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.

Phylogeny, taxonomy and grouping of Trypanosoma rangeli isolates from man, triatomines and sylvatic mammals from widespread geographical origin based on SSU and ITS ribosomal sequences.

Phylogenetic relationships among Trypanosoma rangeli isolates from man, wild mammals and triatomine bugs from widespread geographical origin were inferred by comparison of the small subunit of ribosomal gene sequences. The phylogenetic trees indicated that the subgenus Herpetosoma is polyphyletic and strongly supported division of this group into two monophyletic lineages, one made up of T. rangeli, T. rangeli-like and allied species and other consisting of T. lewisi and related taxa. Based on phylogenetic analysis, morphology, behaviour in vertebrate and invertebrate hosts and epidemiology we propose: a) the validation of Herpetosoma as a taxon comprised only for species of group lewisi and the maintenance of T. lewisi as the type-species of this subgenus; b) the classification of T. rangeli, T. rangeli-like and allied species into a 'T. rangeli-clade' more closely related to Schizotrypanum than to T. lewisi or T. brucei. The phylogenetic tree disclosed at least 4 groups within the clade T. rangeli, all confirmed by polymorphism of the internal transcribed spacer, thus conferring for the first time phylogenetic support to groups of T. rangeli and corroborating the high complexity of this taxon. Grouping was independent of their mammalian host-species and geographical origin, indicating that other factors are determining this segregation.

Persistencia de la transmisión de la enfermedad de chagas sin colonización por el vector conocido, en localidades controladas de Venezuela / Persistent transmission of chagas disease in controlled Venezuelan localities in absence of known domestic vectors

Utilizando tres técnicas serológicas anti-Trypanosoma cruzi se ha estudiado la seroprevalencia para la enfermedad de chagas en ocho comunidades rurales del área endémica del estado Trujillo, Venezuela. De 647 personas investigadas, que representan el 41 por ciento de la población residente en viviendas no colonizadas por Rhodnius prolixus, 124 (19,2 por ciento) resultaron reactivas con al menos dos de las tres técnicas utilizadas. La seropositividad en niños menores de 10 años fue del 2,8 por ciento (7/244). Estos resultados revelan que actualmente persiste la transmisión de la enfermedad de chagas en regiones controladas y en viviendas no localizadas por triatominos, ello plantea la necesidad de revaluar la situación epidemiológica del estado Trujillo

PCR detection of specific Leishmania-DNA in patients with periodontal disease.

This study deals with the detection of Leishmania braziliensis DNA in gingival specimens from 10 individuals who all had suffered from cutaneous leishmaniasis 5-10 years prior to the examination and all had been treated with anti-leishmaniasis drugs. This preliminary study gives an interesting contribution to the oral microbiology of this disease, with the observation that inflamed periodontal tissues can serve as a factor affecting the dispersion of Leishmania parasites in individuals who had suffered from cutaneous leishmaniasis. These finding are corroborated by the results of polymerase chain reaction (PCR) which demonstrated the presence of Leishmania DNA in tissue samples of patients with periodontal diseases.

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.

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.

[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.

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.

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.

The genital atrium as a good taxonomic character to distinguish between species of phlebotomine sandflies (Diptera: Psychodidae) from Venezuela.

The shape and size of the genital atria of 17 phlebotomine sandflies from Venezuela were examined. The atria were found to express constant characters among individuals of the same species and to be sufficiently different between species to allow taxonomic separation. The spines on the genital armature are described and the characters that can be used to classify individual specimens to species level identified. It is suggested that these characters can be of use on specimens where the spermathecae have been lost or where cryptic species are concerned. Dissection procedures to display the atrium are given.

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.

[Clinical, parasitological and histopathologic follow-up studies of acute Chagas patients treated with benznidazole]. / Seguimiento clínico, parasitológico e histopatológico de pacientes chagásicos agudos tratados con benznidazol.

With the purpose of studying their clinical and histopathologic evolution, 10 acute chagasic patients with myocarditis diagnosed by endomyocardial biopsy and positive sero-parasitologic methods were evaluated at 11 months (8-21 months) after treatment with oral benznidazole. Four of them were reevaluated 5 years post-treatment (58-68 months). Study protocol consisted of clinical, hemodynamic, echocardiographic, seroparasitologic and histopathologic evaluations. Results showed evidence of persisting myocarditis in 90% and 75% of patients evaluated at 11 months and 5 years respectively, along with asymptomatic, subclinical left ventricular systolic dysfunction being recognized in 75% of patients evaluated 5 years after treatment. All parasitologic studies became negative during follow-up, but serology remained positive for Trypanosoma cruzi antibodies in 80% and 75% of patients studied at 11 months and 5 years. In conclusion, myocardial damage was constantly found in our acute chagasic patients. Treatment with benznidazole eliminated symptoms and parasitemia, but it does not seem to alter favorably the histopathological evolution of the chagasic cardiac disease.

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.

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.

Comparison of three methods for age-grading of female neotropical phlebotomine sandflies.

Changes in appearance of the accessory glands (AG), ovaries (OV) and the absence or presence of crease marks at the edges of the genital atrium armature (GA) were compared as methods to age-grade female laboratory-bred (Lutzomyia migonei) and wild-caught (Lu.youngi, Lu.spinicrassa, Lu.ovallesi) Neotropical sandflies. Dissections at different physiological stages revealed that all parous and high proportions of nulliparous (4/38) unmated, 12/12 mated) females showed similar residual AG secretions. Hence AG appearance (nullipars with AG translucent, versus yellowish opaque granulated AG previously regarded as signs of parousness) is unreliable for age-grading. Readily recognizable changes in both GA (98% creased) and the OV (100% yellowish with loose ovarioles) indicated that these organs could be employed to discriminate between nulliparous and parous females. Despite finding one misdiagnosed female (a nullipar having creased GA indicative of parity), the GA method of age-grading is recommended because females may be examined satisfactorily at any stage of the gonotrophic cycle. Moreover, the GA characteristics of pickled or dry preserved sandflies as well as fresh slide-mounted specimens can be determined easily. Among sixty-five wild-caught Lutzomyia females dissected (mostly Lu.spinicrassa and Lu.youngi), the proportions of specimens yielding preparations suitable for examination were 97% for GA, 89% for OV, but only 58% for AG, which reinforces GA as the preferred character for age-grading.

The genital atrium as a new character for distinguishing parous and nulliparous Neotropical sandflies.

Changes in the appearance of the membrane delimiting the genital atrium of Lutzomyia migonei are described for the first time. Dissections of laboratory-bred females at different physiological stages (i.e. on emergence, after a bloodmeal and after oviposition) revealed clear and constant differences between the membranes of the parous and nulliparous flies. The state of the membrane can be used as an alternative method for accurately age-grading female sandflies. Dissection methods and a simplified technique to prepare parts of sandflies for examination in a scanning electron microscope are also presented.

Presence of Leishmania braziliensis in blood samples from cured patients or at different stages of immunotherapy.

By using a PCR assay specific for Leishmania braziliensis in blood samples, we detected this parasite in patients cured by immunotherapy or at different stages of treatment. We also found the parasite in subjects who had never suffered leishmaniasis but who had lived in endemic areas and migrated to nonendemic ones many years ago. These results suggest that L. braziliensis infections are difficult to eradicate and that a clinical cure but rarely a complete elimination of the parasite is generally accomplished.