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									Advance Diagnostic of TB and
         MDR-TB


               Muh. Nasrum Massi
        Medical Microbiology Department,
               Faculty of Medicine,
  Hasanuddin University, Makassar, South Sulawesi,
                    Indonesia
CLINICAL MICROBIOLOGY EXAMINATION
 Clinicians



  SUSPECT
  TB PATIENT
                     SPECIMEN
                     VISUAL EXAMINATION
                                          GROWTH MEDIA

 MICROSCOPE EXAMINATION




                          CULTURE


    RESULT BACTERIAL IDENTIFICATION
                 DRUG SUSCEPTIBILITY TEST

    Clinicians               PATIENT
Past:
Diagnosis by smear microscope (ZN is the
   best)
Present:
1. ZN staining, only 30-60% sensitive
2. Only 50% total culture positive with AFS
   positive
3. If only by smear, D/ 50% TB patient will
   be lost
     The problem with smear
            negative

If negative smear result, clinician always
   thinking that patient not infectious,
 it means no need treatment, spread of
   infection – public health impact
 Development of culture and identification

• Culture conventional
Present:
Liquid media more superior than solid media
   because of:
1. Rapid detection
2. More species can growth
3. Mycobacteria can growth well
4. More reliable for DST
      The important of culture
1. Culture still as a gold standard for
   longtime
2. We need culture for ID and DST
   Automated culture methods
1. The BACTEC TB-460 system (Becton
  Dickinson, Sparks, MD)
• A modified Middlebrook 7H9 medium is used, in
  which one of the components, palmitic acid, is
  radiolabeled with 14C
• When viable mycobacteria are present in the
  culture vial, the radiolabeled palmitic acid is
  metabolized and radioactive CO2 is liberated
  into the gaseous phase.
2. The BACTEC MGIT960 system (Becton
  Dickinson, Sparks MD)
• Mycobacteria Growth Indicator Tube (MGIT) is a
  modified Middlebrook 7H9 medium in which a
  supplement OADC and PANTA antibiotic mixture used in
  the radiometric system.
• If viable mycobacteria are present in the tube, oxygen is
  consumed due to their metabolism, the quenching effect
  lowers accordingly, and the bottom of the tube
  fluorescens when exposed to ultraviolet light.
                   TM
   MGIT                  Components
• BBL MGIT Tubes - 7 mL Modified Middlebrook 7H9
            broth and Fluorescent Indicator of base.
• BBL MGIT OADC - Enrichment Supplement
     • Oleic Acid - utilized by tubercle bacilli
     • Albumin - binds free fatty acids
     • Dextrose - energy source
     • Catalase - destroys toxic peroxides
• BBL MGIT PANTA - Mixture of antimicrobial agents
     • Polymyxin B
     • Amphotericin B
     • Naladixic Acid
     • Trimethoprim
     • Azlocillin
    Specimen Processing
• Add specimen to 50 mL plastic centrifuge
  tube.
   –Specimen should not exceed 10 mL.
• Add NALC/NaOH solution in volume
  equal to that of specimen. Mix well.
• Allow specimen to stand for 15-20 minutes
  at room temperature.
      Specimen Processing

• Process specimens using NALC-NaOH
  method as recommended in CDC Manual:
   Public Health Mycobacteriology: A Guide
   for the Level III Laboratory.
• BBL MycoPrep may be used.
             TM
  MGIT            Tube Preparation

• While specimens are in centrifuge:
  – Label MGIT tube with specimen number.
  – Prepare PANTA.
     • Reconstitue with 15 mL OADC
  – For best results, add PANTA solution just
    prior to specimen inoculation.
          TM
MGIT           Tube Preparation

• Tightly recap tube and mix well.
• Wipe tubes and caps with tuberculocidal
  disinfectant.
• Enter MGIT tubes into MGIT 960
  Instrument.
       3. The VersaTREK (previously known as the
             ESP system II) by Trek Diagnostic Systems.
       • a modified Middlebrook 7H9 medium to which the OADC
         enrichment must be added.
       • If viable mycobacteria are present in the bottle, the
         oxygen consumption due to their metabolism reduces
         the internal pressure.




