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Clinical Analyses

Easy diagnosis

In just a few minutes the equipment tests blood for microorganisms linked to 20 diseases

064-067_Multitestes_192_1DrümA diagnostic device capable of detecting up to 20 diseases in just a few short minutes is being developed by a consortium led by three research institutes based in the State of Paraná. Known as the multiplex diagnosis platform, the kit is to go into industrial production in 2014 at Lifemed, a company headquartered in the city of Pelotas (State of Rio Grande do Sul). Initially, the device will be used to diagnose HIV, cytomegalovirus, measles, syphilis, toxoplasmosis and hepatitis A, B and C in pre-natal exams in the Rede Cegonha (Stork Network), a Ministry of Health program to assist mothers and babies. The research for the development of the product is being led by the Carlos Chagas Institute (ICC) of the Oswaldo Cruz Foundation (Fiocruz), and by the Federal (UFPR) and Federal Technological (UTFPR) universities of Paraná, in the city of Curitiba, in addition to seven other institutions from other states, with articulation being provided by the National Institute for Science and Technology (INCT) for Diagnosis in the Public Health sector.

According to Marco Aurélio Krieger, a researcher at ICC and the coordinator of the project, the new equipment was developed based on two concepts, lab-on-a-chip (an entire laboratory contained in a disposable card) and point of care (of simple execution at medical practices or out-patient clinics). Therefore, it is portable, can be battery-powered and enables testing in medical clinics or even in remote locations. “Additionally, the device was developed entirely in Brazil,” explains Krieger.

According to Carlos Gadelha, Health Ministry Secretary for Science, Technology and Strategic Inputs, his department will invest roughly R$ 950 million over five years for the purchase of these kits. The number of such devices produced will rise progressively during this period. According to him, purchases by the Federal Government will lead to savings of more than R$ 177 million over the course of the five years and the unit price of the product should fall some 30% during this period. “In the first year (2014), about 2 million kits will be produced at R$ 30.40 each,” he explains. “In the last year (2019), 10 million units will be made at R$ 21.50 each.” These figures do not include normal taxes on the product, merely those relative to the payment of staff.

064-067_Multitestes_192DrümEssentially, the equipment consists of three parts: microparticles of polystyrene (the same material used to make styrofoam); a polymer disk, similar to a CD, which the researchers call a ‘chip’; and the equipment that makes it spin. The particles were developed by the team of the physicist Cyro Ketzer Saul, from the UFPR Physics Department. Each of these particles is roughly 10 micrometers in diameter (one micrometer equals one-millionth of a meter). “By means of chemical reactions, we coated the particles with the antigens of a specific pathogen, such as a virus or other disease-causing microrganisms,” explains Krieger. The antigen is a protein or part of a protein that is foreign to the organism, provoking an immunological response, with the formation of antibodies.

The second part, namely the chip or disk, was also developed at UFPR by Professor Wido Herwig Schreiner’s team. It is a small disk about three centimeters in diameter, with 40 “mini-wells” divided into two concentric circles, each of which has 20 such mini-wells. Each pair of these (one in the inner circle and one in the outer circle) is linked by a micro-conduit to the center of the disk. On the outer part of the disk, there is a border of absorbent material that the researchers refer to as the big diaper. The third part of the kit is a device developed at UFTPR by a group led by researcher Fabio Kurt Schneider, which makes the disk spin and produces the test results, showing whether a particular patient has the analyzed diseases.

The entire diagnosis process is simple. Thousands of micro-spheres with antigens for a specific type of disease are placed on each inner-circle mini-well. Antibodies that can recognize human antibodies are inserted in the 20 mini-wells of the outer circle. They will therefore identify any type that is linked to the antigens. Their job is to act as a control element. The test is carried out by placing a drop of blood in the center of the disk, which is inserted into the player, which spins it. As it spins, the blood flows along the micro-conduits into the mini-wells. If there is any antibody to a specific disease in the blood being tested, it will stick to the antigen to try to kill it. Thus, one finds out if the person whose blood is being tested has the disease. “However, for this to be visible, another protein is placed automatically in the center of the disk, which is then spun again,” says Saul. “This is called the reporter protein and it can stick to any antibody that has adhered to a particle with antigen.”

