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CHAGAS DISEASE

The doors are still open for the reduviid bug

Wild species are not fought against, but they transmit the parasite

There are reasons in abundance to be worried in relation to Chagas disease, a sickness still incurable, typical of developing countries which infects between 4 and 5 million people in Brazil and between 16 and 18 million in all of Latin America. It is true that there have been many advances. Campaigns which took place during the 80s by the National Health Foundation (Funasa in the Portuguese acronym) reduced by 90% the spread of the disease in Brazil, through the control of the population of the reduviid bug – which is known as barbeiro in Portuguese and also as kissing bug in English – the transmitting insect for the protozoan which causes Chagas sickness, the Trypanosoma cruzi. Now it is being forecast for 2001 the total eradication of the household reduviid bug.

It’s not enough. Contrary to the euphoria which is growing as we seemingly approach the apparent end of the transmitter of Chagas disease in some countries such as Uruguay, Chile, Argentina and Brazil, scientists recognize the success of the campaign, the fastest and cheapest form of epidemiological control. However, they warn: progressive abandon of the combat of the bugs will leave the door open for the disease to return and for the insect to again infest homes. Another reason for concern is that species of the wild “kissing bug”, capable of transmitting the parasite – which were not eliminated by the agents of Funasa – are coming closer and closer to homes.

In February of this year, at a congress at the London School of Hygiene and Tropical Medicine, in London, the biologists Bianca Zingales, of the Chemical Institute of the University of São Paulo (USP), and Marcelo Briones, of the Federal University (Unifesp) of São Paulo, informed that actually there are two, and not just one, strains of Trypanosoma cruzi which cause the sickness. One strain – the more aggressive – is associated with humans, and the other with wild animals (see box). In one way, this conclusion complicates the situation by indicating that there is an enemy which up until now hid in the shadow of the other. On the other hand, it answered a question which had always intrigued scientists: how could a single species, the T. cruzi, provoke symptoms so different of the disease?

Described in 1909 by the sanitary doctor, from the state of Minas Gerais, Carlos Ribeiro Justiniano Chagas (1879-1934), who named the parasite in honor of the doctor Oswaldo Cruz (1872-1917), the disease of Chagas presents an acute phase with high fever, an increase in the spleen and even alterations in the heartbeat. Afterwards it makes a truce – of up to 30 years. Between 60% and 70% of those infected don’t present any symptoms. Between 20% and 30% of the sick develop a heart problem with the risk of sudden death; between 8% and 10% suffer from a dilation of the esophagus or intestine; and 3% present complications in the nervous system.

The only drug presently available, benznidazole , is extremely toxic and only resolves the complications in the acute phase of the disease. To close the circle of combat against T. cruzi, it is not enough, however, to eliminate the reduviid bug. One needs to better understand the parasite and its mechanisms of interaction with the organism, and even more, to insist on the development of new drugs. This is not easy. The researchers themselves recognize that T. cruzi is “a dangerous and intelligent enemy” which manages to invade the cells of hosts without facing any resistance. Worse still: it is welcomed and protected in the interior of the cell where it then multiplies.

To penetrate the tissue of the human organism, the parasite needs to recognize the surface of the cell and at the same time to be itself recognized. For this, it uses mechanisms so subtle that at times it seems to be an invited guest. “The T. cruzi will only survive if it meets a cell, penetrates it and stays there”, explains the medical doctor Walter Colli, of the Chemical Institute of USP, who has been studying the relationship between the parasite and the host cell since the seventies. According to him, discovering how the protozoan enters the cell and how it survives, it would be possible to identify the metabolic pathways which will permit the development of inhibitor drugs for the processes of recognition, adhesion or invasion, on both sides and thus cause the death of Trypanosoma cruzi.

Each time closer
The biochemical parasite laboratory, co-directed by Colli and by the biologist Maria Júlia Manso Alves, discovered a new family of molecules called GIPLs or glycoinositolphospholipideos. They exist in the membrane of parasites in large quantities and are similar to the so-called ‘anchors’, which insert proteins into the membrane cells. By understanding the composition of the membrane of the parasite, it may be easier to understand the chemical reactions which occur when a cell – protozoan – meets another cell of the human body.

Júlia says that they are very close to working out the mechanisms of adhesion and of penetration used by the parasite as the result of the project Trypanosoma cruzi: Interaction Parasite – Host, which has had R$ 480,000 granted by FAPESP to the laboratory of the two researchers during the past four years. It is not the first time that this laboratory has contributed to the combat of the Chagas disease.

