isdh lab tb testing update lixia liu phd mp(ascp) september 16, 2010
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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?