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Detection of Mycobacteria Using Chromatography

#K-1427


Detection and Identification of Mycobacteria Using Chromatography. The Study of the Peculiarities of Composition, Profile of Fatty and Mycolic Acids of Drug-Resistant M. Tuberculosis

Tech Area / Field

  • MED-DID/Diagnostics & Devices/Medicine
  • BIO-MIB/Microbiology/Biotechnology

Status
3 Approved without Funding

Registration date
25.05.2006

Leading Institute
Scientific and Analytical Center Biomedpreparat LLC, Kazakstan, Stepnogorsk

Supporting institutes

  • "Romat" Pharmaceutical Limited Company, Kazakstan, Pavlodar reg., Pavlodar\nKazakhstan National Medical University, Kazakstan, Almaty

Collaborators

  • Centers for Disease Control and Prevention (CDC) / National Center for Infectious Diseases, USA, GA, Atlanta

Project summary

Aim of the project. 1) The investigation by chromatography methods of the details of composition of fatty acids and mycolic acids profile of drug-susceptible and drug-resistant M. tuberculosis strains isolated from patients in the Republic of Kazakhstan. 2) The search for new compounds, inhibiting the synthesis of mycolic acids and the investigation of its influence on structure of components of lipid membrane. 3) Elaboration of standardized methods for the detection, identification and typing of Mycobacterium species circulating in Kazakhstan by chromatography methods and its harmonization with internationally accepted methods.

State of affairs in the field of researches and influence of the proposed project on the progress in this field

During the last decade tuberculosis again has become one of the important public health problems in the world. According to the data of experts of the WHO 1/3 of the world population is infected by M. tuberculosis, 5-10% of them will fall ill with one of the tuberculosis forms during their life. Tuberculosis caused by multidrug-resistant bacteria is also increasing; the acquired forms of drug- resistance are 60%. The problem of tuberculosis is intensified by the development of HIV-infection epidemics. Epidemiological situation on tuberculosis in Kazakhstan during last decade is bad and has a tendency to change for the worse. Kazakhstan according to the data of European bureau of WHO occupies the leading place in the world on the detected tuberculosis cases for the first time and has high mortality rates. Patients may be infected not only with multiple strains of M. tuberculosis but also with other species of Mycobacterium. The data on infections with mycobacterium other than tuberculosis (MOTT) is lacking in part because of the lack of tools to identify species of Mycobacterium, although some work is being done with conventional biochemical tests and polymerase chain reaction (PCR)-based methods. In some part of patients tuberculosis is accompanied by hepatitis C, HIV-infection. Diagnostics of tuberculosis in Kazakhstan are done by traditional methods: cutaneous tuberculin tests (the opportunities of which are limited), X-rays researches of lungs and the final diagnosis is achieved by bacteriological tests (4-8 weeks are required). The method is directed at detecting patients with advanced disease-those who have a cavity of disintegration in their lungs. Treatment of such patients is difficult, expensive and long. In Kazakhstan, the criterion for success of patients’ treatment is negative result of bacterial culture tests, rather than X-ray data demonstrating closing of a cavity of disintegration. Clinical importance of identification of mycobacterium infections is constantly increasing which is connected with the development of secondary immunodeficient conditions in various categories of population which are more frequently become infected by conditionally pathogen mycobacterium (mainly Mycobacterium avium complex, MAC and M. kansasii). Until the present moment the identification of mycobacterium is based on its phenotypical properties, which takes not less than 6 weeks. Identification of polymerase chain reaction (PCR) permitting to conduct the detection in biomaterial from the patients and identification of the appurtenance of isolated cultures to the M. Tuberculosis complex in Kazakhstan is at the stage of formation. Differentiation of M. tuberculosis from other mycobacteria in Kazakhstan is not sufficiently sensitive and specific. Drug resistance of M. tuberculosis isolates is also defined by culture-based tests. M. tuberculosis bacteria grow very slowly, the time to grow on solid media is from 3 weeks to 2, 5 months. Before receipt of drug-susceptibility results, the patients are usually treated with a standard regimen, which may include anti-tuberculosis drugs to which the bacteria has developed the resistance. One of the methods of rapid mycobacterium identification is based on the analysis of lipids of cell wall and mycolic acids with the use of gas (GC) and high performance liquid chromatography with the fluorescent detection (FL-HPLC). The important of this method are that it is a relatively easy method and that a single test can identify most pathogenic Mycobacterium species. Overall, there is a large need in Kazakhstan and the countries of the CIS; we offer the following project, which takes advantage of the medical, microbiological and chemical expertise of laboratories. Applied preparations for tuberculosis treatment were elaborated more than 50 years ago, all of them are toxic and at the present time to all of them there developed drug - resistance. One of the reasons of new preparation absence is a complex, long procedure of in vitro testing (90 days). The modern medicine sharply needs drugs effective for treatment of resistant forms of tuberculosis. The development of chromatography method of detection and identification of mycobacteria species in the combination of BACTEC technology and PCR- analysis is suggested by us. Analytical review showed that the suggested work is distinguished in novelty:

  • In detection the peculiarities of fatty acids composition and mycolic acids profile of clinical strains of M. tuberculosis, isolated from patients from various regions of Kazakhstan;
  • In differentiating the peculiarities of fatty acids composition of lipid membrane and profile of mycolic acids of drug -resistant M. tuberculosis;
  • In search of combinations suppressing the synthesis of mycolic acids of drug -resistant strains of M. tuberculosis.

