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Identities revealed

Santa Catarina progresses with fluorescent trypanosome in the fight to reduce false diagnoses of Chagas's disease

A team from the Federal University of Santa Catarina (UFSC), in Florianópolis, is on the way towards solving one of the problems of the false diagnosis of Chagas’s disease: a mistake about the real causal agent of the problem. Even today, a harmless protozoon, the Trypanosoma rangeli can easily be mistaken for the real agent of the illness of Chagas, Trypanosoma cruzi, as it is transmitted by the same insects and causes similar response in the infected organism.

A false diagnosis, which is also originated by the use of non-standardized reagents or techniques, generates unnecessary treatment and increases expenditure on this public health problem: it is calculated that the costs of each 100,000 infected patients, including the medical treatment and the work absenteeism, exceed US$ 50 million a year. There are in Brazil some 8 to 9 million people infected, but the examination techniques in use do not make it possible to distinguish how many may have been contaminated by T. cruzi and how many by T. rangeli – and therefore who has the risk of developing the disease or not.

The mistake can be cleared up using a fluorescent trypanosome, which may lead to unequivocal diagnoses. The group of researchers coordinated by Edmundo Grisard and Mário Steindel implanted the gene of the Green Fluorescent Protein (GFP) from a jellyfish, Aequorea victoria, into a T. rangeli. When produced by other organisms, the protein causes an intense and stable green fluorescence. Afterwards, through a fluorescence microscope, the researchers track the path of the parasite in the organisms where they are lodged, and, little by little, establish the differences with T. cruzi.

At the beginning of March, this way proved to be the right one, by revealing an unknown detail of the cycle of the parasite in the main transmitting insect, the kissing bug (Triatoma infestans, Panstrongylus spp. or Rhodnius spp.). The team from Santa Catarina observed that one form of reproduction of the T. rangeli without an apparent flagellum (the extension of the cell membrane that helps movement), called amastigote, in the interior of the cells of the kissing bugs, are actually forms with flagella. The results contrast with those obtained with conventional microscopy, which indicated forms without a flagellum at this stage of the parasite. In kissing bugs, T. cruzi does not undergo similar stages.

The green protein was implanted in the genome of T. rangeli by means of a technique called transfection, which is beginning to be used more intensively in Brazil in the study of nucleated microorganisms, such as protozoa. In the UFSC, this technique also served to implant, this time in a T. cruzi, the gene of the beta-galactosidase enzyme, which produces a yellow, blue or red coloring, according to the technique used for detection.

This trick used in the tests of natural compounds against Chagas researched at the university itself: only the live parasites produce the enzyme, which quickly indicates if the candidate medicine is effective or not. Of the 150 or so compounds tested since last year, only two showed an effect against trypanosomes in laboratory cells. Both were extracted from shrubs, one from the Polygala genus (of the Poligalaceae family), and the other from the Trichilia genus (Meliaceae), and are now in the final stage of being characterized chemically.

Chagas’s disease, which in its serious or chronic form causes the progressive destruction of the cardiac or digestive tissues, affects about 18 million persons in the Americas – half of these are in Brazil. “Could they all be Chagas sufferers, or could we have a considerable number of persons infected by T. rangeli?”, Grisard questions. According to him, infection by T. rangeli affects, in a still not quantified way, the group of symptom-free carriers of Chagas’s disease (60% of the total), in particular those who live in the northern and northeastern regions, areas where there are kissing bugs that can transmit T. rangeli to man. The evolution of the disease facilitates the mistake.

After the initial or acute stage, when diagnosis is easier, infection by T. cruzi passes on to a chronic stage, in which it is very difficult to find the parasite. In this stage, called the indeterminate form of the disease, the person may be without any symptoms. From five to 30 years may elapse without the carrier showing any one of the characteristic forms of the disease, which still does not have an effective treatment.

Those infected by T. rangeli may remain all this time thinking that they have been hit by a disease that they actually do not have, or take medicine against a parasite that, although harmless for human beings, unleashes a response from the immune system that is regarded as identical, using the traditional methods, to the response set off by T. cruzi. This is called the serological cross reaction, which happens because the current routine techniques for blood tests do not differentiate the two parasites.

One of the ways of distinguishing between the two species is observing they way that they appear in the blood of the infected individuals, known as trypomastigote of the blood: T. rangeli is larger and longer and has a less voluminous organelle called kintoplast than T. cruzi. Only this form is very rare.The group from the UFSC reconciles the search for a methodology that avoids false diagnoses with the accurate study of the biology and the epidemiology of T. rangeli, the life cycle and its precise geographical distribution of which are hardly known – described in 1920 in Venezuela, it was put aside until research showed its overlapping of T. cruzi.

The sea of science
Added to these lines of research is the work with molecular markers, techniques like polymerase chain reaction (PCR), sequencing of nucleic acids (DNA and RNA, which define the genetic characteristics of an organism) and analysis of enzymes, with which specific targets are sought – an enzyme, a protein, a gene, anything, in short, that is exclusive to this parasite and that allows it to be detected unequivocally.

The objective is to arrive as soon as possible at something simple like a kit for testing malaria: a little strip that is put into the blood serum and takes on a characteristic color if the result is positive. This is the resource that was certainly dreamt of by the doctor from Minas Gerais, Carlos Ribeiro Justiniano Chagas (1878-1934), who in 1909 characterized the disease and took part in a number of scientific expeditions through the hinterland of the country. “With the target identified, developing a kit for diagnosis is becoming a feasible reality”, says Grisard, who comes from Santa Catarina and is used to checking his own routes. He has been a yachtsman since the age of six (he is now 36 years old), and in the rare intervals of time off, he takes the helm of ocean-going yachts, from 8 to 30 meters in length. He was one of Brazil’s representatives at the 1984 Olympic Games, in Los Angeles, in the United States.