During the 80s, Júlia discovered a family of glycoproteins which she named Tc-85, also associated with the entry of the parasite into the cell. Recently identified, in one of the members of the family Tc-85, a fragment which could be blocked to inhibit the cellular adhesion. In other words, Júlia now knows that there is a receptor in the host cell which recognizes a molecule of T. cruzi and allows the parasite to install itself in the organism. She hopes within one year to conclude the work to which she is dedicated, the final mapping of Tc-85.

A DNA vaccine
Little by little the pieces of the intricate jig saw puzzle of the disease of Chagas are falling into place. At Unifesp the group led by the biochemist Nobuko Yoshida managed to advance in the identification and characterization of the molecules involved in the cellular signaling in the relationship parasite to host. One of them present in a specific form of T. cruzi found in the reduviid bug is the gp82, a glycoprotein (protein which contains sugar) which seems to take part in the process of the entrance of the parasite into the cell.

This is one of the results of the project Aspects of Immunobiology of Trypanosoma cruzi: Interaction with Cells of Mammiferous Hosts and Induction of an Immune Response, which began last year and will continue until 2003, with financing of R$ 350,000 and a further US$ 300,000 by FAPESP. The group led by Nobuko managed as well to demonstrate that immunization with DNA is capable of conferring resistance against the T. cruzi and leads to the destruction of the parasite. Besides the production of specific antibodies, this immunization stimulates the cells of the immune system and produces interpheron-gamma, a substance which carries out an important role in the control of infection. The team hopes that these studies help in making possible a vaccine which will improve the treatment of the disease and prevent those who are sick from developing its more serious form.

However, the development of a vaccine and of medicines does not depend solely of scientific findings. There is also the need for a jolt, according to the researchers, in the disinterest of the pharmaceutical industry to produce and sell medicines which would be directed basically to the poorer population of developing countries. The predominant thinking is that, in relation to other diseases such as malaria and dengue fever, Chagas’s disease does not justify the high investments with the production of new chemotherapy..

Infested municipalities
There remains, however, the more practical road: the permanent control of the reduviid bug, considered the “best vaccine”. “Experience accumulated since 1950 demonstrates that Chagas disease transmission can be halted through the adequate use of insecticides, along with improved housing and education”, observes Laboratory of Triatomines and Epidemiology of Chagas Disease of the René Rachou Institute of Belo Horizonte (MG).

Even so, the control of the reduviid bug cannot have a final date, alerts the researcher. According to her, this work should consider two epidemiological situations. The first involves native species of the kissing bug , coming from a wild environment – caatinga ( the semi-arid vegetation of the northeast of Brazil), rain forests, and Cerrado ( wooded savanna) for example – which present a huge capacity of adaptation to the environment constructed by man. These species, which already live around houses, are always trying to colonize new areas. Thus, they can set up new transmission centers.

The second situation is with respect to areas infested by Triatoma infestans, one of the species of kissing bug adapted to houses. T. infestans probably originated in the Andean region of Cochabamba in Bolivia, from where it spread to Peru, Chile, Paraguay, Argentina, Uruguay and Brazil. Away from its natural habitat, it lives exclusively in houses and maintains a very close relationship with man and his domesticated animals.

His team accompany as well the occurrence of other species of the kissing bug insect throughout the country. “We know that today there are human transmitters of Chagas disease in the hinterland of Ceará, by way of Triatoma brasiliensis.” said Liléia. “We checked out as well the infestation by Triatoma pseudomaculata on the outskirts of the city of Sobral, the second largest city of Ceará.”

The specialists recommend: the control of the transmitter must be carried out inclusive in areas where there are recent cases, such as in the Amazon. At the age of 70, 42 of them dedicated to the epidemiology of the disease of Chagas, José Rodrigues Coura, Director of the Institute Oswaldo Cruz (IOC), of Rio de Janeiro, tells that in the region North, in consequence of deforestation, the wild reduviid bug is immigrating to houses and placing at risk the health of those living inside. To give a dimension to the risk of the adaptation of the reduviid bugs to households, Coura built wooden small houses and with thatched roofing in the middle and in high Negro river.

He left the houses unoccupied, returning some time later and found wild reduviid bugs. His work showed that the potential center is the community of the piaçaveiros , makers of ropes from vegetable fibers, which had already been attacked by the wild bug. It is worth remembering that a single insect can create a colony in a house, since each female can lay around 400 eggs throughout its lifetime.

