Impairment of Interleukin-17A Expression in Canine Visceral Leishmaniosis is Correlated with Reduced Interferon-γ and Inducible Nitric Oxide Synthase Expression.

Dogs are the primary urban reservoir of Leishmania infantum and play a crucial role in the transmission of this parasite to man via sandflies. The spleen and liver are the main target organs of L. infantum infection, but few studies have evaluated the immune response to this infection in the canine liver. To identify the immunological mediators involved in resistance and/or susceptibility to canine visceral leishmaniosis (CVL), we selected 21 dogs naturally infected by L. infantum and classified as asymptomatic or symptomatic. Immunological parameters were analysed and correlations with clinical signs were determined. Symptomatic dogs showed higher numbers of parasites and less leucocyte infiltration in the liver compared with asymptomatic dogs. The progression of this disease was characterized not only by the down regulation of T helper (Th) 1-related cytokines, such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, but also by the down regulation of genes encoding interleukin (IL)-17A, inducible nitric oxide synthase (iNOS) and IL-10 in the spleen and liver in symptomatic dogs compared with asymptomatic dogs. Importantly, IL-17A gene transcription level was positively correlated with mRNA expression for iNOS and IFN-γ. Th1- and Th17-related cytokines therefore appear to play a role in restricting parasite growth via iNOS activation and decrease susceptibility of dogs to CVL.

Genetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil.

Trypanosoma cruzi genetic diversity was investigated in 25 isolates (vectors and humans) from the semiarid zone of the State of Rio Grande do Norte, Brazil. Molecular markers (3' region of the 24Salpha rRNA; mitochondrial cytochrome oxidase subunit 2 (COII) gene; spliced leader intergenic region (SL-IR) gene; allelic size microsatellite polymorphism) identified 56% TcIII (100% Panstrongyluslutzi; 50% Triatomabrasiliensis); 40% TcII (91.7% humans; 50% T. brasiliensis) and 4% TcI (human). Microsatellite analysis revealed monoclonal and heterozygous patterns on one or more microsatellite loci in 64% of T. cruzi isolates (92.3% triatomines; 33.3% humans) and 36% putative polyclonal populations (66.7% humans; 7.7% triatomines) by loci SCLE10, SCLE11, TcTAT20, TcAAAT6, all belonging to TcII. Identical T. cruzi polyclonal profiles (88.9%) were detected, mostly from humans. The adaptative natural plasticity of TcII and TcIII and their potential for maintaining human infection in T. brasiliensis were confirmed. Intraspecific and phylogenetic T. cruzi diversity in the sylvatic and domestic transmission cycles in this specific region will provide exclusive control strategies.

Random amplified polymorphic DNA profiles of Trypanosoma cruzi isolates from chagasic patients with different clinical forms.

The genetic variability of 61 Trypanosoma cruzi isolates from 47 chronic chagasic patients of Minas Gerais state was analyzed by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) using M13-40, lambdagt11-F, and L15996 primers. Cluster analysis by unweighted pair group method analysis was applied to RAPD profiles, and cluster analysis used to verify a possible correlation among different clinical forms of the disease from these patients. The T. cruzi isolates showed distinct grouping on tree topology, with the isolates not being possible to establish a correlation to the clinical forms of Chagas' disease. These data showed that the T. cruzi isolates from these patients would compose a group of populations well correlated genetically.

Blood culture and polymerase chain reaction for the diagnosis of the chronic phase of human infection with Trypanosoma cruzi.

In the present study, we evaluated for the first time the profile of blood parasitism in untreated, chronic Chagas' disease. The study was conducted on 60 patients and a control group of nine serologically negative individuals. Analysis of three blood samples showed 70% cumulative positivity for blood culture and 86.7% positivity for PCR. The comparison of the two tests revealed that 41.1% (74/180) of the samples presented positive results for both PCR and blood culture, 22.2% (40/180) were positive for PCR alone, and 4.4% (8/180) were positive for blood culture and negative for PCR. The addition of the second sample raised positivity significantly for both blood culture ( P=0.0000) and PCR ( P=0.0369). Addition of the third sample was also statistically significant for blood culture ( P=0.0001) but not for PCR ( P=0.1186). These data point to the importance of studying the parasitemia of Trypanosoma cruzi-infected individuals before specific treatment. They also suggest that at least two blood samples should be collected and that two tests should be used, if possible--a procedure that considerably improves the parasitologic diagnosis of Chagas' disease and the evaluation of therapeutic efficacy.

