Long term evaluation of etiological treatment of Chagas disease with benznidazole

The aim of this article is to present an investigation of cure rate, after long follow up, of specific chemotherapy with benznidazole in patients with both acute and chronic Chagas disease, applying quantitative conventional serological tests as the base of the criterion of cure. Twenty one patients with the acute form and 113 with one or other of the various chronic clinical forms of the disease were evaluated, after a follow up period of 13 to 21 years, for the acute, and 6 to 18 years, for the chronic patients. The duration of the acute as well as the chronic disease, a condition which influences the results of the treatment, was determined. The therapeutic schedule was presented, with emphasis on the correlation between adverse reactions and the total dose of 18 grams, approximately, as well as taking into consideration precautions to assure the safety of the treatment. Quantitative serological reactions consisting of complement fixation, indirect immunofluorescence, indirect hemagglutination, and, occasionally, ELISA, were used. Cure was found in 76 per cent of the acute patients but only in 8 per cent of those with chronic forms of the disease. In the light of such contrasting results, fundamentals of the etiological therapy of Chagas disease were discussed, like the criterion of cure, the pathogenesis and the role of immunosuppression showing tissue parasitism in long standing chronic disease, in support of the concept that post-therapeutic consistently positive serological reactions mean the presence of the parasite in the patient's tissues. In relation to the life-cycle of T. cruzi in vertebrate host, there are still some obscure and controversial points, though there is no proof of the existence of resistant or latent forms. However, the finding over the last 15 years, that immunosuppression brings about the reappearance of acute disease in long stand chronic patients justifies a revision of the matter. Facts were quoted in favor of the treatment of chronic patients.

Course of infection and histopathological lesions in mice infected with seventeen Trypanosoma cruzi strains isolated from chronic patients

Mice were infected with blood forms of 17 Trypanosoma cruzi strains recently isolated from chronic patients, which were dassified as of low, medium or high virulence on grounds of the prepatent period, parasitemia and mortality at the acute phase. A total of 212 mice were studied after 3, 6, 9 and 12 months of infection. In the chronic phase, intracellular parasites were detected in 11.0%,27.9%and 54.0,% of mice inoculated, respectively, with the low, medium and high virulent strains (r= 0.98, p < 0.005). Heart fibrosis was also related to virulence, affecting 5.7%, 11.6%and30.8% (r = 0.98, p < 0.001) of the mice inoculated with the above strains; a similar relationship was observed between intensity and frequency of the heart inflammatory reaction and the severity of infection at its early stage. Necrotizing arteritis was detected in 12.2% of the inoculated animals and this lesion was related to the infection duration rather than to strain characteristics. Inflammatory lesions and tissue parasitism were stable within the period of observation, whereas fibrosis was Progressive. The findings suggest that mice may reproduce heart lesions resembling human pathology and that organ damage apparently depends on the parasite virulence.

Biological behavior of Trypanosoma cruzi stocks obtained from the state of Amazonas, Western Brazilian Amazon, in mice

INTRODUCTION: The biological diversity of circulating Trypanosoma cruzi stocks in the Amazon region most likely plays an important role in the peculiar clinic-epidemiological features of Chagas disease in this area. METHODS: Seven stocks of T. cruzi were recently isolated in the State of Amazonas, Brazil, from humans, wild mammals, and triatomines. They belonged to the TcI and Z3 genotypes and were biologically characterized in Swiss mice. Parasitological and histopathological parameters were determined. RESULTS: Four stocks did not promote patent parasitemia in mice. Three stocks produced low parasitemia, long pre-patent periods, and a patent period of 1 day or oscillating parasitemia. Maximum parasitemia ranged from 1,400 to 2,800 trypomastigotes/0.1mL blood. Mice inoculated with the T. cruzi stocks studied showed low positivity during fresh blood examinations, ranging from 0% to 28.6%. In hemoculture, positivity ranged from 0% to 100%. Heart tissue parasitism was observed in mice inoculated with stocks AM49 and AM61. Stock AM49 triggered a moderate inflammatory process in heart tissue. A mild inflammatory process was observed in heart tissue for stocks AM28, AM38, AM61, and AM69. An inflammatory process was frequently observed in skeletal muscle. Examinations of brain tissue revealed inflammatory foci and gliosis in mice inoculated with stock AM49. CONCLUSIONS: Biological and histopathological characterization allowed us to demonstrate the low infectivity and virulence of T. cruzi stocks isolated from the State of Amazonas.

