Dengue Virus Infection of Primary Human Smooth Muscle Cells.

Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection.

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.

Exposure of dogs to spotted fever group rickettsiae in urban sites associated with human rickettsioses in Costa Rica.

The zoonotic transmission cycles of Rickettsia rickettsii and other spotted fever group (SFG) rickettsiae in Latin America have usually been associated with rural or sylvatic environments, although domestic dogs can be implicated in more populated settings. In this study, exposure of dogs to SFG rickettsiae in the Greater Metropolitan Area of Costa Rica was investigated. Dogs from sites associated with human cases and from dog shelters were evaluated by indirect immunofluorescence assay (IFA) using antigen of SFG rickettsiae. Rickettsia spp. were detected in ectoparasites by polymerase chain reaction (PCR). A total 18.5% (31/168) of dogs associated with human cases and 6.8% (11/161) of dogs in shelters had IgG end titers≥64 to Rickettsia spp. The odds of being seropositive were greater in dogs from areas associated with human cases when compared to shelters (OR: 3.2; 95% C.I: 1.5-5.6). Rhipicephalus sanguineus sensu lato (s. l.) was present in all sites associated with human cases. Rickettsia felis URRWXCal2 and R. felis-like RF2125 were detected in Ctenocephalides felis, and Rickettsia sp. IbR/CRC in Ixodes boliviensis. Results demonstrate that dogs from the main urban center of Costa Rica have been exposed to SFG rickettsiae, especially in areas with known human infection. Both human and animal health sectors must be aware of possible rickettsial diseases in urban areas, where dogs may also serve as sentinels for human infection.

Pathogenic potential of a Costa Rican strain of 'Candidatus Rickettsia amblyommii' in guinea pigs (Cavia porcellus) and protective immunity against Rickettsia rickettsii.

'Candidatus Rickettsia amblyommii' is a spotted fever group rickettsia that is not considered pathogenic, although there is serologic evidence of possible infection in animals and humans. The aim of this study was to evaluate the pathogenic potential of a Costa Rican strain of 'Candidatus R. amblyommii' in guinea pigs and determine its capacity to generate protective immunity against a subsequent infection with a local strain of Rickettsia rickettsii isolated from a human case. Six guinea pigs were inoculated with 'Candidatus R. amblyommii' strain 9-CC-3-1 and two controls with cell culture medium. Health status was evaluated, and necropsies were executed at days 2, 4, and 13. Blood and tissues were processed by PCR to detect the gltA gene, and end titers of anti-'Candidatus R. amblyommii' IgG were determined by indirect immunofluorescence. To evaluate protective immunity, another 5 guinea pigs were infected with 'Candidatus R. amblyommii' (IGPs). After 4 weeks, these 5 IGPs and 3 controls (CGPs) were inoculated with pathogenic R. rickettsii. Clinical signs and titers of anti-Rickettsia IgG were determined. IgG titers reached 1:512 at day 13 post-infection with 'Candidatus R. amblyommii'. On day 2 after inoculation, two guinea pigs had enlarged testicles and 'Candidatus R. amblyommii' DNA was detected in testicles. Histopathology confirmed piogranulomatous orchitis with perivascular inflammatory infiltrate in the epididymis. In the protective immunity assay, anti-Rickettsia IgG end titers after R. rickettsii infection were lower in IGPs than in CGPs. IGPs exhibited only transient fever, while CGP showed signs of severe disease and mortality. R. rickettsii was detected in testicles and blood of CGPs. Results show that the strain 9-CC-3-1 of 'Candidatus R. amblyommii' was able to generate pathology and an antibody response in guinea pigs. Moreover, its capacity to generate protective immunity against R. rickettsii may modulate the epidemiology and severity of Rocky Mountain spotted fever in areas where both species circulate.

Detection of an undescribed Rickettsia sp. in Ixodes boliviensis from Costa Rica.

Ixodes boliviensis is a tick of carnivores that is common on domestic dogs. The only Rickettsia that has been detected previously in this species is 'Candidatus Rickettsia andeanae'. We report the detection of an undescribed Rickettsia sp., named strain IbR/CRC, in I. boliviensis collected from dogs in Costa Rica. Analyses of gltA, ompA, and htrA partial sequences place Rickettsia sp. strain IbR/CRC in the group of R. monacensis, also close to an endosymbiont of Ixodes scapularis and other undescribed rickettsiae. It was not possible to isolate Rickettsia sp. strain IbR/CRC in Vero E6 or C6/36 cell lines. Isolation and further characterization of Rickettsia sp. strain IbR/CRC and the other undescribed rickettsiae are required to determine their taxonomic status and pathogenic potential.