Cyclophosphamide-induced immunosuppression protects cardiac noradrenergic nerve terminals from damage by Trypanosoma cruzi infection in adult rats.

Trypanosoma cruzi-infected juvenile rats develop severe cardiac sympathetic denervation in parallel with acute myocarditis. This aspect has not been studied in adult rats, thought to be resistant to this infection. The mechanism involved in T. cruzi-induced neuronal damage remains to be completely elucidated. In juvenile rats, the mortality during the acute phase depends on T. cruzi populations, ranging from 30% to 100%. Therefore, studies of mechanisms through hazardous procedures such as immunosuppression are restricted. The current paper shows that adult rats infected with T. cruzi (Y strain) develop severe acute myocarditis and cardiac sympathetic denervation, despite null mortality and virtual absence of patent parasitaemia followed by negative haemoculture. Recovery from the myocarditis and denervation occurred but PCR studies showed persistence of parasite DNA at least until day 111 post inoculation. Immunosuppression by cyclophosphamide treatment increased the parasitaemia, prevented the acute myocarditis and the sympathetic denervation without significant alteration of the myocardial parasitism. These results argue against a direct role for parasite-derived products and implicate the inflammatory cells in the denervation process. As previous studies in juvenile animals have discarded an essential role for radiosensitive cells, the macrophages remain as the possible effectors for the T. cruzi-induced neuronal damage.

Chagas' disease diagnosis: comparative analysis of parasitologic, molecular, and serologic methods.

During the course of chronic chagasic infection, low parasitemia levels prevent parasite detection by current techniques such as hemoculture and xenodiagnosis. Since serologic tests have sensitivity but lack specificity, molecular assays based on the polymerase chain reaction (PCR) have been proposed as alternative tools for parasite detection in individuals with chronic Chagas' disease. A variable degree of PCR efficiency has been reported in the literature and illustrates the need for further evaluation of large numbers of chagasic patients. In this study, we compared an optimized PCR technique with hemoculture and complement-mediated lysis (CoML) in 113 individuals from or living in endemic areas of Brazil who had conventional serologic results that were either positive, negative, or inconclusive. The PCR amplification yielded positive results in 83.5% (66 of 79) of individuals with positive serology, 47.6% (10 of 21) with negative serology, and 46.2% (6 of 13) with inconclusive serology. Of 10 patients with negative serology and positive PCR result, eight (80%) had positive CoML, indicating that they could have been chagasic but were not mounting immune responses. The PCR results were also positive for all individuals who had positive hemoculture, for 37 individuals with negative hemoculture and positive serology, and for two of six individuals with inconclusive serology and negative hemoculture. Thirteen individuals living in nonendemic areas who had negative serology were used as a negative control group: 100% had negative PCR results. Our results show that the optimized PCR protocol used here was very sensitive in detecting the presence of Trypanosoma cruzi in chronic chagasic patients. The PCR and CoML results were well correlated in all of the groups studied, which suggests that our PCR protocol may be effective in the evaluation of cure in patients who receive anti-parasite treatment.

Trypanosoma cruzi: optimization of polymerase chain reaction for detection in human blood.

We have optimized the conditions for DNA extraction and polymerase chain reaction (PCR) amplification to diagnose the presence of Trypanosoma cruzi DNA in the blood and serum of patients with chronic Chagas disease. The approximately 330-bp fragment of the kinetoplast minicircles was used as a target for amplification. The use of chemiluminescence on slot blots with a specific alkaline phosphatase-conjugated oligonucleotide probe detected specific product from as little as 0.1 fg of T. cruzi kDNA. An additional product of approximately 200 bp inadvertently amplified from the human genome was observed in human blood from T. cruzi-negative and -positive samples and served as an internal control of the amplification. Samples from other mammalian hosts were also assayed using the PCR protocol. The higher sensitivity of our PCR method observed in both acute and chronic phases of T.cruzi infections in mice and dog, respectively, could be useful in monitoring the course of infection during experimental drug tests in laboratory animals. Since this procedure showed a higher sensitivity than other protocols in the literature, it may be a suitable routine test in diagnosing Chagas disease, especially for patients presenting very low parasitemia levels.

