Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

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.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil / Avaliação de três métodos de extração de DNA obtidos de amostras de sangue coletadas em papel de filtro em infecções subpatentes de Plasmodium da região Amazônica no Brasil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

Infecção assintomática por Plasmodium é um novo desafio para a saúde pública no Brasil. A reação em cadeia da polimerase (PCR) é o melhor método para detectar baixas parasitemias presentes em pacientes com infecção assintomática. Nas áreas endêmicas, a coleta de sangue total é dificultada pela distancia geográfica, transporte e adequada armazenagem das amostras. A coleta de sangue em papel de filtro pode ser uma alternativa nessas áreas de difícil acesso. Neste estudo foram comparados três diferentes métodos de extração de ADN a partir de papel de filtro usando como controle extração a partir de sangue total. O protocolo Chelex®-Saponina foi o que obteve o melhor resultado quando comparado com os outros três protocolos. No entanto a sensibilidade foi de 66,7% para o P. falciparum e 31,6% para o P. vivax. Conclui-se que em caso de infecção assintomática o papel de filtro não é ainda uma boa alternativa para coleta de amostras.

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

Prevalencia de autoanticuerpos contra receptores autonómicos en pacientes panameños con cardiopatía chagásica crónica y con otras formas de cardiopatía / Prevalence of autoantibodies against autonomic receptors in patients with chronic cardiopathies

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 comparación con 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L 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 ƒÀ-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 ƒÀ-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.

Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6 percent) asymptomatic and 17 acute (65.4 percent) including 5 (19.2 percent) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

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 percent for trypanosome infections, a seroprevalence of 2.9 percent against T. cruzi and a predominance of T. rangeli infection (6.8 percent). The immunological and clinical implications of these findings are discussed.

Eco-epidemiological aspects of Trypanosoma cruzi, Trypanosoma rangeli and their vector (Rhodnius pallescens) in Panama

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.