Information about project participants. The Monitoring Laboratory of joint-stock company “Biomedpreparat-engineering Centre” of former defensive enterprise, which during the USSR specialized on elaboration of technologies and manufacture of the biological weapon. Experts of laboratory have a great experience on identification of especially dangerous species of infections (Bac. anthracis, Ps.malei) with use of various kinds of chromatography (gas and liquid) and capillary electrophoresis. Now the laboratory has highly skilled experts in this field. Pharmaceutical company “Romat” has highly skilled personnel, well equipped microbiological laboratory with a high performance liquid chromatography Perkin Elmer LAS, owned vivary. Microbiological laboratory of Kazakh National Medical University has BACTEC 960 for rapid mycobacterium detection and testing of anti- tuberculosis drugs in vitro, gas chromatography HP 6890 with the computer program on microorganisms’ identification on fatty-acid profile Sherlock MIDI System and data bank on 2500 species of bacteria, PCR- laboratory.

Expected results:

  1. Collection of clinical isolates from patients with tuberculosis and infections with MOTT from of different regions of Kazakhstan will be created;
  2. The rapid, high sensitive, automated method of detection and identification of mycobacterium by HPLC with fluorescent detection from smear-positive sputum specimens will be improved;
  3. A library of chromatograms of methyl esters of fatty acids and mycolic acids of identified clinical isolates and Mycobacterium species will be created. The library will be a critically needed resource for laboratories that use the new method to identify Mycobacterium species and will be harmonized with existing of international colleagues.
  4. A standardized method for the detection and identification of mycobacterium by GC and HPLC will be development for use in diagnostics laboratories in Kazakhstan. The method will be compatible with existing protocols used in other countries.
  5. Criteria to determine resistance of M. tuberculosis to anti-tuberculosis drugs on a basis of fatty acids composition or the content of mycolic acids will be developed;
  6. Features of fatty acids character and of mycolic acids profile mycobacterium isolates with various sensitivity to medical products will be developed;
  7. The assortment the combinations, retarding the synthesis of mycolic acids of M. tuberculosis strains will be established.
  8. The sputum structure and condensate of expirated air of tuberculosis patients by gas and liquid chromatography methods will be investigated, will be given the recommendations for inculcation in laboratory practice:

The expected results will have the perspectives for inculcation of express method of M. tuberculosis species identification in the laboratory and clinical practice from the biomaterials, control of treatment efficiency, identification of drug –susceptibility, drug -resistance of clinical strains and tuberculosis diagnose at the early stages. The detection of combinations, perspective for drug elaboration effective for treatment of resistant tuberculosis forms has a good commercial potency. Project realization solves the main social task as well- to confine the expansion of infection diseases.

Science meaning of the project:

  • Detection the peculiarities of composition of fatty acids and profile of chromatograms of mycolic acids of clinical tuberculosis strains, isolated from patients from different regions of Kazakhstan;
  • Elaboration of the criteria of drug - susceptibility and drug-resistance of M. tuberculosis strains, isolated from patients from different regions of Kazakhstan;
  • Detection of the combinations, effective for treatment of drug- resistant tuberculosis forms.

Direction of the researches:
  • Collection of clinical strains from patients with tuberculosis and infections with MOTT from various regions of Kazakhstan, identification of isolated strains by standard phenotypical and molecular biological methods;
  • Chromatographic analysis (GC and HPLC) of fatty acids ethers and mycolic acids and other mycobacterium components of identified clinical strains, interlaboratory collation of the expected results;
  • Development and evaluation of the accelerated high-sensitive automated method of detection and identification of mycobacterium by the HPLC with fluorescence detection from smear-positive sputum specimens;
  • Comparative research the peculiarities of the composition of fatty acids and mycolic acids profile of drug- susceptible and drug -resistant M. tuberculosis strains;
  • Search of combinations, retarding the synthesis of mycolic acids of drug -resistant M. tuberculosis strains;
  • Creation of a library of chromatograms for fatty and mycolic acids of M. tuberculosis and other well-characterized Mycobacterium species, comparison to existing libraries such as the in of the U.S. HPLC Users group.
  • Distribution of recommendations for development of the Kazakhstan standard for identification of mycobacterium by the methods of GC and UPLC. Harmonization of development methods with the international standards.

The realization of the project will give the opportunity to the scientists and engineers, in past being involved in the elaboration and manufacture of biological weapon of mass destruction, redirect their energy and talent on the solving the problem of bioprotection in Kazakhstan- in the decreasing of tuberculosis infection in the Republic of Kazakhstan.

The collaborator’s role is to discuss and to adjust the research methodology, new directions in this field, review of the account materials on the project. Collaborator will promote the laboratory personnel participation in the international conferences connected with the project subject, in the common publications and research results patent.

Technical approach and methodology are based on the identification and quantitative determination of mycolic acids composition, qualitative and quantitative determination of fatty acids of lipid membrane of M. tuberculosis. Used methods are gas and high performance liquid chromatography. The comparative analysis of expected results with the identification of mycobacteria by PCR methods and by classical bacteriological method.


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