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.

Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14 alpha demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > or = 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.

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.

Cure of short- and long-term experimental Chagas' disease using D0870.

Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.

Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil.

The sensitivities for Chagas disease diagnosis of haemoculture, xenodiagnosis, and polymerase chain reaction (PCR) amplification of Trypanosoma cruzi kinetoplast deoxyribonucleic acid (DNA) were compared for 101 patients living in an endemic region who were serologically positive for T. cruzi. PCR gave 60 positive results (59.4%), while a haemoculture was positive in 26 cases (25.7%) and xenodiagnosis in 36 (35.6%). Four xenodiagnosis-positive but PCR-negative patients were examined in detail. The discrepancies were not due to inhibition of the PCR reactions, as the samples were used successfully to amplify a human sequence. Nor were they due to a variation in kinetoplast DNA sequences, as the kinetoplast DNA of the parasite strains isolated from these patients after xenodiagnosis gave rise to the expected product when amplified by the PCR. We concluded that no parasite was present in the 5 mL of blood used for PCR, while probably a single T. cruzi cell was present in the blood volume ingested by the insects during xenodiagnosis (about 3 mL). This suggests that the total blood quantity collected for the PCR may be important with patients with low parasitaemia.

Trypanosoma cruzi: parasite detection and strain discrimination in chronic chagasic patients from northeastern Brazil using PCR amplification of kinetoplast DNA and nonradioactive hybridization.

Blood samples from 172 individuals from northeastern Brazil were subjected to PCR amplification of Trypanosoma cruzi-specific kDNA sequences. This method enabled us to detect parasite DNA in 21 of 47 patients that were serologically positive. In addition, 1 patient that gave doubtful results with chagasic serology was confirmed as positive by PCR. We applied the same PCR detection method to the feces of wild triatomines captured in the same region, obtaining three positive results that were confirmed by microscopic examination. The 25 amplified products obtained in this study were then reamplified with primers that gave a final amplicon containing sequences from the most variable region of kDNA minicircles. These were used as probes in hybridization experiments aimed at defining the degree of relatedness between the strains infecting humans and insects based on kDNA homologies. We found that the amplification products from the three triatomines were related and showed no cross-hybridization with those obtained from human infections. Eight amplified products from human infections showed no cross-hybridization and did not hybridize with products from other patients. This indicates that the strains of T. cruzi circulating in the region present a high level of genetic heterogeneity. Finally, a number of amplified products hybridized with amplicons that did not hybridize with each other, indicating that infections with a parasite population presenting a mixed kDNA content (either due to different strains of T. cruzi or to a hybrid parasite) are a more frequent event than previously thought.

Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation.

Trypanosoma cruzi specific sequences were amplified by the polymerase chain reaction from total blood of human chagasic patients and normal individuals. A 330 bp fragment originating from kinetoplast DNA was specifically detected in most chagasic individuals. We tested the sensitivity and specificity of this method in normal and affected individuals attending the Evandro Chagas Hospital, Rio de Janeiro. The results of these tests were compared with serological diagnosis performed using standard techniques, and in some cases with xenodiagnosis. We found that none of the serologically negative individuals gave any specific amplification product, whereas 55 out of 61 patients previously serodiagnosed as chagasic were positive using the PCR method (sensitivity: 90%). Xenodiagnosis, which is currently considered to be the most sensitive parasitological technique for Chagas' disease diagnosis, detected only 12 out of 28 serologically positive patients (sensitivity: 43%). The usefulness of the PCR method was further investigated with chagasic patients who had received anti-parasite treatment with benznidazole. It has always been difficult to evaluate the incidence of cure in such cases by serology, since a humoral response against T. cruzi antigens may remain for years even in the absence of the parasite. We observed a positive amplification result in only 9 out of 32 treated patients who remained reactive when tested using classical serology. These observations suggest that PCR is the most sensitive technique available for direct detection of T. cruzi in chagasic patients and that it can be a very useful instrument for the follow-up of patients after specific treatment.

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.

Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area.

The feasibility of using DNA amplification by the polymerase chain reaction (PCR) for specific detection of Trypanosoma cruzi in human blood specimens was investigated. One hundred blood samples were collected in an endemic area of Minas Gerais, Brazil. They were submitted to DNA extraction and PCR amplification with kinetoplast DNA-specific primers using a simplified boiling procedure that linearized most minicircle molecules without the aid of chemical reagents. Samples that gave negative results were checked for possible inhibition of amplification using primers derived from a human-specific sequence, and those showing some level of inhibition were retested after a new DNA extraction. Of 86 patients previously diagnosed as chagasic by serologic techniques, 83 were positive in our PCR test (sensitivity = 96.5%), including all the xenodiagnosis-positive patients and 21 (87.5%) of 24 xenodiagnosis-negative individuals. In addition, four of six patients with doubtful serologic results were confirmed as positive by PCR. Our results suggest that the PCR may be a useful complement to serology in the diagnosis of Chagas' disease, and that it is the most powerful technique available for parasite detection in patients with chronic disease.

Characterization of a Trypanosoma cruzi specific nuclear repeated sequence.

We report the isolation of three different clones from a Trypanosoma cruzi genomic library bearing a common repeated sequence. This sequence is not tandemly repeated, and is dispersed on many chromosomes. All of the T. cruzi strains tested share this element. On the other hand, it is absent from the genome of other Kinetoplastida. The size of this element is about 10-12 kb, and its copy number is 220 in the T. cruzi Dm 28c genome. A transcript homologous to this sequence is detected in epimastigote forms of the parasite.