Myocardial parasite persistence in chronic chagasic patients.

The persistence of Trypanosoma cruzi tissue forms was detected in the myocardium of seropositive individuals clinically diagnosed as chronic chagasic patients following endomyocardial biopsies (EMBs) processed by immunohistochemical (peroxidase-anti-peroxidase [PAP] staining) and molecular (polymerase chain reaction [PCR]) techniques. An indirect immunofluorescent technique revealed antigenic deposits in the cardiac tissue in 24 (88.9%) of 27 patients. Persistent T. cruzi amastigotes were detected by PAP staining in the myocardium of 22 (84.6%) of 26 patients. This finding was confirmed with a PCR assay specific for T. cruzi in 21 (91.3%) of 23 biopsy specimens from the same patients. Statistical analysis revealed substantial agreement between PCR and PAP techniques (k = 0.68) and the PCR and any serologic test (k = 0.77). The histopathologic study of EMB specimens from these patients revealed necrosis, inflammatory infiltrates, and fibrosis, and made it possible to detect heart abnormalities not detected by electrocardiogram and/or cineventriculogram. These indications of myocarditis were supported by the detection of T. cruzi amastigotes by the PAP technique or its genome by PCR. They suggest that although the number of parasites is low in patients with chronic Chagas' disease, their potential for heart damage may be comparable with those present during the acute phase. The urgent necessity for testing new drugs with long-term effects on T. cruzi is discussed in the context of the present results.

Detection and significance of inapparent infection in Chagas disease in western Venezuela.

Inapparent infections of Trypanosoma cruzi were detected in symptomless seropositive people living in close proximity, and under the same conditions of risk, to patients with acute Chagas disease. Similar infections were also detected in sera samples of people from 25 villages of western Venezuela where Chagas disease is endemic. Seropositivity in all the 1,251 studied samples was established by use of 3 serological methods (direct agglutination test, indirect immunofluorescence antibody test, and enzyme-linked immunosorbent assay). Each seropositive sample was tested for detection of anti-T. cruzi-specific immunoglobulin (Ig) M and IgG levels and specific T. cruzi infection by molecular methodology (polymerase chain reaction assay). The combined analysis of the serologic (IgM and IgG levels), molecular (specific T. cruzi DNA), and statistical findings demonstrated the existence of a different stage of T. cruzi infection in asymptomatic patients, which is suggested to be recognized as inapparent infection. Its definition, significance, and comparison with typical Chagas disease phases are presented, and its potential epidemiological importance is discussed.

Acute Chagas' disease in western Venezuela: a clinical, seroparasitologic, and epidemiologic study.

A clinical, parasitologic, and serologic study carried out between 1988 and 1996 on 59 acute-phase patients in areas of western Venezuela where Chagas' disease is endemic showed 19 symptomatic patterns or groups of symptoms appearing in combination with different frequencies. The symptomatic pattern with the highest frequency was that showing simultaneously fever, myalgia, headache, and Romaña's sign, which was detected in 20% of the acute-phase patients. Asymptomatic individuals and patients with fever as the only sign of the disease made up 15% and 11.9% of the total acute cases, respectively. Statistical correlation analysis revealed that xenodiagnosis and hemoculture were the most reliable and concordant of the five parasitologic methods used; these two methods also showed the highest proportions in detecting any clinical symptomatic pattern in acute-phase patients. A similar high reliability and concordance was obtained with a direct agglutination test, an indirect immunofluorescent antibody test, and an ELISA as serologic tests, which also showed a higher proportion of positive detection of clinical patterns than parasitologic methods (P < 0.001). It is recommended that individuals coming from endemic areas showing mild and/or severe clinical manifestations should be suspected of being in contact or having been in contact with Trypanosoma cruzi, be referred for parasitologic and serologic evaluations to confirm the presumptive clinical diagnosis of acute Chagas' disease, and start specific treatment. The epidemiologic implications of the present findings are discussed and the use of similar methodology to evaluate other areas where Chagas' disease is endemic is suggested.

Detection of Leishmania causing visceral leishmaniasis in the Old and New Worlds by a polymerase chain reaction assay based on telomeric sequences.

