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Tuberculosis: Diagnosis Will Be More Precise

11.11.2005


A diagnostic approach will allow to quickly and precisely identify the enemy - tuberculosis culture pathogene, the approach being based on the so-called subtraction hybridization. How it can help to identify ‘personality’ of a dangerous bacterium was discussed by researchers from Moscow with their colleagues at the II International Conference “Molecular Medicine and Biosafety” in late October this year.



For smatterers the notion of tuberculosis – is necessary and sufficient for definition of the disease and its pathogene, tubercle bacillus or, in other words, Koch’s bacillus, and has long ago lost romantic veil of Chekhov’s and Dostoyevsky’s hectic heroines. However, specialists know well that considerable genetic variability is typical of the Mycobacterium tuberculosis bacteria population. Simply speaking, these pathogenes may be very different. On the one hand, according to the force of influence on human beings: some pathogens are more, figuratively speaking, ‘wicked’ (virulent), others – are less wicked. It is necessary to know where the difference lies – what changes in the microorganism’s genome cause changes in its properties, including changes in its virulence.

Furthermore, tuberculosis pathogens, like cockroaches, are able to adapt themselves to the circumstances. If they are exterminated by people, part of them dies, but the remaining ones produce posterity resistant to the applied poison. In case of tuberculosis, this is becomes apparent in occurrence of cultures resistant to this or that kind of drugs. Therefore, to treat for sure physicians use several drug substances at once – they fight, so to say, through extended front. On top of the fact that the patient gets high doses of ‘redundant’ drugs, which are far from innocuous for a patient, as a result of such mass attack there appear cultures with multiple drug resistance, and this is a real headache for those who is trying to cure the disease.


To recognize what particular culture caused a certain patient’s disease, to study properties of this culture and, finally, to choose the most efficient and safe drug will be possible with the help of the method being developed under the guidance of Academician Evgeni Sverdlov by specialists of the Shemyakin and Ovchinnikov Institute of Bio-organic Chemistry (Russian Academy of Sciences) and their colleagues from the State Research Center for Applied Microbiology (Obolensk). Being concerned by tuberculosis attack on Russia and worldwide, the International Science and Technology Center, Ministry of Industry, Science and Technology, Russian Academy of Sciences (in the framework of the “Physicochemical Biology” program) and the Foundation of the President of the Russian Federation sponsored the researchers’ effort.

“The subtraction hybridization method is rather difficult for explanation, says Nikolai Gvozdevsky, one of the participants to the project. In a general way, the concept is that DNA molecules of the known and investigated culture are ‘clipped’ into parts in a certain way at full length, these parts are marked on both sides and they are mixed in a certain ratio. Then each part is divided into two parallel nucleotide sequences (DNA, as we know from times of Watson and Crick, is a spiral consisting of two complementary, i.e. fitting to each other in a unique way, nucleotide chains), and then these two untwisted parts are allowed to join again. Thanks to some experimental techniques, the results allow to identify which DNA sections distinguish one culture from the other and to single out these sections. A vast electronic card file already available for DNA sequences of some widespread mycobacterial cultures, for its part, may help to determine which section of the genome was found as a result and which protein product this gene (DNA section) may code.

Then the researchers use the found genomic sequences (unique for certain mycobacteria) for testing a large population sampling of pathogens for presence of these particular sequences. Unknown clinical isolate of M. tuberculosis may contain or not contain such a genetic marker in its genome composition. To answer the question “yes or no” allows another method called the PCR - polymerase chain reaction, perhaps one of the most widespread experimental approaches now in molecular clinical diagnostics. As a result of such analysis a set of final “yes” and “no” is obtained relating to all genes found in the course of subtraction hybridization, it allows like a binary code to group mycobacterial isolates and to recognize what particular culture got into the researcher’s test-tube.”

In the course of their investigation the researchers not only studied about two hundred clinical isolates educed from samples of patients’ phlegm, but also discovered three genes in the genome of one of the cultures, which were not present in the genomes of other cultures. These genes are interesting by the fact that they are typical of the isolate which is the most virulent of all. The researchers assume that these gene specifically are responsible for unusually high malicious ‘sociability’ of this culture – its ability to interact with the host organism and correspondingly its special danger (virulence) to the host.

Sergey Komarov | alfa
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