A role for extracellular amastigotes in the immunopathology of Chagas disease

In spite of the growing knowledge obtained about immune control of Trypanosoma cruzi infection, the mechanisms responsible for the variable clinico-pathological expression of Chagas disease remain unknown. In a twist from previous concepts, recent studies indicated that tissue parasitism is a pre-requisite for the development of chronic myocarditis. This fundamental concept, together with the realization that T. cruzi organisms consist of genetically heterogeneous clones, offers a new framework for studies of molecular pathogenesis. In the present article, we will discuss in general terms the possible implications of genetic variability of T. cruzi antigens and proteases to immunopathology. Peptide epitopes from a highly polymorphic subfamily of trans-sialidase (TS) antigens were recently identified as targets of killer T cell (CTL) responses, both in mice and humans. While some class I MHC restricted CTL recognize epitopes derived from amastigote-specific TS-related antigens (TSRA), others are targeted to peptide epitopes originating from trypomastigote-specific TSRA. A mechanistic hypothesis is proposed to explain how the functional activity and specificity of class I MHC restricted killer T cells may control the extent to which tissue are exposed to prematurely released amastigotes. Chronic immunopathology may be exacerbated due the progressive accumulation of amastigote-derived antigens and pro-inflammatory molecules (eg. GPI-mucins and kinin-releasing proteases) in dead macrophage bodies.

The search for new antimalarial drugs from plants used to treat fever and malaria or plants ramdomly selected: a review

In this review we discuss the ongoing situation of human malaria in the Brazilian Amazon, where it is endemic causing over 610,000 new acute cases yearly, a number which is on the increase. This is partly a result of drug resistant parasites and new antimalarial drugs are urgently needed. The approaches we have used in the search of new drugs during decades are now reviewed and include ethnopharmocology, plants randomly selected, extracts or isolated substances from plants shown to be active against the blood stage parasites in our previous studies. Emphasis is given on the medicinal plant Bidens pilosa, proven to be active against the parasite blood stages in tests using freshly prepared plant extracts. The anti-sporozoite activity of one plant used in the Brazilian endemic area to prevent malaria is also described, the so called "Indian beer" (Ampelozizyphus amazonicus, Rhamnaceae). Freshly prepared extracts from the roots of this plant were totally inactive against blood stage parasites, but active against sporozoites of Plasmodium gallinaceum or the primary exoerythrocytic stages reducing tissue parasitism in inoculated chickens. This result will be of practical importance if confirmed in mammalian malaria. Problems and perspectives in the search for antimalarial drugs are discussed as well as the toxicological and clinical trials to validate some of the active plants for public health use in Brazil.

Genitourinary changes in hamsters infected and reinfected with Trypanosoma cruzi

Authors describe genitourinary changes in male hamsters infected and reinfected with Trypanosoma cruzi. Changes in genital organs have been described in human and in experimental chagasic infection. Genital dysfunctions in chronic chagasic patients affect ejaculation, libido and sexual potency, and testis biopsies may show arrested maturation of germ cells, oligozoospermia and azoospermia. Sixty-five male hamsters were inoculated and reinoculated with 2x10³ trypomastigotes of T. cruzi VIC strain, and 22 non-infected animals constituted the control group. Animals were necropsied and fragments from testis, epididymis, seminal vesicle and bladder were collected and stained with hematoxylin-eosin. Peroxidase anti-peroxidase procedure was utilized to detect tissue parasitism. T. cruzi nests were found in testis, epididymis and seminal vesicle of these hamsters. Such parasitism plays a role in the origin of genital lesions observed in humans and laboratory animals during chronic chagasic infection.

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x10(5) "P" strain oocysts, GII, 5.0x10(6) "RH" strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.

