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)

High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area.

The detection of Trypanosoma cruzi kinetoplast DNA by polymerase chain reaction (PCR) amplification is a potentially powerful tool for the parasitological diagnosis of Chagas' disease. We have applied this technique in a field situation in Bolivia, where 45 children from a primary school were subjected to serological testing, buffy coat analysis and PCR diagnosis. 26 of the 28 serology-positive individuals were also positive by PCR. In addition, two serology-negative children gave a positive result by PCR, including one who was positive in the buffy coat test. These results suggest that PCR detection of T. cruzi DNA in blood can be a very useful complement to serology in Chagas' disease diagnosis in Bolivia.

Low probability of transmission of Trypanosoma cruzi to humans by domiciliary Triatoma sordida in Bolivia.

The role of Triatoma sordida in the domestic transmission of Trypanosoma cruzi was assessed in 7 rural localities in Velasco Province, Department of Santa Cruz, Bolivia. Tri. sordida, the only triatomine species identified in these localities, was found inside 58.0% of houses but not in large numbers (3.1 bugs per infested house on average). A total of 220 faecal samples from domiciliary bugs was examined microscopically and by the polymerase chain reaction for the presence of trypanosomes: 21.4% were infected. Analysis of blood meals of domiciliary Tri. sordida showed that humans were the commonest host (70.4%), followed by chickens and dogs. Four of 418 persons tested were seropositive for Tryp. cruzi. Only 2 of a second group of 62 persons living in dwellings infested by Tri. sordida were seropositive. Tryp. cruzi infection was demonstrated in dogs and domestic rats. Three other species of small mammals were found to be infected with trypanosomes. In our study area, domestic Tri. sordida are mainly incriminated in the transmission of Tryp. cruzi to synanthropic animals, whereas transmission to humans is very rare. The presence in houses of small populations of Tri. sordida infected with Tryp. cruzi is therefore currently insufficient for this insect to constitute a major epidemiological risk factor.

Trypanosoma cruzi genotypes associated with domestic Triatoma sordida in Bolivia.

Triatoma sordida is the second species of Triatominae considered of epidemiological significance in Bolivia. Associated with Triatoma infestans in various regions, it is as yet the only triatomine species established in human dwellings in localities of Velasco province, Department of Santa Cruz. This domestication is considered as primary. Flagellate parasites were detected in 16.2% of domiciliary T. sordida and the kDNA-PCR confirmed the presence of Trypanosoma cruzi. Frequencies of T. cruzi clonets 20 and 39, common clonets in Bolivian domestic cycle (T. infestans), were established by their direct detection in feces using PCR and hybridization. These clonets present low frequencies in T. sordida and synanthropic mammals. Forty-six stocks were isolated and analysed by multilocus enzyme electrophoresis (MLEE). The MLEE showed a higher clonal diversity than in T. infestans domestic cycle and the genotypes were clustered in the two principal lineages of T. cruzi. Within each lineage, a broad variability was observed. Mixture of genotypes was mostly observed in mammals. The large diversity of T. cruzi in this cycle should be related to its sylvatic origin. Moreover, the current limited sample of stocks suggests a lineage association with specific hosts.

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.

Smallness of the panmictic unit of Triatoma infestans (Hemiptera: Reduviidae).

The population genetic structure of Triatoma infestans (Klug), the principal vector of the causative agent of Chagas disease in Bolivia, was investigated by enzyme electrophoresis at 15 loci, of which 3 were polymorphic. A total of 1,286 adults and nymphs was collected from 19 localities of the Cochabamba (high endemicity) and La Paz (low endemicity) departments. Previous results were confirmed, including a low level of polymorphism (0.20), low genetic distance between geographic areas, and a population structure compatible with an isolation by distance model. However, a high proportion (26.3%) of the surveyed localities showed a significant excess of homozygotes, disputing previous conclusions that considered the village as the probable panmictic unit. The excess of homozygotes was reduced when smaller subunits, such as individual houses or chicken coops, were considered, indicating a Wahlund effect.

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.

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.

Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay.

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard's distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.

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.

Leucemia neutrofílica crónica asociada a leucemia cutánea: reporte de un caso clinico / Vhronic neutrophilic leukemia asociated with leukemia cutis: case report

Se describe un caso de leucemia neutrofílica crónica asociada a leucemia de piel, en una paciente de 49 años de edad, sexo femenino, diagnosticada en julio de 2001; con presencia de 43.605/mm3 neutrófilos en sangre periférica y médula ósea hipercelular con serie granulocítica hiperplástica. el estudio histopatológico de la piel reveló infiltrado del 70 porciento de blastos tipo mieloide. Fue tratada con hidroxiurea, siendo favorable la resuesta clínica y laboratorial. el hemograma de diciembre de 2001 presentó 5.656/mm3 neutrófilos.

Característica bio-celulares de las leucemias agudas en Bolivia y su diversidad en relación con otros paises: reporte de 100 caos de leucemias agudas / Biocell characteristic of the acute leukemia in Bolivia and its diversity in the relationship with another countries: report of the 100 cases of acute leukemia

Pregunta de investigación. ¿Cuáles son las características bio-celularesde las leucemias agudas en Bolivia, y presentará diferencias comparando con otros países?. Objetivos: 1. Determinar las caracteristicas bio-celulares de las leucemias agudas en Bolivia. 2. Comparar la frecuencia y distribución de las características bio-celualres de las leucemias agudas en otros países. Diseño: Descriptivo, corte transversal.Lugar: Unidad de Biología Molecular Paolo Belli, Instituto de Genética, Facultad de Medicina. UMSA. La Paz, Bolivia. Poblacióo: 100 Pacientes con leucemias agudas, 48 niños y 52 adultos provenientes de diferentes Centros Hospitalarios del país. Métodos: Las muestras de sangre periférica y médula ósea fueron estudiados con lossiguientes métodos de análissi: a)Estudio morfológico con tinción May Grunwald-Giemsa, B9 Estudio citoquímico con Mieloperoxidasa y Alfa Nalfta Naftil Butirato Esterasa c) Estudio Inmunocitoquímico utilizando anticuerpos monoclonales. Los resultados fueron analizados según el paquete estadísitco EPIDAR versión 2.0. Resultados: De los 100 casos de leucemias agudas, 48 fueron pediatricos (LLA 83.3 porciento y LMA 16.7 porciento); 95 porciento fueron LLA-B y 5 porciento LLA-T; por ora parte las LMA se distribuyeron como sigue: 12.5 porciento MO, 37.5 M1, 37.5 porciento M2 y 12.5 porciento M4. De los 52 adultos (LLA 61.5 porciento y LMA 38.5 porciento); 87.5 porciento fueron LLA-B y 12.5 porciento LLA-T. La LMA se distribuyeron de la siguiente manera: 10 porciento MO, 35 porciento M1, 15 porciento M2, 35 porciento M3 y 5 porciento M5. Conclusiones: Los valores porcentuales obtenidos en el presente estudio reflejan un patrón característico para nuestra población y confirman su diversidad epidemiológica de las leucemias agudas en comparación con otras regiones.

Baja probabilidad de transmisión de trypanosoma cruzi a humanos por triatoma sordida domiciliado en el departamento de Santa Cruz / Low probability of transmission od Trypanosoma cruzi to humans by triatoma sordid domiciled in the department of Santa Cruz

Entre más de 100 especies de triatominae (hemiptera: ruduviidae) descritas en el Nuevo Mundo, triatoma sordida es considerada de significancia epidemiológica como vector de trypanosoma cruzi por su amplia área de distribución y su tendencia para invadir ambientes domésticos.