Luciamáre Perinetti Alves Martins

Caracterização biológica, histopatológica e análise de ácido nucléico de uma cepa Trypanosoma cruzi da região de Marília, SP

The aim of this report was to study the biological and histopathological behavior of a Trypanosoma cruzi strain, which is found in the region of Marilia. The strain was isolated in 1997, by artificial xenodiagnosis. Twenty-five swiss mice were intraperitoneally inoculated. Eleven were used for observation of parasitemia and trypomastigotes morphology and 14 were sacrificed after 17, 23, 30, 60 and 180 days post-infection. Heart, esophagus, liver, colon, and skeletal muscle (fragment of the right thigh) were collected for histopathological study. LIT culture medium was accomplished for DNA analysis. The results showed predominance of broad forms, low parasitemia with mean peaks of 860 trypomastigotes/5ml of blood of the 20th day of infection. No animal died in the acute phase of infection. Histopathological analysis showed several pseudocysts of amastigotes in heart, rare in skeletal muscle and colon with discreet inflammatory process. When Famema strain was compared with Y strain, which was isolated from a patient who lived in the same area, a distinct behaviour and biological characteristics were observed. However, DNA analysis placed them in same group, hence displaying the proximity of these strains.

Triatomíneos rupestres infectados por Trypanosomatidae, coletados em Quaraí, Rio Grande do Sul, 2003

Rupestrian triatomines were captured in six Quarai city localities, RS, to verify the level of Trypanosomatidae infection, as well as the animal reservoir. The capture occurred in a wild environment and 453 samples were collected, which were identified and separated by nymphal instar. 421 (92.9%) samples of Triatoma rubrovaria, 26 (5.7%) of Triatoma circummaculata and 6 (1.3%) of Panstrongylus tupynambai were collected. Only 13 samples (4.2%) of Triatoma rubrovaria presented Trypanosomatidae infection. After mice and LIT culture inoculation, five strains of Trypanosoma cruzi were isolated. Of these triatomines, 4 (30.8%) displayed no reagent precipitin for the antiserum tested, 4 (30.8%) were positive for rodent antiserum, 4 (30.8%) were positive for goat antiserum and 1 (7.7%) were positive for human and pig antiserum.

Susceptibilidade de Rhodnius neglectus, Rhodnius robustus e Triatoma infestans (Hemiptera, Reduviidae, Triatominae) à infecção por duas cepas de Trypanosoma cruzi (Kinetoplastidae, Trypanosomatidae) utilizando xenodiagnóstico artificial

The susceptibility of 3rd instar nymph of Triatominae Rhodnius neglectus, R. robustus and Triatoma infestans to Trypanosoma cruzi Y and AMJM strains was verified using artificial xenodiagnosis. After the accomplishment of the xenodiagnosis, the faeces of the Triatominae were analyzed on two-day intervals from day 5 until day 31 post infection, using the abdominal compression technique. The results showed differences in the susceptibility of the Triatominae for the two strains studied, and the optimal period reading differed from day 11 to day 19 for the Y strain and from day 11 to day 15 for the AMJM strain. For the Y strain, all three Triatominae species showed good susceptibility, whereas in the AMJM strain, the highest susceptibility was observed with R. neglectus, followed by T. infestans and R. robustus.

EFFECTS OF VITAMIN C SUPPLEMENTATION ON THE CHRONIC PHASE OF CHAGAS DISEASE

Introduction: In order to examine the effectiveness of vitamin C (ascorbic acid) in combating the oxidative insult caused by Trypanosoma cruzi during the development of the chronic phase of Chagas disease, Swiss mice were infected intraperitoneally with 5.0 × 104 trypomastigotes of T. cruzi QM1strain. Methods: Mice were given supplements of two different doses of vitamin C for 180 days. Levels of lipid oxidation (as indicated by thiobarbituric acid reactive substances-TBARS), total peroxide, vitamin C, and reduced glutathione were measured in the plasma, TBARS, total peroxide and vitamin C were measured in the myocardium and histopathologic analysis was undertaken in heart, colon and skeletal muscle. Results: Animals that received a dose equivalent to 500 mg of vitamin C daily showed increased production of ROS in plasma and myocardium and a greater degree of inflammation and necrosis in skeletal muscles than those that received a lower dose or no vitamin C whatsoever. Conclusion: Although some research has shown the antioxidant effect of vitamin C, the results showed that animals subject to a 500 mg dose of vitamin C showed greater tissue damage in the chronic phase of Chagas disease, probably due to the paradoxical actions of the substance, which in this pathology, will have acted as a pro-oxidant or pro-inflammatory.

