Aspectos ecoepidemiológicos, detección natural e identificación molecular de Leishmania spp. en Lutzomyia reburra, Lutzomyia barrettoi majuscula y Lutzomyia trapidoi / Eco-epidemiological aspects, natural detection and molecular identification of Leishmania spp. in Lutzomyia reburra, Lutzomyia barrettoi majuscula and Lutzomyia trapidoi

Resumen Introducción. La provincia de Pichincha, Ecuador, es un área endémica de leishmaniasis cutánea, en donde se han determinado como vectores los flebotomíneos antropofílicos con infección natural por Leishmania spp. Sin embargo, no se ha evaluado el papel en la transmisión de las especies zoofílicas. Objetivo. Evaluar la infección natural por Leishmania en dos especies de flebotomíneos zoofílicos, Lutzomyia reburra y Lu. barrettoi majuscula, y en una antropofílica, Lu. trapidoi, así como la endofagia y la sinantropía de estas especies en el noroccidente de Pichincha. Materiales y métodos. Los flebotomíneos se recolectaron en trampas de luz CDC colocadas en diferentes hábitats y altitudes en sitios que son focos de leishmaniasis cutánea. La infección con Leishmania spp. se detectó en el ADN genómico de hembras de las especies de flebotomíneos de interés. Se amplificó el gen espaciador interno de la transcripción del ARN ribosómico, unidad I (ITS1), y los genes de las topoisomerasas mitocondrial II (mtTOPOII) y nuclear II (TopoII). Se determinaron los porcentajes de positividad para Leishmania a escala espaciotemporal, la proporción de endofagia y el índice de sinantropía. Resultados. Se determinó la presencia de infección natural por Le. amazonensis en Lu. reburra (9,5 %) y Lu. b. majuscula (23,8 %); en Lu. trapidoi se detectaron Le. amazonensis, Le. brazilienis y Le. naiffilainsoni. Los flebotomíneos eran asinantrópicos y con baja endofagia. Conclusión. Se registró por primera vez la presencia de infección natural en Lu. reburra y Lu. barrettoi majuscula por Le. amazonensis, y se demostró la importancia de los flebotomíneos zoofílicos en el mantenimiento del ciclo de transmisión de Leishmania spp. en focos endémicos.

Abstract Introduction: The province of Pichincha in Ecuador is an endemic area of cutaneous leishmaniasis, where anthropophilic sand flies with natural infection by Leishmania, have been reported as vectors. However, the role in transmission of zoophilic species has not been evaluated. Objective: To evaluate natural infection by Leishmania in two zoophilic phlebotomine sand fly species, Lutzomyia reburra and Lu. barrettoi majuscula, and one anthropophilic species, Lu. trapidoi, as well as the endophagy and synanthropism of these species in the northwest of Pichincha. Materials and methods: Phlebotomines were collected using CDC light traps in different habitats and altitudes with presence of cutaneous leishmaniasis. Leishmania infection was detected using genomic DNA from females of the collected sand flies. We amplified the internal transcribed spacer gene of ribosomal RNA I (ITS1), the mitochondrial topoisomerase II gene (mtTOPOII), and the nuclear topoisomerase II gene (TopoII). Percentages of positivity for Leishmania, at spatio-temporal scale, proportion of endophagy and synanthropism index were calculated. Results: Natural infection was determined for Le. amazonensis in Lu. reburra (9.5%) and Lu. b. majuscula (23.8%), while in Lu. trapidoi we detected Le. amazonensis, Le. brazilienis and Le. naiffilainsoni. Phlebotomines were asynanthropic and with low endophagy. Conclusion: Natural infection with Le. amazonensis was recorded for the first time in Lu. reburra and Lu. b. majuscula, demonstrating the importance of zoophilic phlebotomines in the maintenance of the Leishmania transmission cycle in endemic foci.

Abundance, composition and natural infection of Anopheles mosquitoes from two malaria-endemic regions of Colombia / Abundancia, composición e infección natural de mosquitos Anopheles en dos regiones endémicas para malaria en Colombia

