A new approach for direct identification and characterization of Tiypaiiosoiiia cruzi stocks in biological samples was tested for field applicability on an extensive sample of feces collected from triatomine vectors from four different species found in Bolivia. The first step of the technique is polymerase chain reaction (PCR) amplification of the hypervnriable region of kinetoplast DNA minicircles of T. cruzí parasites. In this report, 345 fecal samples were analyzed and the PCR results were compared with microscopic examination. For Triatoina iilfesraizs, the principal Bolivian vector, both techniques were in concordance 85.3% of the time. For the three other species, RIzodrzius pictipes, Erciyi-us m m " u s , and Triaroiiia sordida, the fecal samples were all negative by microscopic examination whereas PCR results showed several T. crirzi-infected insects in each species. The second step of the procedure is the characterization of the T. ci-uzi clones by means of hybridization of the PCR products with clone-specific probes generated by the PCR. We used two probes corresponding to major clones circulating in high frequency in Bolivia (as shown by previous population genetic studies using isoenzyme characterization). We obtained four primary results: 1) we confirm the importance of two major clones in Bolívia in two distinct regions; 2) we report high rates of mixed infections (multiple clones in a single vector) in Triatoina iilfestaizs, up to 22% and 35% in Cochabamba and La Paz departments, respectively; 3) the results favor the absence of interaction between different clones; and 4) we find, for the first time, evidence of the major clones circulating in three species of triatomines that are known as mainly sylvatic species. The origin of these clones, sylvatic or domestic, is also discussed.

Abstract. We performed serological and pathological studies on 495 patients with Chagas' disease from different areas of Bolivia. Eighty-nine Trypanosoma cruzi strains, isolated by xenodiagnosis, were characterized by 12 isoenzyme loci and were related to the presence of cardiac changes and enteric disease with megacolon. There was a high heterogeneity of human zymodemes, presenting evidence of 2 predominant zymodemes genetically dissimilar from each other and ubiquitous in Bolivia. The frequencies of these predominant zymodemes among strains from patients were compared to strains from triatomine bugs previously studied. We observed mixtures of different zymodemes within the same patient, a phenomenon seen previously in Bolivian patients. There was no apparent difference of pathogenicity between the 2 more frequent zymodemes isolated from humans.Trypanosoma cruzi, the agent of Chagas' disease, is associated with 2 main clinical forms, cardiac and intestinal. These clinical forms are not equally distributed within the geographical range of the disease, and it has been proposed that T. cruzi zymodeme distribution could explain this fact.' We present here pathological, serological, and parasitological studies, involving 495 patients from Bolivia, showing the diversity of the clinical forms and the distributions of these clinical forms and T. cruzi zymodemes isolated from human. The relationships are presented and discussed. MATERIALS AND METHODS PatientsTwo groups were examined at different times and under different conditions. Group I, consisting of 364 patients, was from the villages of Chiwisivi (Department La Paz) at 2,800 meters above sea level (mas), Salinas (Department Santa-Cruz) at 800 mas, and Camiri (Department Santa-Cruz) at 800 mas ( Table 1) The second group was composed of 13 1 patients, all exhibiting a positive serology (serodiagnosis as noted above). They originated from different endemic regions of Bolivia, although 108 had been living in La Paz (3,600 mas) for at least 5 years (average time spent in La Paz: 10 years). The endemic areas where the patients had spent their childhoods or the first endemic area where they had spent > 1 year were considered the geographical origins of infection ( Table 2). The following tests were conducted ECG, x-ray of the right anterior side of the esophagus, and x-ray of colon after barium washing (4 x-ray fìlms. Group II patients were then classified as follows:Asymptomatic-positive serology, normal ECG, normal esophagus and colon.Cardiopathy-positive serology, ECG with 52 1

The risk of domestic transmission of Trypanosoma cruzi (Chagas) by sylvatic triatomines was assessed in an isolated area of the subandean region of Bolivia. None of the 390 residents examined had serological evidence of infection. Two sylvatic triatomine species, Eratyrus mucronatus (Stål) and Triatoma sordida (Stål), were found in houses and in peridomestic structures. The collection of nymphal instars of both species from some houses indicated possible domesticity. Microscopic examination of feces from 92 insects showed no parasites, and cultures from the guts of 30 insects were negative. Nevertheless, a polymerase chain reaction (PCR) test performed on the same fecal samples showed the presence of T. cruzi DNA in 19.1 and 12.5% of E. mucronatus and T. sordida, respectively. These 16 PCR-positive samples were hybridized with 2 T. cruzi-specific probes known from the domestic cycle in Bolivia (clones 20 and 39). At least 1 of these clones was identified in 7 bugs (5 E. mucronatus and 2 T. sordida). Moreover, no hybridization was observed with these probes in S E. mucronatus and 1 T. sordida samples that showed an amplified band by PCR. These data indicated that T. cruzi clones, genetically unrelated to clones 20 and 39, also were circulating in this area. Based on these results, the 2 sylvatic triatomine species encountered in Apolo should not be overlooked as possible local vectors of T. cruzi.

Trypanosoma cruzi infection was studied in 1,298 sera samples of blood banks from 7 capital departments of Bolivia, using the immunofluorescence test (IFI) and Enzyme Linked Immunosorbent Assay (ELISA). The percentages of positivity in these 7 departments have an average of 28% and are distributed as follows: Sta. Cruz 51%, Tarija 45%, Cochabamba 28%, Sucre 39%, La Paz 4.9%, Oruro 6% and Potosi 24%. The prevalence is related with the altitude levels of the different departments. However in Potosi (3,945 m) we found a 24% of prevalence, probably due to the proximity of endemic valleys to the city. The authors suggest a strict control in blood donors since there exists a great risk of infection.

Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined in chagasic patients and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti-T.cruzi response was observed, both IgM and IgG anti-T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas' disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.