Evaluation of antioxidant therapy in experimental Chagas disease

INTRODUCTION: Stimulation of inflammatory mediators such as cytokines and chemokines may cause oxidative stress in Chagas disease. In this study, we evaluated the merit of vitamins C and E as antioxidant therapy to minimize the oxidative stress-induced damage in an experimental model of Chagas disease. METHODS: Ninety-six Swiss mice were infected with Trypanosoma cruzi QM2 and treated with vitamins C, E, or both (C/E) for 60 and 120 days, and their effects compared to placebo administration were evaluated in the acute and chronic disease phases. RESULTS: There was no difference in parasitemia among treatment groups. However, histological analysis showed more severe inflammation in the skeletal muscle in the vitamin supplementation groups at both the acute and chronic phases. Biochemical analyses during the acute phase showed increased ferric-reducing ability of plasma (FRAP) and glutathione (GSH) levels in the vitamin C and C/E groups. In the chronic phase, a decrease in GSH levels was observed in the vitamin E group and a decrease in thiobarbituric acid reactive substances (TBARS) was observed in the vitamin C/E group. Moreover, there was a decrease in TBARS in the cardiac tissues of the vitamin C and C/E groups compared to that of the placebo group, although this level was greater in the vitamin E group than in the vitamin C group. CONCLUSIONS: The antioxidant action of vitamins C and E reduced oxidative stress in both the acute and chronic phases of Chagas disease, with a marked effect from joint administration, indicating their inherent synergism.

Biological and Molecular Characterization of Trypanosoma cruzi Strains from Four States of Brazil

Chagas disease affects between six and seven million people. Its etiological agent, Trypanosoma cruzi, is classified into six discrete typing units (DTUs). The biological study of 11 T. cruzi strains presented here included four parameters: growth kinetics, parasitemia curves, rate of macrophage infection, and serology to evaluate IgM, total IgG, IgG1, IgG2a, and IgG3. Sequencing of small subunit of ribosomal RNA (SSU rRNA)was performed and the T. cruzi strains were classified into three DTUs. When their growth in liver infusion tryptose medium was represented in curves, differences among the strains could be noted. The parasitemia profile varied among the strains from the TcI, TcII, and TcIII groups, and the 11 T. cruzi strains produced distinct parasitemia levels in infected BALB/c. The TcI group presented the highest rate of macrophage infection by amastigotes, followed by TcII and TcIII. Reactivity to immunoglobulins was observed in the TcI, TcII, and TcIII; all the animals infected with the different strains of T. cruzi showed anti-T. cruzi antibodies. The molecular study presented here resulted in the classification of the T. cruzi strains into the TcI (Bolivia, T lenti, Tm, SC90); TcII (Famema, SC96, SI8, Y); and TcIII (QMM3, QMM5, SI5) groups. These biological and molecular results from 11 T. cruzi strains clarified the factors involved in the biology of the parasite and its hosts. The collection of triatomine (vector) species, and the study of geographic distribution, as well as biological and molecular characterization of the parasite, will contribute to the reporting and surveillance measures in Brazilian states.

INTESTINAL AND PULMONARY INFECTION BY Cryptosporidium parvum IN TWO PATIENTS WITH HIV/AIDS

We describe two patients with HIV/AIDS who presented pulmonary and intestinal infection caused by Cryptosporidium parvum, with a fatal outcome. The lack of available description of changes in clinical signs and radiographic characteristics of this disease when it is located in the extra-intestinal region causes low prevalence of early diagnosis and a subsequent lack of treatment.

