M.kansasii M.marinum M.avium M.simiae M.szulgai M.gordonae M.xenopi M.intermedium M.intracellulare M.scrofulaceum M.avium sp.

Transcription

1 Molecular-genetic methods for species identification of mycobacteria Smirnova T.G., Chernousova L.N., Vorobyeva A.V., Larionova E.E., Andreevskaya S.N. Central TB Research Institute of RAMS, Moscow 2010

2 Species of nontuberculous mycobacteria M.kansasii M.marinum M.avium M.simiae M.szulgai M.gordonae M.xenopi M.intermedium M.intracellulare M.scrofulaceum M.avium sp. paratuberculosis M.vaccae M.chelonei M.fortuitum M.phlei M.peregrinum M.smegmatis M.terrae M.termoresistible

3 Species of nontuberculous mycobacteria Infection by nontuberculous mycobacteria (NTM) is the cause of mycobacteriosis M.kansasii M.marinum M.avium M.simiae M.szulgai M.gordonae M.xenopi M.intermedium M.intracellulare M.scrofulaceum M.avium sp. paratuberculosis M.vaccae M.chelonae M.fortuitum M.phlei M.peregrinum M.smegmatis M.terrae

4 Species identification of mycobacteria More attention is being paid worldwide to infections induced by NTM This is linked to the Increase in numbers of mycobacteriosis registered.

5 Species identification of mycobacteria The reasons for increased numbers of mycobacteriosis registered in countries with traditionally low incidence are: Mycobacteriosis is a frequent opportunistic infection in AIDS ( up to 50% of AIDS patients are infected with NTM) Improvement of detection due to introduction of liquid culture based automatic systems (Bactec MGIT 960) Implementation of molecular-genetic systems for species identification of NTM

6 Methods and test-systems systems for speсies identification of NTM and MTBC Microbiological methods

7 Methods and test-systems systems for speсies identification of NTM and MTBC Sequencing PCR restriction fragment length polymorphism analysis

8 Methods and test-systems systems for species identification of NTM and MTBC Commercial test-systems based on PCR AMPLICOR Mycobacterium tuberculosis test (Roche Diagnostic Systems, Inc., Indianapolis, IN). Amplified Mycobacterium tuberculosis Direct test (MTD, Gen-Probe, Inc., San Diego, CA) Biochip Technology

9 Methods and testtest-systems for species identification of NTM and MTBC M.avium ssp., M.chelonae, M.abscessus, M.fortuitum, M.gordonae, M.intracellulare, M.scrofulaceum, M.interjectum, M.kansasii, M.malmoense, M.peregrinum, M.marinum/M.ulcerans, M. xenopi, M.tuberculosis complex, M.simiae, M.mucogenicum, M.goodii, M.celatum, M.smegmatis, M.genavense, M.lentiflavum, M.heckeshornense, M.szulgai, M.intermedium, M.phlei, M.haemophilum, M.kansasii, M.ulcerans, M.gastri, M.asiaticum, M.shimoidei GenoTypeCM and GenoTypeAS (HAIN Lifescience, Lifescience, Germany)

10 Methods and test-systems systems for species identification of NTM and MTBC BD MGIT TBc Identification TEST (TBc ID), Becton Dickinson

11 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry Mass-spectrometry is a method to determine molecular structure of substances based on registration of massspectrum of ions which form as a result of sample ionization First mass-spectrometer was constructed in The basis of the technique : 1. Neutral molecules of investigated material are ionized 2. Ions are transmitted in a vacuum tube. Inside the tube they are moved in electromagnetic field at a speed inversely proportionate to their mass. 3. Dynode secondary emission electron multipliers detect the time of flight of ions 4. Computer program plots mass-spectrum of molecules of the investigated material.

12 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry E.coli protein mass-spectrum

13 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry We aimed at developing a novel technique for species identification of NTM by direct protein profiling and further collecting totally characterized NTM strains. The basis for the proposed method of NTM identification is obtaining mass-spectrums of ribosomal proteins. It is known that amino acid sequences are conservative within species and differ from species-to-species Accumulation of mass-spectral data for conservative proteins will allow to create data base and perform species identification of NTM

14 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry Nonmycobacterial strains

15 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry At present our laboratory set out to create a data base of mass-spectrums of ribosomal proteins derived from nontuberculous mycobacteria as well as Mycobacterium tuberculosis complex This work is carried out in collaboration with Scientific Research Institute of Physical-Chemical Medicine

16 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry By now we managed to collect 35 strains of NTM belonging to following species: M.avium, M.intracellulare, M.scrofulaceum, M.chelonae, M.abscessus, M.gordonae, M.kansasii, M.deirnhoferi, M.fortuitum, M.peregrinum, M.smegmatis, M.gastri, M.phlei, M.lentiflavum, M.simiae as well as 40 strains of M.tuberculosis complex: M.bovis Bovinus, M.bovis BCG, M.tuberculosis H37Rv, M.tuberculosis H37Ra and M.tuberculosis clinical isolates from TB patients in Central TB Research Institute

17 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry Each strain was characterized using several techniques: microbiological (Ziehl- Neelsen staining, growth on TCH medium and sodium salicilate and nitratereductase test), PCR with primers, complementary to IS6110, Genotype Mycobacterium CM/AS (Hain-Lifescience, Germany) M.avium M.chelonae M.fortuitum M.kansasii Strains with consistent results of these diagnostic techniques were used for mass-spectrometry study.

