ISDH Lab TB Testing Update

Lixia Liu PhD MP(ASCP)

September 16, 2010

Mycobacterium Assays Microscopy Examination Real Time PCR (Polymerase Chain Reaction) Identification

Growth Characteristic

Biochemicals

HPLC (High Performance Liquid Chromatography)

AccuProbe Drug Susceptibility Test (DST)

Samples

Decontamination (non-sterile)

AFB Smear Culture

Real Time PCR* HPLC

DST** Biochemical

Send to CDC

Simplified Testing Algorithm

* It is performed on one sputum sample of each new patients only** It is performed on first TB isolates of new patients only

AccuProbe Assay

MGIT +, AFB+ LJ Slant +, AFB+

STAT testing using AccuProbe Assay*

AFB+, 7H11 plate

*Request for this test should be approved by TB program prior to submission

Microscopic Examination Results

Microscopic Examination

Acid Fast BacteriaNot Found

Found< 1 per field1-10 per field

> 10 per field

Real Time PCR Results

Real Time PCR Assay • TB Complex

Positive Negative Inconclusive

Identification Results Identification

Mycobacterium tuberculosis complex avium complexchelonaekansasiixenopifortuitum

gordonaeother species

Not isolated Culture is contaminated, more time required Overgrown Culture sent to CDC

DST Results

Drug Susceptibility

• Streptomycin 2.0 µg/ml Susceptible Resistant

• Isoniazid 0.1 µg/ml Susceptible Resistant

• Rifampin 2.0 µg/ml Susceptible Resistant

• Ethambutol 2.5 µg/ml Susceptible Resistant

• Pyrazinamide 100 µg/ml Susceptible Resistant

Other Results

Unsatisfactory • Leak in Transit • Quantity Not Sufficient • Unacceptable Container • No Specimen • No Name/Patient ID on Tube

Samples Submitted for TB Genotyping

Freezes aliquots of isolates submitted for genotyping

Forward isolates to TB genotyping center. Genotyping results are entered to ISDH-TB

program database.

TB Genotyping

Spoligotyping (Spacer Oligonucleotide typing)

MIRU (Mycobacterial Interspersed Repetitive Units)

RFLP (Restriction Fragment Length Polymorphism)

Spoligotyping

a hybridization assay that detects variability in the direct repeat (DR) region in the DNA of M. tuberculosis.

Cited from CDC’s TB Genotyping Website

MIRU Analysisanalysis of DNA segments containing “tandem

repeated” sequences in which the number of copies of the repeated sequence varies among strains.

12 loci

24 loci

Sample MIRU dataExample 1

 MIRU locus name

02 04 10 16 20 23 24 26 27 31 39 40

No. of repeats 2 3 2 2 3 4 2 5 3 3 2 2

MIRU designation: 232234253322

Example 2

  MIRU locus name

02 04 10 16 20 23 24 26 27 31 39 40

No. of repeats 1 4 3 2 2 4 0 4 3 5 4 11

MIRU designation: 14322404354b

Cited from CDC’s TB Genotyping Website

RFLP Analysis

RFLP genotyping detects variations generated by the insertion element IS6110.

Data example

Cited from CDC’s TB Genotyping Website

CULTURE!

CULTURE!!

CULTURE!!!

Submit all TB isolates to ISDH Lab!

New Changes

Sputum Collection Algorithm

Non Tuberculosis Isolate Submission

Time Span and Number of Specimens Collected for

Selected Active Cases

Case 1 Case 2 Case 3 Case 4 Case 5

Number of Specimens

Period of Collection (Days)

21

42

21

53

23

47

28

57

46

77

* Non Tuberculous Mycobacteria

M. tuberculosis24.2%

NTM*70.0%

Other Identification5.8%

Identification for Culture Samples Received N=398

The Indiana State Department of Health provides laboratory support for tuberculosis (TB)control and prevention by isolating, identifying, and performing susceptibility testing on clinical

specimens from submitters pre-approved by our TB program.  The lab also performs TB rule in/out testing on mycobacterium isolates from Laboratories lacking this capability.  However, speciation on non-tuberculosis (NTB) isolates

will not be performed.  

Non-tuberculosis Isolate Submission

New Laboratory Information System (LIS)

LIMSNET & STARLIMS

LIMSNET STARLIMS LIMSNETSystem Flow

Data Flow Web-based Report Generation Report Receiving data entry

Work Flow Submitters ISDH Lab Submitters

What Does LIMSNET Offer?

No more paper forms to fill out for submission.

Sample status checking Real time result reporting Retrieve TB results at submitters’

sites

How Do I Sign Up for LIMSNET?

http://limsnet.isdh.in.gov

Call the help desk line at 888-535-0011 or 317-921-5506

Contact Information TB Control Program

• 317-233-7545 TB Laboratory

• 317-921-5892 (phone)• 317-927-7804 (fax)

Shipping Container and Submission Form• 317-921-5875

Acknowledgement

ISDH TB Lab Crew and others• Ed Harris• Laura Taube• Avia Boss• Jessica Gentry

…

QUESTIONS?