Detection of Rickettsia spp. in ticks of wildlife fauna from Costa Rica: First report of Rickettsia rhipicephali in Central America.

In the past two decades, new species of Rickettsia have been detected and described worldwide, some of them considered pathogenic for humans. Although Costa Rica is considered a biodiversity hotspot, the knowledge about rickettsiae in sylvatic ecosystems and wild animals is scarce. The aim of this preliminary study was to detect and identify species of Rickettsia in ticks collected from wild animals in Costa Rica. A total 119 ticks were collected from 16 animal host species belonging to diverse vertebrate families (Didelphidae, Procyonidae, Felidae, Choloepodidae, Bradypodidae, Myrmecophagidae, Tayassuidae, Tapiridae, Phyllostomidae, Bufonidae, Geoemydidae, Boidae, Colubridae), and they were grouped into 43 pools to detect the presence of Rickettsia spp. DNA by PCR targeting the gltA gene. In positive pools, amplicons of the ompA, sca5 (ompB), and/or htrA genes were also amplified to identify the species present. The identification of some ticks was also confirmed by molecular methods. Four species of Rickettsia were detected in eight (19%) tick pools: Rickettsia amblyommatis in four pools of Amblyomma geayi (host: Caluromys derbianus) and one pool of Amblyomma cf. parvum (host: Nasua narica), Rickettsia rhipicephali in one pool of Dermacentor latus (host: Tayassu pecari), 'Candidatus Rickettsia colombianensi' in one pool of Amblyomma sp. nymphs (host: Boa constrictor), and Rickettsia sp. genotype IbR/CRC in one pool of Ixodes cf. boliviensis (host: Puma concolor). This is the first molecular detection of R. rhipicephali in Central America, and of 'Candidatus R. colombianensi' in Costa Rica. Results show that diverse wild animals and their ticks are associated with several species of rickettsiae in Costa Rica, which may come in contact with humans and other domestic animals in sylvatic environments.

New records and phylogenetic position of Ornithodoros knoxjonesi (Ixodida: Argasidae).

Larvae of Ornithodoros knoxjonesi collected at five localities in three countries were studied using morphological and molecular methods to confirm this species' taxonomic validity. The larva of O. knoxjonesi is characterized as having 14 pairs of dorsal setae, eight pairs of ventral setae, plus a posteromedian seta; an elongate dorsal plate, tapered anteriorly; and a hypostome that is narrower near its midlength, with posteriorly projecting denticles. Although the larvae of O. knoxjonesi and Ornithodoros peropteryx are morphologically quite similar, the larva of O. knoxjonesi is characterized as having dorsal setae that are wider at the tip than at the base, while in O. peropteryx these setae are narrower at the tip than at the base; moreover, the dorsal setae are shorter in O. knoxjonesi (Al 0.037-0.065; Pl 0.035-0.059) than in O. peropteryx (Al 0.120-0.132; Pl 0.080-0.096). These species also differ in that O. knoxjonesi possesses only the Al seta on tarsus I, whereas O. peropteryx has both Al and Pl setae. And while both species have two setae on coxae I-III, in O. knoxjonesi the anterior seta is tapering and smooth and the posterior is fringed, while both setae are fringed in O. peropteryx. At the molecular level, based on a maximum likelihood analysis using approximately 400 bp of the mitochondrial 16S rDNA gene, O. knoxjonesi appears as an independent lineage, separated from O. peropteryx by a genetic distance of 16.28 %. Balantiopteryx plicata is a common host of O. knoxjonesi; however, in this work we report Pteronotus personatus and Pteronotus gymnonotus as new hosts of this tick species.

