BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

ABSTRACT Cutaneous leishmaniasis (CL) is an endemic disease in the Republic of Panama, caused by Leishmania (Viannia) parasites, whose most common clinical manifestation is the presence of ulcerated lesions on the skin. These lesions usually present a chronic inflammatory reaction, sometimes granulomatous, with the presence of lymphocytes, plasma cells and macrophages. This study describes the histopathological characteristics found in the skin lesions of patients with CL caused by Leishmania (V.) panamensis in Panama. We analyzed 49 skin biopsy samples from patients with clinical suspicion of CL, by molecular tests (PCR for subgenus Viannia and HSP-70) and by Hematoxylin-Eosin staining. Samples were characterized at the species level by PCR-HSP-70/RFLP. From the 49 samples studied, 46 (94%) were positive by PCR and were characterized as Leishmania (V.) panamensis. Of these, 48% were positive by Hematoxylin-Eosin staining with alterations being observed both, in the epidermis (85%) and in the dermis (100%) of skin biopsies. The inflammatory infiltrate was characterized according to histopathological patterns: lymphohistiocytic (50%), lymphoplasmacytic (61%) and granulomatous (46%) infiltration, being the combination of these patterns frequently found. The predominant histopathological characteristics observed in CL lesions caused by L. (V.) panamensis in Panama were: an intense inflammatory reaction in the dermis with a combination of lymphohistiocytic, lymphoplasmacytic and granulomatous presentation patterns and the presence of ulcers, acanthosis, exocytosis and spongiosis in the epidermis.

Chagas disease, caused by Trypanosoma cruzi infection, is a zoonosis of humans, wild and domestic mammals, including dogs. In Panama, the main T. cruzi vector is Rhodnius pallescens, a triatomine bug whose main natural habitat is the royal palm, Attalea butyracea. In this paper, we present results from three T. cruzi serological tests (immunochromatographic dipstick, indirect immunofluorescence and ELISA) performed in 51 dogs from 24 houses in Trinidad de Las Minas, western Panama. We found that nine dogs were seropositive (17.6% prevalence). Dogs were 1.6 times more likely to become T. cruziseropositive with each year of age and 11.6 times if royal palms where present in the peridomiciliary area of the dog’s household or its two nearest neighbours. Mouse-baited-adhesive traps were employed to evaluate 12 peridomestic royal palms. All palms were found infested with R. pallescens with an average of 25.50 triatomines captured per palm. Of 35 adult bugs analysed, 88.6% showed protozoa flagellates in their intestinal contents. In addition, dogs were five times more likely to be infected by the presence of an additional domestic animal species in the dog’s peridomiciliary environment. Our results suggest that interventions focused on royal palms might reduce the exposure to T. cruzi infection.

Introducción. La implementación de los programas de vigilancia, prevención y control de la enfermedad de Chagas requiere una aproximación integral. La sostenibilidad de los programas depende de la participación comunitaria amparada en un conocimiento básico del problema. Objetivo. Evaluar los conocimientos de los entrevistados que facilitan o limitan la vigilancia, prevención y control de la enfermedad de Chagas en dos comunidades endémicas de Panamá donde Rhodnius pallescens es el vector principal. Materiales y métodos. Mediante un cuestionario se evaluaron los conocimientos y los factores de riesgo relacionados con la enfermedad de Chagas en 201 personas mayores de 10 años de las comunidades endémicas de Las Pavas y Lagartera Grande, ubicadas en la ribera oeste del Canal de Panamá. Con ayuda de los moradores también se evaluó la presencia de chinches triatominos en 93 viviendas a lo largo de un año. Resultados. De las personas entrevistadas, 69,2 % (139/201) tenía pocos o muy pocos conocimientos sobre la enfermedad de Chagas y 93 % (187/201) estaba expuesto a factores de riesgo moderados o altos. Se capturaron chinches triatominos en 20,4 % (19/93) de las viviendas evaluadas, de los cuales, el 86,8 % (66/76) era R. pallescens. Se encontró asociación significativa entre la presencia de chinches dentro de las viviendas y las viviendas catalogadas como precarias (p<0,01). Conclusión. A pesar de que en estas dos comunidades se han desarrollado programas de educación sanitaria, es necesario reforzar los conocimientos básicos sobre la enfermedad de Chagas antes de establecer medidas de prevención y vigilancia que involucren la participación activa de sus habitantes.