A fatal urban case of rocky mountain spotted fever presenting an eschar in San Jose, Costa Rica.

This study reports the first urban human case of Rocky Mountain spotted fever caused by Rickettsia rickettsii, in Costa Rica. An 8-year-old female who died at the National Children's Hospital 4 days after her admission, and an important and significant observation was the presence of an "eschar" (tache noire), which is typical in some rickettsial infections but not frequent in Rocky Mountain spotted fever cases.

Rickettsia felis in Ctenocephalides felis from Guatemala and Costa Rica.

Rickettsia felis is an emerging human pathogen associated primarily with the cat flea Ctenocephalides felis. In this study, we investigated the presence of Rickettsia felis in C. felis from Guatemala and Costa Rica. Ctenocephalides felis were collected directly from dogs and cats, and analyzed by polymerase chain reaction for Rickettsia-specific fragments of 17-kDa protein, OmpA, and citrate synthase genes. Rickettsia DNA was detected in 64% (55 of 86) and 58% (47 of 81) of flea pools in Guatemala and Costa Rica, respectively. Sequencing of gltA fragments identified R. felis genotype URRWXCal(2) in samples from both countries, and genotype Rf2125 in Costa Rica. This is the first report of R. felis in Guatemala and of genotype Rf2125 in Costa Rica. The extensive presence of this pathogen in countries of Central America stresses the need for increased awareness and diagnosis.

An update on the detection and treatment of Rickettsia felis.

Rickettsia felis was described as a human pathogen almost two decades ago, and human infection is currently reported in 18 countries in all continents. The distribution of this species is worldwide, determined by the presence of the main arthropod vector, Ctenocephalides felis (Bouché). The list of symptoms, which includes fever, headache, myalgia, and rash, keeps increasing as new cases with unexpected symptoms are described. Moreover, the clinical presentation of R. felis infection can be easily confused with many tropical and nontropical diseases, as well as other rickettsial infections. Although specific laboratory diagnosis and treatment for this flea-borne rickettsiosis are detailed in the scientific literature, it is possible that most human cases are not being diagnosed properly. Furthermore, since the cat flea infests different common domestic animals, contact with humans may be more frequent than reported. In this review, we provide an update on methods for specific detection of human infection by R. felis described in the literature, as well as the treatment prescribed to the patients. Considering advances in molecular detection tools, as well as options for as-yet-unreported isolation of R. felis from patients in cell culture, increased diagnosis and characterization of this emerging pathogen is warranted.

First report of the isolation and molecular characterization of Rickettsia amblyommii and Rickettsia felis in Central America.

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

Determinación de subtipos del virus respiratorio sincicial en muestras positivas por el virus, aisladas en el Hospital Nacional de Niños / Detection of human respiratory syncytial virus subtypes A and B in hospitalized children in Costa Rica

Objetivo: El virus respiratorio sincicial, VRS, es un pneumovirus de la familia Paramyxovridae, que causa enfermedad severa del tracto respitaroio inferior en neonatos y niños pequeños, especialmente en los primeros años de vida. Es responsable de constantes hospitalizaciones y visitas a los servicios de emergencias. Se han identificado dos subtipos: VRS-A y VRS-B, mediante anticuerpos monoclonales y técnicas moleculares. El objetivo de este estudio fue establecer por primera vez la circulación de ambos subtipos del VRS, en muestras positivas de niños hospitalizados durante el pico estacional de 2008, en el Hospital Nacional de Niños, HNN. Métodos: Se analizaron 49 muestras de aspirados nasofaríngeos de niños hospitalizados, de un total de 578, de las cuales 197 fueron previamente positivas para VRS por inmunofluorescencia directa. Se realizó cultivo celular, y un RT-PCR múltiple, estandarizado en el laboratorio, para detectar VRS-A y VRS-B. Resultados: La frecuencia del VRS fue del 34 por ciento en el HNN, para agosto y septiembre de 2008. De las 49 muestras analizadas por RT-PCR, 41, 84 por ciento, fueron positivas, 34, 83 por ciento, por el subtipo A y 7, 17 por ciento, por el B; 8 fueron negativas. Ningún paciente presentó infección mixta y no hubo diferencia entre los síntomas, la edad o el origen geográfico de los niños. El cultivo fue positivo solo en el 30 por ciento de las muestras. Conclusión: La frecuencia del VRS para el periodo en estudio fue del 34 por ciento de las muestras analizadas en aspirados nasofaríngeos. Este es el primer reporte de la detección de los subtipos A y B del VRS, en una pequeña cohorte del HNN, confirmados por un RT-PCR múltiple estandarizado en el laboratorio.

