty samples were randomly submitted to DNA sequencing of the aforementioned fragment. By direct microscopic examination, frequency of the complex E. histolytica/E. dispar/E. moshkovskii was 18.8% (34/181). PCR showed a frequency of 49.1% (89/181), discriminated as 23.2% (42/181) that were positive for E. dispar, 25.4% (46/181) for E. moshkovskii and 0.55% (1/ 181) for E. histolytica. Also, mixed infections were detected between E. dispar and E. moshkovskii at 4.42% (8/181) of the samples. Molecular barcoding confirmed the diagnosis depicted by the multiplex PCR assay. Conclusions/Significance This is the first description of E. moshkovskii in Colombia and the second report in South-America to our knowledge. Our results suggest the need to unravel the true epidemiology of Entamoeba infections around the world, including the real pathogenic role that E. moshkovskii may have.

Risk factors associated with Chagas disease in pregnant women in Santander a highly endemic Colombian area.

To determine the prevalence and risk factors associated with Chagas disease in pregnant women in an endemic area of Santander, Colombia.

Purification of Trypanosoma cruzi metacyclic trypomastigotes by ion exchange chromatography in sepharose-DEAE, a novel methodology for host-pathogen interaction studies

Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures. A simple, easy-to-execute and effective protocol based on ion exchange chromatography on Sepharose-DEAE resin for the purification of T. cruzi trypomastigotes is described. T. cruzi strains from the Discrete Typing Units (DTUs) I and II were used. The strains were harvested in LIT medium at a concentration of 1×107epimastigotes/mL. We calculated the time of trypomastigotes increment (TTI). Based on the data obtained, Ion exchange chromatography was performed with DEAE-sepharose resin. To verify the purity and viability of the trypomastigotes, a culture was carried out in LIT medium with subsequent verification with giemsa staining. To evaluate if the technique affected the infectivity of trypomastigotes, in vitro assays were performed in Vero cells and in vivo in ICR-CD1 mice. The technique allowed the purification of metacyclic trypomastigotes of other stages of T. cruzi in a percentage of 100%, a greater recovery was observed in cultures of 12days. There were differences regarding the recovery of metacyclic trypomastigotes for both DTUs, being DTU TcI the one that recovered a greater amount of these forms. The technique did not affect parasite infectivity in vitro or/and in vivo.

Diversity patterns, Leishmania DNA detection, and bloodmeal identification of Phlebotominae sand flies in villages in northern Colombia

Leishmaniases are neglected tropical diseases exhibiting complex transmission cycles due to the number of parasite species circulating, sand fly species acting as vectors and infected mammals, including humans, which are defined in the New World as accidental hosts. However, current transmission scenarios are changing, and the disease is no longer exclusively related to forested areas but urban transmission foci occur, involving some species of domestic animals as suspected reservoirs. The aim of this study was to determine the transmission cycles in urban environments by evaluating sand fly diversity, detection of Leishmania DNA, and bloodmeal sources through intra and peridomestic collections. The study was carried out in Colombia, in 13 municipalities of Cordoba department, implementing a methodology that could be further used for the evaluation of vector-borne diseases in villages or towns. Our sampling design included 24 houses randomly selected in each of 15 villages distributed in 13 municipalities, which were sampled in two seasons in 2015 and 2016. Sand flies were collected using CDC light traps placed in intra and peridomestic habitats. In addition to the morphological identification, molecular identification through DNA barcodes was also performed. A total of 19,743 sand flies were collected and 13,848 of them (10,268 females and 3,580 males) were used in molecular procedures. Circulation of two known parasite species–Leishmania infantum and Leishmania panamensis was confirmed. Blood source analyses showed that sand flies fed on humans, particularly in the case of the known L. infantum vector, P. evansi; further analyses are advised to evaluate the reservoirs involved in parasite transmission. Our sampling design allowed us to evaluate potential transmission cycles on a department scale, by defining suspected vector species, parasite species present in different municipalities and feeding habits.