Papel potencial de Aedes albopictus Skuse en la transmisión de virus dengue (DENV) en una zona de actividad piñera de Costa Rica

RESUMEN Objetivo: Evaluar, bajo una perspectiva ecológica, la presencia de Aedes albopictus y su infección natural por virus dengue (DENV) en una zona de actividad piñera de Costa Rica. Método: Se colectaron mosquitos adultos en galerías forestales colindantes con piñeras, viviendas en proximidad a cultivos (<1 km) y viviendas en lejanía (110 km). Se empleó el índice de Shannon-Wiener para estimar biodiversidad. La infestación larvaria se evaluó en plantas de piña y viviendas y se calcularon índices aédicos de viviendas (IV) y de contenedores (IC). La detección de DENV en adultos (cuerpos y cabezas) y en larvas de Ae. albopictus se efectuó mediante RT-PCR y secuenciación. Resultados: Se colectaron 1376 adultos en total: Ae. albopictus (5,81%), Anopheles apicimacula (5,01%), Culex coronator (11,55%), Cx. inflictus (6,1%), Cx. nigripalpus (48,11%), Cx. quinquefasciatus (23,34%) y Limatus durhamii (0,07%). El índice de biodiversidad fue mayor en galerías forestales. Ae. albopictus adultos fueron colectados principalmente en el área de piñeras (73/80), aunque sólo dos larvas en las plantas de piña. Los índices aédicos en proximidad (IV: 40,7%, IC: 26,9%) y en lejanía (IV: 51,7%, IC: 29,6%) no mostraron diferencias significativas (IV Z=0,56, p=0,58; IC Z=0,16, p=0,87). Se detectó DENV-2 y DENV-3 en 2/20 grupos de cabezas y DENV-1 en 2/74 grupos de larvas de Ae. albopictus. Discusión: Las galerías forestales próximas a cultivos de piña podrían considerarse "islas ecológicas" adecuadas para el refugio de Ae. albopictus. La presencia de DENV en adultos y larvas sugiere un papel activo de Ae. albopictus en la transmisión de virus en este ecosistema.

ABSTRACT Objective To evaluate, under an ecological perspective, the presence of Aedes albopictus and the wild infection by dengue viruses (DENV) in an area of pineapple activity in Costa Rica. Materials and methods Adult mosquitoes were collected in forest galleries limiting pineapple plantations, houses adjacent to plantations (<1 km), and distant houses (1-10 km). Shannon-Wiener index was used to estimate biodiversity. Larval infestation was evaluated in pineapple plants and houses, and aedic house (HI) and container (CI) indices were calculated. Detection of DENV in Ae. albopictus adults (bodies and heads) and larvae was performed by RT-PCR and sequencing. Results A total 1376 adult mosquitoes were collected: Ae. albopictus (5.81%), Anopheles apicimacula (5.01%), Culex coronator (11.55%), Cx. inflictus (6.1%), Cx. nigripalpus (48.11%), Cx. quinquefasciatus (23.34%), and Limatus durhamii (0.07%). Biodiversity index was higher in forest galleries. Most adult Ae. albopictus were collected in forests close to pineapple fields (73/80), although only 2 larvae were detected in pineapple plants. Larval indices in adjacent houses (HI: 40.7%, CI: 26.9%) and distant houses (HI: 51.7%, CI: 29.6%) were similar (HI Z=0.56, p=0.58; CI Z=0.16, p=0.87). DENV-2 and DENV-3 were detected in 2/20 "pools" of Ae. albopictus heads and DENV-1 in 2/74 "pools" of larvae. Conclusion Forest galleries that are in proximity to pineapple plantations could be considered "ecological islands" that are suitable for refuge of Ae. albopictus. Presence of DENV in adults and larvae suggests an active role for Ae. albopictus in virus transmission within this ecosystem.

Evidence for Widespread Associations between Neotropical Hymenopteran Insects and Actinobacteria.