We present a new polymerase chain reaction assay based on telomeric sequences of Leishmania donovani. When this assay was used in dilutions of purified L. donovani DNA, a strong amplification signal was observed with 1 fg of DNA. In a specificity test that used purified DNA from Old World and New World Leishmania, the assay recognized all parasites isolated from patients with visceral leishmaniasis, except for 2 isolates of Leishmania colombiensis from Venezuela and 1 isolate from Brazil. All Leishmania major and Leishmania tropica isolates tested were negative, except for one isolate in each species. We also used the assay on fresh and archive bone marrow samples recovered from Giemsa-stained slides and from dried blood stains.

Green fluorescent protein-tagged Leishmania in phlebotomine sand flies.

In this work we have used for the first time green fluorescent protein (GFP) tagged cells of the human parasite Leishmania donovani to observe its development in the gut of phlebotomine sand flies. Low numbers of GFP-tagged L. donovani were more easily detected than nontagged Leishmania, suggesting that GFP-tagged Leishmania could be used to efficiently study the biology of Leishmania in their vectors, and open the possibility of using nonaxenic flies. Using this method, we found that GFP-tagged L. donovani, the ethiological agent of Old World Kala-azar, were able to establish an infection within the gut of Lutzomyia species, which are vectors of New World Leishmania. The GFP-tagged parasites divide successfully in the gut of colonized and in wild caught Lu. longipalpis (Lutz & Neiva, 1912), Lu. ovallesis (Ortiz, 1952), and Lu. youngi (Feliciangeli & Murillo, 1985). In the case of Lulongipalpis the labeled parasite exhibited a normal anterior development as the one observed in its natural vector.

Molecular analysis of surface glycoprotein multigene family TrGP expressed on the plasma membrane of Trypanosoma rangeli epimastigotes forms.

Trypanosoma rangeli, a non-pathogenic hemoflagelate that in Central and South America infects humans, shares with Trypanosoma cruzi reservoirs and triatomine vectors, as well as geographical distribution. Recently, we have described in T. rangeli a truncated gene copy belonging to the group II of the trans-sialidase superfamily (TrGP). This superfamily, collectively known in T. cruzi as gp85/TS, includes members that are involved in host cell invasion and infectivity. To confirm the presence of this superfamily in the genome of T. rangeli and obtain a better knowledge of its characteristics, we designed a PCR and RT-PCR cloning strategy to allow sequence analysis of both genomic and transcribed copies. We identified two full-length copies of TrGP, some pseudogenes, and N- and C-terminal sequences of several genes. We also analyzed the expression and cellular localization of these proteins in epimastigote forms of a Venezuelan T. rangeli isolate using polyclonal antibodies made against a recombinant peptide from the N-terminal region of a TrGP member. We confirmed that TrGP is a multigenic family that shares many features with T. cruzi gp85/TS, including the telomeric location of some of its members, and by immunofluorescence analysis that its location is at the surface of the parasite.

Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors.

To make reliable interpretations about evolutionary relationships between Trypanosoma rangeli lineages and their insect vectors (triatomine bugs of the genus Rhodnius) and, thus, about the determinant factors of lineage segregation within T. rangeli, we compared phylogenies of parasite isolates and vector species. Sixty-one T. rangeli isolates from invertebrate and vertebrate hosts were initially evaluated in terms of polymorphism of the spliced-leader gene (SL). Further analysis based on SL and SSUrRNA sequences from 33 selected isolates, representative of the overall phylogenetic diversity and geographical range of T. rangeli, supported four phylogenetic lineages within this species. By comparing the phylogeny of Rhodnius species with that inferred for T. rangeli isolates and through analysis of the geographical range of the isolates, we showed that there is a very significant overlap in the distribution of Rhodnius species and T. rangeli lineages. Congruence between phylogeographical analysis of both T. rangeli lineages and complexes of Rhodnius species are consistent with the hypothesis of a long coexistence of parasites and their vectors, with lineage divergence associated with sympatric species of Rhodnius apparently without association with particular vertebrate hosts. Separation of T. rangeli isolates from vectors of distinct complexes living in sympatry favours the absence of gene flow between the lineages and suggests evolution of T. rangeli lineages in independent transmission cycles, probably associated to specific Rhodnius spp. ecotopes. A polymerase chain reaction assay based on SL intergenic sequences was developed for simultaneous identification and lineage genotyping of T. rangeli in epidemiological surveys.