What Happens to Sputum After it Gets to the Lab? La Vonda Benbow, BS, MLT(ASCP)cm Mycobacteriology Supervisor North Carolina State Laboratory of

Transcription

1 What Happens to Sputum After it Gets to the Lab? La Vonda Benbow, BS, MLT(ASCP)cm Mycobacteriology Supervisor North Carolina State Laboratory of Public Health 1

2 Objectives Discuss the process for receiving and processing sputum Explain the various technologies available to identify M. tuberculosis Explain technologies and methodologies available to obtain M. tuberculosis drug susceptibilities 2

3 Digestion/Decontamination Kinyoun stain SPUTUM Solid culture media 3

4 Safety first! Biosafety Level 2 and Biosafety Level 3 suite Standard precautions include negative air flow, hands-free hand washing sinks, double-door containment Bioaerosol containing equipment Autoclaved waste Personal Protective Equipment (PPE) Liquid impermeable lab coats and isolation gowns Double gloves and shoe covers PAPRs (Powered-air Purifying Respirator) or N-95 masks 4

5 First things first! CDC Public Health Image Library, Image ID#15485, Debora Cartagena 5

6 Specimen Submission Specimens should be sent to the laboratory within 24 hours of collection o o Refrigerate if mailing will be delayed more than a few hours Avoid batching of samples 6

7 Specimen Receipt Specimen accessioning o o o Opening the mail Data entry Test request Reagent and media preparation Process can take minutes 7

8 Specimen Processing Decontamination and concentration Acid-Fast Bacillus (AFB) smear o Fluorescent stain Inoculation of media o Solid media o Liquid media 8

9 Centrifugation, Digestion and Decontamination 4-5 step process o o o o Centrifugation N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) liquify sputum and decontaminate Neutralization buffer + Centrifugation Resuspension of pellet Process takes approximately minutes 9

10 Acid Fast Bacteria (AFB) Smear Fluorescent dye (staining reagent) Non-specific o o All mycobacteria are acid fast Does not distinguish between viable and dead organisms Limited sensitivity o 5,000 10,000 AFB/mL required for detection Takes approximately 90 minutes 4.5 hours to complete CDC Image #10033/ Hsi Liu, Ph.D., MBA, James Gathany 10

11 Inoculation of Media Lowenstein-Jensen (LJ) solid media 7H10/7H11 Middlebrook solid media Liquid media Media inoculation 11

12 Inoculation of Media Lowenstein-Jensen (LJ) Gold standard Solid media formulated to support the growth of mycobacteria Inoculated with digested/decontaminated sputum Observed for 6-8 weeks for growth 12

13 Inoculation of Media Middlebrook 7H10/7H11 Inoculated with digested/decontaminated sputum Grows fewer contaminants than egg-based media Observed for 4-6 weeks for growth CDC Image #2904/ Dwight Lambe 13

14 Inoculation of Media 7H9 Broth (Liquid) Inoculated with digested/decontaminated sputum Used in automated systems Growth can be detected is as few as days from specimen receipt Observed for 4-6 weeks for growth 14

15 Mycobacterium spp. Identification- Visual Identification HPLC DNA Probes Polymerase Chain Reaction (PCR) DNA Sequencing Matrix assisted light desorption time of flight (MALDI-tof) 15

16 Mycobacterium spp. Identification Visual Identification Solid media o Magnifying lamps o Stereomicroscopes o Trained personnel Liquid media o Flakes o Kinyoun stain CDC, Public Health Image Library, #

17 Mycobacterium spp. Identification High-performance Liquid Chromatography HPLC Can be extracted from any medium High biomass requirement Expertise required to read chromatograms Old technology 48 hour process 17

18 Mycobacterium spp. Identification DNA Probes Can be performed from any medium Nucleic acid hybridization 2-3 hours process Expensive 18

19 Mycobacterium spp. Identification - PCR Polymerase Chain Reaction Molecular test - amplifies a single copy or a few copies of a piece of DNA making thousands-billions of copies of a DNA sequence (amplification/detection) Rapid detection 2 to 4 hours 19

20 Mycobacterium spp. Identification- DNA Sequencing Determines the precise order of nucleotides within a DNA molecule Labor intensive Requires expertise in molecular methodologies (PCR, agarose gels) 48 hour process 20

21 Mycobacterium spp. Identification MALDI-tof Matrix Assisted Laser Desorption Ionization time of flight Not FDA cleared; for research use only Rapid identification approx. 1 hour 21

22 Drug Susceptibility Testing Automated liquid detection Conventional Molecular Detection for Drug Resistance 22

23 Mtb Drug Susceptibilities Liquid Automated system First line drugs day turn-around time (barring no contamination or other issues) 23

24 Mtb Drug Susceptibilities Conventional CDC Image #16464/ Dr. George Kubica Drug added to solid media Performed when resistance seen to first line drugs Performed only on Mtb Manual method requiring colony counting 24

25 Mtb Drug Susceptibilities DNA Sequencing Determines the precise order of nucleotides within a DNA molecule Looking for mutations Labor intensive Requires expertise in molecular methodologies (PCR, agarose gels) 48 hour process 25

26 Summary Sample Processing Decontamination and concentration Inoculate media Lowenstein- Jensen 7H10 plate Liquid media Fluorescent smear Performed on concentrated clinical samples Identification Culture Kinyoun staining Highperformance Liquid Chromatography (HPLC) Identification DNA Probes PCR Sequencing MALDI-tof Drug Susceptibility Testing Liquid First line drugs Conventional drug susceptibility testing Second-line drugs Sequencing 26

27 Thank you! 27