The evolutionary success of hymenopteran insects has been associated with complex physiological and behavioral defense mechanisms against pathogens and parasites. Among these strategies are symbiotic associations between Hymenoptera and antibiotic-producing Actinobacteria, which provide protection to insect hosts. Herein, we examine associations between culturable Actinobacteria and 29 species of tropical hymenopteran insects that span five families, including Apidae (bees), Vespidae (wasps), and Formicidae (ants). In total, 197 Actinobacteria isolates were obtained from 22 of the 29 different insect species sampled. Through 16S rRNA gene sequences of 161 isolates, we show that 91% of the symbionts correspond to members of the genus Streptomyces with less common isolates belonging to Pseudonocardia and Amycolatopsis. Electron microscopy revealed the presence of filamentous bacteria with Streptomyces morphology in brood chambers of two different species of the eusocial wasps. Four fungal strains in the family Ophiocordycipitacea (Hypocreales) known to be specialized insect parasites were also isolated. Bioassay challenges between the Actinobacteria and their possible targeted pathogenic antagonist (both obtained from the same insect at the genus or species level) provide evidence that different Actinobacteria isolates produced antifungal activity, supporting the hypothesis of a defensive association between the insects and these microbe species. Finally, phylogenetic analysis of 16S rRNA and gyrB demonstrate the presence of five Streptomyces lineages associated with a broad range of insect species. Particularly our Clade I is of much interest as it is composed of one 16S rRNA phylotype repeatedly isolated from different insect groups in our sample. This phylotype corresponds to a previously described lineage of host-associated Streptomyces. These results suggest Streptomyces Clade I is a Hymenoptera host-associated lineage spanning several new insect taxa and ranging from the American temperate to the Neotropical region. Our work thus provides important insights into the widespread distribution of Actinobacteria and hymenopteran insects associations, while also pointing at novel resources that could be targeted for the discovery of active natural products with great potential in medical and biotechnological applications.

'Candidatus Rickettsia nicoyana': A novel Rickettsia species isolated from Ornithodoros knoxjonesi in Costa Rica.

Rickettsiae are intracellular bacteria commonly associated with hematophagous arthropods. Most of them have been described in hard ticks, but some have been found in soft ticks. Here we report the detection and isolation of a new Rickettsia from Ornithodoros knoxjonesi larvae collected from Balantiopteryx plicata (Emballonuridae) in Nicoya, Costa Rica. Two ticks were processed to detect Rickettsia spp. genes gltA, ompA, ompB, and htrA by PCR. Part of the macerate was also inoculated into Vero E6 and C6/36 cell lines, and cells were evaluated by Giménez stain, indirect immunofluorescence assay (IFA), and PCR. Both ticks were positive by PCR and rickettsial growth was successful in Vero E6 cells. Amplification and sequencing of near full length rrs, gltA, sca4 genes, and fragments of ompA and ompB showed that the Rickettsia sp. was different from described species. The highest homologies were with 'Candidatus Rickettsia wissemanii' and Rickettsia peacockii: 99.70% (1321/1325) with both sequences for rrs, 99.58% (1172/1177) and 99.76% (1246/1249) for gltA, 99.26% with both sequences (2948/2970 and 2957/2979) for sca4, 98.78% (485/491) and 98.39% (2069/2115) for ompA, and 98.58 (1453/1474) and 98.92% (1459/1475) for ompB; respectively. Bat blood, spleen, liver, and lung samples analyzed for Rickettsia detection were negative. Results demonstrate that the Rickettsia isolated from O. knoxjonesi is probably an undescribed species that belongs to the spotted fever group, for which 'Candidatus Rickettsia nicoyana' is proposed. Considering that B. plicata inhabits areas where contact with humans may occur and that human parasitism by Ornithodoros has been reported in the country, it will be important to continue with the characterization of this species and its pathogenic potential.

Detection of rickettsiae in fleas and ticks from areas of Costa Rica with history of spotted fever group rickettsioses.

Outbreaks of spotted fevers have been reported in Costa Rica since the 1950s, although vectors responsible for transmission to humans have not been directly identified. In this study, species of Rickettsia were detected in ectoparasites from Costa Rica, mostly from five study sites where cases of spotted fevers have been reported. Ticks and fleas were collected using drag cloths or directly from domestic and wild animals and pooled according to species, host, and location. Pools were analyzed initially by PCR to detect a fragment of Rickettsia spp. specific gltA gene, and those positive were confirmed by detection of htrA and/or ompA gene fragments. Partial sequences of the gltA gene were obtained, as well as at least one ompA and/or ompB partial sequence of each species. Rickettsia spp. were confirmed in 119 of 497 (23.9%) pools of ticks and fleas analyzed. Rickettsia rickettsii was identified in one nymph of Amblyomma mixtum and one nymph of Amblyomma varium. Other rickettsiae present were 'Candidatus Rickettsia amblyommii' in A. mixtum, Amblyomma ovale, Dermacentor nitens, and Rhipicephalus sanguineus s. l.; Rickettsia bellii in Amblyomma sabanerae; Rickettsia felis in Ctenocephalides felis; and Rickettsia sp. similar to 'Candidatus R. asemboensis' in C. felis, Pulex simulans, A. ovale, and Rhipicephalus microplus. Results show the presence of rickettsiae in vectors that may be responsible for transmission to humans in Costa Rica, and evidence suggests exposure to rickettsial organisms in the human environment may be common. This is the first study to report R. rickettsii in A. varium and in A. mixtum in Costa Rica.