The enigmatic role of cholinergic reflex in the pathogenesis of Chagas disease

This study evaluated the inflammatory process in the colons of mice infected with Trypanosoma cruzi QM2 strain, through the analysis of muscle reactivity and the measurement of butyrylcholinesterase (BuChE) in plasma. “Swiss” mice were infected with T. cruzi QM2 strain and after 15 (G15), 30 (G30), 60 (G60), 90 (G90), and 210 (G210) days, each group had blood collected for the measurement of butyrylcholinesterase plasma concentrations ([BuChE]), a measure which functioned as an indicator of plasmatic Ach levels. All groups, except G15, had a segment of proximal colon removed to assess muscle reactivity to acetylcholine (Ach) and noradrenaline (NA) stimulation. After reactivity tests, the tissues were then fixed and stained with hematoxylin and eosin (HE) for histological evaluation of inflammatory response. The QM2 strain did induce inflammatory process in mice colon, and demonstrated differences in muscular contraction between the G60 and G210 groups, with p < 0.05. Plasma [BuChE] increased during the acute phase of infection (p < 0.05) with subsequent heterogeneous decline in the late chronic phase. These results show that the QM2 strain has tropism to the colon of mice and causes damage characteristic of megacolon; also, Ach has an enigmatic importance in the anti-inflammatory reflex over the course of T. cruzi infection.

A Highly Sensitive and Specific Conventional Molecular Diagnosis for Leishmania infantum Chagasi Based on a Single Copy Gene

Leishmaniasis are zoonotic diseases caused by a protozoa from Trypanosoma family and of the genus Leishmania, being transmitted by sandfly vectors. Leishmania genus comprise 30 species, including 20 species able to cause disease with different clinical manifestations in humans, ranging from asymptomatic, cutaneous and mucocutaneous lesions, to the severe visceral form. According to the World Health Organization, visceral leishmaniasis, caused by Leishmania infantum chagasi in the Americas, is the most severe form of the disease, and is lethal if not treated. Brazil is part of the group of countries with the highest prevalence of this disease, concentrating 90% of the cases registered in Latin America. A rapid and accurate diagnostic method is of great importance to detect and treat specifically L. i. chagasi in the American continent where others trypanosomiasis circulate, making a specific diagnostic difficult, due to cross reaction. In this work, a new conventional molecular diagnostic method was developed, based on the single copy L. infantum chitinase encoding gene, which presented high specificity and showed increased sensitivity when compared to the method based on the gene encoding the internal transcribed spacer 1 of the Leishmania rRNA (rDNA ITS-1) to diagnose L. i. chagasi on human clinical samples.

A highly sensitive Leishmania infantum chagasi isolation method from bone marrow and peripheral blood of adults and children

Dear Editor, Visceral leishmaniasis (VL) is the most severe form of a neglected tropical diseases group transmitted by phlebotomine sandflies harboring promastigotes forms of donovani complex Leishmania species with distinct eco-epidemiological patterns. The anthroponotic L. donovani in East Africa and India; and the zoonotic L. infantum in Europe and North Africa and in America after its introduction during European colonization, being named L. infantum chagasi [1]. Promastigotes regurgitated by sandfly vector into the mammalian host dermis are phagocytosed by monocyte cells lineage, replicating as amastigotes [2]. Infected macrophages migrate through lymphatic and vascular systems infiltrating lymph nodes (LN) bone marrow (BM), spleen (S) and liver, causing a strict fatal systemic disease, if untreated. According to World Health Organization last review on global VL incidence, 90% of 0.2 to 0.4 million cases per year, worldwide, occur in six countries: India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil (90% of American human cases); with a mortality rate of 10-20% [3]. In endemic and new affected areas, early and accurate VL diagnosis are shown to decrease mortality rates [4]. VL classical diagnosis correspond to microscopic examination of amastigotes in BM, LN and S cells smears, Giemsa or Leishman stained, obtained through aspiration; a painful, invasive and risky procedure, principally in children. Accuracy of this method depends on the tissue examined, the quality of reagents used and the technician identification ability [5]. Serologic and nucleic acid based VL diagnostic methods present several drawbacks like cross reaction with others American endemic trypanosomiasis including tegumentar leishmaniasis species [6]. Presence of macrophages in peripheral blood (PB) makes culture isolation suitable, as showed for children and adult patients infected with L. infantum from Mediterranean basin [7-9]. Most prominent L. infantum culture isolation techniques from PB presenting high sensitivity depends on sample washing with culture medium and concentration, or isolation of peripheral blood mononuclear cells (PBMC) [7,9], which are hard to be performed in laboratory routine, principally due to contamination risk during sample manipulation. Low efficiency of L. infantum isolation methods are probably related to immune complexes mediated parasite lysis [10,9]. Therefore, by using a BM and PB cells extensive saline washing approach, we obtained a 100% sensitive L. i. chagasi isolation method directly from adult and children patients samples.