Palomino et al Tuberculosis 2007; From basic science and patient care
     4. BacT/Alert 3D (previously known as
       MB/BacT) by bioMérieux
     • A modified Middlebrook 7H9 medium is used in which a supplement,
       OADC and polymyxin B, amphotericin B, nalidixic acid,
       trimethoprim, vancomycin and azlocillin.
     • If viable mycobacteria are present in the bottle, the CO2 produced
       by their metabolism causes a change in the color of the sensor, from
       green to yellow, which alters the intensity of the reflected light ray




Palomino et al Tuberculosis 2007; From basic science and patient care
Identification of Mycobacterium
             Colony morphology and biochemical
              characteristics of species in the M
                    tuberculosis complex




Palomino et al Tuberculosis 2007; From basic science and patient care
      Molecular Detection
       of Tuberculosis

• Commercial, FDA-approved assays:
  – PCR (Roche), TMA (GenProbe)
• User-developed (“home brew”) assays
  – Single Copy Targets
    • Pab, dnaJ, MBP 70, MBP 64
  – Multicopy Targets
    • Repeated Sequences (IS6110 (IS986), rRNA)
     The Amplicor MTB Test (Roche Molecular
       Systems, Basel, Switzerland)
     • A 584 bp fragment of the 16S ribosomal RNA gene,
       comprising a species-specific region flanked by genus-
       specific sequences, is amplified using biotinylated
       primers.relies on standard PCR.




Palomino et al Tuberculosis 2007; From basic science and patient care
     Amplified Mycobacterium tuberculosis Direct Test
       (AMTD), developed by Gen-Probe (San Diego, CA,
       USA), is an isothermal (42°C) transcriptase-mediated
       amplification system.
     • A M. tuberculosis complex-specific region of the 16S
       ribosomal RNA gene produces double-stranded
       ribosomal DNA, due to the combined action of reverse
       transcriptase and ribonuclease.




Palomino et al Tuberculosis 2007; From basic science and patient care
     The BD ProbeTec ET (Becton Dickinson, Sparks, MD)
           uses DNA polymerase and isothermal strand
           displacement amplification to produce multiple copies of
           IS6110, an insertion element unique to M. tuberculosis
           complex.




Palomino et al Tuberculosis 2007; From basic science and patient care
AccuProbe (Gen-Probe, San Diego, CA)
• The probe is a ssDNA oligonucleotide, complementary to
  a short, species-specific sequence within a hypervariable
  region of the 16S rDNA. It is labeled with an acridinium
  ester, a chemiluminescent molecule, which gives light
  when properly excited.
                                   Line probe assays

       • Three commercial methods are available, INNO-LiPA
         MYCOBACTERIA (Innogenetics, Ghent, Belgium),
         GenoType Mycobacterium (Hain, Germany), and
         GenoType MTBC (Hain, Germany).




Palomino et al Tuberculosis 2007; From basic science and patient care
                           Genetic identification methods

     The PCR restriction-enzyme analysis (PRA) method is
       based on the amplification of a 441-bp fragment of the
       hsp65 gene by PCR, followed by the digestion of the
       amplified product with two restriction enzymes BstEII
       and HaeIII according to the procedure first described by
       Telenti (Telenti 1993).




Palomino et al Tuberculosis 2007; From basic science and patient care
 M. tuberculosis DNA Targets
• 16S rRNA
  – multicopy RNA target – TMA
  – only a single copy DNA target – one rRNA operon
• 65 kD antigen (HSP)
  – single copy chromosomal DNA target
• IS6110 (IS986)
  – multicopy, mobile repetitive DNA element
  - 15-20 copies per chromosome
  – specific for M tuberculosis
DST
Drug Susceptibility Test should be done in


- Patient with history of irregular treatment.
- Patient with treatment failure.
- Patient with relapse.
- Patient with contact to drug resistant patient.
- Patient with smear positive at the end of
  intensive phase.
     Conventional Method.


1. Resistance Ratio Method
               (Mitchison, 1954)
2. Absolute Concentration Method
               (Meissner,1964)
3. Proportional Method
               (Canetti, 1969)
 Factors should be avoided in DST.
 - Error in preparation of drug solution or drug
    containing media.
- Improper storage of drug powder, or drug
   solution or drug containing media.
- Failure to recognized the simultaneous presence
   of M.tuberculosis.
Factors Influencing the Outcome
 of Drug Susceptibility Test

     1. Media
     2. Stability of the drugs
     3. Inoculation
     4. Incubation
     5. Quality Control
             MAS-PCR Method
The method is based on mismatch between nucleotide on the
3’ end of a primer with a mutated nucleotide in the DNA
template. Under stringent annealing condition, the “wild-type”
primer will not anneal to the mutated template because the
temperature of annealing exceeds the melting temperature of the
primer.
          Line Probe Assays: HAIN Test

• Rapid molecular method to detect MDR- TB
• It detects genetic mutations, and is based on DNA extraction,
  multiplex polymerase chain reaction (PCR) amplification, and
  reverse hybridization :
     # Rif resistance: the rpoB gene
     # INH resistance: katG gene and inhA gene
• directly from smear-positive sputum,
• results available within 1 day
• High sensitivity and specificity for HR resistance : High correlation
  with conventional culture and DST.
• Costs: 50% of conventional methods
• Reduces workload for conventional/culture
               Method: GenoType® – DNA•STRIP®
                         Technology




