MYCOBACTERIOLOGY 2004 What laboratories are doing and where they are headed

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MYCOBACTERIOLOGY 2004 What laboratories are
doing and where they are headed

• Current identification techniques Molecular
• E. Tortoli

ASM 2004 New Orleans, May 26
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Conserved genetic regions, the Rosetta stone of
molecular identification
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Conserved genetic regions, the Rosetta stone of
molecular identification
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variable genus-specific region
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Conserved genetic regions, the Rosetta stone of
molecular identification
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variable genus-specific region hypervariable
species-specific region
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Conserved genetic regions, the Rosetta stone of
molecular identification
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variable genus-specific region hypervariable
species-specific region hypervariable
intraspecies-specific region
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DNA probes

• Liquid-phase hybridization
• Solid-phase reverse hybridization

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Liquid-phase hybridization

• AccuProbe
• Target 16S rRNA
• Selection hybridization protection assay
• Detection chemiluminescence
• Available for
• 2 complexes
• 4 species

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Solid-phase reverse hybridization
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Solid-phase reverse hybridization 1

• INNO LiPA
• Target ITS
• Probes for
• Mycobacterium genus
• 2 complexes
• 15 (1) species
• Intraspecific differentiation of 3 species

• GenoType
• Target 23S rDNA
• Probes for
• Mycobacterium genus ???
• 1 complex
• 11 species
• Intraspecific differentiation of 1 species
• New version double strip with 1312 species

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Solid-phase reverse hybridization 2

• GenoType MTBC
• Targets
• 23S rDNA
• M. tuberculosis complex-specific region
• gyrB
• 4 regions containing base-substitutions specific
  for M. tuberculosis (M. africanum II, M.
  canettii), M. africanum I, M. microti, BCG, M.
  caprae
• RD1
• 9,500 bp region present in all members of M.
  tuberculosis complex other than BCG

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DNA-probes discrepant results

• M. kansasii types iii-iv AccuProbe-MK NEG
• M. palustre AccuProbe-MAC pos
• M. paraffinicum LiPA-MAIS pos
• M. thermoresistibile, M. agri, M. mageritense, M.
  senegalense, M. alvei LiPA-MFO pos
• MAC non avium, non intracellulare GenoType-MI
  pos or GenoType NEG
• M. interjectum GenoType-MSC pos
• M. saskatchewanense AccuProbe-MAC pos
• M. chimaera AccuProbe-MI pos GenoType-MI pos
  LiPA-MIN2 pos

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PCR-RFLP analysis

• Target amplification
• Restriction enzymes digestion
• Electrophoresis
• Determination of size of fragments 40 bp

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hsp65 PRA

• Bst EII
• no digestion (440 bp)
• 2-3 major fragments production (320-80 bp)
• Hae III
• 2-5 major fragments production (265-40 bp)

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hsp65 PRA algorithm

• a proper algorithm allows the differentiation of
  species, within each pattern of Bst EII
  fragments, on the basis Hae III pattern

• many species with multiple patterns (up to 9)
• few patterns shared by several species

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Genetic sequencing

• 16S rDNA
• Internal transcribed spacer
• 23S rDNA
• hsp65
• sod

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16S rDNA

• About 1,500 bp
• Hypervariable regions A and B
• Species-specificity
• Several sequevars
• Phylogenetic markers
• Short helix 18 characterizing ancestral, rapidly
  growing, mycobacteria
• Evolution of slow growers associated with a 12
  nucleotide insertion in helix 18
• Development of two new branches, among slow
  growers, characterized by
• further insertion of 2 nucleotides in helix 18
• lost of the acquired 12 nucleotide insertion
• Development of a new branch, among rapid growers,
  characterized by a cytosine insertion in helix 10

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ITS

• About 300 bp
• No hypervariable region recognizable
• Many mycobacteria characterized by a high number
  of sequevars
• MAC
• M. fortuitum complex
• M. gordonae
• M. kansasii

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23S rDNA

• About 3,000 bp
• 250 bp variable region
• Species-specificity, several species with
  overlapping sequence, no intraspecific variability

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Sequencing limitations

• Limited portion of genome surveyed
• Investigation of highly conserved genes make
  species variability less evident than in the
  DNA-DNA homology investigations
• Species with overlapping sequence
• Species with multiple sequevars
• Lack of quality control of sequence entries in
  public databases
• best match sometimes favoring ragged or outdated
  entries

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Microarray-microchip technology

• Microarray miniaturized multiprobe system
• electronically controlled
• non electronic
• Multiple step identification of many strains
  (amplicon down)
• Single step identification of one strains
  (capture down)

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Amplicon down

• PCR-products from various unidentified
  mycobacteria are bound to different pads of a
  micro-array

• In subsequent steps, one or more (differently
  marked) probes are added

• At each step the monitoring of pads presenting
  hybridization allows, on the basis of the
  specificity of the probe used, the identification
  of the corresponding amplicon

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Capture down

• Different species-specific probes are bound to
  the various pads of a micro-array

• Addition of PCR-product from an unidentified
  strain

• Revelation of the hybridization by adding a
  reporter (marked probe) aiming to a non
  species-specific trait of the amplified region

• Identification on the basis of the specificity of
  the probe bound to the pad presenting the
  hybridization

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Future prospects

• Use of microchip technology for
• Identification
• Genotypic detection of resistances
• Molecular epidemiology