TB Lab Methods and Their Limitations

Transcription

1 TB Lab Methods and Their Limitations Alla Ostash Supervisor of TB, STD and Central Accessioning Units WA Public Health Laboratory June 2018 Objectives Upon completion of this training, participants will be able to: describe how various laboratory tests confirm active TB disease to improve patient outcome describe available tests, how to request them, and how to more accurately interpret test results identify the limitations of some lab methods when additional testing may be warranted 1

2 Microscopy The most cost effective and rapid test available BUT Microscopy does not differentiate between dead or live organisms There must be 5,000 to 10,000 of AFB organisms per 1ml of sputum for routine detection in stained smears * Culture only needs viable AFB organisms per ml of sputum * o AFB smear negative but culture positive specimens * Public Health Mycobacteriology. A Guide to the Level III Laboratory. US Dept of Health and Human Services/Public Health Services/Center for Disease Control. Patricia T. Kent, George P. Kubica, 1985 Fluorescent microscopy Ziehl Neelsen stain depicting cording 2

3 NAAT Nucleic Acid Amplification Test An umbrella name for any amplification type assay PCR, GeneXpert, etc. WAPHL NAAT uses IS6110 for DNA target Insertion Sequence number 6110 Present in all MTB complex species Can have as few as 0 or as many as 36 repeats per cell less than 0.1% of Beijing strain have no IS6110* The higher the number of repeats per cell the more sensitive the test *Manual of Clinical Microbiology. Patrick Murray, Ellen Baron, James Jorgensen, Marie Louise Landry, Michael Pfaller. 9 th edition NAAT Specificity and Sensitivity WAPHL NAAT 200 organisms/ml of specimen* Can not distinguish between live or dead organisms Detects TB DNA in patient specimen Overall Sensitivity 84.2% Specificity 98.8% GeneXpert Identification of Mycobacterium tuberculosis Screens for Rifampin Resistance directly in clinical specimens Overall Sensitivity 93.8% Specificity 98.7% Tanya A. Halse, Justine Edwards, Phyllis L. Cunningham, William J. Wolfgang, Nellie B. Dumas,Vincent E. Escuyer, and Kimberlee A. Musser J Clin Microbiol Apr;48(4):

4 Molecular Detection of Drug Resistance MDDR CDC offers an MDDR for an extended array of mutations associated with resistance to: Isoniazid (INH) Rifampin (RIF) Ethambutol (EMB) Pyrazinamide (PZA) Fluoroquinolones (FQ) Kanamycin, Amikacin and Capreomycin (KN, AM, CAP) To request this test, please contact the State TB Laboratory for details and instructions MDDR Targets, Sensitivity, and Specificity If mutation is detected in inha, consider the resistance to Ethionamide 4

5 What Do We Have So Far? After these 3 laboratory tests in conjunction with clinical findings, the clinician can determine the following How infectious the patient is Confirm an active TB disease The most effective treatment regimen AFB Smear NAAT Confirmation of TB disease and preliminary sensitivities MDDR 5

6 TB Culture The gold standard and final identification confirming active TB disease Takes 2 4 weeks to grow Can support growth not only of TB but other mycobacteria as well Essential step for TB sensitivities Growth on Solid Media M. tuberculosis on Middlebrook plate M. tuberculosis on LJ slant M. gordonae on LJ slant 12 6

7 Discussion False positive case Consider clinical presentation of the patient and request expedite genotyping If the patient genotyping results match to a current case with no epi link, most likely this is a case of laboratory cross contamination Low level of TB organisms TB culture only requires organisms per ml of sputum for detection* Specimen quality! Genotyping should confirm a recent transmission Lab error Specimens may have been labeled incorrectly during multiple steps of testing and sample transfers among various laboratories * Public Health Mycobacteriology. A Guide to the Level III Laboratory. US Dept of Health and Human Services/Public Health Services/Center for Disease Control. Patricia T. Kent, George P. Kubica, 1985 Culture Based Sensitivities Provide information on sensitivity pattern of MTB (Mycobacterium tuberculosis) BUT Sensitivities require pure MTB isolate Can not be performed on clinical samples Take 2 3 weeks for results False resistance may be detected 7

8 Sensitivity Methods FDA Approved Methods MGIT First Line DST o Mycobacterium Growth Indicator Tube Available but NOT FDA approved VersaTrek Sensititre Laboratory developed methods o MDDR CDC o Whole Genome Sequencing Wadsworth Center, NY State DOH MGIT Sensitivities Detect MTB growth by depletion of Oxygen in the MGIT tube The same instrument is used for culture First line DST Streptomycin (1.0 µg/ml) Isoniazid (0.1µg/mL) Rifampin (1.0 µg/ml) Ethambutol (5.0 µg/ml) Pyrazinamide (100 µg/ml) SIRE and PZA WAPHL, SHMC(PAML), Harborview (except for Strep & PZA), private labs (Quest) 8

9 MGIT Sensitivities Performed on every first time positive MTB isolate from a patient Re checked after 3 months from the original collection date if culture conversion hasn t occurred May be done earlier if suspecting resistance Set up at least once a week at WA PHL SIRE requires days incubation PZA may require up to 21 days Challenges of Drug Sensitivity Testing (DST) MTB is a very slow growing organism Poor competitor, easily overgrown by normal flora or other Mycobacteria Very easily over or under inoculated during set up False results o Requires extensive training for the personnel PZA drug is active in low acidic environment Very hard to achieve in vitro MTB grows very poorly in PZA medium 9

10 MGIT PZA sensitivity Growth Control Strep INH=R Rif EMB MGIT SIRE sensitivity Growth Control PZA Contamination 10

11 Agar Proportion Method or Plate Sensitivities Any resistance to SIRE is confirmed by agar proportion method PZA is not available on plates Disks saturated with known concentration of drugs are imbedded into agar on the plates The drug diffuses throughout the agar providing proper drug concentration MTB cultures are diluted to acceptable concentration of organism and then inoculated Plate Sensitivities Once the plates are inoculated, they are incubated for 3 weeks to achieve sufficient growth A batch of sensi plates ready for inoculation Sensi plates with sufficient MTB growth 11

12 WAPHL Plate Sensitivities Drugs Tested First Line (mcg/ml) Streptomycin (2.0) Streptomycin (10.0) Isoniazid (0.1) Second line (mcg/ml) Ethionamide (5.0) p Aminosalicylic Acid (2.0) Ofloxacin (1.0) Amikacin (6.0) Isoniazid (0.2) Rifampin (1.0) Ethambutol (5.0) Ethambutol (10.0) Summary Every lab test gives you one piece of a puzzle Important to understand the lab report No standardized language TB has one of the most complex testing algorithm Multiple steps and laboratories, requires experienced staff and expensive facilities, convoluted specimen transfer among reference labs WE (WAPHL) ARE YOUR RESOURCE! 12

13 THANK YOU! QUESTIONS? WAPHL TB Lab 13