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
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Mycobacterium Assays Microscopy Examination Real Time PCR (Polymerase Chain Reaction) Identification
Growth Characteristic
Biochemicals
HPLC (High Performance Liquid Chromatography)
AccuProbe Drug Susceptibility Test (DST)
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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
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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
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Microscopic Examination Results
Microscopic Examination
Acid Fast BacteriaNot Found
Found< 1 per field1-10 per field
> 10 per field
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Real Time PCR Results
Real Time PCR Assay • TB Complex
Positive Negative Inconclusive
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Identification Results Identification
Mycobacterium tuberculosis complex avium complexchelonaekansasiixenopifortuitum
gordonaeother species
Not isolated Culture is contaminated, more time required Overgrown Culture sent to CDC
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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
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Other Results
Unsatisfactory • Leak in Transit • Quantity Not Sufficient • Unacceptable Container • No Specimen • No Name/Patient ID on Tube
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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.
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TB Genotyping
Spoligotyping (Spacer Oligonucleotide typing)
MIRU (Mycobacterial Interspersed Repetitive Units)
RFLP (Restriction Fragment Length Polymorphism)
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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
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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
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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
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RFLP Analysis
RFLP genotyping detects variations generated by the insertion element IS6110.
Data example
Cited from CDC’s TB Genotyping Website
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CULTURE!
CULTURE!!
CULTURE!!!
Submit all TB isolates to ISDH Lab!
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New Changes
Sputum Collection Algorithm
Non Tuberculosis Isolate Submission
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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
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* Non Tuberculous Mycobacteria
M. tuberculosis24.2%
NTM*70.0%
Other Identification5.8%
Identification for Culture Samples Received N=398
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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
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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
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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
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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
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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
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Acknowledgement
ISDH TB Lab Crew and others• Ed Harris• Laura Taube• Avia Boss• Jessica Gentry
…
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QUESTIONS?