Introduction: The implementation of surveillance, control and prevention measures for Chagas´ disease requires an integrated approach. The sustainability of programs depends on community participation supported on a basic understanding of the problem. Objective: To evaluate the knowledge of people interviewed which facilitates or limits the surveillance, prevention and control of Chagas´ disease in two endemic Panamanian communities where Rhodnius pallescens is the main vector. Materials and methods: A questionnaire assessed the knowledge and risk factors related to Chagas´ disease in 201 people over 10 years of age from the endemic communities of Las Pavas and Lagartera Grande located on the western shore of Panama Canal. Additionally, with the help of the residents, the presence of triatomine bugs in 93 dwellings was evaluated during a year. Results: Of those interviewed, 69.2% (139/201) had poor or very poor knowledge about Chagas´ disease. Nearly 93% (187/201) was exposed to moderate or high risk factors. Triatomine bugs were collected in 20.4% (19/93) of the evaluated homes; 80% (66/76) of them were R. pallescens . Significant association was found between the presence of triatomines inside the houses and precarious housing (p<0.01). Conclusion: Despite having been exposed to health education-related programs, it is necessary to strengthen the basic knowledge about Chagas´ disease in the two communities before establishing prevention and surveillance measures which involve active participation of its inhabitants.

Introducción. La evaluación de la sensibilidad de las poblaciones de Rhodnius pallescens permite detectar y vigilar los grados de resistencia en el transcurso del tiempo, con el fin de evaluar los efectos de las estrategias de control y manejo de la resistencia. Objetivo. Determinar la línea base de sensibilidad de los principios activos deltametrina y lambdacihalotrina en ninfas de estadios I y V, en dos poblaciones de campo de R. pallescens. Materiales y métodos. Los bioensayos se realizaron tomando como referencia el protocolo estandarizado de aplicación tópica para R. prolixus y adaptado para R. pallescens. Resultados. Con la aplicación tópica del insecticida lambdacihalotrina en ninfas de estadio I en R. pallescens (Chilibre) y R. pallescens (Cerro Cama), los valores de la dosis letal 50 (DL50) expresados en nanogramos por insecto fueron de DL50=0,129 y DL50=0,109, respectivamente. En ninfas de estadio V los valores fueron de DL50=1,712 y DL50=3,478, respectivamente. Por su parte, la aplicación tópica de deltametrina en ninfas de estadio I, registró valores de DL50=0,022 y DL50=0,021 y, en ninfas de estadio V, de DL50=2,110 y DL50=1,548. Conclusiones. En ninfas de estadios I y V de ambas poblaciones de R. pallescens, se observó mediante los valores obtenidos de factor de resistencia que no existen ninguna diferencia significativa en el efecto tóxico de los insecticidas estudiados. Esto permite establecer que las dos cepas de R. pallescens son sensibles a los insecticidas deltametrina y lambdacihalotrina.

Introduction. Systematic evaluation of the susceptibility of disease vectors to insecticides permits the detection of the development of insecticide resistance over time. This is necessary to evaluate the effectiveness of control methods and to plan management strategies of the resistance. Objective. The baseline susceptibility was determined for I and V instar nymphs of Rhodnius pallescens to the active ingredients of the insecticides deltamethrin and lambdacyhalothrin. Materials and methods. The bioassays were applied to two field populations of R. pallescens collected in Chilibre and Cerro Cama, Panamá. A standard protocol for topical application was adapted from that developed for Rhodnius prolixus. Bioassays were performed using topical applications on the dorsal abdominal surface, with volumes of 0.1µl and 0.5µl acetone solution of insecticide for nymphs of stage I and V respectively, using 5µl and 25µl Hamilton microsyringes with a repeating dispenser. Ten nymphs were used for each insecticide concentration. Results. With the topical application of lambdacyhalothrin onfirst-instar nymphs from Chilibre and Cerro Cama, the LD50 values expressed in ng/insect were 0.13 and 0.11 respectively. In fifth-instar nymphs the LD50 values were 1.71 and 3.48, respectively. For deltamethrin, the topical application on first-instar nymphs resulted in LD50 values of 0.02 and 0.02, and in fifth-instar nymphs the LD50 values were 2.11 and 1.55, respectively. Conclusions. In I and V instar nymphs from the two R. pallescens populations, resistance factor values demonstrated no significant difference in the toxic effects of the two insecticides and indicated that the R. pallescens populations were susceptible them.