Coura didn’t stop there. “If the remaining centers are not eradicated in the states of Bahia, Tocantins, Minas Gerais, Pernambuco, Piauí and Rio Grande do Sul, where there are more than 100 municipalities infested with the reduviid bug, the disease could come back as it has happened with dengue fever and malaria”, he says. In other words, it is possible to lose what has already been done in the controlled areas.

Genes reveal new lineage

Bianca Zingales and Marcelo Briones worked intensely during the 90s, researching the biological, biochemical and phylogenetic (relative to the evolution history of the species) differences between the strains or lineage of Trypanosoma cruzi. In the end, they concluded that Chagas disease may have different origins. The existence of different groups of T. cruzi have already been suspected, but they were the ones who verified what the other researchers had tried and failed before them.

During the 80s, the English professor Michael Miles, of the London School of Hygiene and Tropical Medicine, gave them a lead when he analyzed some strains of T. cruzi and suggested that they might be divided into two groups. In 1993, it fell to the doctorate student, Ricardo Peres do Souto, at that time 26 years of age, a postgraduate at Chemistry Institute of USP, and advised by Bianca, to shift the direction of the facts. On analyzing 16 strains through PCR (polymerase chain reaction) Souto obtained only two amplification patterns of a region of the gene of RNA ribosome, a material considered reliable for phylogenic studies.

“The sequencing of these genes permit us to establish the history of the species”, instructs Bianca. It was the missing step. Ricardo Souto thus confirmed the hypothesis of Miles, the first to suggest the presence of two groups. In 1996, studies carried out by Fiocruz and the University of California, in Los Angles, established indefinitely the presence of two phylogenetic lineages through the analysis of a larger sample of 90 strains, isolated from human and wild kissing bugs from five South American countries.

These results come from two projects, the Genome of Trypanosoma cruzi: Optical Molecular Cariotype, Gene Mapping and Genic Disruption and Phylogenetic Lineage of Trypanosoma cruzi: Epidemiological, Evolutionary and Biochemical Characterization, which relied upon, respectively, financing of: R$ 26,800 and R$ 58,000 from FAPESP. Since March of 1998, Bianca and Briones – with the help of two specialists of the Oswaldo Cruz Foundation (Fiocruz), José Rodrigues Coura and Octavio Fernandes – analyzed this time close to 300 insect stocks coming from 12 Brazilian states.

What they observed was that one strain is associated with humans, and the other with wild animals. Each one of them is associated respectively with the two cycles of the sickness, one domestic and the other wild. The linkage between one and the other is made when the wild kissing bugs come into homes and bring the parasite into the home. “In patients of endemic zones there has been an elevated predominance of one strain, the T. cruzi 2, with a high incidence of heart sickness”, tells Bianca “On the other hand, patients in the Amazon, which is not an endemic region, have T. cruzi of a lineage which is milder, namely the T. cruzi 1.” The analysis of insect stocks from counties such as Colombia, Venezuela, Bolivia and Mexico confirm the association of the two lineages.

The picture became more clear last year through a study carried out in the Atlantic Rain Forest: all the 26 samples of T. cruzi collected from infected armadillos and golden lion tamarin were of the same lineage, the same one found in human strains, whereas 85% of the skunk were effected with the lineage related to the wild cycle. There are already kissing bugs with the origins of T. cruzi of the two lineages.

The result was clear: there was a preferential association of the lineage T. cruzi 1 with a determined class of mammals, the skunks (very primitive marsupials), while the other, the T. cruzi 2, was associated with primates, placental mammals – and both were present in the wild cycle. However: the two lineages presented different epidemiological characteristics. One associated with the disease is much more virulent. The other causing an indeterminate form of the disease.

What came afterwards was even more fascinating. Marcelo Briones, after sequencing genes of RNA ribosomes of trypanosomes, interwove the evolutionary history of the two species and concluded: both had a common ancestor between 88 million to 40 million years in the past. According to him, an ancestor of T. cruzi had spread widely 250 million years in the past when all the continents were linked.

When the Americas separated, between 170 and 100 million years ago, the two lineages of T. cruzi also separated: one, the T. cruzi 1, remained in South America, infecting predominantly marsupials, and the other, in North America. living with the placental mammals. Between 2 and 5 million years ago the two Americas again came together via the isthmus of Panamá. The result: there was a monumental movement of animals from one place to another. With the invasion of placental animals coming from the North, arrived the lineage of the parasite T. cruzi 2, and both get mixed.

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