Kinetoplast DNA signatures of Trypanosoma cruzi strains obtained directly from infected tissues.

We report here a polymerase chain reaction (PCR)-based DNA profiling technique that permits Trypanosoma cruzi strain characterization by direct study of infected tissues. This is based on application of a recently developed method of DNA fragment identification, called low-stringency single specific primer PCR (LSSP-PCR), to the study of the variable region of kinetoplast DNA (kDNA) minicircles from T. cruzi Thus, we can translate the intraspecific polymorphism in the nucleotide sequence of kDNA minicircles into a specific and highly reproducible kDNA signature. Comparison with the phenogram obtained by DNA fingerprinting analysis of a set of T. cruzi strains showed good qualitative correlation between the degree of divergence of the LSSP-PCR profiles and the genetic distance between the strains. kDNA signatures of heart tissue from acutely or chronically infected animals revealed perfect concordance with the patterns obtained from cultured parasites for the CL and Colombiana strains but not for the Y strain, which is known to be multiclonal. However, the match was perfect for studies with two clones of the Y strain. We take this as evidence that in some multiclonal strains there is heterogeneity among the clones in the degree of tropism for the heart tissue. Finally, we showed that it is possible to obtain a T. cruzi kDNA signature from the heart of a human patient with chronic Chagasic myocardiopathy. kDNA signatures obtained by LSSP-PCR of sequences amplified from infected tissues constitute a new tool to study the molecular epidemiology of Chagas' disease.

Influence of parasite presence on the immunologic profile of peripheral blood mononuclear cells from chagasic patients after specific drug therapy.

The aim of this study is to evaluate the effects of parasite clearance on the immunological profile of peripheral blood mononuclear cells (PBMC) from chagasic patients submitted to specific drug therapy. PBMC were examined by flow cytometry and proliferative responsiveness to Trypanosoma cruzi-related stimuli. Three groups of patients were studied: not treated (NT), treated not cured (TNC) and cured (C). All data were compared to values from uninfected individuals (NI). NT displayed a lower percentage of CD3+ cells as compared to NI, while TNC and C had mean values that were between those from NI and NT. Infected patients had double the percent of CD3+ HLA-DR+ cells, independent of the efficacy of the treatment. Thus, absence of circulating parasites did not reduce T cell activation in Chagas' disease. NT displayed a higher percentage of CD5+ B cells as compared to NI, while TNC and C had mean values between those from NI and NT. In contrast to the phenotypic data, the in vitro mean proliferative responses to parasite-related stimuli of PBMC from C were reduced to the low mean levels observed in NI. These striking differences were statistically different from the high responses seen in NT and TNC. Our data suggest that proliferative responses of PBMC from C reflect immunological changes due elimination of parasite. However, successful treatment did not alter the levels of peripheral T cell activation.

Potential use of WR6026 as prophylaxis against transfusion-transmitted American trypanosomiasis.

Since transmission of Chagas' disease by the insect vector is under control in Brazil, transmission by blood transfusion is acquiring special relevance in areas where the disease is endemic and also in countries whose populations are free of infection but that are receiving immigrants from areas where the disease is endemic. Gentian violet, a phenylmethane dye, was the first agent used for the chemical prophylaxis of blood destined for transfusion. A concentration of 0.6 mmol of this dye per liter is effective at eliminating trypomastigotes from blood after 24 h of incubation at 4 degrees C. It is the only effective trypanosomicidal agent available. In the search of alternate compounds, we examined a number of synthetic compounds. They were screened for their activities against blood trypomastigotes of the Y, CL, and B229 strains of Trypanosoma cruzi by using two or more dilutions of each compound. We found that compound Q45, a 6-methoxy-8(diethylaminohexylamino)lepidine dihydrochloride, was highly effective at clearing parasites from infected blood. Doses of 65 and 130 micrograms of this compound eliminated trypomastigotes from blood experimentally contaminated with T. cruzi parasites. These results indicate that Q45 is remarkably active against circulating trypomastigotes. Further studies evaluating Q45 as a prophylactic agent for preventing the transmission of T. cruzi by blood transfusion are of interest.

Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease.

A 24-kDa recombinant protein from Trypanosoma cruzi (rTc24) was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) tests to identify treated chagasic patients considered parasitologically cured on the basis of persistently negative tests of hemocultures and lytic antibodies. Some of these patients were termed dissociated because their sera, although negative by the complement-mediated lysis test, were positive by conventional serology. The negative lysis test indicates the absence of active infection after specific treatment, but this assay requires live and infectious parasites and cannot be used easily in a laboratory routine. Here we tested rTc24 by ELISA and Western blotting as an alternative for the complement-mediated lysis test. For the group of patients with active infection despite the treatment (uncured patients), all the sera tested recognized rTc24 in both tests. For the dissociated patients, approximately 80% of the sera did not react with rTc24 in the ELISA or in Western blots, in agreement with the negative complement-mediated lysis tests. Thus, the 24-kDa T. cruzi recombinant antigen, when used for initial trials to evaluate cure of chagasic patients submitted to specific treatment, will allow the identification of most, but not all, cases.

[Soluble antigens released by Trypanosoma cruzi trypomastigotes used in ELISA to detect cure in chagasic patients following specific treatment]. / Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de ELISA para detectar cura em pacientes chagásicos após tratamento específico.

Two soluble antigens isolated from T. cruzi were evaluated by ELISA for the diagnosis of chronic infection and assessment of cure after specific chemotherapy, namely, supernatants from parasite cell-cultures (SpAg) and trypomastigote excretory/secretory antigens (ESAg). Among the treated patients, a group defined as "dissociated" had been monitored for 3 to 10 years and displayed negative hemocultures and tests of trypomastigote complement-mediated lysis persistently negative although positive by conventional serology (indirect fluorescence with epimastigote, mainly). A negative lysis test indicates parasite elimination by the patient. Our ELISA results showed that among the non-treated chagasic controls the SpAg e ESAg detected 93% and 100%, of the cases, respectively. However, only 28% of sera from the dissociated group, considered as cured, were positive by ELISA using SpAg whereas all of such sera were negative using ESAg. Therefore ELISA with ESAg seems to be ideal to replace the complement-mediated lysis test with the aim to define cure after treatment of the chronic infection by T. cruzi.

Humoral immune response to the Trypanosoma cruzi complement regulatory protein as an indicator of parasitologic clearance in human Chagas' disease.

Immunoprecipitation of the purified 160-kDa complement regulatory protein of Trypanosoma cruzi by Chagas' disease patient sera was examined as a possible correlate of the complement-mediated lysis test and as an indicator of parasite clearance. The results presented demonstrate that assessment of the humoral response to this antigen is a useful indicator of parasite clearance and may be particularly helpful in the assessment of some patients for whom other serological tests produce ambiguous results.

Use of Trypanosoma cruzi purified glycoprotein (GP57/51) or trypomastigote-shed antigens to assess cure for human Chagas' disease.

With the exception of assays for the detection of antibodies promoting complement-mediated lysis of Trypanosoma cruzi, serologic tests have generally failed to assess the effectiveness of chemotherapy for Chagas' disease. Conventional serology, although useful for the diagnosis of infection, is not capable of determining which patients have been cured. Here we demonstrate that a high proportion of antibodies detected by conventional serology (using fixed epimastigotes or trypomastigotes or crude extracts obtained therefrom) are directed against the carbohydrate residue galactosyl alpha 1- > 3 galactose (Gal alpha 1- > 3 Gal), a determinant also recognized by antibodies from noninfected healthy volunteers. In a study of 14 cured patients with long-term followup, we found that the persistently positive reactions detected using conventional serology were largely eliminated following immunoadsorption with melibiose. Because of their wide distribution among microorganisms of intestinal and pulmonary microflora, these carbohydrate determinants may keep stimulating lymphocytes previously primed by T. cruzi Gal alpha 1- > 3 Gal epitopes, thereby accounting for false-positive results in cured patients. Consistent with this proposition, enzyme-linked immunosorbent assays performed with two distinct T. cruzi antigen preparations that lack the Gal alpha 1- > 3 Gal epitope, namely purified GP57/51 and trypomastigote-shed antigens, were indeed capable of determining a cure after chemotherapy, albeit to a different degree. Collectively, the data indicate that conventional immunoassays prepared with highly specific T. cruzi antigens can be useful in the assessment of a cure after chemotherapy.

