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Clinical analysis

On the trail of the Trypanosome

With greater precision, new test detects presence of antibodies of Chagas's disease in the blood

MIGUEL BOYAYANElectrode immobilizes protozoon’s antigensMIGUEL BOYAYAN

Researchers from the São Paulo State University (Unesp) in Araraquara and from the University of São Paulo (USP) have just developed an unprecedented methodology for the serological diagnosis of Chagas’s disease, an ailment that affects 6 million Brazilians and another 10 million persons in the Americas. To make a diagnosis, the main advantage of the method, which uses an amperometric immunosensor (the result appears in the reading of the electric current), is its precision: the device detects in blood serum very small quantities of antibodies of the parasite that causes the illness, the protozoon Trypanosome cruzi .

These antibodies are produced by the person’s organism when infected. Studies carried out by the researchers from Unesp and USP have shown that the new methodology is far more sensitive and precise than the test called Elisa (Enzyme Linked Immunosorbent Assay), widely used for this kind of diagnosis.

Another advantage of the new test is that it is processed quickly. The analysis takes some 40 minutes, a far shorter time than the conventional serological tests. Done on a polystyrene board, the Elisa immunoassay takes up to two hours. The new processing can also be done at room temperature, while its rival kits have to be processed in a sterilizer. Moreover, the use of the immunosensor in serological tests facilitates repetition, in the case of doubtful samples.

“The immunosensor will be an important public health instrument in countries where the disease is endemic, explains chemist Hideko Yamanaka, a professor from Unesp’s Chemistry Institute and the coordinator of the team of inventors. Besides being able to be used in clinical analysis laboratories for diagnosing patients, the new method will also be able to be used in blood banks. According to Hideko, the immunosensor will bring greater agility in the screening of donors, reducing the cases of the ailment being transmitted by transfusion.

The other members of the team that developed the immunosensor are biochemist Antonio Aparecido Pupim Ferreira, who is studying for a doctorate under the supervision of Hideko; Professor Walter Colli, from USP’s Chemistry Institute; and biomedical specialist Paulo Inácio da Costa, from Unesp’s School of Pharmaceutical Sciences. The last two took part in the development of the immunosensor as collaborators. The researchers’ estimate is that the new technology will be launched at a price similar to the commercial Elisa kits.

It will, however, have a great comparative advantage: the commercial kit has a board with 96 tests, and once the packaging is opened, the ones that are not used have to be thrown away. “In our case, we will be able to configure the immunosensor in the way that is convenient to meet the demands of the laboratories”, says Hideko. This will make it possible for the unitary price of the immunosensor to be lower than Elisa.

As a result of its unprecedented nature, the new technology was registered with the National Institute for Industrial Property (INPI), with support form FAPESP’s Nucleus for Patenting and Licensing Technology (Nuplitec). With this, negotiations have now started with industrial concerns that supply kits for clinical analysis laboratories for them to produce the new test. “The talks are at a very preliminary stage. As the majority of these companies are multinationals, we are applying for an international patent for the immunosensor, to facilitate negotiations”, explains Hideko.

Electrochemical transducer
The amperometric immunosensor developed by the researchers was chosen as the second most important work at Unesp’s 1st Technology Display, held between October 28th and 30th in São Paulo. Something that called attention was the fact that the device is based on the interaction between antigen and antibody, in the form of a biosensor with an active biological component (T. cruzi antigens) closely coupled to the surface of a transducer which converts a biological signal into an electrical signal.

To create the new test, the researchers used an electrochemical transducer (or electrode) that is available on the market, and normally used in chemical analyses of several substances in the biological, clinical and industrial areas, besides being used in the monitoring and control of some pollutants of the environment. “Our work consisted of making some modifications to this electrode, altering its surface chemically, for it to be capable of immobilizing the protozoon’s antigens and to become selective for the anti-Trypanosoma cruzi antibodies“, says pharmacist-biochemist Antonio Pupim Ferreira. “That is to say, to have the capacity to react with the antibodies present in the serum of the victims of Chagas’s disease”.

Illness transmitted in transfusions

Described in 1908 by the Brazilian scientist Carlos Chagas, Chagas’s disease, also know as American trypanosomiasis (the terminology adopted by the International Classification of Diseases – ICD) continues without any means for a definitive cure and without any vaccines. It is an infectious parasitic disease caused by the Trypanosoma cruzi protozoon, transmitted by hematophagous insects of the Triatoma infestans species, popularly known as kissing bugs. Nowadays, transmission occurs mainly in blood transfusions.

“According to the World Health Organization (WHO), vectorial transmission has been controlled as a result of successful public policies in the countries affected by the ailment”, explains Professor Walter Colli, from USP. The actions are concentrated in works of basic sanitation, in combating the insect that transmits it, and in improving housing in the countryside, as the kissing bug usually hides in cracks in clay or wooden walls.

Classic transmission happens when the kissing bug bites a person who has been infected by the disease. The insect then becomes a host for the parasite. Next, feeding on the blood of a healthy person, it defecates and deposits the protozoon on the victim’s skin, and when this is scratched, the parasite is taken into the blood stream.

Unless it is quickly diagnosed, still in the acute stage, when there is a cure, the ailment enters into the chronic stage, with the Trypanosoma installing itself in the muscles, in particular in the heart, and bringing about cardiac insufficiency and arrhythmia, which can cause death. Another means of infection occurs in transmission of mother to child, during pregnancy. And the only way of diagnosing the protozoon’s presence in the organism is by means of blood tests.

The project
Immunosensor for Chagas’s Disease (nº 01/14397-2); Modality Intellectual Property Support Program (PAPI); Coordinator Hideko Yamanaka – Chemistry Institute at Unesp Araraquara; Investment R$ 12,000.00