Virginia Maria Barros de Lorena

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.

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.

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.

Evaluation of an interferon gamma assay in the diagnosis of latent tuberculosis infection in patients with rheumatoid arthritis.

The tuberculin skin test is not an ideal screening test for the patients with rheumatoid arthritis to identify cases of latent tuberculosis infection (LTBI) prior to the start of treatment with anti-TNFs, as it responds inadequately to late hypersensitivity, which is fundamental for producing a response to the inoculated antigen. Assays based on detection of the production of IFNγ in vitro by mononuclear peripheral cells stimulated by specific antigens are more specific than PPD in detecting LTBI. The aim of this study was to evaluate the performance of T-SPOT.TB in diagnosis of LTBI in patients with rheumatoid arthritis, comparing with the PPD. The specificity of the T-SPOT.TB varied from 87 to 90% and the negative-predictive value (NPV) from 94.4 to 100%. It can be concluded that the T-SPOT.TB showed high specificity and NPV, proving the capability of identifying false-negative cases of PPD, raising the level of safety for the use of anti-TNFs.

Humoral and cellular immune responses in BALB/c and C57BL/6 mice immunized with cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens, in acute experimental Trypanosoma cruzi infection

In previous studies, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins induced specific humoral and cellular immune responses in susceptible and resistant mice in the absence of Trypanosoma cruzi infection with a significant induction of the Interferon-gamma (IFN-γ) production in those animals. In this follow-up paper, the immunostimulatory and protective effects of these proteins were evaluated by immunizing with CRA or FRA antigens, BALB/c and C57BL/6 mice and challenging with a T. cruzi (Y strain). Both proteins induced humoral response with high levels of IgG isotypes as well as cellular immunity with high levels of IFN-γ when compared to controls. However, the lymphocyte proliferative response was minimal. The survival rate at 30 days post-infection was significant in CRA (60%) or FRA (50%) - immunized BALB/c mice and CRA (83.3%) - immunized C57BL/6 mice. Taken as a whole these findings indicate that CRA and FRA are immunogenic and potentially important for protective immunity.

Prevalência de infecção chagásica em doadores de sangue no estado de Pernambuco, Brasil

A doenca de Chagas e uma infeccao sistemica de evolucao cronica cujo agente etiologico e o parasita Trypanosoma cruzi. O ultimo relato encontrado sobre a soroprevalencia da doenca em doadores de sangue realizado na capital pernambucana, Recife, data de 1970, onde foi encontrada uma prevalencia de 4,4% em doadores de um hospital local. Devido a falta de informacoes divulgadas sobre a infeccao por T. cruzi e sendo Pernambuco uma regiao endemica para esta enfermidade, o presente estudo se propos a analisar o perfil dos doadores de sangue do Hemocentro de Pernambuco (Hemope), que apresentaram reatividade para doenca de Chagas, no periodo de 2002 a 2007. O perfil dos doadores inaptos foi avaliado de acordo com genero, idade e procedencia segundo as mesorregioes de Pernambuco. Foi encontrada uma prevalencia de 0,17% para doenca de Chagas e 6,89% das bolsas descartadas deveram-se a essa reatividade. Em relacao ao genero dos doadores, foi significativamente maior a contribuicao dos homens (p<0,0001). A faixa etaria de 18-30 anos apresentou menor quantidade de sorologias reativas (20,21%). Foi verificado tambem que, na Regiao Metropolitana do Recife, a quantidade de reacoes inconclusivas foi estatisticamente maior que a quantidade de sorologias reagentes (p=0,0440). Desta forma, estudos epidemiologicos fornecem dados importantes no sentido de se avaliar diretamente o risco de transmissao de uma doenca por transfusao sanguinea e permitem que tambem em regioes endemicas se avalie a eficacia das medidas para o controle vetorial.

Usefulness of real time PCR to quantify parasite load in serum samples from chronic Chagas disease patients.

BackgroundInconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening.MethodsIn order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction.ResultsThe comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 105 to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes – Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples.ConclusionsTaken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.

IL-10 and IFN-γ gene expression in chronic Chagas disease patients after in vitro stimulation with recombinant antigens of Trypanosoma cruzi.

