Yara de Miranda Gomes

Elevated Concentrations of CCL2 and Tumor Necrosis Factor—α in Chagasic Cardiomyopathy

Chronic myocarditis is the main pathological finding associated with Chagas disease-related morbidity. Concentrations of CCL2, CCL3, tumor necrosis factor (TNF)-alpha, and brain natriuretic peptide (BNP) were evaluated in plasma samples obtained from patients with different clinical forms of chronic chagasic cardiomyopathy. Patients with more-severe Chagas disease had elevated plasma concentrations of TNF-alpha, CCL2, and BNP, and there was a good correlation between levels of these proteins (especially TNF-alpha ) and the degree of heart dysfunction. Indeed, TNF-alpha level was an excellent predictor of heart failure. Peripheral blood mononuclear cell samples obtained from patients with mild or severe chagasic cardiomyopathy produced greater amounts of TNF-alpha and CCL2 than did those obtained from noninfected individuals. The elevation of TNF-alpha and CCL2 levels in the plasma of patients appears to be secondary to the degree of heart dysfunction, whereas spontaneous production of TNF-alpha and CCL2 by mononuclear cells is secondary not only to heart dysfunction, but also to the underlying inflammation in the heart of chagasic patients. Measurement of the TNF-alpha level could be a useful tool in the identification of patients with heart dysfunction who may benefit from further investigation and treatment.

Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies?

In the acute phase and in the chronic forms of Chagas disease, the etiological diagnosis may be performed by detection of the parasite using direct or indirect parasitological methods and by the presence of antibodies in the serum by way of serological tests. Several techniques are easily available, ranging from the simplest wet smear preparation to immuno-enzymatic assays with recombinant antigens that will meet most diagnostic needs. Other tests under evaluation include a molecular test using polymerase chain reaction, which has shown promising results and may be used as a confirmatory test both in the acute and chronic phases of the disease. Better rapid tests are needed for diagnosis, some of which are already under evaluation. Additionally, there is a need for tools that can identify patients cured shortly after specific treatment. Other needs include a marker for prognosis and early diagnosis of congenital transmission.

The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood.

Recent research has demonstrated the high sensitivity of real time PCR (qPCR) in the diagnosis of Leishmania infantum infection. The goal of this study was to develop and evaluate a qPCR detection system for the diagnosis of visceral leishmaniosis (VL) in dogs. Specific primer sets were developed for the Leishmania donovani complex, in which a fragment of 132 bp of kDNA from L. infantum was amplified. The reaction was performed using the ABI PRISM 7000 system with ABI PRISM software used to carry out the analysis. When canine blood samples were assessed using this system the detection limit of the method was found to be 0.07 parasites per reaction, the efficiency was 94.17% (R(2)=0.93, slope=-3.47) and the sensitivity and specificity were 100% and 83.33% respectively. The use of such a sensitive, reproducible and rapid qPCR-based assay will be useful in the diagnosis and control of L. infantum infection in endemic areas, where serological surveys often underestimate true disease prevalence.

Identification of potentially diagnostic Leishmania braziliensis antigens in human cutaneous leishmaniasis by immunoblot analysis.

ABSTRACT The antibody response in patients with American cutaneous leishmaniasis was analyzed by immunoblotting with soluble and insoluble antigens of Leishmania braziliensis. The recognition of the 27- and/or 30-kDa soluble antigens was considered relevant for the diagnosis of cutaneous leishmaniasis. Immunoblotting was found to be significantly more sensitive and specific than indirect immunofluorescence and enzyme-linked immunosorbent assay.