Difference in susceptibility to lysis between clones of the Y strain of Trypanosoma cruzi.

Three clones isolated from the Y strain of Trypanosoma cruzi--YP1, YP2 and YP3--were adapted to in vitro cultivation in VERO cells. The recovery of the parasites from the Y strain and clone YP3 was similar after 24 hr of contact with cells (3.2% and 2.7%, respectively) and much lower than the recovery of clones YP1 and YP2 (56.7% and 60.0% of inoculum, respectively). After five days incubation, the ratio Trypomastigotes/Amastigotes released into the supernatants was about 90/10 for clone YP1, YP3 and Y strain, and 50/50 for clone YP2. After nine days, the ratio was 62/38 for clone YP1, 97/3 for clone YP3, 81/19 for Y strain and 50/50 for clone YP2. The susceptibility of tissue culture derived trypomastigotes (TCT) to lysis in the presence of chronic chagasic human sera and human complement was assessed using Complement Mediated Lysis reaction (CML). Trypomastigotes from clone YP2 were consistently less susceptible to CML (% lysis less than 20), than parasites from the other clones and Y strain. Parasites of clone YP3 had susceptibility to CML comparable to that of the Y strain (about 70%), while TCT of clone YP1 had intermediary susceptibility (40%).

Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study.

A complement-mediated lysis test (CoML) using living trypomastigotes was compared with conventional serological methods and with haemoculture. Over a 10 years follow-up period evidence was obtained which supported the view that chagasic patients, treated with nitroheterocyclic drugs, in whom CoML had reverted to negative, might be considered cured despite conventional serology remaining positive.

An experimental and clinical assay with ketoconazole in the treatment of Chagas disease.

Ketoconazole, an azole antifungic drug which is already in the market has also been demonstrated to be active against Trypanosoma cruzi experimental infections. In this paper we confirmed the drug effect and investigated its range of activity against different T. cruzi strains naturally resistant or susceptible to both standard drugs Nifurtimox and Benznidazole used clinically in Chagas disease. Moreover, we have shown that the association of Ketoconazole plus Lovastatin (an inhibitor of sterol synthesis), which has an antiproliferative effect against T. cruzi in vitro, failed to enhance the suppressive effect of Ketoconazole displayed when administered alone to infected mice. Finally, administration in chronic chagasic patients of Ketoconazole at doses used in the treatment of deep mycosis also failed to induce cure as demonstrated by parasitological and serological tests. The strategy of identify and test drugs which are already in the market and fortuitously are active against T. cruzi has been discussed.

Hemocultures from chronic Chagasic patients using EDTA or heparin as anticoagulants.

Hemoculture tests, a method for the detection of Trypanosoma cruzi, were used to investigate the effects of the anticoagulants heparin or EDTA on the parasite growth in culture medium (liver infusion tryptose, LIT). Hemocultures from 13 patients with positive serology for chronic Chagas' disease performed in parallel with both anticoagulants resulted in a total of seven (54%) positive hemocultures, three positive with blood samples collected with EDTA (23%), two with heparin (15%) and two with both anticoagulants (15%). There was no significant difference between the number of positive tubes in blood samples collected with either heparin (11%) or with EDTA (13%), an indication that heparin does not block the growth of T. cruzi. However, the simultaneous use of both anticoagulants may improve the positivity index of the hemocultures.

Hemocultures from chronic chagasic patients using EDTA or heparin as anticoagulants

Hemoculture tests, a method for the detection of Trypanosoma cruzi, wre used to investigate the effects of the anticoagulants heparin or EDTA on the parasite growth in culture medium (liver infusion tryptose, LIT). Hemocultures from 13 patients with positive serology for chronic Chagas' disease performed in parallel with both anticoagulants resulted in a total of seven (54%) positive hemocultures, three positive with blood samples collected with EDTA (23%), two with heparin (15%) and two with both anticoagulants (15%). There was no significant difference between the number of positive tubes in blood samples collected with either heparin (11%) or with EDTA (13%), an indication that heparin does not block the growth of T. cruzi. However, the simultaneous use of both anticoagulants may improve the positivity index of the hemocultures