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The life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, involves different forms of the parasite, which alternates between insect and vertebrate hosts. One critical process in the parasite's life cycle is metacyclogenesis, in which the replicative non-infective forms present in the insect midgut differentiate into non-dividing vertebrate-infective forms. It is known that proline (Pro) is important for this process and that leucine (Leu) and isoleucine (Ile) can act as inhibitors of metacyclogenesis. In this study, we investigated further the role of branched-chain amino acids (BCAAs) as negative modulators of parasite differentiation and infection capability in vitro. We found that BCAAs can down-regulate metacyclogenesis, inhibiting Pro-dependent differentiation. Furthermore, we evaluated the ability of all three BCAAs to influence the differentiation of intracellular stages and found that they could modulate the release of trypomastigotes from infected host cells. These findings suggest that BCAAs may have an important role in the complex life cycle of T. cruzi. Thus, enzymes of their metabolism and other interacting proteins could be potential targets for the development of new therapeutic strategies for Chagas disease., Competing Interests: Declaration of competing interest On the behalf of the authors of the manuscript entitled ‘The Branched Chain Amino Acids (BCAAs) modulate the intracellular differentiation processes in Trypanosoma cruzi’, Ludmila N. Rapado, Janaina F. Nascimento, N. Carolina Manchola, Flavia S. Damasceno, Renan W. Achjian I declare that there is no financial/personal interest that could affect the interpretations of our results., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein-protein inhibitors., (open access.)

Among the scarce validated drug targets against Chagas disease (CD), caused by Trypanosoma cruzi , the parasite's nucleoside salvage system has recently attracted considerable attention. Although the trypanocidal activity of tubercidin (7-deazapurine) has long been known, the identification of a class of 7-substituted tubercidin analogs with potent in vitro and in vivo activity and much-enhanced selectivity has made nucleoside analogs among the most promising lead compounds against CD. Here, we investigate the recently identified TcrNT2 nucleoside transporter and its potential role in antimetabolite chemotherapy. TcrNT2, expressed in a Leishmania mexicana cell line lacking the NT1 nucleoside transporter locus, displayed very high selectivity and affinity for thymidine with a K m of 0.26 ± 0.05 µM. The selectivity was explained by interactions of 2-oxo, 4-oxo, 5-Me, 3'-hydroxy and 5'-hydroxy with the transporter binding pocket, whereas a hydroxy group at the 2' position was deleterious to binding. This made 5-halogenated 2'-deoxyuridine analogues good substrates but 5-F-2'-deoxyuridine displayed disappointing activity against T. cruzi trypomastigotes. By comparing the EC 50 values of tubercidin and its 7-substituted analogues against L. mexicana Cas9, Cas9 ΔNT1 and Cas9 ΔNT1+TcrNT2 it was shown that TcrNT2 can take up tubercidin and, at a minimum, a subset of the analogs.

Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6-8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 "chimeric" molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 μM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 μM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi., Competing Interests: The authors have declared that no competing interests exist.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi , a hemoflagellate parasite, is the etiological agent of Chagas disease that affects about 6-7 million people worldwide, mostly in Latin America. The parasite life cycle is complex and alternates between an invertebrate host-Triatominae vector-and a mammalian host. The parasite adaptation to the several microenvironments through which it transits is critical to success in establishing infection. Moreover, environmental cues also play an important role on the parasite development, and it can modulate the infection. In the present study, we discussed how the temperature oscillations and the nutritional state of the invertebrate host can affect the parasite development, multiplication, and the differentiation process of epimastigote forms into metacyclic trypomastigotes, called metacyclogenesis. The impact of oxidative imbalance and osmotic stresses on the parasite-vector relationship are also discussed., (Copyright © 2020 Melo, Guarneri and Silber.)

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC 50(Epi) = 23.1 ± 1.75 μM] and intracellular forms of trypomastigotes [IC 50(Tryp) = 8.5 ± 2.9 μM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs., (Copyright © 2020 American Society for Microbiology.)

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5-6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer's disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma , the etiological agent of Chagas disease, is dependent on proline for a variety of processes, such as energy metabolism, host cell invasion, differentiation, and resistance to osmotic, metabolic, and oxidative stress. On this basis, we investigated a possible relationship between prolinemia and severity of cruzi , the etiological agent of Chagas disease, is dependent on proline for a variety of processes, such as energy metabolism, host cell invasion, differentiation, and resistance to osmotic, metabolic, and oxidative stress. On this basis, we investigated a possible relationship between prolinemia and severity of T. -infected patients and 29 age-matched healthy volunteers (control) of both sexes, recruited at the Chagas Disease Service from the Department of Cardiology, Hospital Provincial del Centenario de Rosario (Rosario, Argentina). Chagasic patients were separated into three groups: chronic asymptomatic, mild/moderate, and severe chronic chagasic cardiomyopathy (CCC) subjects. We observed a significant decrease of 11.7% in prolinemia in chagasic patients when compared to controls. Further analysis within the three groups of chagasic patients also revealed a statistically significant decrease of prolinemia in severe CCC patients compared to controls, showing a relative difference of 13.6% in proline concentrations. These data point to the possibility that collagen-which participates in the healing process of cardiac tissue-and proline metabolism in the myocardium could constitute new factors affecting the evolution of Chagas disease.cruzi infection in chronic patients, as reported here. The study population consisted of 112 subjects, separated into 83 chronically T. cruzi -infected patients and 29 age-matched healthy volunteers (control) of both sexes, recruited at the Chagas Disease Service from the Department of Cardiology, Hospital Provincial del Centenario de Rosario (Rosario, Argentina). Chagasic patients were separated into three groups: chronic asymptomatic, mild/moderate, and severe chronic chagasic cardiomyopathy (CCC) subjects. We observed a significant decrease of 11.7% in prolinemia in chagasic patients when compared to controls. Further analysis within the three groups of chagasic patients also revealed a statistically significant decrease of prolinemia in severe CCC patients compared to controls, showing a relative difference of 13.6% in proline concentrations. These data point to the possibility that collagen-which participates in the healing process of cardiac tissue-and proline metabolism in the myocardium could constitute new factors affecting the evolution of Chagas disease.

