Lutzomyia nuneztovari anglesi (Le pont & Desjeux, 1984) as a vector of Leishmania amazonensis in a sub-Andean leishmaniasis focus of Bolivia.

Recently, a new Leishmania amazonensis focus was described in a sub-Andean region (1,450-2,100 meters above sea level) of Bolivia. In this area, three anthropophilic sandfly species were identified: Lutzomyia nuneztovari anglesi Le Pont & Desjeux, 1984, which represented 86-99% of the captures, Lu. galatiae Le Pont et al., 1998, and Lu. shannoni Dyar 1929. Only Lu. nuneztovari anglesi was found naturally infected by flagellates (16 of 1,715 females). Three Leishmania stocks were isolated and analyzed by isoenzyme electrophoresis at 11 loci. No significant isoenzymatic differences were demonstrated between them and 7 stocks isolated from patients from the same area, and previously characterized as L. amazonensis. Moreover, in a simplified protocol, the experimental infection of Lu. nuneztovari anglesi by L. amazonensis was successful in 92% of the surviving specimens. These data are discussed in relation to the Killick-Kendrick criteria. These results strongly suggest that Lu. nuneztovari anglesi is the vector of L amazonensis at Cajuata, Inquisivi, La Paz, Bolivia.

Identificación de los clonet 20 y 39 en heces de triatoma infestans por la reacción de la polimerasa en cadena (PCR) / Identification of the clonet 20 and 39 in stools of triatoma infestans by the reaction of the chain polymerase (PCR)

Actualmente, la caracterización genética de las cepas de Trypanosoma cruzi consiste en el análisis isoenzimático o análisis del ADN del genoma e implica el aislamiento y cultivo masivo de cada cepa.

En Bolivia, los pacientes chagasicos son mas infectados por el clonet 39 de trypanosoma cruzi / In Bolivia, the Chagas patients are but infected by the clonet 39 of trypanosoma cruzi

Los clones de trypanosoma cruzi exhiben una gran heterogeneidad biológica y se propuso que sus propiedades biológicas puedan estar relacionadas a su contitución genética.

A new focus of cutaneous leishmaniasis due to Leishmania amazonensis in a Sub Andean region of Bolivia.

We detected a new outbreak focus with high incidence of cutaneous leishmaniasis in the Sub Andean region of La Paz. This area was never considered previously as an endemic zone of leishmaniasis. Leishmania stocks from human lesions were isolated: three stocks were explored by pulse field gradient electrophoresis, showing evidence for their affiliation to the L. mexicana complex. Eight stocks were submitted to isoenzyme electrophoresis and compared with five reference strains: L. amazonensis, L. braziliensis, L. chagasi, L. mexicana and L. pifanoi. Close genetic proximity was evidenced between newly isolated parasites and the reference stock of L. amazonensis, whereas high divergence was observed between them and either the L. pifanoi, L. mexicana, L. braziliensis and L. chagasi reference strains.

Different behavior of two Trypanosoma cruzi major clones: transmission and circulation in young Bolivian patients.

Specificity of two widespread Trypanosoma cruzi clonal genotypes or "clonets" (20 and 39) was first analyzed by hybridization with a large set of T. cruzi stocks characterized by multigenic study relying on both MLEE and RAPD. Then, these clonets were detected in the blood of Chagasic children from a Bolivian endemic area by a combination of polymerase chain reaction and clonet-specific DNA hybridization. The distribution of these clonets in patients was significantly different from that observed in the vectors of the same area (Triatoma infestans). In vectors, clonets 20 and 39 are found with comparable frequencies (0.69 and 0.67, respectively) in contrast with patients, in whom clonet 20 and mixed infections exhibit low frequencies. The Chagasic population can be divided into acute infections and latent infections above the accepted criterion of parasitemia (direct microscopic examination). The results suggest a limited selection in the transmission of the two clonets and a further drastic control of clonet 20 parasitemia by the immune system of children patients.

Immune response to Trypanosoma cruzi shed acute phase antigen in children from an endemic area for Chagas' disease in Bolivia.

