LABORATORY DIAGNOSTIC TOOLS

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LABORATORY DIAGNOSTIC TOOLS
Presented by Jason A. Lee
1999
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New Technology in TB Testing
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TB is a Deadly Infectious Disease Worldwide

• 1.7 billion people infected with TB
• 10 will develop active disease
• 8 million new cases/year
• 3.3 million deaths/year

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CDC RECOMMENDATIONS FOR TB LABORATORIES

• Report AFB results within 24 hours of
  receipt in Lab Fluorescent stain (PHL
  confirms with Kinyoun stain)
• Detect growth in liquid medium within 10
  days PHL uses MB/BacT system and L - J -
  slants
• Identify MTB isolates within 14-21 days of
  receipt in Lab
• Mycolic acid patterns by HPLC or nucleic
  acid probes

Cont
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CDC RECOMMENDATIONS FOR TB LABORATORIES (cont)

• Determine susceptibility of new MTB isolates
  to primary drugs and report results within
  21-28 days of receipt in Lab. When
  Multi-drug resistance is found, test isolate to
  another methodology for confirmation.
• Isoniazid, rifampin, streptomycin, ethambutol
  and pyrazinamide
• Submit all isolates to a fingerprinting Lab
• This helps to identify cross-contamination
  and provides EPI information

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Clinical Significance ofRapid TB Testing

• Isolate patients to prevent spread of disease
• Treatment decisions Is it MTB or MOTT?
• Reduce health care cost
• Cost of patient isolation/treatment
• Cost of further spread of disease

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AVERAGE TB LABORATORY
Sputum or Other Sample
Na0H - NALC Centrifugation
Sediment
Solid Culture
Liquid Culture
Acid- Fast Microscopy
Report within 24 hrs
Identify - HPLC or Probes
No Growth Report within 6 wks
Report within 21 days
MTB
MOTT
Drug Susceptibility
Report within 28 days
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PHL - TB LABORATORY
Sputum or Other Sample
Na0H - NALC Centrifugation
Sediment
LJ
BacT (MB/BacT
AFB
Fluorochrome Confirmed by Kinyoun
Report within 24 hrs
No Growth Report Within 6 wks
Growth
New Patients Not on Treatment
Amplified Test Gen Probe
Identify by Mycolic Acid Pattern using HPLC or by
Nucleic Acid Probes.
Emergency Reports can be reported within 6hrs
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MTB
MOTT
Results Reported 7 - 21 days
All isolates Are Sent For Fingerprinting
Drug Susceptibilities BacTec
Conventional Plate Susceptibilities Performed
for Confirmation
Non - resistant
Resistant
Results Reported 21 - 35 days and decreasing)
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GETTING READY TO PROCESS
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SORTING SPECIMENS
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CHECKING THE INFORMATION
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PROCESSING BEGINS
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SAFE CENTRIFUGATION
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CHECKING AND RE-CHECKING
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PAYING ATTENTION PREVENTS MISTAKES
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INOCULATING THE BacT BOTTLE
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INOCULATING THE LJ TUBE
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MAKING SMEARS
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CYTOSPIN CLEANS UP SMEARS
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CYTOSPIN FUNNEL
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CYTOSPIN - FINAL STEP
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DAYS END
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BEING CAREFUL WITH A MESSY JOB
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READING SMEARS
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AFB FLUOROCHROME
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AFB POSITIVE SMEAR
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MAKING SMEARS
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AFB POSITIVE - CORDING
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BIGGER IS BETTER
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Bac T ---- The Future
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THE PRINCIPLE OF GROWTH DETECTION
CO2 Sensor
From Green To Yellow
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BacT TO THE FUTURE
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THIS IS A POSITIVE
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POSITIVE BOTTLES ARE REMOVED IMMEDIATELY
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WORKING HERE IS GREAT
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YES, IT IS YELLOW!
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
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THE PATTERN FOR MTB
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THE PATTERNS FOR MOTTs
M. fortuitum
M. chelonae
M. terrae complex (M. nonchromogenium
M. gordonae
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A DINOSAUR!
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THATS A LOT OF LJs!
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LOOKING FOR GROWTH
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DRUG SUSCEPTIBILITY PLATES
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MORE DRUG SUSCEPTIBILITY PLATES
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TB TESTING --- THE AMPLIFIED WAY
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Amplified Mycobacterium Tuberculosis Direct (MTD)
Assay

• Amplified molecular diagnostic assay detects
  M. tuberculosis directly in sputum samples in
  3 hours
• Utilizes a Transcription-Mediated Amplification
  system (TMA)
• Transcription of RNA amplicon from ribosomal RNA
  targets
• Amplicon is detected by DNA probes in the
  Hybridization Protection Assay format (HPA)
• Single tube format

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Amplified MTD Clinical Utility Algorithm
AFB Smears Positive Samples

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MTD Test
TB Unlikely 86.3 NPV, MOTT Likely
MTD-Positive 100 PPV for TB
Verify MOTT by Alternate Testing Method
Repeat MTD Test with New Specimen
Test for Inhibition
Positive M. tuberculosis Identification
Confirmation with Culture
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AMPLIFICATION TEST PERFORMED BY THE PHL
This sample was digested by the submitter,
referred to the PHL, we digested it again!
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Amplified Test Reliability
Culture
-

TP 43
FP 1

Gen-Probe Amplified Test

FN 0
TN 31
-
Sensitivity TP x 100
100
TP FN
Specificity TN x 100
97
TP FN
TP x 100 98
Positive Predictive Value
TP FP
Negative Predictive Value
TN x 100 100
TP FN
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BACK TO THE FUTURE
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TB OR NOT TB?
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Dr. Jason Lee
Cathy Walker
TECHNICAL SUPPORT
We Thank You