Identification of Mycobacteria

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Identification of Mycobacteria in the Clinical
Laboratory by Nucleic Acid Sequencing
Raymond P. Podzorski, Ph.D., D(ABMM) Waukesha
Memorial Hospital
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Methods Used by Laboratories for Mycobacterial
Identification

• 1. Biochemical testing only
• 2. Biochemical testing and probes
• 3. High performance liquid chromatography
• 4. Nucleic acid sequencing
• 5. Line-probe assay
• 6. Refer cultures to state or reference laboratory

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Problems That We have With Mycobacterial
Identification

• At times common methods do not provide an
  accurate identification- best fit basis
• Most common methods are not rapid
• Most common methods often cannot identify new
  species
• Most smaller laboratories do not have access to
  some of the newer technologies

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Problems That We have With Mycobacterial
Identification

• Nucleic acid probes are excellent tools however,
  they are limited by the number of species that
  they can identify
• Referral laboratories are helpful but
  identifications may require an extended time and
  they can be expensive
• It seems as if the names are always changing or
  new names are being introduced

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How Many Valid Species of Mycobacteria are There
Today?

• 1. 35-40
• 2. 60-75
• 3. 76-85
• 4. 86-95
• 5. 96-105

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The Mycobacterial Maze
lentiflavum
heckeshornense
neoaurum
triplex
bohemicum
kubicae
septicum
komossense
botiense
confluentis
elephantis
chubense
doricum
moriokaense
wolinskyi
immunogenum
goodii
hodleri
agri
gilvum
mageritense
alvei
austroafricanum
rhodesiae
heidelbergense
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Nucleic Acid Sequencing of Mycobacteria in the
Clinical LaboratoryTargets

• 16S rDNA gene
• Heat shock protein 65
• 23S- 16S intergenic spacer region
• rpoB gene
• Others

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16S rDNA Sequencing for Bacterial ID

• Molecular Chronometers Woese et. al., The
  phylogeny of prokaryotes, Science, 1980
• Regions of the 16S rDNA gene conserved among all
  Eubacteria, regions conserved among genera of
  bacteria, regions conserved among species of
  bacteria
• 16S rDNA gene analysis is now considered the gold
  standard for bacterial identification
• 16S rDNA gene ? 1,500 bp - 5 500 bases
  (hypervariable) commonly used for identification

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16S rDNA Cycle SequencingTemplate Synthesis by
PCR
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16S rDNA Cycle Sequencing

• A, C, G, T

• PCR product
• Specific primer
• DNA polymerase
• Buffer

• diA, diC, diG, diT

Thermal Cycling
Fragment Separation and Base Calling
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O
Base (A, C, G, or T)
5
p
O
CH2
HO
O-
1
4
H
H
2
3
Normal deoxynucleoside triphosphate
5
1
4
2
3
Chain terminator dideoxynucleoside triphosphate
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Mycobacterial Sequencing Time Line Waukesha
Memorial Hospital
Day 1
Day 2
Day 3
800 AM
830 AM
1115 AM
1200 PM
300 PM
1000 AM
230 PM
300 PM
PCR
Cycle Sequencing
Data Analysis
Ship via FedEx
Place On Sequence Analyzer
Cell Lysis
Amplicon Purification and Product Determination
Sequencing Purification
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Where To Get DNA Sequencing?

Numerous companies will run your sequencing
reactions on their equipment - Make sure their
equipment is complementary to the sequencing
chemistry you are using - Find one that will
allow you to ship sequencing reactions at
ambient temperature and in water - Be clear on
price and period of time the price is good for -
Be sure you have software to process the DNA
sequence data your receive
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What Do You Think the Reagents Cost to Sequence a
Mycobacterial Isolate?

• 1. 25.00-30.00
• 2. 35.00-50.00
• 3. 60.00-75.00
• 4. 80.00-100.00
• 5. ?100.00
• 6. Who cares, everything in medicine is
  expensive.

