Identification of new world Leishmania using ribosomal gene spacer probes.

DNA probes from the nontranscribed ribosomal spacer (NTS), of Leishmania garnhami and Leishmania braziliensis were constructed and tested for sensitivity and specificity against different Leishmania isolates. The L. garnhami probes were species-specific under hybridization conditions of high stringency, but displayed specificity for the mexicana complex under conditions of intermediate stringency. The L. braziliensis probes showed 'complex' specificity. RFLP for the nontranscribed spacer within the braziliensis complex revealed very homogeneous patterns even for organisms currently accepted as different species. A PCR assay for the detection of Leishmania from the braziliensis complex is presented.

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.

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 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.

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.

Cardiac involvement is a constant finding in acute Chagas' disease: a clinical, parasitological and histopathological study.

During the last 8 years 58 acute cases of Chagas' disease were studied. Patients from an endemic area of the state of Barinas, Venezuela, showed fever (98%) and circulating forms of T. cruzi (100%), and were treated with oral benznidazole. The recorded mortality was 8.6%. Acute myocarditis was constantly found either in myocardial biopsies or at necropsy, even in patients without any other sign of cardiac compromise (36%), which was detected by chest X-ray in 58%, by 2D echocardiography in 52%, by resting ECG in 41% and by clinical findings in 27.5% of the patients. Cardiomegaly was due to pericardial effusion rather than ventricular dilatation in most instances. Treatment eliminated parasitemia but negativized serology in only 20% of patients. It also appeared to have little influence on the ongoing myocarditic process, emphasizing the need for better therapeutic schedules, able to avoid or control the early appearance of immunologic mechanisms and microcirculatory damage involved in the future development of chronic chagasic myocarditis.

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.

Multivariate analysis to discriminate species of phlebotomine sand flies (Diptera:Psychodidae): Lutzomyia townsendi, L. spinicrassa, and L. youngi.

Multivariate discriminant analysis was employed to discriminate on a morphological basis females of 3 closely related sand fly species, Lutzomyia townsendi (Ortiz), L. spinicrassa Morales, Osorno-Mesa, Osorno & Hoyos, and L. youngi Feliciangeli & Murillo. Principal component and canonical discriminant analysis compared a set of 31 morphological characters measured from known specimens to detect differences among the 3 species. A subset of 6 characters separated the 3 species with a high level of confidence. A simple method is presented to identify an unknown specimen as L. townsendi, L. spinicrassa, or L. youngi using these 6 morphological characters.

Changes in the total content of iron, copper, and zinc in serum, heart, liver, spleen, and skeletal muscle tissues of rats infected with Trypanosoma cruzi.

The effects of the Trypanosoma cruzi infection on the total content of the essential trace elements iron (Fe), copper (Cu), and zinc (Zn) in serum, heart, liver, spleen, and skeletal muscle were determined in "Wistar" rats inoculated with reticulotropic "Y" strain trypanosomes (Tryps) in their slender blood form. The 250 rats were divided in two groups of 80 rats (L-1 and L-2) and one of 90 (C) used as controls. L-1 and L-2 were inoculated with 2 x 10(5) and 5 x 10(2) Tryps, respectively. Ten rats of the C group were killed the inoculation day (i), and ten rats of each group chosen at random were killed and blood parasitemia determined at 5, 10, 15, 20, 25, 30, 60, and 90 post-i days covering the infection acute-phase myocarditis. Previously cryohomogenized and lyophilized tissues were digested in an HNO3- H2O2 mixture with the aid of a microwave oven, and the elements Fe, Cu, and Zn were determined by Flame Atomic Absorption Spectrometry. Generally, more intense changes were observed in the L-1 group. Serum Fe and Zn levels are lower and Cu levels higher in groups L-1 and L-2 than in C. However, Fe is not significantly sequestered in the liver during the acute phase of the infection as expected, but of the tissues studied, the spleen was the main site of Fe binding. Zn tended to increase in all tissues, except in the spleen, where during the acute phase of the infection, the total content of Zn in groups L-1 and L-2 was lower than in group C. Cu increased mainly in the spleen and muscle. In general, each tissue presented its own pattern of redistribution related to its nature, functions, and number of parasites inoculated, and these patterns may have been altered by the tropism of the parasite.

[Effect of intralesional treatment with emetine hydrochloride on Leishmania (Viannia) braziliensis in hamsters]. / Efecto del tratamiento intralesional con clorhidrato de emetina sobre Leishmania (Viannia) braziliensis en hámsteres.

The therapeutic effect of the emetine hydrochloride alkaloid administered intralesionally was compared with that of standard parenteral treatment with Glucantime in outbred male hamsters experimentally infected with 4 x 10(3) amastigotes of Leishmania (Viannia) braziliensis. Both chemotherapeutic agents reduced significantly (P < 0.01) the average lesion sizes in experimental animals in comparison with those untreated. The alkaloid infiltration was found to be as effective as the antimonial injection for clinical resolution. The ultrastructural effects on the Leishmania parasites exposed to emetine were observed mainly in the inner cytoplasm, which appeared disorganized, pycnotic and with loss of morphological definition; however, any known emetine hydrochloride action mechanism factor could not be directly related with ultrastructure effects detected on leishmanial parasites. Smears, conventional histopathology, culture in NNN medium and indirect immunoperoxidase method showed viable amastigotes in nodules and/or scars of all the evaluated hamsters 75 to 230 days after the end of treatment. These findings suggest that measurement of the size of cutaneous leishmania lesions does not appear to be a valid criterion for evaluating the efficiency of chemotherapy in experimental LT. Detection of leishmania parasites in the lesion scars, supports the hypothesis that man could be considered as an domestic reservoir.

Phenol red method for measuring the pH of the gut contents in Lutzomyia longipalpis (Psychodidae:Diptera).

AIM: To develop a method to estimate the pH of the gut contents of female sandflies using a microcapillary feeding technique. METHODS: Female Lutzomyia longipalpis were fed with a small quantity of phenol red solution (indicator range pH 6.8-8.4) before and after a bloodmeal. The colour patterns of the gut contents were recorded by video microscopy immediately after the alimentary canal was dissected out of the sandfly body, and used to determine the pH level. RESULTS: In unfed flies the thoracic mid-gut (TMG) is normally neutral, with the pH ranging between 7.0 to 7.3; and the abdominal mid-gut(AMG) is mildly alkaline from pH 7.1 to 8.4 with the maximum pH observed at the junction with the hind-gut. The presence of sugar in the crop reduced the pH of the TMG to 6.8, and the presence of a recently ingested bloodmeal raised the pH of the TMG to 7.4. However, as bloodmeal digestion proceeded the pH of the TMG was reduced to acidic levels, pH 6.8 or below. CONCLUSION: The new method could be integrated with the investigation of metacyclogenesis of Leishmania parasites in vivo.

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.

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa.

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed.

Studies on Trypanosoma rangeli Tejera, 1920. VI. Developmental pattern in the haemolymph of Rhodnius prolixus.

The morphological sequence of Trypanosoma rangeli development in the body cavity of Rhodnius prolixus is described. The metacyclic trypanosome is the product of successive division and transformation during the intra and extracellular development in the haemocoele. The significance of the early invasion of T. rangeli into the haemolymph is discussed. The epidemiological importance of the developmental pattern of T. rangeli in the vectors haemolymph and the host-response to the parasite are considered.