Helen Rodrigues Martins

Development of a label-free immunosensor based on surface plasmon resonance technique for the detection of anti-Leishmania infantum antibodies in canine serum.

In this work, a surface plasmon resonance (SPR) immunosensor was developed using an 11-mercaptoundecanoic acid (11-MUA) modified gold SPR sensor chip for the detection of anti-Leishmania infantum antibodies. The soluble antigens of L. infantum were securely immobilized on an SPR gold disk by an 11-MUA self-assembled monolayer. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques were employed in the characterization of the antigen immobilization. After the immunosensor construction, canine serum positive for visceral leishmaniasis was added to its surface and showed significant variation in the SPR angle, indicating excellent sensitivity of the technique for antigen-antibody interaction detection. Moreover, the addition of negative serum was accompanied by a smaller response, demonstrating that the immunosensor shows good specificity against anti-L. infantum antibodies. Therefore, this work demonstrates the successful development of an SPR sensor for anti-L. infantum antibodies detection in short time, showing a great perspective as a sensing system of visceral leishmaniasis in endemic regions.

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment.

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1% of CDt patients (23/28) remained clinically stable and 95.4% of the INDt (21/22) and 33.3% of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2%/year and was especially low in INDt patients (0.5%/year) relative to CARD/DIGt patients (7.4%/year). Positive haemoculture in treated patients was observed in 7.1% of the cases. None of the INDt (0/21) and 33.3% of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2%, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7% of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution were selectively observed in the group of INDt patients and did not occur for CARD/DIGt patients.

Trypanosoma cruzi: Effect of benznidazole therapy combined with the iron chelator desferrioxamine in infected mice

Iron chelators have been employed in various studies aimed at evaluating the relationship between the iron status of the host and the development of infection. In the present study, the effects of benznidazole (BZ) therapy in combination with the iron chelator desferrioxamine (DFO) on the development of infection in mice inoculated with Trypanosoma cruzi Y strain have been investigated. Infected mice treated with DFO presented lower levels of parasitemia compared with infected untreated animals. Therapy with BZ for 21 days, with or without DFO, led to decreased parasitemia and reduced mortality, but BZ in combination with DFO treatment for 35 days (BZ/DFO-35) gave 0% mortality. All infected groups presented lower levels of iron in the liver, but serum iron concentrations were greater in DFO-35 and BZ/DFO-35, whereas hemoglobin levels were higher in BZ/DFO-35 and lower in DFO-35 compared with other treated groups. The percentage cure, determined from negative hemoculture and PCR results in animals that had survived for 60 days post-infection, was 18% for BZ and BZ/DFO-35, 42% for BZ combined with DFO for 21 days, and 67% for DFO-35. The results demonstrate that modification in iron stores increases BZ efficacy.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Spatial distribution of triatomines in domiciles of an urban area of the Brazilian Southeast Region

Reports of triatomine infestation in urban areas have increased. We analysed the spatial distribution of infestation by triatomines in the urban area of Diamantina, in the state of Minas Gerais, Brazil. Triatomines were obtained by community-based entomological surveillance. Spatial patterns of infestation were analysed by Ripley’s K function and Kernel density estimator. Normalised difference vegetation index (NDVI) and land cover derived from satellite imagery were compared between infested and uninfested areas. A total of 140 adults of four species were captured (100 Triatoma vitticeps, 25Panstrongylus geniculatus, 8 Panstrongylus megistus, and 7 Triatoma arthurneivai specimens). In total, 87.9% were captured within domiciles. Infection by trypanosomes was observed in 19.6% of 107 examined insects. The spatial distributions ofT. vitticeps, P. geniculatus, T. arthurneivai, and trypanosome-positive triatomines were clustered, occurring mainly in peripheral areas. NDVI values were statistically higher in areas infested by T. vitticeps and P. geniculatus. Buildings infested by these species were located closer to open fields, whereas infestations of P. megistus andT. arthurneivai were closer to bare soil. Human occupation and modification of natural areas may be involved in triatomine invasion, exposing the population to these vectors.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes.

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.

Applicability of a novel immunoassay based on surface plasmon resonance for the diagnosis of Chagas disease

BACKGROUND We defined the methodological criteria for the interpretation of the results provided by a novel immunoassay based on surface plasmon resonance (SPR) to detect antibodies anti-Trypanosoma cruzi in human sera (SPRCruzi). Then, we evaluated its applicability as a diagnostic tool for Chagas disease. METHODS To define the cut-off point and serum dilution factor, 57 samples were analyzed at SPRCruzi and the obtained values of SPR angle displacement (ΔθSPR) were submitted to statistical analysis. Adopting the indicated criteria, its performance was evaluated into a wide panel of samples, being 99 Chagas disease patients, 30 non-infected subjects and 42 with other parasitic/infectious diseases. In parallel, these samples were also analyzed by ELISA. RESULTS Our data demonstrated that 1:320 dilution and cut-off point at ∆θSPR=17.2 m° provided the best results. Global performance analysis demonstrated satisfactory sensitivity (100%), specificity (97.2%), positive predictive value (98%), negative predictive value (100%) and global accuracy (99.6%). ELISA and SPRCruzi showed almost perfect agreement, mainly between chagasic and non-infected individuals. However, the new immunoassay was better in discriminate Chagas disease from other diseases. CONCLUSION This work demonstrated the applicability of SPRCruzi as a feasible, real time, label free, sensible and specific methodology for the diagnosis of Chagas disease.

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

INTRODUCTION The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. METHODS Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. RESULTS The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2 ± 0.25 to 13.7 ± 3.08 days), whereas the patent period ranged from 3.3 ± 1.08 to 34.5 ± 3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. CONCLUSIONS The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis.

Genetic modulation in Be-78 and Y Trypanosoma cruzi strains after long-term infection in Beagle dogs revealed by molecular markers.

The genetic profile of Trypanosoma cruzi was evaluated in parasite populations isolated from Beagle dogs experimentally infected with Be-78 and Y strains that present distinct biological and genetic characteristics. Molecular characterization of the isolates obtained 30days and 2years after infection was carried out. For typing MLEE, sequence polymorphisms of the mitochondrial cytochrome oxidase subunit II gene (COII) and RAPD profiles were used. The profiles of MLEE were the same for the parental Be-78 strains as their respective isolates. However, changes of MLEE profile were observed in two T. cruzi isolates from dogs inoculated with Y strain. Changes in the mitochondrial DNA (COII) and RAPD profiles of the Y strain were also observed. The dendogram constructed by UPGMA with RAPD results indicated two major branches. Global data show that the genetic modulation in polyclonal strains during the long-term infection occurred and was strain-dependent. This study still suggests that each host (here each dog) harbors a determinate T. cruzi population that may change or be modulated throughout long-term infection. This might to hinder the observation of correlation between the genetics of T. cruzi and their biological properties and behavior in different host species due to the complexity of the parasite-host interaction in which probably the genetic background of both should be considered.