Aplicación de un método analítico revalidado por cromatografía líquida para la cuantificación de permetrina 1 por ciento / High performance liquid chromatography-validated analytical method for 1 percent permethrin quantification

INTRODUCCIÓN: en Cuba, al igual que en otros países, la infestación por Pediculus capitis se ha incrementado notablemente a partir de la década de los setenta, por lo que se desarrolla un intenso programa dirigido a su control y prevención, en el cual se incluyen estudios sobre la calidad de la permetrina, que es un piretroide sintético, activo frente a una amplia gama de parásitos, con actividad pediculicida a 1 por ciento y escabicida a 5 por ciento. Dada su baja toxicidad y por responder favorablemente a la relación riesgo/beneficio, constituye uno de los pediculicidas de elección para disminuir la infestación por piojos. OBJETIVO: revalidación de una metódica analítica por cromatografía líquida de alta resolución y su aplicación en la cuantificación de 12 muestras de 2 lotes de loción de permetrina 1 por ciento, de producción nacional. MÉTODOS: los parámetros evaluados fueron linealidad, precisión y exactitud. Se prepararon 7 estándares de calibración y soluciones de trabajo con concentraciones que se encontraban entre 100 y 15 000 µg/mL. Estos parámetros de validación fueron verificados a través del coeficiente de correlación, el coeficiente de variación y el error relativo porcentual. RESULTADOS: la especificidad del método fue corroborada a través de los cromatogramas realizados, en los cuales los tiempos de retención fueron entre 8,8 y 9,2 min para el isómero trans y entre 10,7 y 11,2 min para el isómero cis. El coeficiente de correlación determinado para ambos isómeros fue mayor que 0,99; el coeficiente de variación fue menor que 3 por ciento y el error relativo porcentual estuvo entre -3 y 1 por ciento, tanto para los estudios interdía como intradía. Se determinó que la media de la concentración de permetrina en lotes de solución tópica 1 por ciento resultó de 6 780,14 µg/mL. CONCLUSIONES: el método revalidado fue lineal, preciso y exacto dentro del rango de concentraciones estudiadas. La concentración de permetrina calculada estuvo por debajo de lo esperado según la información de la composición de la solución de permetrina 1 por ciento (9 100-10 900 µg/mL). Este método resulta útil para la cuantificación de solución de permetrina 1 por ciento en los estudios de calidad del producto farmacológico que se distribuye a la población.

INTRODUCTION: in Cuba, as well as in other countries, Pediculus capitis infestation has increased since the 70´s; this is the reason why a program aimed at controlling and preventing this infestation was designed. This program includes quality control studies of the active synthetic pyrethroid Permethrin and its activity at 1 percent on lice and 5 percent on scabies. Due to its low toxicity and positive risk/benefit ratio, Permethrin is a pediculicide of choice to decrease Pediculus capitis infestation. OBJECTIVE: to validate an analytical method using high performance liquid chromatography and its application in the quantification of 12 samples from 2 batches of Cuban-made 1 percent permethrin pediculicidal preparation. METHODS: linearity, precision and accuracy parameters were determined. Seven calibration standards and working solutions with concentrations ranging from 100 to 15 000 µg/mL were prepared. These validation parameters were verified through the correlation coefficient, the variation coefficient and the percentage relative error. RESULTS: the specificity of this method was proved in the chromatograms that showed retention times of 8.8 to 9.2 min for the isomer trans and 10.7 to 11.2 min for the isomer cis. The correlation coefficient for both isomers was higher than 0.99; the variation coefficient was under 3 percent and the percentage relative error was -3 and 1 percent for the inter-day and intraday studies, respectively. The mean permethrin concentration in 1 percent solution batches was estimated at 6 780.14 µg/mL. CONCLUSIONS: the validated method was linear, precise and exact within the range of the studied concentrations. The estimated concentration of permethrin was below the expected rate according to the composition of 1 percent permethrin solution (9 100-10 900 µg/mL). This method may be useful for the quantification of 1 percent permethrin solutions in the quality studies of the product sold to the population.

