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Genetics

Fine sieve

Fiocruz test detects slight infections of schistosomiasis

Thirty years after a group of researchers from Minas Gerais created the methods for detecting schistosomiasis that has been adopted all over the world, another team, also from Minas Gerais and from the same institution, has found a way of identifying slight infections, which elude the technique in use. Researchers from René Rachou Research Center of the Oswaldo Cruz Foundation (Fiocruz) in Belo Horizonte have developed a DNA (deoxyribonucleic acid) diagnostic test that registers traces of Schistosoma mansoni, the worm that causes the disease, ten times more precisely – in samples with up to two eggs per gram of feces, while the usual method only detects something when there are 24 eggs or more per gram.

In the first field test, carried out with 194 inhabitants from Comercinho, an endemic area for the disease in the Jequitinhonha Valley, in the north of the state of Minas Gerais, the new test showed a sensibility of up to 97% and showed signs of the parasite in samples where the three tests of feces carried out by the traditional technique – counting the eggs using microscopy – had not registered anything. The researchers also used the conventional technique, and, as a standard for comparison, of another 20 samples of feces from inhabitants of Belo Horizonte not exposed to situations of risk and, in theory, free from the worm.

The exam gave a negative result for only two cases where there were traces of the worm, which had been detected by the traditional technique. “The DNA may have deteriorated while being transported to the laboratory, or substances may have appeared that inhibit its amplification in the PCR (polymerase chain reaction, a technique that copies specific strands of the genetic material)”, ponders Ana Rabello, a doctor at Fiocruz and one of the authors of the new test. The result does not invalidate what for her is the clearest indication for the application of the new kind of diagnosis: the cases in which the feces test fails.

The authors of the new method believe that the two approaches may be complementary and allow a wider ranging treatment of schistosomiasis, known popularly in Brazil as ‘water belly’, which affects 10 million people in Brazil, above all in the rural areas. Transmitted by Biomphalaria glabrata snails, the disease affects 200 million people in the world – and 20 million of these develop a severe clinical condition, with irreversible damage to the liver and the gall bladder. “For the time being, it is impossible to eradicate schistosomiasis”, Ana Rabello comments. “With the methods of diagnosis and treatment in use, we have managed to reduce by as much as 70% the number of persons infected”. No matter how much the contaminated individuals are medicated, total elimination is impossible, because transmission is carried on by unidentified or uncured cases.

Now, by detecting slight infections, the hope is to take a leap forward in combating the disease, identifying up to 90% of the people who really are infected – with luck, up to 100%, depending on the quantity and the intensity of the infection, in an endemic area. Described in an article published in February in the American Journal of Tropical Medicine, the method may also be applied to blood samples, but it is neither simple nor cheap. While the common exam is done with an optical microscope, which can be carried to the field and is ready on the spot, the new test, which uses PCR in the laboratory, starts with the extraction of the parasite’s DNA from the human feces, and ends when it is observed in a sort of radiography if the sample analyzed displays a sequence of nucleotides (adenine, guanine, cytosine and thymine) specific to Schistosoma.

This sequence, which has 120 nucleotides, is a sort of fingerprint of the worm. Who made the discovery was also from Minas Gerais – Emmanuel Dias-Neto – a molecular biologist who is currently working at the Institute of Psychiatry of the School of Medicine at the University of São Paulo (USP). From that point, Dias-Neto set up a smaller sequence, with 20 nucleotides, called aprimer , which fits that specific region of the worm’s DNA. It was left to biologist Luís André Pontes, during his studies for a doctorate under the supervision of Ana Rabello and Dias-Neto, to perfect the method. It would be easier to develop this today, with the sequencing of the worm’s genome, now at a very advanced stage (see Pesquisa FAPESP nº 77).

Pontes has been testing the primers since 1996, until reaching the final version and verifying that the DNA used would not be mistaken with the DNA of other worms that are common in human feces. With the same test, the researchers recently succeeded in detecting fragments of other species, such as Schistosoma haematobium and S. japonicum, which infect man in Africa and Asia. The sequencing of the DNA fragments of these species, now under way, makes it possible to imagine even more sensitive tests, capable of differentiating between each one of them.

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