Antônia Cláudia Jácome da Câmara

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays

Background The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA. Methodology By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain. Findings and Conclusions A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

Genome-Wide Screening and Identification of New Trypanosoma cruzi Antigens with Potential Application for Chronic Chagas Disease Diagnosis

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.

Troponin T autoantibodies correlate with chronic cardiomyopathy in human Chagas disease

To evaluate the potential involvement of anti‐Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease.

Naturally Leishmania infantum-infected dogs display an overall impairment of chemokine and chemokine receptor expression during visceral leishmaniasis

Dogs are the primary reservoir for Leishmania parasites. The immune response induced by Leishmania infantum infection in these animals has not been completely elucidated, and few studies have investigated the relationship between the expression levels of chemokines and chemokine receptors and the clinical status of dogs with canine visceral leishmaniasis (CVL). The aim of this study was to correlate the clinical status of naturally L. infantum-infected dogs (from rural areas of Mossoró city, State of Rio Grande do Norte, Brazil) with the expression levels of chemokines (ccl1, ccl2, ccl3, ccl4, ccl5, ccl17, ccl20, ccl24, ccl26, cxcl9, cxcl10) and chemokine receptors (cxcr3, ccr3, ccr4, ccr5, ccr6, ccr8) in the liver and spleen determined using real-time PCR. Twenty-one dogs were clinically evaluated and classified as asymptomatic (n=11) or symptomatic (n=10). Splenomegaly, weight loss and onychogryphosis were the most pronounced symptoms. In the liver, the mRNA expression levels of ccl1, ccl17, ccl26, ccr3, ccr4, ccr5, ccr6, and ccr8 were lower in symptomatic animals than in asymptomatic animals. Compared with uninfected animals, symptomatic dogs had lower expression levels of almost all molecules analyzed. Moreover, high clinical scores were negatively correlated with ccr5 and ccr6 expression and positively correlated with cxcl10 expression. We conclude that the impairment of the expression of chemokines and chemokine receptors results in deficient leukocyte migration and hampers the immune response, leading to the development of disease.

Seroepidemiology of Trypanosoma cruzi infection in the semiarid rural zone of the State of Rio Grande do Norte, Brazil

INTRODUCTION A seroepidemiological survey was carried out to evaluate Trypanosoma cruzi infection in an endemic area of the State of Rio Grande do Norte, Brazil, involving rural residents. METHODS Sixteen municipalities were randomly selected, 15 from the west mesoregion and one from the central, with an estimated population of 83,852 individuals. A total of 1,950 blood samples were collected in the west mesoregion and 390 in Caicó. Anti-T. cruzi antibodies were detected using the Chagatest® ELISA HAI-hemagglutination kits and indirect immunofluorescence. As sera presented indeterminate results, TESAcruzi® western blot was performed to confirm reactivity. RESULTS An estimated seroprevalence of 6.5% was determined for the west mesoregion and 3.3% for Caicó. Seropositivity rises progressively with the age of individuals, up to 40 years in Caicó and up to 50 years in the west mesoregion. Only educational level and knowledge regarding the triatomine were associated with seropositivity. No seroreactive individuals under 18 years of age were identified. CONCLUSIONS Infection by T. cruzi remains high and is concentrated in municipalities in the central western area of the west mesoregion; however, evidence suggests a decline in vector transmission in this mesoregion and in Caicó. Epidemiological variables appear not to influence seropositivity, with the exception of education and knowledge concerning the triatomine, among seroreactive individuals from the west mesoregion.

Inflammation Enhances the Risks of Stroke and Death in Chronic Chagas Disease Patients.