5-Lipoxygenase plays a role in the control of parasite burden and contributes to oxidative damage of erythrocytes in murine Chagas` disease

Chagas` disease is accompanied by severe anemia and oxidative stress, which may contribute to mortality. In this study, we investigated the role of 5-lipoxygenase (5-LO) in the control of parasitism and anemia associated with oxidative damage of erythrocytes in experimental Trypanosoma cruzi infection. Wild-type C57BL/6, 129Sv mice treated or not with nordihydroguaiaretic acid (NDGA, 5-LO inhibitor), mice lacking the 5-LO enzyme gene (5-LO(-/-)) and inducible nitric oxide synthase gene (iNOS(-/-)) were infected with the Y strain of T cruzi. impairment of 5-LO resulted in increased numbers of trypomastigote forms in the blood and amastigote forms in the heart of infected mice. We assessed oxidative stress in erythrocytes by measuring oxygen uptake, induction time and chemiluminescence following treatment with tert-butyl hydroperoxide (TBH). Our results show that 5-LO metabolites increased lipid peroxidation levels in erythrocytes during the early phase of murine T cruzi infection. NDGA treatment reduced oxidative damage of erythrocytes in C57BL/6 T cruzi-infected mice but not in C57BL/6 iNOS-/- infected mice, showing that the action of NDGA is dependent on endogenous nitric oxide (NO). In addition, our results show that 5-LO metabolites do not participate directly in the development of anemia in infected mice. We conclude that 5-LO products may not only play a major role in controlling heart tissue parasitism, i.e., host resistance to acute infection with T cruzi in vivo, but in the event of an infection also play an important part in erythrocyte oxidative stress, an NO-dependent effect. (C) 2009 Elsevier B.V. All rights reserved.

In vivo infection by Trypanosoma cruzi: The conserved FLY domain of the gp85/trans-sialidase family potentiates host infection

Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas` disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 mu g/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7.6-fold), heart (3-fold) and small intestine (3.6-fold). Moreover, an intense inflammatory response and increment of CD4(+) T cells (1.7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4(+)CD25(+)FoxP3(+) T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas` disease.

Toxoplasma gondii: Evidence for the transmission by semen in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Dezoito bovinos foram inoculados com Toxoplasma gondii e distribuídos aleatoriamente em três grupos de seis bovinos cada: GI (2,5x10(5) oocistos da cepa P), GII (5,0x10(6) taquizoítos da cepa RH) e GIII (controle). Exames clínicos, sorológicos e parasitêmicos foram realizados. Pesquisas do parasito, por meio da bioprova e pela técnica de Reação em Cadeia pela Polimerase (PCR), foram realizadas no sêmen e em fragmentos de musculatura esquelética, linfonodos, cérebro, retina, baço, fígado, pulmão, testículo, epidídimo e vesícula seminal. Amostras de sangue e sêmen foram colhidas nos dias -2, -1, 1, 3, 5, 7, 14 e, semanalmente, até o 84º dia pós-infecção (DPI). Os bovinos inoculados (GI e GII) apresentaram hipertermia do 3º ao 16º DPI. Anticorpos contra T. gondii foram detectados (IFI) no 5º DPI (1:16), em ambos grupos inoculados (oocistos e taquizoítos), atingindo picos de 1:4096 no 7º DPI. Surtos parasitêmicos ocorreram em todos os bovinos infectados, principalmente do 7º ao 28º DPI, independente da cepa e inóculo utilizados. O bioensaio revelou a presença do parasito em amostras seminais dos bovinos infectados com oocistos (GI) e taquizoítos (GII), em diversas datas experimentais, entre o 7º e 84º DPI. Parasitismo tissular por T. gondii foi diagnosticado por meio da bioprova e pela técnica da PCR, em vários fragmentos de tecidos e/ou órgãos. Os achados sugerem a possibilidade da ocorrência da transmissão sexual do T. gondii na espécie bovina.

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n= 24) and asymptomatic (n= 14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p= 0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p= 0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p= 0.027) and was directly correlated with antibody levels (p= 0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas. © 2013 Elsevier B.V.

Aspectos da infecção toxoplasmática no sistema reprodutor de ovinos (Ovis aries) machos experimentalmente infectados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Transmissão congênita de toxoplasma gondii (Nicolle & Manceaux, 1909), em cabras experimentalmente reinfectadas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serological and infection statuses of dogs from a visceral leishmaniasis-endemic area

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)