ISDH Lab TB Testing Update Lixia Liu PhD MP(ASCP) September 16, 2010

Mycobacterium Assays Microscopy Examination Microscopy Examination Real Time PCR (Polymerase Chain Reaction) Real Time PCR (Polymerase Chain Reaction) Identification Identification Growth Characteristic Biochemicals HPLC (High Performance Liquid Chromatography) AccuProbe Drug Susceptibility Test (DST) 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 Microscopic Examination Acid Fast Bacteria Not Found Found < 1 per field 1-10 per field Acid Fast Bacteria Not Found Found < 1 per field 1-10 per field > 10 per field > 10 per field

Real Time PCR Results Real Time PCR Assay Real Time PCR Assay TB ComplexTB Complex Positive Positive Negative Negative Inconclusive Inconclusive

Identification Results Identification Mycobacterium tuberculosis complex avium complex chelonae kansasii xenopi fortuitum Identification Mycobacterium tuberculosis complex avium complex chelonae kansasii xenopi fortuitumgordonae other species Not isolated Not isolated Culture is contaminated, more time required Culture is contaminated, more time required Overgrown Overgrown Culture sent to CDC Culture sent to CDC

DST Results Drug Susceptibility Drug Susceptibility Streptomycin2.0 µg/ml SusceptibleResistantStreptomycin2.0 µg/ml SusceptibleResistant Isoniazid 0.1 µg/mlSusceptibleResistant Isoniazid 0.1 µg/mlSusceptibleResistant Rifampin2.0 µg/mlSusceptibleResistant Rifampin2.0 µg/mlSusceptibleResistant Ethambutol2.5 µg/mlSusceptibleResistant Ethambutol2.5 µg/mlSusceptibleResistant Pyrazinamide100 µg/mlSusceptibleResistant Pyrazinamide100 µg/mlSusceptibleResistant

Other Results Unsatisfactory Unsatisfactory Leak in TransitLeak in Transit Quantity Not SufficientQuantity Not Sufficient Unacceptable ContainerUnacceptable Container No SpecimenNo Specimen No Name/Patient ID on TubeNo Name/Patient ID on Tube

Samples Submitted for TB Genotyping Freezes aliquots of isolates submitted for genotyping Freezes aliquots of isolates submitted for genotyping Forward isolates to TB genotyping center. Forward isolates to TB genotyping center. Genotyping results are entered to ISDH-TB program database. Genotyping results are entered to ISDH-TB program database.

TB Genotyping Spoligotyping ( Spacer Oligonucleotide typing ) Spoligotyping ( Spacer Oligonucleotide typing ) MIRU ( Mycobacterial Interspersed Repetitive Units) MIRU ( Mycobacterial Interspersed Repetitive Units) RFLP ( Restriction Fragment Length Polymorphism) RFLP ( Restriction Fragment Length Polymorphism)

Spoligotyping 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 Cited from CDC’s TB Genotyping Website

MIRU Analysis MIRU Analysis analysis of DNA segments containing “tandem repeated” sequences in which the number of copies repeated” sequences in which the number of copies of the repeated sequence varies among strains. of the repeated sequence varies among strains. 12 loci 12 loci 24 loci 24 loci

Sample MIRU data Sample MIRU data Example 1 MIRU locus name No. of repeats MIRU designation: Example 2 MIRU locus name No. of repeats MIRU designation: b Cited from CDC’s TB Genotyping Website

RFLP Analysis RFLP Analysis RFLP genotyping detects variations generated by the insertion element IS6110. Data example Cited from CDC’s TB Genotyping Website Cited from CDC’s TB Genotyping Website

CULTURE! CULTURE!! CULTURE!!! Submit all TB isolates to ISDH Lab!

New Changes Sputum Collection Algorithm Sputum Collection Algorithm Non Tuberculosis Isolate Submission Non Tuberculosis Isolate Submission Case 1Case 2Case 3Case 4Case 5 Number of Specimens Period of Collection (Days) Time Span and Number of Specimens Collected for Selected Active Cases The numbers on the blue bars represent the numbers of specimen collected from each patient. The numbers on the red bars represent the numbers of day the period of collection spanned. Case 1Case 2Case 3Case 4Case 5 Number of Specimens Period of Collection (Days) Time Span and Number of Specimens Collected for Selected Active Cases The numbers on the blue bars represent the numbers of specimen collected from each patient. The numbers on the red bars represent the numbers of day the period of collection spanned. Case 1Case 2Case 3Case 4Case 5 Number of Specimens Period of Collection (Days) Time Span and Number of Specimens Collected for Selected Active Cases The numbers on the blue bars represent the numbers of specimen collected from each patient. The numbers on the red bars represent the numbers of day the period of collection spanned. Case 1Case 2Case 3Case 4Case 5 Number of Specimens Period of Collection (Days) Time Span and Number of Specimens Collected for Selected Active Cases The numbers on the blue bars represent the numbers of specimen collected from each patient. The numbers on the red bars represent the numbers of day the period of collection spanned.

Case 1Case 2Case 3Case 4Case 5 Number of Specimens Period of Collection (Days) Time Span and Number of Specimens Collected for Selected Active Cases The numbers on the blue bars represent the numbers of specimen collected from each patient. The numbers on the red bars represent the numbers of day the period of collection spanned. Time Span and Number of Specimens Collected for Selected Active Cases Time Span and Number of Specimens Collected for Selected Active Cases Time Span and Number of Specimens Collected for Selected Active Cases Case 1Case 2Case 3Case 4Case 5 Number of Specimens Period of Collection (Days)

* Non Tuberculous Mycobacteria M. tuberculosis 24.2% NTM* 70.0% Other Identification 5.8% Identification for Culture Samples Received N=398 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 LIMSNETSTARLIMS LIMSNET System 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. No more paper forms to fill out for submission. Sample status checking Sample status checking Real time result reporting Real time result reporting Retrieve TB results at submitters’ sites Retrieve TB results at submitters’ sites

How Do I Sign Up for LIMSNET? Call the help desk line at Call the help desk line at or or

Contact Information TB Control Program TB Control Program TB Laboratory TB Laboratory (phone) (phone) (fax) (fax) Shipping Container and Submission Form Shipping Container and Submission Form

Acknowledgement ISDH TB Lab Crew and others ISDH TB Lab Crew and others Ed HarrisEd Harris Laura TaubeLaura Taube Avia BossAvia Boss Jessica GentryJessica Gentry …

QUESTIONS?