[High performance liquid chromatography-validated analytical method for 1% permethrin quantification]. / Aplicación de un método analítico revalidado por cromatografía líquida para la cuantificación de permetrina 1%.

INTRODUCTION: In Cuba, as well as in other countries, Pediculus capitis infestation has increased since the 70's; this is the reason why a program aimed at controlling and preventing this infestation was designed. This program includes quality control studies of the active synthetic pyrethroid Permethrin and its activity at 1% on lice and 5% on scabies. Due to its low toxicity and positive risk/benefit ratio, Permethrin is a pediculicide of choice to decrease Pediculus capitis infestation. OBJECTIVE: To validate an analytical method using high performance liquid chromatography and its application in the quantification of 12 samples from 2 batches of Cuban-made 1% permethrin pediculicidal preparation. METHODS: Linearity, precision and accuracy parameters were determined. Seven calibration standards and working solutions with concentrations ranging from 100 to 15000 microg/mL were prepared. These validation parameters were verified through the correlation coefficient, the variation coefficient and the percentage relative error. RESULTS: The specificity of this method was proved in the chromatograms that showed retention times of 8,8 to 9,2 min for the isomer trans and 10,7 to 11,2 min for the isomer cis. The correlation coefficient for both isomers was higher than 0,99; the variation coefficient was under 3% and the percentage relative error was--3 and 1% for the inter-day and intraday studies, respectively. The mean permethrin concentration in 1% solution batches was estimated at 6 780,14 microg/mL. CONCLUSIONS: The validated method was linear, precise and exact within the range of the studied concentrations. The estimated concentration of permethrin was below the expected rate according to the composition of 1% permethrin solution (9 100-10 900 microg/mL). This method may be useful for the quantification of 1% permethrin solutions in the quality studies of the product sold to the population.

Efecto del Vimang® en pacientes VIH/sida / Effect of Vimang® in HIV/AIDS patients

ANTECEDENTES: el estrés oxidativo ha sido considerado como cofactor de la progresión de la infección por el VIH al estado sida. OBJETIVOS: evaluación del posible efecto antioxidante y de las funciones de diversos sistemas en el organismo, que resultan en la seguridad toxicológica para su empleo. MÉTODOS: considerando lo anterior se estudiaron 68 individuos seropositivos al VIH de modo aleatorio, a doble ciegas en dos grupos: a uno se le suministró Vimang® durante 6 meses y al otro placebo. Se hicieron determinaciones del estado redox, índices bioquímicos de función renal, hepática y hematológica, y se realizó una evaluación de la ingesta dietaria a través de encuestas durante 7 d al inicio y al final del estudio. RESULTADOS: se evidenció de manera estadísticamente significativa (p< 0,05), un cambio beneficioso en los índices redox en el grupo con Vimang® respecto al placebo. No se encontraron diferencias significativas en la comparación de los índices dietarios evaluados, ni en los índices bioquímicos de función renal, hepática y hematológica entre los grupos, ni al final del estudio. CONCLUSIONES: las evidencias demuestran el efecto antioxidante del Vimang® sin influencias tóxicas en el período de 6 meses en los individuos VIH/sida estudiados.

INTRODUCTION: the oxidative stress (OS) has been recognized as a co-factor of HIV infection evolution to AIDS condition. OBJECTIVES: to evaluate the possible antioxidant effect and the impact on the functioning of several systems in the body, resulting in the toxicological safety of Vimang® use. METHODS: sixty eight HIV-seropositive patients were double-blind randomized in two groups; the first was supplied with Vimang® during six months and the other with placebo. Redox condition and the biochemical indexes of the hematological, renal and hepatic functions were measured. Also, dietary intake was assessed through surveys administered for 7 days at the beginning and at the end of the study. RESULTS: there were statistically significant differences between the groups regarding positive change in redox figures in Vimang® group compared to those of the placebo group. No significant difference was found either in the evaluated dietary intake indexes or in the biochemical indexes of the renal, hematological and hepatic functions at the end of the study. CONCLUSIONS: Vimang® antioxidant effect was shown, without any toxic influence during the six month-study conducted in HIV/AIDS patients.