Abstract Introduction: In Colombia there are three Anopheles species implicated in malaria transmission as primary vectors; however, the local role of some Anopheles species must still be defined. Objective: To determine the abundance, composition and natural infection rates for Anopheles mosquitoes with Plasmodium spp. in two malaria-endemic regions of Colombia. Materials and methods: Anopheles mosquitoes were collected using the human-landing catches and while resting in livestock corrals in nine localities of two malaria-endemic regions of Colombia. Mosquitoes were morphologically identified and confirmed by PCR-RFLP-ITS2. Identified mosquitoes were processed and tested for Plasmodium parasite infection by ELISA and ssrRNA-based nested PCR. Results: We collected 1,963 Anopheles mosquitoes corresponding to nine species. The most abundant species were Anopheles nuneztovari (53.5%) and A. darlingi (34.5%), followed by A. triannulatus s.l. (6%), and other species (˜5.9%). Three species were naturally infected with Plasmodium spp.: A. darlingi, A. nuneztovari and A. triannulatus s.l. Conclusions: Natural infection of A. darlingi and A. nuneztovari indicate that these malaria vectors continue to be effective carriers of Plasmodium in the localities under study in Valle del Cauca and Chocó. Additionally, the infected A. triannulatus s.l. collected in livestock corrals in the locality of the department of Córdoba suggests the need for further studies to define the epidemiological importance of this species given its abundance and opportunistic anthropophilic behavior.

Resumen Introducción. En Colombia hay tres especies de mosquitos Anopheles implicadas como vectores primarios en la transmisión de la malaria o paludismo; sin embargo, el rol local de algunas especies de Anopheles aún debe determinarse. Objetivo. Determinar la abundancia, la composición y la infección natural de mosquitos anofelinos con Plasmodium spp. en dos regiones endémicas de malaria en Colombia. Materiales y métodos. Se recolectaron mosquitos del género Anopheles usando los métodos de recolección con cebo humano y en reposo en corrales de ganado vacuno, en nueve localidades de dos regiones endémicas para malaria en Colombia. Los especímenes se identificaron morfológicamente y se confirmaron por reacción en cadena de la polimerasa (PCR) de los polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP) en el espaciador intergénico ribosómico nuclear 2 (Internal Transcribed Spacer, ITS-2) (PCR-RFLPITS2). Los especímenes se procesaron y analizaron mediante ELISA y PCR anidada basada en la subunidad pequeña del ARN ribosómico (small subunit ribosomal RNA, ssrRNA) para determinar la infección por Plasmodium. Resultados. Se recolectaron 1.963 mosquitos Anopheles correspondientes a nueve especies. Anopheles nuneztovari fue la especie predominante (53,5 %), seguida por A. darlingi (34,5 %), A. triannulatus s.l. (6 %) y por otras especies (˜5,9 %). Tres especies se encontraron naturalmente infectadas con Plasmodium spp.: A. darlingi, A. nuneztovari y A. triannulatus s.l. Conclusiones. La infección natural de A. darlingi y A. nuneztovari indica que estos vectores primarios siguen siendo actores principales en la transmisión de malaria en las localidades estudiadas de los departamentos del Valle del Cauca y Chocó. Además, el espécimen A. triannulatus s.l. infectado, recolectado en corrales de animales de la localidad estudiada en el departamento de Córdoba, indica que existe la necesidad de estudios futuros para establecer la importancia epidemiológica de esta especie dada su abundancia y comportamiento antropofílico oportunista.

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

Especies de Anopheles presentes en el departamento del Putumayo y su infección natural con Plasmodium / Anopheles species present in the department of Putumayo and their natural infectivity with Plasmodium

Introducción. El departamento del Putumayo es una región endémica para malaria, o paludismo, causada principalmente por Plasmodium vivax . Los vectores en esta región incluyen Anopheles darlingi , el cual se ha encontrado solamente en el municipio de Puerto Leguízamo, y recientemente se incriminaron como vectores en Puerto Asís a las especies An. rangeli y An. oswaldoi . Objetivo. El propósito del trabajo fue determinar el papel de An. benarrochi B en la transmisión de malaria en este departamento, ya que se reporta como la especie más abundante que pica a los humanos. Materiales y métodos. Se recolectaron larvas y adultos de Anopheles spp. entre el 2006 y el 2008 en los municipios Puerto Leguízamo y Puerto Asís, y se obtuvieron secuencias del gen ITS-2 y del gen mitocondrial COI para confirmar las determinaciones taxonómicas por morfología. Se practicó la prueba ELISA para establecer la infección por P. vivax y P. falciparum. Resultados. Se identificaron 6.238 individuos correspondientes a 11 especies: An. albitarsis s.l. (1,83 %), An. benarrochi B (72,35 %), An. braziliensis (0,05 %), An. costai (0,06 %), An. darlingi (19,37 %), An. mattogrossensis (0,08 %), An. neomaculipalpus (0,13 %), An. oswaldoi s.l. (0,64 %), An. punctimacula (0,03 %), An. rangeli (5,12 %) y An. triannulatus s.l. (0,34 %). Se evaluaron 5.038 adultos por ELISA y 5 se encontraron positivos para P. vivax 210 y VK 247, todos pertenecientes a la especie An. benarrochi B. Conclusión. Los resultados sugieren que An. benarrochi B juega un papel en la transmisión de P. vivax en el departamento de Putumayo, dada su alta atracción por los humanos y su infección natural con Plasmodium spp.

