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LABORATORY DIAGNOSTIC TOOLS

LABORATORY DIAGNOSTIC TOOLS. Presented by Jason A. Lee. 1999. New Technology in TB Testing. 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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LABORATORY DIAGNOSTIC TOOLS

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  1. LABORATORYDIAGNOSTICTOOLS Presented by Jason A. Lee 1999

  2. New Technology in TB Testing

  3. 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

  4. 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 daysPHL 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 Con’t

  5. CDC RECOMMENDATIONS FOR TB LABORATORIES (con’t) • 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

  6. 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

  7. AVERAGE TB LABORATORY Sputum or Other Sample Na0H - NALC Centrifugation Sediment Liquid Culture Solid 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

  8. PHL - TB LABORATORY Sputum or Other Sample Na0H - NALC Centrifugation Sediment BacT (MB/BacT LJ AFB Fluorochrome Confirmed by Kinyoun Reportwithin 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

  9. 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)

  10. GETTING READY TO PROCESS

  11. SORTING SPECIMENS

  12. CHECKING THE INFORMATION

  13. PROCESSING BEGINS

  14. SAFE CENTRIFUGATION

  15. CHECKING AND RE-CHECKING

  16. PAYING ATTENTION PREVENTS MISTAKES

  17. INOCULATING THE BacT BOTTLE

  18. INOCULATING THE LJ TUBE

  19. MAKING SMEARS

  20. CYTOSPIN CLEANS UP SMEARS

  21. CYTOSPIN FUNNEL

  22. CYTOSPIN - FINAL STEP

  23. DAY’S END

  24. BEING CAREFUL WITH A MESSY JOB

  25. READING SMEARS

  26. AFB FLUOROCHROME

  27. AFB POSITIVE SMEAR

  28. MAKING SMEARS

  29. AFB POSITIVE - CORDING

  30. BIGGER IS BETTER

  31. “Bac T ---- The Future”

  32. THE PRINCIPLE OF GROWTH DETECTION FromGreenTo Yellow CO2 Sensor

  33. BacT … TO THE FUTURE

  34. THIS IS A POSITIVE

  35. POSITIVE BOTTLES ARE REMOVED IMMEDIATELY

  36. “WORKING HERE IS GREAT”

  37. YES, IT IS YELLOW!

  38. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

  39. THE PATTERN FOR MTB

  40. THE PATTERNS FOR MOTTs M. fortuitum M. chelonae M. terrae complex (M. nonchromogenium M. gordonae

  41. A DINOSAUR!

  42. THATS A LOT OF LJs!

  43. LOOKING FOR GROWTH

  44. DRUG SUSCEPTIBILITY PLATES

  45. MORE DRUG SUSCEPTIBILITY PLATES

  46. TB TESTING --- THE AMPLIFIED WAY

  47. 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

  48. Amplified MTD Clinical Utility Algorithm AFB Smears Positive Samples + - MTD Test MTD-Positive 100% PPV for TB TB Unlikely 86.3% NPV, MOTT Likely Verify MOTT by Alternate Testing Method Repeat MTD Test with New Specimen Test for Inhibition Positive M. tuberculosis Identification Confirmation with Culture

  49. AMPLIFICATION TEST PERFORMED BY THE PHL * This sample was digested by the submitter, referred to the PHL, we digested it again!

  50. 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 TPx 100 = 98% Positive Predictive Value = TP+ FP Negative Predictive Value = TN x 100 = 100% TP+ FN

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