Objectives: To develop an alcohol-free solution suitable for children of benznidazole, the drug of choice for treatment of Chagas disease., Methods: In a quality-by-design approach, a systematic optimisation procedure was carried out to estimate the values of the factors leading to the maximum drug concentration. The formulations were analysed in terms of chemical and physical stability and drug content. The final preparation was subjected to an in vivo palatability assay. Mice were infected and treated orally in a murine model., Results: The results showed that benznidazole solubility increased up to 18.38 mg/ml in the optimised co-solvent system. The final formulation remained stable at all three temperatures tested, with suitable drug content and no significant variability. Palatability of the preparation was improved by taste masking of BZL. In vivo studies showed that both parasitaemia and mortality diminished, particularly at a dose of 40 mg/kg/day., Conclusion: Quality by design was a suitable approach to formulate a co-solvent system of benznidazole. The in vivo studies confirmed the suitability of the optimised such solutions to diminish both parasitaemia and mortality. Thus, this novel alternative should be taken into account for further clinical evaluation in all age ranges., (© 2017 John Wiley & Sons Ltd.)

Trypanosoma cruzi is the causative agent of Chagas' disease, which affects some 8 - 10 million people in the Americas. The only two drugs approved for the etiological treatment of the disease in humans were launched more than 40 years ago and have serious drawbacks. In the present work, we revisit the unique characteristics of T. cruzi mitochondria and mitochondrial metabolism. The possibility of taking advantage of these peculiarities to target new drugs against this parasite is also discussed.

* Introduction * Chagas' disease * Chemotherapy * Immune response in experimental T. cruzi infection * Immune response in human beings infected with T. cruzi * Immune response in the treatment of chagasic infection * The need for new therapeutic alternatives for Chagas' disease * Conclusions The final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order. Among the pathogenic trypanosomatids, some species are of particular interest due to their medical importance. These species include the agent responsible for Chagas' disease, Trypanosoma cruzi. Approximately 8 to 10 million people are infected in the Americas, and approximately 40 million are at risk. In the present review, we discuss in detail the immune mechanisms elicited during infection by T. cruzi and the effects of chemotherapy in controlling parasite proliferation and on the host immune system.

Chagas' disease is routinely diagnosed by detecting specific antibodies (Abs) using serological methods. The methodology has the drawback of potential cross-reactions with Abs raised during other infectious and autoimmune diseases (AID). Fusion of DNA sequences encoding antigenic proteins is a versatile tool to engineer proteins to be used as sensitizing elements in serological tests. A synthetic gene encoding a chimeric protein containing the C-terminal region of C29 and the N-terminal region of TcP2beta was constructed. A 236-serum panel, composed of 104 reactive and 132 nonreactive sera to Chagas' disease, was used to evaluate the performance of the chimera. Among the nonreactive sera, 65 were from patients with AID (systemic lupus erythematosus and rheumatoid arthritis) or patients infected with Leishmania brasiliensis, Brucella abortus, Streptococcus pyogenes, or Toxoplasma gondii. The diagnostic performances of the complete TcP2beta (TcP2betaFL) and its N-terminal region (TcP2betaN) were evaluated. TcP2betaFL showed unspecific recognition toward leishmaniasis (40%) and AID Abs (58%), while TcP2betaN showed no unspecific recognition. The diagnostic utility of the chimera was evaluated by analyzing reactivity and comparing the results with those obtained with TcP2betaN. The chimera reactivity was higher than that of the peptide fractions (0.874 versus 0.564 optical density, P = 0.0017). The detectability and specificity were both 100% for the whole serum panel tested. We conclude that the obtained chimera shows an improved selectivity and sensitivity compared with other ones previously reported, therefore displaying an optimized performance for Trypanosoma cruzi infection diagnosis.

Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.

Chagas' disease is a zoonosis caused by the parasite Trypanosoma cruzi, a haematic protozoan, transmitted by insects from the Reduviidae family. This constitutes a relevant health and socio-economic problem in the Americas, with 11 - 18 million people infected, and approximately 100 million people at risk. The therapeutic possibilities rely into two drugs, nifurtimox and benznidazole, that were discovered more than thirty years ago, and are mainly successful during the acute phase of the disease. In the majority of the cases the disease is diagnosed in the chronic phase, when the therapy is inefficient and the probability of cure is low. In addition, these drugs are highly toxic, with systemic side effects on patients. Trypanosoma cruzi has a metabolism largely based on the consumption of amino acids, mainly proline, aspartate and glutamate, which constitute the main carbon and energy sources in the insect stage of the parasite life cycle. These amino acids also participate in the differentiation process of the replicative non-infective form (epimastigote) to the non-replicative infective form (trypomastigote). In particular, the participation of proline in the intracellular differentiation cycle, which occurs in the mammalian host, was recently demonstrated. In addition, an arginine kinase has been described in T. cruzi and T. brucei, which converts free arginine to phosphoarginine, a phosphagen with a role as an energy reservoir. Arginine kinase seems to be an essential component of energy management during stress conditions. Taken together, these data indicate that amino acid metabolism may provide multiple as yet unexplored targets for therapeutic drugs.

In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.

In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas' disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively.

The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in Trypanosome cruzi-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa T. cruzi antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections.

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T. cruzi DNA fragment (sensitivity >95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radiolabeled PCR amplification product as probe (sensitivity 100%).