Along with several other aspects of Chagas disease, the mechanisms responsible for the different clinical outcomes observed in chronic infected individuals have not yet been clarified. It is believed that the host immune response to the parasite plays an important role in the development of the pathology. Therefore, the aim of this study was to evaluate the relationship between IL-10 and IFN-γ gene expression profile, after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with Trypanosoma cruzi recombinant antigens CRA (cytoplasmatic repetitive antigen) and FRA (flagellar repetitive antigen), and the clinical forms of chronic Chagas disease. Twenty patients with the cardiac form of the disease (CARD), of whom 10 had the mild cardiac form (CARD 1) and 10 the severe cardiac form (CARD 2), and 20 patients with the indeterminate form (IND), were selected at the Chagas Disease Unit of the Oswaldo Cruz University Hospital, University of Pernambuco, Recife, Pernambuco, Brazil. The PBMCs of these individuals were cultured in the presence of CRA or FRA for 3 days and IL-10 and IFN-γ gene expression was evaluated by detection of its messenger RNA using Real Time Quantitative PCR. Although no significant difference was observed between the groups of individuals studied, we found that most patients with IND displayed high levels of IFN-γ gene expression, while the majority of patients with CARD 1 presented high levels of IL-10. The results of this study thus highlight the important role that inflammatory cytokines play in patients with the IND group controlling for parasite replication, and that anti-inflammatory cytokines play in determining susceptibility to progression to symptomatic clinical forms of the disease.

Chagas disease in the State of Pernambuco, Brazil: analysis of admissions and mortality time series

INTRODUCTION A time series study of admissions, deaths and acute cases was conducted in order to evaluate the context of Chagas disease in Pernambuco. METHODS Data reported to the Information Technology Department of the Brazilian National Health Service between 1980 and 2008 was collected for regions and Federal Units of Brazil; and microregions and municipalities of Pernambuco. Rates (per 100,000 inhabitants) of hospitalization, mortality and acute cases were calculated using a national hospital database (SIH), a national mortality database (SIM) and the national Information System for Notifiable Diseases (SINAN), respectively. RESULTS The national average for Chagas disease admissions was 0.99 from 1995 to 2008. Pernambuco obtained a mean of 0.39 in the same period, with the highest rates being concentrated in the interior of the state. The state obtained a mean mortality rate of 1.56 between 1980 and 2007, which was lower than the national average (3.66). The mortality rate has tended to decline nationally, while it has remained relatively unchanged in Pernambuco. Interpolating national rates of admissions and deaths, mortality rates were higher than hospitalization rates between 1995 and 2007. The same occurred in Pernambuco, except for 2003. Between 2001 and 2006, rates for acute cases were 0.56 and 0.21 for Brazil and Pernambuco, respectively. CONCLUSIONS Although a decrease in Chagas mortality has occurred in Brazil, the disease remains a serious public health problem, especially in the Northeast region. It is thus essential that medical care, prevention and control regarding Chagas disease be maintained and improved.

Resposta atenuada ao PPD no diagnóstico de infecção tuberculosa latente em pacientes com artrite reumatoide

INTRODUCTION: With the introduction of Tumor Necrosis Factor Inhibitors (anti-TNFs) into rheumatological practice, it has become obligatory to identify cases of latent tuberculosis infection (LTBI) prior to the start of treatment, using PPD, chest radiography and clinical history of tuberculosis contact. Patients with Rheumatoid Arthritis (RA) have an abnormality of the cellular immune function, characterized by decreasing responsiveness of peripheral mononuclear cells (T Reg lymphocytes), leading to a loss in delayed hypersensitivity, which is fundamental for the recognition of antigens, such as PPD. OBJECTIVES: The purpose of our study was to evaluate the response to PPD in patients with RA, compared with healthy people, in an area where tuberculosis is endemic, as is the state of Pernambuco. METHODOLOGY: We studied 96 patients, 48 with RA and 48 healthy subjects, most of them females. All patients were given an interdermic injection of 0.1 mL PPD RT-23. The reading of the PPD result was carried out 72 hours after application, by way of palpation of maximum transverse diameter of induration, and the result was expressed in millimeters. RESULTS:In the RA group, the average time of diagnosis was 10.2 years, the average dosage of methotrexate was 15.5 mg / week, the average dosage of prednisone 12.7 mg / day and the average activity of the disease, measured using CDAI, was 30.4. In the healthy subjects group there was a greater number of positive PPD results (33.3%) when compared with the results for the RA group (14.6%), with a statistically significant difference (p = 0.034). CONCLUSION:The performance of PPB in LTBI diagnosis is poor in patients with RA. These results suggest that more careful screening needs to be undertaken before treatment with an anti-TNF drug.