Ischemic strokes have been implicated as a cause of death in Chagas disease patients. Inflammation has been recognized as a key component in all ischemic processes, including the intravascular events triggered by vessel interruption, brain damage and repair. In this study, we evaluated the association between inflammatory markers and the death risk (DR) and stroke risk (SR) of patients with different clinical forms of chronic Chagas disease. The mRNA expression levels of cytokines, transcription factors expressed in the adaptive immune response (Th1, Th2, Th9, Th17, Th22 and regulatory T cell), and iNOS were analyzed by real-time PCR in peripheral blood mononuclear cells of chagasic patients who exhibited the indeterminate, cardiac, digestive and cardiodigestive clinical forms of the disease, and the levels of these transcripts were correlated with the DR and SR. Cardiac patients exhibited lower mRNA expression levels of GATA-3, FoxP3, AHR, IL-4, IL-9, IL-10 and IL-22 but exhibited higher expression of IFN-γ and TNF-α compared with indeterminate patients. Digestive patients showed similar levels of GATA-3, IL-4 and IL-10 than indeterminate patients. Cardiodigestive patients exhibited higher levels of TNF-α compared with indeterminate and digestive patients. Furthermore, we demonstrated that patients with high DR and SR exhibited lower GATA-3, FoxP3, and IL-10 expression and higher IFN-γ, TNF-α and iNOS mRNA expression than patients with low DR and SR. A negative correlation was observed between Foxp3 and IL-10 mRNA expression and the DR and SR. Moreover, TNF-α and iNOS expression was positively correlated with DR and SR. Our data suggest that an inflammatory imbalance in chronic Chagas disease patients is associated with a high DR and SR. This study provides a better understanding of the stroke pathobiology in the general population and might aid the development of therapeutic strategies for controlling the morbidity and mortality of Chagas disease.

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.

Trypanosoma cruzi: Biodistribution of technetium-99m pertechnetate in infected rats

With the aim of investigating the biodistribution of technetium-99m pertechnetate ((99m)TcO4-) in rats infected with Y strain of Tripanosoma Cruzi, at the peak of parasitemia, (14th day of infection), we injected Wistar rats with 0.1 ml of (99m)TcO4- (3.7MBq). After 60 min, the percentage of radioactivity per gram was counted in several isolated organs and blood, using a gamma counter (1470 Wizard, PerkinElmer Finland). The uptake of (99m)TcO4- increased significantly in blood and decreased in the colon of infected animals (p<0.05). A significant reduction in serum iron and red blood cells and a significant increase in total proteins, leukocytes and lymphocytes in the infected rats were observed, compared with controls (p<0.05). A reduction in muscle layer thickness of the colon and mononuclear inflammation were observed. These results conclusively demonstrate that T. cruzi infection would be associated with changes in the biodistribution of (99m)TcO4- and in colon morphology, with potential clinical implications.

Chagas disease: morbidity profile in an endemic area of Northeastern Brazil

INTRODUCTION This study evaluated the clinical forms and manifestation severities of Chagas disease among serologically reactive individuals from Western Rio Grande do Norte (Northeastern Brazil). METHODS This cross-sectional study included 186 adults who were evaluated using electrocardiography, echocardiography, chest radiography, and contrast radiography of the esophagus and colon. A clinical-epidemiological questionnaire was also used. RESULTS The indeterminate, cardiac, digestive, and cardiodigestive clinical forms of Chagas disease were diagnosed in 51.6% (96/186), 32.2% (60/186), 8.1% (15/186) and 8.1% (15/186) of the participants, respectively. Heart failure (functional classes I-IV) was detected in 7.5% (14/186) of the participants, and 36.4% (24/66), 30.3% (20/66), 15.2% (10/66), 13.6% (9/66), and 4.5% (3/66) of the patients were at stage A, B1, B2, C, and D, respectively. Dilated cardiomyopathy and electrocardiographic changes were detected in 10.2% (19/186) and 48.1% (91/186) of the participants, respectively. Apical aneurysm was diagnosed in 10.8% (20/186) of the participants, and other changes in the segmental myocardial contractility of the left ventricle were diagnosed in 33.9% (63/186) of the participants. Megaesophagus (groups I-IV) was observed in 7% (13/186) of the participants, megacolon (grades 1-3) was detected in 12.9% (24/186) of the participants, and both organs were affected in 29.2% (7/24) of the megacolon cases. CONCLUSIONS We detected various clinical forms of Chagas disease (including the digestive form). Our findings indicate that clinical symptoms alone may not be sufficient to exclude or confirm cardiac and/or digestive damage, and the number of patients with symptomatic clinical forms may be underestimated.