Introducción. La enfermedad de Chagas es la principal causa de cardiomiopatía crónica en Centroamérica. Existe controversia sobre los mecanismos causantes de la patología cardiaca observada durante la fase crónica de esta parasitosis. Varios estudios han detectado la presencia de autoanticuerpos circulantes dirigidos contra receptores beta-adrenérgicos y colinérgicos muscarínicos del miocardio en pacientes chagásicos, que pueden desencadenar señales intracelulares y alterar la función cardiaca durante el curso de la enfermedad. Objetivo. Nuestro objetivo principal fue comparar la frecuencia sérica de estos autoanticuerpos en pacientes chagásicos crónicos con la observada en pacientes con otras formas de cardiopatía y en controles sanos. Materiales y métodos. Se determinó la prevalencia de autoanticuerpos contra receptores beta-adrenérgicos y colinérgicos muscarínicos en cuatro grupos de pacientes panamelos: 53 pacientes chagásicos, 25 pacientes seronegativos con insuficiencia cardiaca, 25 pacientes con diferentes tipos de arritmia cardiaca y 25 controles sanos. Resultados. Los autoanticuerpos contra receptores autonómicos fueron más frecuentes en el grupo de pacientes con cardiopatía chagásica crónica (24,5%) comparados con el grupo de insuficiencia cardiaca (20,0%) y con el grupo con arritmias cardiacas (16,0%). Al comparar la proporción de autoanticuerpos entre el grupo de pacientes con cardiopatía chagásica crónica y los controles sanos, se detectaron diferencias muy significativas (24,5% versus 0%; p=0,0015). De los 53 pacientes con infección crónica, 48 (90,6%) presentaron algún grado de alteración cardiaca. Conclusiones. En comparacióncon el grupo de controles sanos, la frecuencia de los autoanticuerpos contra receptores autonómicos se encuentra significativamente aumentada en pacientes con enfermedad de Chagas crónica y con otras formas de cardiopatía.

Introduction. Chagas´ disease is the main cause of chronic myocardiopathy in Central America. The mechanisms proposed for this cardiac pathology during the chronic phase remain controversial. Several studies have detected the presence of circulating autoantibodies against β-adrenergic and cholinergic muscarinic receptors of the myocardium in patients with Chagas disease. These autoantibodies can trigger intracellular signals and modify the cardiac function during the progression of the disease. Objectives. The serological frequency of these autoantibodies was compared among patients with chronic Chagas disease, patients with other cardiopathies and healthy controls. Materials and methods. The prevalence of autoantibodies against β-adrenergic and cholinergic muscarinic receptors was determined in four groups of Panamenian patients: 53 chagasic patients, 25 serologically negative patients with cardiac insufficiency, 25 patients with cardiac arrhythmia and 25 healthy individuals. Results. The antibodies against autonomic receptors were more frequently observed in patients with chronic chagasic cardiomyopathy (24.5%) compared to the cardiac insufficiency group (20.0%) and the cardiac arrhythmia group (16.0%). The proportion of autoantibodies was significantly different between the groups with chronic chagasic cardiomyopathy and healthy controls (24.5% versus 0%; p=0.015). Of the 53 chronically infected chagasic patients, 48 (90%) showed some degree of cardiac dysfunction. Conclusions. The frequency of autoantibodies against autonomic receptors is significantly increased in patients with chronic Chagas disease and in patients with other cardiopathies.

O triatomíneo silvestre Rhodnius pallescens é considerado o mais importante vetor do Trypanosoma cruzi e Trypanosoma rangeli no Panamá. Entretanto, seu comportamento e características biológicas são pouco estudados. Para controlar a doença de Chagas no Panamá é necessário melhorar a compreensão dos aspectos eco-biológicos do R. pallescens. Neste estudo, investigaram-se as fontes de alimentação de R. pallescens usando dot-blot e o índice de infecção por Trypanosoma por metodologia molecular, em área endêmica da doença de Chagas na região central do Panamá. Foi observado que 20,3% dos barbeiros coletados em palmeiras peridomésticas se alimentavam de gambás (Didelphis marsupialis). Contudo, barbeiros coletados dentro das residências apresentaram antropofagia aumentada (25,4%). Considerando o ambiente doméstico e peridoméstico juntos, o percentual de R. pallescens infectados com Trypanosoma foi de 87,4 %. Nos ambientes doméstico e peridoméstico, a infecção por T. cruzi foi de 80,4% e 90%; a infecção por T. rangeli foi de 47% e 51%, respectivamente. Observou-se infecção mista em 43% dos triatomíneos coletados em ambiente doméstico e em 51% dos triatomíneos peridomésticos. Estes achados fornecem embasamento para a implementação de um controle adequado e um programa de vigilância para a doença de Chagas em regiões onde o R. pallescens é endêmico.

The sylvatic triatomine Rhodnius pallescens is considered to be the most important and widespread vector of Trypanosoma cruzi and Trypanosoma rangeli in Panama. However, its behavior and biological characteristics have only been partially investigated. Thus, to achieve sustainable and efficient control over Chagas disease in Panama, a better understanding of the ecology and biology of R. pallescens is essential. In this study we evaluated R. pallescens host feeding sources using a dot-blot assay, and the trypanosome infection index by PCR analysis in a Chagas disease endemic area of central Panama. It was found that in peridomestic palm trees, 20.3% of the examined bugs had fed on opossums (Didelphis marsupialis). However, we observed an increased anthropophagy (25.4%) for those bugs collected inside houses. Considering the domestic and peridomestic habitats as a whole, the proportion of collected R. pallescens infected with trypanosomes was 87.4%. In the two habitats the predominant infection was with T. cruzi (80-90%). Between 47-51% of the analyzed triatomines were infected with T. rangeli. Mixed infections (40-51%) were also detected. These findings provide a better basis for the implementation of a rational control and surveillance program for Chagas disease in regions where R. pallescens is endemic.