Introduction: Putumayo is considered an endemic region for malaria transmission, mainly due to Plasmodium vivax. The vectors in this region are Anopheles darlingi , which has been found only in the municipality of Puerto Leguízamo, and An. rangeli and An. oswaldoi s.l. , which were recently incriminated as vectors in Puerto Asís. Objective: The purpose of this study was to determine the role of An. benarrochi B in malaria transmission in Putumayo, given that it is the most abundant species biting humans. Materials and methods: Collections of immature and adult stages of Anopheles spp. were made between 2006 and 2008 in the municipalities of Puerto Leguízamo and Puerto Asís in Putumayo, and sequences of internal transcribed spacer 2 ( ITS-2 ) of ribosomal DNA and the mitochondrial gene COI were obtained to confirm the morphological determinations. ELISA was carried out for P. vivax and P. falciparum infectivity. Results: A total of 6,238 specimens were identified, distributed in 11 species: An. albitarsis s.l. (1.83%), An. benarrochi B (72.35%), An. braziliensis (0.05%), An. costai (0.06%), An. darlingi (19.37%), An. mattogrossensis (0.08%), An. neomaculipalpus (0.13%), An. oswaldoi s.l. (0.64%), An. punctimacula (0.03%), An. rangeli (5.12%), and An. triannulatus s.l. (0.34%). A total of 5,038 adults were assessed by ELISA and 5 were found positive for P. vivax 210 and VK 247, all belonging to An. benarrochi B. Conclusion: The results suggest that An. benarrochi B plays a role in the transmission of P. vivax in Putumayo due to its high human contact and natural infection with Plasmodium sp.

Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications / Detecção de DNA de Bartonella henselae em amostras clínicas, incluindo sangue periférico, de pacientes imunocompetentes e imunodeprimidos por meio de três amplificações duplas

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

Bactérias do gênero Bartonella constituem patógenos emergentes detectados em biópsias de linfonodos e secreções de gânglios provavelmente devido a maior concentração de bactérias. Vinte e três amostras de 18 pacientes com dados clínicos, laboratoriais e/ou epidemiológicos sugestivos de bartonelose foram submetidas a três amplificações duplas para a detecção de fragmento da proteína de choque térmico de 60-kDa (HSP), do espaçador interno 16S-23S rRNA (ITS) e da proteína de divisão celular (FtsZ) de Bartonella henselae, para melhorar a detecção em amostras clínicas. Na primeira amplificação, uma, quatro e cinco amostras, respectivamente, foram positivas pelo HSP (4,3%), FtsZ (17,4%) e pelo ITS (21,7%). Com a segunda amplificação foram identificadas seis amostras positivas pelo nested-HSP (26%), oito pelo nested-ITS (34,8%) e 18 pelo nested- FtsZ (78,2%), correspondentes a 10 amostras de sangue periférico, cinco biópsias de linfonodos, duas biópsias de pele e um aspirado de gânglio. A nested-FtsZ foi mais sensível que a nested-HSP e a nested-ITS (p < 0,0001), possibilitando a detecção de DNA de Bartonella henselae em 15 de 18 pacientes (83,3%). No presente estudo, três nested-PCR, consideradas específicas para a amplificação da Bartonella henselae, foram desenvolvidas, porém somente a nested-FtsZ não amplificou o DNA de Bartonella quintana. Concluímos que amplificações duplas aumentaram a detecção de DNA de B. henselae, e que a nested-FtsZ foi a mais sensível e a única específica para B. henselae em diferentes amostras biológicas. Como todas as amostras detectadas pelo HSP-nested e nested-ITS foram também pela nested-FtsZ, inferimos que, em nossa casuística, as infecções foram causadas por Bartonella henselae. A elevada positividade de amostras de sangue chamou a atenção para a utilização deste material biológico na investigação de bartoneloses, independentemente do estado imune dos pacientes. Este fato é importante no caso de pacientes criticamente enfermos e crianças pequenas para evitar procedimentos mais invasivos, como biópsias e punções de gânglios.

Detection and genotyping of Giardia intestinalis isolates using intergenic spacers(IGS)-based PCR

Giardia intestinalis infections arise primarily from contaminated food or water. Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was performed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176 bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.