A field study of the immune response to the shed acute phase antigen (SAPA) of Trypanosoma cruzi was carried out in the locality of Mizque, Cochabamba department, Bolivia. Schoolchildren (266), with an average of 8.6 +/- 3.6 years, were surveyed for parasitological and serological diagnosis, as well as antibodies directed against SAPA using the corresponding recombinant protein in ELISA. The antibodies against SAPA were shown in 82% of patients presenting positive serological diagnosis (IgG specific antibodies). The positive and negative predictive values were 0.88. Antibodies anti-SAPA were shown in 80.8% of the chagasic patients in the initial stage of the infection (positive IgM serology and/or positive buffy coat (BC) test) and in 81.4% of the patients in the indeterminate stage of the infection (positive IgG serology with negative BC and IgM tests). These results show that the anti-SAPA response is not only present during the initial stage of the infection (few months) but extends some years after infection.

PCR-based diagnosis for Chagas' disease in Bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis.

A large field study has been performed in the Cochabamba region of Bolivia with the aim of comparing the polymerase chain reaction (PCR) with other diagnostic methods for Chagas' disease. The amplification of Trypanosoma cruzi-specific kinetoplast DNA sequences in blood samples was compared with classical serological methods, specific IgM detection and direct parasite visualization for 268 school children in a single village where Chagas' disease transmission is active. Of 113 children positive by classical serology or buffy coat examination, 106 were detected by PCR (sensitivity: 93.8%). We did not observe any significant difference of PCR sensitivity between initial (IgM and/or buffy coat positive) and indeterminate stage (only IgG positive) patients. Among the remaining 155 children unconfirmed as chagasic (who were either only IgM positive, IgG-, IgM-, and buffy coat-negative) only 1 case was PCR positive. This case may be due to DNA contamination, or to a very recent infection not detected otherwise, or to specific immune depression. These results show that PCR is a very sensitive parasitological test for Chagas' disease in active transmission regions. The future follow-up of the possibly infected patients who were only IgM-positive should clarify the interest of PCR and IgM tests in the detection of starting infections.

Trypanosoma cruzi: study of the distribution of two widespread clonal genotypes in Bolivian Triatoma infestans vectors shows a high frequency of mixed infections.

The detection of two widespread Trypanosoma cruzi clonal genotypes (20 and 39) in feces of Bolivian specimens of the vector Triatoma infestans was performed by a combination of polymerase chain reaction and clone-specific DNA hybridization. The hybridization pattern of 186 PCR positive samples cf T. infestans feces collected in two Bolivian departments identified clone 20 in 74.2% and clone 39 in 63.4% of the triatomine bugs. For the first time, a high percentage (mean: 43.2 +/- 26%) of mixed infections (presence of both clones in a given fecal sample) in various localities was recorded. Results were in agreement with the two assumptions of independent transmission of clones 20 and 39 and of the absence of selection in the natural cycles under survey. Statistical analysis of the geographical distribution of clones 20 and 39 favored the hypotheses that the frequencies of T. cruzi natural clones are different among localities and that these differences are not proportional to the distances that separate the localities. The epidemiological significance of these results is discussed.

Sylvatic triatomines (Hemiptera: Reduviidae) in Bolivia: trends toward domesticity and possible infection with Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae).

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.

Field application of polymerase chain reaction diagnosis and strain typing of Trypanosoma cruzi in Bolivian triatomines.

A new approach for direct identification and characterization of Trypanosoma 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 hypervariable region of kinetoplast DNA minicircles of T. cruzi parasites. In this report, 345 fecal samples were analyzed and the PCR results were compared with microscopic examination. For Triatoma infestans, the principal Bolivian vector, both techniques were in concordance 85.3% of the time. For the three other species, Rhodnius pictipes, Eratyrus mucronatus, and Triatoma sordida, the fecal samples were all negative by microscopic examination whereas PCR results showed several T. cruzi-infected insects in each species. The second step of the procedure is the characterization of the T. cruzi 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 Bolivia in two distinct regions; 2) we report high rates of mixed infections (multiple clones in a single vector) in Triatoma infestans, 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.(ABSTRACT TRUNCATED AT 250 WORDS)