Genotyping of Mycobacterium Tuberculosis Isolates

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Genotyping of Mycobacterium Tuberculosis Isolates
Thomas M. Shinnick, Ph.D. Tuberculosis/Mycobacteri
ology Branch Division of AIDS, STD, and TB
Laboratory Research
SAFER HEALTHIER PEOPLE
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History of TB Genotyping Efforts

• IS6110 fingerprinting -- 1990
• CDC-funded regional labs -- 1992
• AR, CA, PHRI (NYC), NY, MI, CDC
• NTGSN project -- 1996-2002
• Added AL, TX, dropped CDC
• Contract genotyping labs -- 2003
• CA, MI

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National Tuberculosis Genotyping and Surveillance
Network

• IS6110-RFLP fingerprinting
• Spoligotyping as secondary typing method
• 10,883 isolates - 6,128 distinct patterns
• IS6110 fingerprinting is difficult to apply on a
  large scale
• technically demanding, cumbersome
• pattern comparison is difficult
• Turnaround time is too long to impact contact
  investigations significantly

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New National Genotyping Strategy

• Primary goal is to support TB Control Program
  Activities
• provide information in a timely manner
• provide typing information on all isolates
• Secondary goals are to
• assist in program evaluation
• characterize circulating strains
• create repository of strains

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Genotyping Strategy - Methods

• Primary typing methods will be PCR-based
• Spoligotyping
• MIRU-VNTR
• Produce easily compared, digital results
• Target turnaround time is 7 days from receipt
• Spoligotype MIRU-type distinguishes about 66
  of strains
• Secondary typing of clustered isolates when
  needed IS6110 RFLP

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Flow Chart of Genotyping Activities
Acquire isolates
Analyze use results
Genotype isolates
TB Program local Labs
Genotyping Lab
TB Program
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Challenges to TB Control Program

• Collect one isolate for each new case
• a variety of labs isolate M. tuberculosis
• public health labs, hospital labs
• commercial labs in and out of state
• obtain isolates in a timely manner
• Find resources to ship isolates
• genotyping is free, program pays shipping
• Develop a protocol for optimal use of the
  genotype information
• criteria for requesting secondary typing
• cluster investigation protocol

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Value of Real-Time Genotyping

• Refine contact and outbreak investigations
• Confirm or refute suspected links between
  patients
• e.g., family members infected with different
  strains
• e.g., casual contacts infected with the same
  strain
• Define magnitude of an outbreak
• e.g., is a new strain part of an on-going
  outbreak?
• Investigate suspected false cultures
• e.g., laboratory cross-contamination

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Value of Universal Genotyping

• Link patients in absence of epi data
• Identify new modes or venues of transmission
• Characterize circulating strains
• Identify highly successful strains
• Identify strains likely to become drug resistant
• Analyze transmission patterns over time
• Evaluate program effectiveness at preventing
  transmission

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Genotyping Services

• Contracts have been awarded
• Michigan and California State Laboratories
• Funds provided to type 8,000 isolates
• Have capacity to type 12,000 isolates
• Labs are ready to accept cultures
• DTBE is developing check list and manual to
  assist State Programs

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Reporting of Results and Program Considerations
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Cluster analysis by Genotyping Lab

• Will identify clusters of isolates in a TB
  Programs jurisdiction
• Where movement of patients is common, programs
  can request cross-jurisdictional cluster analysis
  
• CDC will maintain national databases, mostly
  limited to spoligotype and MIRU-VNTR results

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Reporting Results

• Isolates will come from a number of labs
• Reports will go to the state TB control program
• Exception will be for suspected false culture
  also report to the relevant lab

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Program Analysis of Genotype Results

• Distribute results to local programs as needed
• Compare genotyping results and results of contact
  investigations, demographics, risk factors,
  geographic distribution, patient information,
  etc.
• Determine if additional typing by IS6110 RFLP is
  warranted

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Some Possible Outcomes

• Most isolates will be distinct
• Known/suspected relationship confirmed by
  matching genotypes
• Matching genotypes suggest unsuspected
  transmission
• IS6110 typing may be requested if program plans
  to follow-up on result.

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IS6110 Fingerprinting

• When requested, isolates with matching spoligo
  and MIRU-VNTR genotypes will be typed by IS6110
  RFLP
• Analysis will be limited to comparing IS6110
  patterns for that specific set of isolates and
  will be reported as clustered, similar (/- 1
  band or 1 shifted band), or unclustered

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M. tuberculosis Typing Methods
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Typing of M. tuberculosis Strains

• Commonly Used Methods
• Fingerprinting using IS6110
• Spoligotyping
• MIRU-VNTR typing
• Alternative Methods
• Phage typing
• Pulsed-field electrophoresis
• Randomly amplified polymorphic DNA

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IS6110-DNA Fingerprinting
IS6110
PvuII
PvuII
Probe
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Spoligotyping

• Spacer oligonucleotide typing
• Reverse dot-blot hybridization method
• Detects presence of 43 spacer sequences in the
  direct repeat locus

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Direct Repeat (DR) Region
4
5
1
2
3
36 bp direct repeat
Unique spacer sequences
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Spoligotyping
H37Rv M. bovis Neg. Ctrl
Samples
4
15
1
43
Oligonucleotides
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Typing Using Tandem Repeats

• The Mtb genome contains 41 loci with direct
  tandem repeats of 50-70 bp
• The number of repeats per locus varies between
  strains
• Strains can be typed based on the number of
  repeats per locus

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MIRU-VNTR

• Variable Number of Tandem Repeats
• Frothingham et al
• Measures variability in six loci
• Mycobacterial Interspersed Repetitive Units
• Supply et al
• Measures variability in 12 loci

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MIRU locations
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Example of a MIRU-VNTR
MIRU4
CDC1551
Egypt
Brazil
Paris
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PCR Amplification of MIRU4
PCR product
CDC1551
Egypt
Brazil
Paris
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Digital result
MIRU pattern 232234253322
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Mtb Strain Typing Methods

• IS6110-based fingerprinting
• Most discriminatory method
• Slowest method -- 3-6 weeks
• Difficult to compare large numbers of patterns
• Spoligotyping and MIRU-VNTR typing
• Less discriminatory than IS6110 typing
• PCR-based ? rapid turnaround
• Yield digital results, facilitates comparisons
• Do not require viable cultures
• Amenable to high throughput approaches
• Spoligotyping Luminex-based systems
• MIRU-VNTR automated sequence analyzers

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Universal genotyping in WI 2000 2002 (n181
isolates)
No. clustered isolates ()
No. clusters (size)
Typing method
spoligotype
MIRU-VNTR
IS6110-RFLP
spoligotype MIRU-VNTR
all three methods