FIG. 1. GenoType MTBDRplus work flow
Hain Lifescience. Genotype® MTBDRplus product insert. Version 1 http://
www.hain-lifescience.de/en/products/microbiology/mycobacteria/genotypemtbdrplus.
html.
                             rpoB gene: rifampicin resistance
                                          rpoB WT2                 rpoB WT4             rpoB WT6
                      rpoB WT1                            rpoB WT3         rpoB WT5                 rpoB WT7    rpoB WT8

                505           508 509       511      513 514 515 516        518       522          526         531   533



                                                         rpoB MUT1 (D516V)         rpoB MUT2B (H526D)
                                                                                                    rpoB MUT3 (S531L)
                       FIG. 3. Locations of probes within the 81-bp hot spot cluster of the
                       rpoB gene.


          rpoB-Wildtype-probes: WT 1 to WT 8
          rpoB-Mutation-probes: MUT D516V, H526Y, H526D, S531L

          Resistance
          •Detection of mutations through missing of wildtype signals
                                      together with
           Detection of mutations through presence of mutation signals

          • Detection of mutations through missing of wildtype signals
Hain Lifescience. Genotype® MTBDRplus product insert. Version 1 http://
www.hain-lifescience.de/en/products/microbiology/mycobacteria/genotypemtbdrplus.
html.
                                                          Evaluation
                                                                             katG
                                                                                                        RMP INH




                                                                                            r poB MUT

                                                                                            katG MUT

                                                                                            inhA MUT
                       r poB MUT2A
                       r poB MUT2B




                                                                         inhA MUT3A
                                                                         inhA MUT3B




                                                                                            katG WT

                                                                                            inhA WT
       #



                       r poB MUT1


                       r poB MUT3



                                                            katG MUT1
                                                            katG MUT2




                                                                         inhA MUT1
                                                                         inhA MUT2




                                                                                                        sensitive


                                                                                                                                 sensitive
                                                                                                                    r esistant


                                                                                                                                             r esistant
                       r poB WT1
                       r poB WT2
                       r poB WT3
                       r poB WT4
                       r poB WT5
                       r poB WT6
                       r poB WT7
                       r poB WT8




                                                                         inhA WT1
                                                                         inhA WT2
                                                            katG WT




                                                                                      TUB
                                                            katG
                       r poB




                                                                         inhA
             TUB
             CC
             AC




        1                                                                             ++ - + - + - + +
        2                                                                             + - + - ++ - + +
        3                                                                             + +- +- - + + +
       4                                                                              + - + - +- + + +
        5                                                                             + - - - - +- + +
                                                         rpoB
                                                                                                inhA
Susceptibility: Hybridization to all the WT probes + no hybridization to a MUT probe
Resistance: Lack of hybridization to one WT probe OR/and present of hybridization to a MUT probe
Mixed populations: Hybridization to both WT and MUT probes
Hain Lifescience. Genotype® MTBDRplus product insert. Version 1 http://
www.hain-lifescience.de/en/products/microbiology/mycobacteria/genotypemtbdrplus.
html.
Genotype MTBDRplus: Evaluations
            results
            Molecular Basis of drug resistant in M.
                    tuberculosis complex
      Drug                     Gene Locus               Gene function                         Percent of
                                                                                              Resistance
      Isoniazid                katG                     Catalase-Peroxidase                   40 - 100 %
                               inhA                     Enoyl-ACP-Reduktase                   appr. 25 %
                               ahpC-Promoter Alkyl-Hydroxid-Peroxidase                        appr. 10 %
      Rifampicin               rpoB                     ß-Subunit of RNA-                     > 90 %
                                                        Polymerase
      Pyrazinamide             pncA                     Pyrazinamidase                        appr. 95 %
      Streptomycin             rpsL                     ribosomal Protein S12                 appr. 60 %
                               rrs                      16S rRNA                              appr. 20 %
      Ethambutol               embB                     Arabinosyl-Transferase                appr. 60 %
      Quinolone                gyrA                     DNA-Gyrase A                          appr.80-90%

Ashok Rattan, et al; Multidrug-Resistant Mycobacterium tuberculosis: Molecular Perspectives
        GeneXpert:Introducing High Tech
                   in ‘’Low Tech’’ settings




Jan Voskens, joint symp NEHCRI-Eijkman,2010
                Conclusion
• Although direct amplification methods for
  identification of Mycobacteria and AST are used
  worldwide, they are far from having
  revolutionized clinical mycobacteriology.
• Culture, supported by microscopy, still remains
  the gold standard, and molecular methods only
  represent a useful support in some cases, to
  speed up the diagnosis of TB.
Thank
 you

								
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