New Scenarios of Chagas Disease Transmission in Northern Colombia

Chagas disease (CD) is a systemic parasitic infection caused by the flagellated form of Trypanosoma cruzi. Córdoba department, located in the Colombian Caribbean Coast, was not considered as a region at risk of T. cruzi transmission. In this article, we describe the first acute CD case in Salitral village in Sahagún, Córdoba, confirmed by microscopy and serological tests. Our results draw attention to a new scenario of transmission of acute CD in nonendemic areas of Colombia and highlight the need to include CD in the differential diagnosis of febrile syndromes in this region.

Coriocarcinoma mediastínico. A propósito de un caso

Sr. Director: Hemos leído con mucho interés el artículo recientemente publicado en su revista “Carcinoma broncogénico asociado a neumotórax espontáneo y bullas” de Martín et al, en el que se destaca una asociación entre la enfermedad bullosa pulmonar y un carcinoma broncogénico de célula no pequeña, cuyo síntoma inicial fue un neumotórax espontáneo. A este respecto nos gustaría realizar algunas consideraciones que creemos interesantes, tanto en el manejo diagnóstico como terapéutico del caso. En primer lugar nos parece correcto que, ante la falta de reexpansión pulmonar completa tras la inserción del drenaje pleural, se realice una tomografía axial computarizada (TAC), como método diagnóstico de elección, previa comprobación de la correcta localización y funcionamiento del drenaje pleural, causa habitual del fracaso. En su artículo en la TAC se describe la existencia de un infiltrado o atelectasia del lóbulo pulmonar inferior derecho, que no se refiere que haya sido estudiado con otros métodos diagnósticos, como la fibrobroncoscopia, esencial en el análisis histológico y para descartar lesiones endobronquiales que pudieran explicar una posible etiología de la falta de reexpansión pulmonar. Por este motivo, suponemos que al paciente, que fue tratado mediante una videotoracoscopia (VTC) por un neumotórax espontáneo sin reexpansión pulmonar, se le realizó previamente una fibrobroncoscopia. En relación con el tratamiento, no tenemos constancia del aspecto macroscópico de las bullas resecadas, pero en los casos en los que existe una mínima duda está justificado realizar un estudio anatomopatológico intraoperatorio. La existencia de un hábito tabáquico (2 paquetes/día) y una evolución tórpida del proceso apoyan el hecho de que se pudiera tratar de un neumotórax espontáneo secundario a alguna enfermedad pulmonar, aparte del enfisema, y por tanto el estudio histológico intraoperatorio estaría justificado. Por último, el descubrimiento accidental de un carcinoma broncogénico dentro de una zona pulmonar bullosa es poco frecuente, pero no excepcional, y su tratamiento debe seguir el patrón establecido de resección pulmonar con fines curativos, es decir, lobectomía pulmonar y linfadenectomía mediastínica. La segmentectomía atípica en el carcinoma broncogénico solamente está justificada en los pacientes que presentan una función respiratoria límite, que no permite una resección pulmonar mayor, puesto que se ha descrito un aumento de la probabilidad de sufrir una recurrencia tumoral local con esta resección. Por estas razones creemos que en este paciente joven, con un carcinoma broncogénico no células pequeñas y sin criterios que contraindicaran la resección mayor, debería haberse tratado mediante una lobectomía pulmonar y una linfadenectomía mediastínica, aunque su realización, como es el caso, pueda estar dificultada por la pleurodesis con talco. En resumen, proponemos nuestro protocolo de actuación para los casos de un neumotórax espontáneo sin reexpansión pulmonar completa. Tras comprobar la correcta localización y funcionamiento del drenaje debe realizarse una TAC y una fibrobroncoscopia. Establecida la necesidad de una exploración quirúrgica con las pruebas anteriores, la VTC con biopsia y estudio anatomopatológico intraoperatorio debe ser el tratamiento de elección. Si el diagnóstico es de bullas subpleurales ha de procederse a una pleurodesis mediante abrasión pleural mecánica. En el caso de confirmarse la existencia de un carcinoma broncogénico, con criterios de operabilidad oncológica, se debe completar la lobectomía pulmonar y una linfadenectomía mediastínica, que es el tratamiento actual que ofrece mayores posibilidades de curación.