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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• Cook et. al., compared the cost of 16S sequence
  ID to biochemical methods for probe-negative
  Mycobact.
• higher cost always associated with biochemical
  methods
• biochemical ID cost varied with organism
  characteristics
• biochemical testing took 2-6 weeks versus 1-2
  days for sequencing

Biochemical 16S sequencing slow/active 59.89
35.45 slow/inert 128.19 35.45 rapid
grower 95.76 35.45
Cook et al, J. Clin. Micro.41(3) 1010-1015,
March, 2003
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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• Patel and Nachamkin studied 113 isolates 89
  (100) gave correct ID using MicroSeq 500
• 7 isolates had incorrect identification using
  biochemicals
• 9 isolates no way of knowing if MicroSeq correct
  or not
• 2 failed phenotypic identification one was sent
  out as M. species
• Patel et al, J. Clin. Micro.38(1) 246-251,
  January, 2000

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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• Cloud et al evaluated the MicroSeq system using
  using 94 clinical isolates and 25 ATTC strains
• Sequencing identified 95 (113/119) of the
  isolates to species
• 15 had incorrect ID by HPLC (16)
• 11 had 100 match with MicroSeq, RIDOM, or
  private databases
• Others most likely absent from databases
• Cloud et al. J. Clin. Micro.40(2)400-406,
  February, 2002

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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• 328 clinical isolates and 59 (45 species) from
  ATCC were used in evaluation 387
• MicroSeq system was used included 83 species.
• 95.1 of clinical isolates gave concordant
  results (group or species level)
• 98.3 of ATCC strains gave concordant results
  (group or species level)

Hall et al. J. Clin. Micro.41(4)1447-1453,
April, 2003
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Number of Unidentifiable Mycobacteria in
Reference Laboratory

• 1,035 mycobacterial isolates submitted to
  Regional Mycobacterial Reference Center,
  Florence, Italy
• All were probe negative had some level of
  identification
• 72 isolates were unable to be identified by
  nucleic acid sequencing- 6.96
• Important to report unusual isolates for later
  study
• Tortolli et al. J. Clin. Micro.
  39(11)4058-4065, November, 2001

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Databases Available for Mycobacterial
Identification

• MicroSeq 500, Applied Biosystems-
• www.appliedbiosystems.com
• RIDOM- Ribosomal Differentiation of Medical
  Microorganisms-
• www.ridom.com
• GenBank
• www.ncbi.nlm.nih.gov
• Ribosomal Database Project
• www.rdp.msu.edu/html/

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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• Turenne et al sequenced 121 ATCC isolates (92
  species) of mycobacteria
• Sequenced almost entire 16S gene (1400 bp)
• Data analyzed by multiple databases including own
  reference strains GenBank and RDP databases and
  RIDOM
• Turenne et al. J. Clin. Micro.
  39(10)3637-3648, October, 2001

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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• Type strain of 80 species was included,18 others
  and 12 strains of non-established species- RIDOM-
  100, GenBank-23
• --------------------------------------------------
  -------------
• Also sequenced 122 clinical isolates
• 61 had 100 similarity with type strains
• 11 had sequences corresponding to uncharacterized
  strains in public databases
• 31 had unique sequences

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Nucleic Acid Sequencing of Mycobacteria in the
Clinical Laboratory

• Public databases offered an erroneous species as
  best match in some cases
• Chose to combine MicroSeq, RIDOM and some
  sequences from GenBank
• Included strains without definitive ID for later
  characterization
• Databases need extreme quality control

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Nucleic Acid Sequencing in the Clinical
LaboratoryBenefits

• More accurate identification
• Objective identification
• Standardized method
• Identification of unusual isolates
• Can identify new species
• Levels the playing field

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Nucleic Acid Sequencing in the Clinical
LaboratoryBenefits

• If MicroSeq is available, can identify aerobic
  and anaerobic bacteria, mycobacteria, and fungi
• Improved patient care
• Shorter turn around time

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Limitations Of Nucleic Acid Sequencing

• Cost---??!!
• Some public databases not quality-controlled-
  RIDOM appears accurate
• Some sequences are not specific for a single
  organism
• Need to include different genotypes in databases

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Limitations Of Nucleic Acid Sequencing Organisms
Having 100 Similarity

• M. bovis and M. microti and M. tuberculosis
  tuberculosis and M. africanum
• M. chelonae and M. abscessus (500 bp)
• M. genavensae and M. simiae
• M. kansasii and M. gastri
• M. peregrinum and M. septicum
• M. fortuitum and M. farcinogenes and M. porcinum
• M. murale and M. tokaiense

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Limitations Of Nucleic Acid Sequencing

• Not all institutions share sequences so that they
  are universally available to all
• Unique sequences will be seen in all laboratories
  and are important and should be included in
  databases

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Algorithm for Identification of AFB
AFB Isolate
Sequence
DNA Probes
Match Report
No Match Group, Supp. Tests, etc
Report
-- Sequence
Match Report
No Match Group, Supp. Tests, etc