Rhodnius pallescens is the main vector of Trypanosoma cruzi in Panama and one of the most relevant secondary vectors in Colombia. Despite the importance of this species, there is limited knowledge about the genetic variability along its geographical distribution. In order to evaluate the degree of karyotype variability we analyzed the meiotic behavior and banding pattern of the chromosomes of 112 males of R. pallescens coming from different regions of Colombia and Panama. Using the C-banding technique we identified two chromosomal patterns or cytotypes characterized by differences in the amount, size and distribution of constitutive heterochromatic regions in the chromosome complement (2n = 20 autosomes plus XY in males). The individuals can be easily classified in each cytotype by the analysis of the chromosomes during first meiotic prophase. The frequencies of the cytotypes are variable according to the geographic origin of the populations. This chromosomal divergence together with morphological data supports the existence of three genetically different populations of R. pallescens and provides new information to understand the distribution dynamics of this species.

A total of 206 serum samples from children (3-14 years old) living in the Amador County (La Chorrera District, Province of Panama) were screened by indirect immunofluorescence antibody test (IFAT) for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR) followed by dot blot hybridization. The results indicated a prevalence of 9.7% for trypanosome infections, a seroprevalence of 2.9% against T. cruzi and a predominance of T. rangeli infection (6.8%). The immunological and clinical implications of these findings are discussed.

A total of 33 crude and cloned Trypanosoma rangeli stocks found as natural infections in human from Panama and other endemic areas of Central and South America were evaluated as producers of sialidase (SA) activity through the MU-NANA fluorescence test. Negative results were observed in 6 of the isolates: Panama (4), Honduras (1), and Brazil (1). In addition, an immunoblotting analysis confirm the presence of the SA antigen in these stocks without enzymatic activity. These findings must be considered in the interpretation of the biological significance of T. rangeli SA and in the proper characterization and identification of this parasite.

A epidemiologia da infecção do T. cruzi foi investigada na margem oriental do canal do Panamá, na região central da Republica do Panamá. A informação obtida durante o estudo avaliou fatores de risco da doença de Chagas nesta área. Entre 1999 e 2000, 1110 triatomíneos foram coletados: 1050 triatomíneos (94,6%) em palmeiras, 27 (2,4%) em habitats periurbanos e 33 (3,0%) no interior de casas. Todos os espécimens foram identificados como R. pallescens. Não havia nenhuma evidência de domiciliação do vetor. O exame de glândulas salivares de 380 R. pallescens revelaram taxa de infecção natural por Trypanosoma de 7,6%, mas o conteúdo da ampola rectal de 373 triatomíneos mostrou 45% de positividade. Os perfis de isoenzimas em Trypanosomas isolados demonstraram que 85,4% (n = 88) eram T. cruzi e 14,6% (n = 15) eram T. rangeli. A análise da refeição de sangue de 829 R. pallescens demonstrou comportamento zoofílico do vetor, sendo os gambás a fonte preferencial de sangue. Soroprevalência nos seres humanos de ambos locais de estudo foi menos que 2%. Nossos resultados demonstram que T. cruzi sobrevive na área em associação equilibrada com R. pallescens e com diversas espécies diferentes de mamíferos em seus nichos naturais. Entretanto, a área é um risco eminente de infecção para sua população, pelo que é importante executar um programa educacional na comunidade a respeito das medidas, do conhecimento e do controle da doença.

The eco-epidemiology of T. cruzi infection was investigated in the Eastern border of the Panama Canal in Central Panama. Between 1999 and 2000, 1110 triatomines were collected: 1050 triatomines (94.6%) from palm trees, 27 (2.4%) from periurban habitats and 33 (3.0%) inside houses. All specimens were identified as R. pallescens. There was no evidence of vector domiciliation. Salivary glands from 380 R. pallescens revealed a trypanosome natural infection rate of 7.6%, while rectal ampoule content from 373 triatomines was 45%. Isoenzyme profiles on isolated trypanosomes demonstrated that 85.4% (n = 88) were T. cruzi and 14.6% (n = 15) were T. rangeli. Blood meal analysis from 829 R. pallescens demonstrated a zoophilic vector behavior, with opossums as the preferential blood source. Seroprevalence in human samples from both study sites was less than 2%. Our results demonstrate that T. cruzi survives in the area in balanced association with R. pallescens, and with several different species of mammals in their natural niches. However, the area is an imminent risk of infection for its population, consequently it is important to implement a community educational program regarding disease knowledge and control measures.