[Effect of Vimang in HIV/AIDS patients]. / Efecto del Vimang en pacientes VIH/sida.

INTRODUCTION: the oxidative stress (OS) has been recognized as a co-factor of HIV infection evolution to AIDS condition. OBJECTIVES: to evaluate the possible antioxidant effect and the impact on the functioning of several systems in the body, resulting in the toxicological safety of Vimang use. METHODS: sixty eight HIV-seropositive patients were double-blind randomized in two groups; the first was supplied with Vimang during six months and the other with placebo. Redox condition and the biochemical indexes of the hematological, renal and hepatic functions were measured. Also, dietary intake was assessed through surveys administered for 7 days at the beginning and at the end of the study. RESULTS: there were statistically significant differences between the groups regarding positive change in redox figures in Vimang group compared to those of the placebo group. No significant difference was found either in the evaluated dietary intake indexes or in the biochemical indexes of the renal, hematological and hepatic functions at the end of the study. CONCLUSIONS: Vimang antioxidant effect was shown, without any toxic influence during the six month-study conducted in HIV/AIDS patients.

Estudio de bioequivalencia: formulaciones genéricas y comerciales de estaduvine, lamiduvine, zidovudine e indinavir en pacientes cubanos infectados con VIH / Bioequivalence study: generic and trade formulations of stavudine, lamivudine, zidovudine and indinavir in Cuban HIV-infected subjects

Objetivo: El presente estudio describe los estudios de bioequivalencia de los genéricos cubanos antirretrovirales estavudina, lamivudina, zidovudina e indinavir con respecto a sus productos innovadores. Diseño: Los estudios de bioequivalencia de cada antirretroviral estudiado (formulación de prueba, P y referencia, R), se realizaron en 13 ó 14 sujetos, según el caso con la utilización de las medias en un estudio aleatorio, cruzado y a dosis única con un período de lavado de 10 días. Métodos: Las concentración de los antirretrovirales en plasma se monitorearon durante un período de 12 h después de la administración de estos, mediante el uso de métodos validados de cromatografía líquida de alta resolución (HPLC). Los parámetros farmacocinéticos se determinaron con el uso el software profesional WinNolin, versión 2.1. La comparación de los parámetros farmacocinéticos se realizó con un intervalo de confianza del 95 por ciento con la utilización del software NCSS 2000 PASS 2000 (hipótesis nula). Para el estudio de bioequivalencia se usó el software Equiv Test de soluciones estadísticas, empleando ANOVA con el 90 por ciento de intervalo de confianza. Resultados: El presente estudio mostró que no se observaron diferencias significativas en las medias de AUC0-12, AUC0-¥, Cmax y Tmax de ambas formulaciones de estavudina, lamivudina, zidovudina e indinavir. Se concluye que los parámetros farmacocinéticos estudiados se encuentran dentro de los rangos establecidos siendo 80-125 por ciento para Cmax, AUC0-12, y AUC0-¥. En el caso de Cmax de la zidovudina el rango fue 70-143 por ciento. Conclusión: Estos resultados avalan que los genéricos antirretrovirales cubanos son bioequivalentes con sus respectivos productos innovadores en términos de velocidad y magnitud de la absorción

Lecciones de nutrición

Nutrición: Requerimientos, recomendaciones y carencias; Alimentos; Nutrición en las distintas etapas de la vida y durante el estrés fisiológico. Dietoterapia: Obesidad; Desnutrición; Diabetes sacarina; Tratamiento diabetológico; Insulinoterapia; Hipoglucemiantes bucales; Acidosis diabética; Hiperlipoproteinemias; Diabetes y embarazo; Aterosclerosis; Alimentación en afecciones renales; Gota; Estrés patológico; Nutrición y salud pública