1. Laura Lane, Epidemiologist
GENOTYPING 101
Laura Lane, Epidemiologist
Genotype Coordinator
August 2014
Introductions- Lana Jones, Tracie Gardner

2. Initial Self-Assessment
How would you rate your current overall knowledge of genotyping?
Geno-what?
I have heard the term thrown around a few times.
My knowledge is solid but not applied regularly.
I view results for patients and clusters on a regular basis, but still have a few questions.
I am an expert and am interested in teaching this course!

3. Initial Self-Assessment
TB genotyping requires a:
Smear positive lab result
Culture positive lab result
Abnormal chest X-ray
Positive NAAT result
I don’t know
Correct answer is B.

4. Initial Self-Assessment
You just exported data from TBGIMS and want to evaluate GENType results for a patient. Select the correct value for the result:
PCR00002
14RF4835
East Asian (L2)
TX_0010
G08448
I don’t know
Correct answer is E

5. Initial Self-Assessment
Genotyping results:
Support contact investigations
Can identify false positive results
Look like= G10509
Are also referred to as DNA fingerprinting
All of the above
I don’t know
Correct answer is E

6. Objectives Describe overall purpose of genotyping in a TB program
Demonstrate examples of GENType results
Communicate “Best Practices” per CDC guidelines

7. TB Program Genotyping Objective
To reduce TB in Texas by identifying TB transmission among cases and contacts using genotyping.

8. Overview
Genotyping: Lab approach used to analyze genetic material of M. tuberculosis
Genotyping uses portions of the genome to identify different strains of M. tuberculosis
Tool to understand transmission of tuberculosis
When viewing initial gentoyping results focus on GENType

9. TB Genotyping Results
When combined with epidemiological data, can identify persons with TB disease involved in same chain of recent transmission
Do not function as a diagnostic measure
Once linked to patient data by State Coordinator (Laura Lane), results are found in the CDC database called Tuberculosis Genotyping Information Management System (TB GIMS)

10. Uses of genotyping Detect false-positive culture results
Enhance investigations
Distinguish relapse from new infection
Monitor trends and evaluate TB control program
Detect M. bovis and M. bovis BCG
- Reinfection: usually, though it is possible to be reinfected with the same strain if the person continues to be exposed after cure.

11. Universal Genotyping
Submitting one isolate from every patient with a culture positive specimen in a TB program’s jurisdiction.

12. What is the difference between a specimen and an isolate?
A specimen is a clinical sample
Sputum, bronchial wash, urine, blood, cerebrospinal fluid; tissues from organs or gastric aspirates
Collected from patients suspected of having TB
Specimens may or may not contain M. tuberculosis
If M. tuberculosis grows in culture media from a specimen, it is called an isolate
Only isolates identified as M. tuberculosis can be genotyped

13. Best Practices Practices All Programs Should Implement
Ensure each patient with a positive Mycobacterium tuberculosis culture result has a genotyped isolate
Best Practice #2
Link genotyping results to surveillance data promptly
Practices Programs Should Implement as Resources Allow
Best Practice #3
Integrate genotyping information into routine case management, contact investigation, and cohort review activities
- When an isolate or clinical specimen is submitted to DSHS lab, genotyping is automatically performed on first specimen
-Linking is performed by Genotyping coordinator; speed is based upon a complete RVCT being submitted to our surveillance department
Best Practice #4
Examine concerning genotyping clusters
Best Practice #5
Communicate with other jurisdictions and CDC
Best Practice #6
Develop and maintain capacity for using genotyping information in routine TB control

14. Spoligotype and MIRU Analysis
Two different types of analytic methods:
Mycobacterial Interspersed Repetitive Units (MIRU)
Spaceroligonucleotide (Spoligotype)

15. GENType Results
Each unique combination of spoligotype + MIRU + MIRU2 is assigned a GENType
Indicated as a “G” followed by 5 digits,
i.e. G00010
No GENTypes were automatically assigned prior to 2009
-GENTpes were not automatically assigned prior to 2009, however some isolates will have GENType results because the state or jurisdiction requested additional testing (usually in the context of an outbreak)

16. How to make a GENType Spoligotyping & MIRU
Spoligotype
MIRU
MIRU2
GENTYPE = discriminatory power **added discriminatory power with 24 locus MIRU**
Spoligotyping & MIRU
These are combined to create the 12-loci result indicating the PCRType
MIRU2
Added in 2009 to expand result to a 24-loci result indicating the GENType
PCRType
PCR00016
GENType
G00013

17. GENType vs PCRType

18. One PCRType = Many GENTYpes One GENTYpe = One PCRTYPE
MIRU
MIRU2
PCR00002
G00010
G00012
G01045
G01046
G01048
G01049
G01050
G01053
G01055
G01061
G01063
G01066
G01083
G01106
Common PCRType
107 different GENTypes for this PCRType!
GENType = greater distinction between the results
Nationally have moved away from PCRType
Clustering algorithms based on GENType
Gentype is more specific

19. MIRU-VNTR
What is meant by a dash(-) or percent sign (%) in a MIRU-VNTR result?
A dash (-) at a particular locus means that the result is unknown or cannot be determined (e.g., the MIRU-VNTR result would be: ).
A percent sign (%) at a particular locus means that there are two valid results for a particular loci (both are accurate) and this indicates mixed infection.

20. Objectives Describe overall purpose of genotyping in a TB program
Demonstrate examples of GENType results
Communicate “Best Practices” per CDC guidelines

21. Questions? Laura.Lane@dshs.state.tx.us 512-533-3162