Use of sentinel animals to demonstrate active leishmanial transmission in an area with low frequency of human lesions in Western Venezuela

One-hundred healthy animal of differents species, including dog (8), fox (1), donkey (1), goat (1), opossum (3), rabbit (8), hamster (33) and guinea pig(45), kept under natural conditions, were used as sentinel animals (SA) to prove active and constant leishmanial transmission, in an area where human cutaneous lesions are rarely observed. The investigation was carried out in a field station located at the Andean region of Western Venezuela, where both sand flies species and Leishmania-parasites have been perviously reported. The study consisted of a follow-up using serological techniques. Blood samples from the SA were taken monthly and the sera processed to demonstrate seroconversion by detecting anti-Leishmania circulating antibodies (Abs). In 56 percent of the used animals belonging to 8 species of susceptible mammals, seroconversion was detected during the time of observation. To corroborate the serological results, 68 serum samples were selected for a PCR assay with 32 (47 percent) of them showing positive results. The results indicate that combination of seroconversion and PCR in SA are useful tools to demonstrate constant and active Leishmania transmission in areas where clinical manifestation is uncommonly observed in the human population. The potential of using SA as a promising method to investigate leishmanial activity under field conditions is stressed and the epidemiological implications of the present findings is discussed.

Association of trauma with Leishmania spp. parasite recall. clinical and experimental evidences

Se dan ciertas evidencias sobre el efecto que traumas los procesos inflamatorios inducidos tienen sobre la reactivación y migración de Leishmania en pacientes clínicamente curados, individuos con infecciones asintomáticas, o modelos experimentales.El estudio de muestra de área inflamada de la piel de pacientes con las características antes mencionadas, reveló la presencia de parásitos leishmánico o parte de su genoma mediante la utilización de métodos parasitológo, inmunohistoquímicos (PAP) y moleculares (PCR). Asi mismo, se logró la detención de ADN específico de leishmania (Viannia) en muestras orales tomadas de pacientes con gingivitis y/o periodintitis, que habia sufrido leishmaniasis 5 a 10 años previo al presente estudio. La dispersión observada en los casos humanos fue corroborada en un modelo animal,provocando un proceso inflamatorio artificial a hámsteres infectados con leishmania. En este caso, observaciones llevadas a cabo entre 4 y 12 días mediante cultivos in vitro, extendidos en láminas coloreadas, o secciones histólogicas, revelaron la presencia de parásitos en el sitio donde se provocó la inflamación (cavidad peritoneal) y en el higado,el bazo, ganglio linfático, médula osea y sangre periférica circulante. Los resultados presentados apoyan el punto de vista de que el caso de leishmaniasis, paciente clinicamente curados con distintos esquemas terapéuticos, individuos con lesiones activas o en caso de infecciones inaperentes o asintomáticas, pueden experimentar una reactivación a distancia como consecuencia de un trauma que induzca a un proceso inflamatorio localizado

Evolución clínica, parasitológica e histopatológica de pacientes chagásicos agudos tratados con benznidazol / Histopathologic, parasitologic and clinic evolution in acute chagasic myocarditis patients treated with benzinidazole

Diez y seis pacientes con miocardiopatía chagásica aguda comprobada con biopsia endomiocárdica y estudio seroparasitológico, tratados con benznidazol, fueron reevaluados a los 12 meses y a los 5 años (5 pacientes). El protocolo consistió en estudio clínico hemodinámico, ecocardiográfico y sero-parasitológico, más evaluación histológica, inmunohistoquímica (inmunofluorescencia indirecta IIT e inmunoperoxidasa PAP) y molecular (PCR) de las biopsias miocárdicas. Los resultados mostraron miocarditis persistente en el 100 por ciento (16/16) y 60 por ciento (3/5) de los pacientes al año y a los 5 años, respectivamente, con evidencias de disfunción ventricular izquierda leve, sin correlato clínico, en el 75 por ciento de los casos a los 5 años. Los métodos parasitológicos directos se negativizaron durante el seguimiento, pero la serología permaneció positiva en el 80 por ciento y 75 por ciento de los pacientes a los 12 meses y 5 años. Además, se pudo demostrar la presencia de parásitos tisulares (PAP), de su genoma (PCR) o de sus antígenos (IIT) en 91 por ciento (10/11) y 75 por ciento (3/4) de las biopsias estudiadas al año y a los 5 años de seguimiento. Concluimos que existe daño miocárdico constante en los pacientes chagásicos agudos, cuya evolución no parece ser modificada favorablemente por el tratamiento con benznidazol, porque a pesar de erradicar la parasitemia, no eliminó los parásitos tisulares, cuyo papel etiopatogenético en la fase crónica debe ser reconsiderado

Evaluation of DNA recombinant methodologies for the diagnosis of Plasmodium falciparum and their comparison with the microscopy assay

Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for the parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21 assay was compared against Giemsa stained thick blood from samples collected at a malaria endemic area of the Bolivar State, Venezuela, at the field station of Malariologia in Tumereno. Coupled to non-radioactive hybridization the pf-21 performed better than the tradicional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitema levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies.

Persistent infections by Leishmania (viannia) braziliensis

Here we review the phenomon of persistency in Leishmania (Viannia) braziliensis infections. In other Leishmania species where appropriate animal models exist, considerable advances in the understanding of basic immunologic mechanisms of persistency have been made: for a review see Aebisher (1994). On the contrary, the evidences of persistence in infections with L. braziliensis rest on studies of human clinical cases many of which we summarized and discussed in this study.

In situ hybridization and immunoperoxidase for quick detection and identification of leishmania braziliensis in tissue

The combined used of in situ hybridization and immunoperoxidase techniques applied to histological sections of the biopsy allowed an increment of the efficiency in the detection of Leishmania diagnosis.The molecular basis for DNA diagnosis is to allow labelled single-stranded species or strain-specific DNA sequences, selected from well characterised reference species, to find and hybridize with homologous DNA from or in, the unknown isolates of parasites. The use of immunoperoxidase assay was useful in disclosing the antigen pathways and revealed amastigotes and antigenic material in various morphological patterns

Homogeneous restriction fragment length polymorphism analysis of the ribosomal DNA repeating unit in New World Leishmania

We have studied the Sau 3AI restriction length polymorphisms (RFLP) of the non-transcribed ribosomal spacer of Leishmania isolates from the mexicana and braziliensis complexes, using cloned sequences of Leishmania garnhami and Leishmania braziliensis. The L. garnhami probe produced very complex but conserved patterns in the homologous organisms, and these were shared by all the mexicana complex isolates at intermediate stringency conditions. The small subunit rRNA coding region within the probe also revealed a polymorphic Sau 3AI site exclusive of the braziliensis isolates. The braziliensis probe, containing only spacer sequences, yielded simple and very homogeneous patterns in all braziliensis isolates regardless of their geographical origin. Two main groups are identified in the New World isolates by the RFLP analysis in coincidence with the accepted mexicana and braziliensis complexes

Trypanosomatidae codon usage and GC distribution

A study od Typanosomatidae GC distribution and codon usage is presented. The codon usage patterns in coincidence with the phylogenetical data are similar in Crithidia and Leishmania, whereas they are more divergent in Trypanosoma brucei and T. cruzi. The analyisis of the GC mutational pressure in these organisms reveals that T. brucei, and to a lesser extent T. cruzi, have envolved towards a more balanced use of all bases, whereas Leishmania and Crithidia retain features of a primeval genetic apparatus. Tables with approximated GC mutational pressure in homologous genes, and codon usage in Trypanosomatidae are presented