Simulation of the operation of the disk under ultraviolet light

SOURCE ICCSimulation of the operation of the disk under ultraviolet lightSOURCE ICC

The final step is to shine ultraviolet light onto the disk. The reporter protein will glow in the mini-wells with antibodies of both the outer control circle and of the inner circle with blood contaminated by a disease, and this image is picked up by a camera in the device. “The control circle plays an important role,” says Krieger. “If any of the mini-wells don’t glow, then the test is not valid. It is proof that there was some problem in its execution.”

So far, the project has undergone the laboratory bench-test stage, which proved its viability. “We already have what is known as proof of concept,” explains Saul. “We know that it works and how it works. Now we want to produce the equipment industrially.” Therefore, in January, an agreement was signed between Finep (the Studies and Projects Finance Agency), Fiocruz and the State of Parana’s Molecular Biology Institute (IBMP), for the release of R$ 8 million for the next phases of development, validation and registration of the kit. The ICC will coordinate this stage, to be conducted with the participation of two of Fiocruz’s other units, the Aggeu Magalhães Institute, based in the State of Pernambuco, and Rio de Janeiro’s Immunobiology Technology Institute (Bio-Manguinhos), besides UFPR, UTFPR and IBMP.

Prototype trials
This phase will also include the participation of Lifemed. According to its national commercial manager, Carlos Passos, first, the project will involve the company’s R&D division. “Based on an initial configuration and the characteristics that meet the Ministry of Health’s requirements, we will first develop a prototype,” he explains. “It will be laboratory bench-tested, before any tests whatsoever are done on patients. After this phase, which normally takes a long time, clinical trials will be carried out. Production as such is the final stage and is likely to occur in accordance with the contract with the Ministry of Health.”

There will probably be no lack of market for these kits. According to the figures from the Ministry of Health, roughly 3 million children are born a year in Brazil. Taking into account that 75.5% of the Brazilian population has no private health plan, it is estimated that about 2.3 million pregnant women depend on SUS (government’s Single Health System) for their check-ups and pre-natal exams. Also according to the Ministry, bearing in mind that the minimum number of check-ups in Brazil is four per pregnancy during the pre-natal phase, whereas the World Health Organization (WHO) recommends a minimum of six, and that at each check-up the recommended tests for HIV, measles, syphilis, toxoplasmosis and hepatitis B should be carried out, this yields some 9.1 million tests a year.

064-067_Multitestes_192_2DrümThere are no figures available as to the amount spent by SUS to carry out these tests, but taking into account that for the ministry’s other programs, an immunoassay test on the Elisa platform costs about R$ 2.00, one can estimate spending of about R$247.8 million a year on testing.

Other figures from the ministry also reveal the importance of investments in order to develop domestic health care technologies, particularly for to the pre-natal area. The figures show that in 2009, for instance, 19.4 million check-ups were carried out with pregnant women, reflecting a 125% increase in terms of access to healthcare services for pre-natal check-ups vs. 2003.

With the full implementation of the Rede Cegonha program in 2014, one of the objectives of which is to expand the access to healthcare of pregnant women, the Ministry of Health expects that there will be a marked increase in the number of appointments. For Gadelha, who is with the health ministry, agreements with Brazilian research institutions and companies for the development of products and technologies have a systemic innovation character, involving advances in new biotechnological approaches and efforts to produce medical equipment and devices in Brazil.

One of the project’s aims is to maximize the possible domestic content of the equipment in so far as possible. Devices such as the chip and the camera have to be imported. However, the other kit components, such as the proteins (antigens and antibodies), the polymer disk of the base of the chip, the polystyrene particles that bind with the proteins and the chip’s on-off switch should be made in Brazil. “In the case of the microparticles, Fiocruz already has a patent application underway,” says Saul. In addition to Lifemed, which is to produce the chip’s on-off switch as well as assemble and market the product, Fiocruz is supporting the setting up of spin-off enterprises to produce the plastic components that will be used to make the chip’s disk.