Trypanosoma cruzi I genotype among isolates from patients with chronic Chagas disease followed at the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, Brazil)

ABSTRACT INTRODUCTION: Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, mainly in Latin America. Trypanosome stocks were isolated by hemoculture from patients followed at Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ) and studied using different approaches. METHODS: For species and genotype identification, the stocks were analyzed by parasitological techniques, polymerase chain reaction assays targeted to specific DNA sequences, isoenzyme patterns, besides sequencing of a polymorphic locus of TcSC5D gene (one stock). RESULTS: The isolates presented typical T. cruzi morphology and usually grew well in routine culture media. Metacyclic trypomastigotes were found in cultures or experimentally infected Triatoma infestans. All isolates were pure T. cruzi cultures, presenting typical 330-bp products from kinetoplast DNA minicircles, and 250 or 200-bp amplicons from the mini-exon non-transcribed spacer. Their genetic type assignment was resolved by their isoenzyme profiles. The finding of TcI in one asymptomatic patient from Paraíba was confirmed by the sequencing assay. TcVI was found in two asymptomatic individuals from Bahia and Rio Grande do Sul. TcII was identified in six patients from Pernambuco, Bahia and Minas Gerais, who presented different clinical forms: cardiac (2), digestive with megaesophagus (1), and indeterminate (3). CONCLUSIONS: The main T. cruzi genotypes found in Brazilian chronic patients were identified in this work, including TcI, which is less frequent and usually causes asymptomatic disease, unlike that in other American countries. This study emphasizes the importance of T. cruzi genotyping for possible correlations between the parasite and patient’ responses to therapeutic treatment or disease clinical manifestations.

Leishmania major: parasite interactions suggesting sexuality

In five experiments, Leishmania (Leishmania) major (MRHO/SU/59/P-strain) grew poorly when seeded in FYTS medium supplemented with 15 per cent fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5 per cent). As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occured at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect ß-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2 per cent). Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process.

Cytological findings suggesting sexuality in Phytomonas davidi (Protozoa: Kinetoplastida)

On few occasions, Phytomonas davidi (McGhee & Postell isolate) cultures in LIT (liver infusion-tryptose) medium around 27ºC presented, as seen in Giemsa-stained smears, a set of pecular morphological features, among them being noticeable the pairs of apposed cells attached by their posterior ends, where occured a stained line and/or a dilatation, usually bulb-like in shape; sometimes this dilatation could occupy one of the cells or hold both together. In some pairs, the nucleous of each parasite seemed migrating towards the other, entering into such dilatation; in others, both nuclei were inside it, sometimes in close proximity or seeming fused; peculiar chromatin arrangements involving both nuclei were occasionally observed. Several mono or binucleate round forms bearing one or two flagella, as well as flagellate slender cells without nucleous were concomitantly seen there. In some instances, an intriguing small stained body, occurred beside a single large nucleous, either in pairs presenting the bulb-like structure or in round cells. There cytological findings seemed steps of a dynamic process suggesting sexuality, since in several of them nuclear interactions following fusion of two parasites appeared to occur.