18 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry NTM strains

19 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry MAIS complex

20 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry M.tuberculosis complex

21 Species identification of mycobacteria using MALDI-ToF mass-spectrometry spectrometry reproducibility of results

22 Species identification of mycobacteria Problems of mycobacteriosis diagnostics in Russia: The problem of mycobacteriosis is hardly covered by special literature and statistical data on the disease could hardly be found. Species identification of mycobacterial cultures is not performed in bacteriology labs over Russia. No local molecular-genetic test-systems are manufactured for time saving and cheap identification of NTM.

23 Nontuberculous mycobacteria species identified in samples from patients in CTRI 889 positive samples (Bactec MGIT 960) 35 samples contained NTM strains

24 Open questions: to laboratory staff: How to perform species identification of NTM? How to perform DST for NTM??? to physicians: Who must treat the patients with mycobacteriosis? Where mycobacteriosis patient should be treated? How to treat mycobacteriosis???

25 DST of NTM (Trek Diagnostic Systems, Magellan Biosciences, England)

26 DST of NTM (Trek Diagnostic Systems, Magellan Biosciences, England) Slow growth mycobacteria Clarithromycin Rifabutin Ethambutol Isoniazid Moxifloxacin Rifampin Trimethoprim/Sulfamethoxazole Amikacin Linezolid Ciprofloxacin Streptomycin Doxycycline Ethionamide Rapid growth mycobacteria Trimethoprim/Sulfamethoxazole Linezolid Ciprofloxacin Impinem Moxifloxacin Cefepime Cefoxitin Amoxicillin/Clavulanic acid Amikacin Ceftriaxone Doxycycline Minocycline Tigecycline Tobramycin Clarithromycin

27 DST of NTM strains was performed for 5 patients with confirmed diagnosis MYCOBACTERIOSIS

28 DST results (Trek Diagnostic Systems, Magellan Biosciences, England) Patient 4294 M.fortuitum Trimethoprim/Sulfameth. RES Linezolid RES(sense to high conc) Ciprofloxacin Impinem RES Moxifloxacin Cefepime RES Cefoxitin RES Amoxicillin/Clavulanic ac.res Amikacin Ceftriaxone RES Doxycycline RES Minocycline RES Tigecycline RES Tobramycin RES Clarithromycin RES Patient 4066 M.chelonae Trimethoprim/Sulfameth. RES Linezolid RES Ciprofloxacin RES Impinem RES Moxifloxacin RES Cefepime RES Cefoxitin RES (sens to high conc) Amoxicillin/Clavulanic RES Amikacin RES (sens to high conc) Ceftriaxone RES Doxycycline RES Minocycline RES Tigecycline RES Tobramycin RES Clarithromycin RES Patient 521 M.abscessus Trimethoprim/Sulfameth. RES Linezolid Ciprofloxacin RES Impinem RES Moxifloxacin RES (sens to high conc Cefepime RES Cefoxitin RES (sens to high conc) Amoxicillin/Clavulanic RES Amikacin Ceftriaxone RES Doxycycline RES Minocycline RES Tigecycline RES (sens to high conc) Tobramycin RES (sens to high conc) Clarithromyc RES (sens to high conc

29 DST results (Trek Diagnostic Systems, Magellan Biosciences, England) Patient 4422 M.kansasii Clarithromycin Rifabutin Ethambutol RES (sens to high conc) Isoniazid (sens to high conc) Moxifloxacin Rifampin Trimethoprim/Sulfamethoxazole RES Amikacin Linezolid Ciprofloxacin RES (sens to high conc) Streptomycin RES (sens to high conc) Doxycycline RES Ethionamide Patient 3377 M.avium Clarithromycin RES (sens to high conc) Rifabutin Ethambutol RES Isoniazid RES Moxifloxacin RES Rifampin Trimethoprim/Sulfamethoxazole RES Amikacin RES (sens to high conc) Linezolid RES Ciprofloxacin RES (sens to high conc) Streptomycin RES Doxycycline RES Ethionamide RES

30 Open questions: to laboratory staff: How to perform species identification of NTM? How to perform DST for NTM? to physicians: Who must treat the patients with mycobacteriosis? Where mycobacteriosis patient should be treated? How to treat mycobacteriosis???

31 Open questions: to laboratory staff: How to perform species identification of NTM? How to perform DST for NTM? to physicians: Who must treat the patients with mycobacteriosis? Where mycobacteriosis patient should be treated? How to treat mycobacteriosis???

32 Open questions: questions:

33 Open questions: The physicians are insufficiently educated in diagnosis and treatment of patients with mycobacterioses which results in delayed detection of the infection. Timely diagnosis of mycobacteriosis depends on highly qualified bacteriological testing and time necessary for identification of obtained cultures. Delay in diagnosis and consequent late start of specific drug therapy may lead to severe progressing disease with lung destruction Т.F.Otten A.V. Vasiliev «Mycobacteriosis», С-Пб., 2005

34 Thank you