Molecular characterization of Rickettsia rickettsii isolated from human clinical samples and from the rabbit tick Haemaphysalis leporispalustris collected at different geographic zones in Costa Rica.

Five strains of spotted fever group (SFG) rickettsiae previously isolated from human clinical cases and from the tick Haemaphysalis leporispalustris were used for molecular characterization in this study to establish their genetic relationship compared with the prototype Rickettsia rickettsii strain Sheila Smith. Samples were tested by polymerase chain reaction (PCR) targeting the rickettsial genes gtlA, ompA, and ompB. PCR products of the latter two genes were DNA sequenced and compared with available sequences in GenBank. The ompA partial sequences of the five Costa Rican isolates showed 100% identity to several R. rickettsii sequences available in GenBank, including the sequence of the virulent reference strain Sheila Smith, whereas the ompB partial sequences of the five Costa Rican isolates showed 99.8-100% identity to R. rickettsii sequences from GenBank. This study showed the first molecular detection of R. rickettsii isolates from Rocky Mountain Spotted Fever patients and from the rabbit tick H. leporispalustris in different geographical zones in Costa Rica.

Alteraciones ultraestructurales inducidas por virus dengue en cultivos celulares / Ultra structural induced alterations for dengue virus in cultured cells

Los cuatro serotipos del virus dengue inducen un efecto citopático (ECP) leve en cultivos celulares provenientes de mamíferos o mosquitos, como son las células Vero y las C6/36, respectivamente. Este ha sido descrito al microscopio electrónico de transmisión (MET); pero las posibles alteraciones a nivel de la superficie de las células infectadas no han sido descrita. Por tal razón investigamos el ECP al MET y al ME de rastreo (MER). Ambas líneas celulares (Vero y C6/36) fueron infectadas con Den-1, se fijaron y procesaron para MET y MER. Las células Vero mostraron vacuolización y algunas veces fusión celular; pero su superficie fue similar en las células sin infectar. Las células C6/36 mostraron un citoplasma totalmente vacuolizado y en el cual sólo se identificó el núcleo y microtúbulos. Al MER esa células pesentaban abundantes filipodia con cadenas de cuerpos esferoides, similares a un rosario, que podrían corresponder a las vacuolas

Tres nuevos casos de Fiebre Manchada de las Monta¿as Rocosas en Costa Rica / Three new cases of Rocky Mountain Spotted Fever in Costa Rica

Se informa sobre tres nuevos casos de Fiebre Manchada de las Monta¿as Rocosas ocurridos en Costa Rica, dos en El Bosque, Limón y el otro en la Virgen, Sarapiquí, Heredia. Se incluyen, además, algunas consideraciones de orden epidemiológico que han caracterizado a esta rickettsiosis en Costa Rica tales como distribución geográfica, clínica, patología, diagnóstico de laboratorio y vectores

Diagnóstico del laboratorio de virus herpes simplex en Costa Rica / Laboratory diagnosis of herplex simplex virus in Costa Rica

El presente trabajo aporta informacion sobre los metodos de uso corriente usados en el laboratorio de Virologia de la Universidad de Costa Rica para el diagnostico por aislamiento y serologia de virus herpes simplex uno y dos, en pacientes que han acudido para su estudio. Se diagnostico 28 pacientes, 4 asintomaticos, 6 con lesiones orales, 12 con lesiones genitales, 3 con lesiones en piel y 3 con encefalitis. Con los resultados obtenidos, se pudo observar en estos pacientes la caracteristica de recurrencia y la localizacion anatomica de ambos serotipos