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the referred kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

Dynamics of the antibody response in patients with therapeutic or spontaneous cure of American cutaneous leishmaniasis

The antigen specificity and the level of the antibody response were analysed in Perambuco State, Brazil, in sera collected in 1995-96 from 58 patients with clinical American cutaneous leishmaniasis (ACL), 25 ACL patients with apparent cure after chemotherapy with meglumine antimonate, and 10 ACL patients with spontaneous cure. Assessment was by immunoblot analysis, ELISA and indirect immunofluorescence, with Leishmania (Viannia) braziliensis antigens, with a particular interest in evaluating whether the dynamics of the antibody response could be useful to monitor clinical cure. A clear decrease of IgG antibody reactivity was noticed after clinical healing, for all of the antigens analysed, with the exception of the 19 kDa antigen, whose recognition frequency in fact increased in the spontaneously cured patients, suggesting that this antigen may play a role in protective immunity against cutaneous leishmaniasis. The recognition frequencies of the most frequently recognized antigens (27 and 30 kDa antigens) diminished approximately 2-fold in patients clinically healed, suggesting that they could be useful as a marker of cure of ACL. In addition, some of the healthy individuals living in endemic areas presented the same immunoblotting pattern of reactivity observed in active ACL, possibly representing asymptomatically infected individuals.

Chagas’ disease diagnosis: comparative analysis of recombinant ELISA with conventional ELISA and the haemagglutination test

Background and Objectives  Serological screening for Chagas’ disease in the blood banks of South America is carried out by using two different assays that generally show a high number of inconclusive results. To establish a combination of two tests that can minimize the number of inconclusive results, we compared a recombinant enzyme‐linked immunosorbent assay (ELISA) with two conventional tests.

Excretory-Secretory Antigens of Trypanosoma cruzi Are Potentially Useful for Serodiagnosis of Chronic Chagas' Disease

ABSTRACT The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA–enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.

Molecular Variations Linked to the Grouping of β- and α-Globin Genes in Neonatal Patients with Sickle Cell Disease in the State of Pernambuco, Brazil

Various factors have been described as phenotypic modulators of sickle cell disease, such as levels of fetal hemoglobin (Hb F), presence of α-thalassemia (thal), and haplotypes of the β-globin genes. In order to characterize and determine the frequency of the βS and βC mutations and the prevalence of −α3.7-thal, 74 patients with sickle cell disease detected during neonatal screening in the State of Pernambuco, Brazil, were studied. The haplotypes of the β gene and −α3.7-thal were determined using polymerase chain reaction (PCR), and specific restriction endonucleases were used to establish the polymorphic sites of the haplotypes. The results showed the high frequency of the Central African Republic (CAR) or Bantu haplotype in the State of Pernambuco, Brazil. The low frequency of the Benin haplotype recorded in this study, in comparison with other states in northeast Brazil, suggests the diversity of origins of Afro-Brazilians in this region.

Cytokine Levels in Serious Cardiopathy of Chagas Disease After In Vitro Stimulation with Recombinant Antigens from Trypanosoma cruzi

The clinical manifestations of human Chagas disease are associated with distinct and complex host–parasite interactions that directly involve the host’s immune system. In this study, we analysed the relationship between the production of intracytoplasmic cytokines after in vitro stimulation with the recombinant antigens CRA (cytoplasmatic repetitive antigen) or FRA (flagellar repetitive antigen) from Trypanosoma cruzi and the chronic cardiac or indeterminate clinical forms of Chagas disease. The chagasic patient groups consisted of 39 individuals, selected at the Chagas Disease Unit of the Oswaldo Cruz University Hospital, whom presented either a cardiac form without cardiac dilatation (CARD 1), cardiac form with cardiac dilatation (CARD 2) or indeterminate form (IND). Blood samples were obtained from these patients and cultured in the presence of CRA or FRA. The cytokines produced by lymphocytes and monocytes after antigen stimulation were analysed by flow cytometry. Our results showed that the IFN‐γ and TNF‐α, produced by CD8+ T lymphocytes after in vitro stimulation with CRA, differed among chagasic patients with CARD 1, CARD 2 or IND. We propose that these cytokines could be utilized as immunological markers for clinical cardiac forms of Chagas disease. In a prospective study of patients presenting IND and CARD 1, the assay performed in this paper could serve as a tool to monitor therapeutic interventions, thus improving the patient’s quality of life.