THE ROLE OF THE LABORATORY

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1 TB CASE MANAGEMENT AND CONTACT INVESTIGATION INTENSIVE NOVEMBER 1-4, 2016 THE ROLE OF THE LABORATORY LEARNING OBJECTIVES Upon completion of this session, participants will be able to: 1. Describe three laboratory methods used in the diagnosis and control of TB resulting in a better understanding of laboratory results and improved communication between the clinician, the laboratory, and the patient INDEX OF MATERIALS 1. The slide outline Presented by: Mark Pandori, Ph.D., H.C.L.D. (A.B.B.) PAGES 1-33 SUPPLEMENTAL MATERIAL None, UCSF 300 Frank H. Ogawa Plaza, Suite 520 Oakland, CA; Office (510)

2 TB CASE MANAGEMENT AND CONTACT INVESTIGATION INTENSIVE NOVEMBER 1-4, 2016 ADDITIONAL REFERENCES CDC. Availability of an Assay for Detecting Mycobacterium tuberculosis, Including Rifampin- Resistant Strains, and Considerations for Its Use United States, MMWR 2013;62 (No.41) October 18, CDC. Guide to the application of genotyping to tuberculosis prevention and control. Page last updated September 1, CDC. Report of an expert consultation on the uses of nucleic acid amplification tests for the diagnosis of tuberculosis. Page last updated September 1, WHO. Drug-resistant tuberculosis. Frequently asked questions. January (Accessed November 10, 2014). CDC. Updated Guidelines for Using Interferon Gamma Release Assays to Detect Mycobacterium tuberculosis Infection, United States. MMWR 2010; 59 (No.RR-5) See CDC factsheet Interferon-Gamma Release Assays (IGRAs). Page last updated September 1, Chang-Hong, S., Xiao-Wu, W., Hai, Z., et al. Immune responses and protective efficacy of the gene vaccine expressing Ag85B and ESAT6 fusion protein from Mycobacterium tuberculosis. DNA Cell Biol Apr;27(4): Honscha, G., Von Groll, A., Valenca, M., et al. The laboratory as a tool to qualify tuberculosis diagnosis. Int. J. Tuberc Lung Dis. 2008;12(2): Perez-Martinez, I., Ponce-De-Leon, A., Bobadilla, M., et al. A novel identification scheme for genus Mycobacterium, M.tuberculosis complex, and seven mycobacteria species of human clinical impact. Eur. J. Clin. Microbiol. Infect. Dis. 2008;27(6): Barman, P., Gadre, D., A study of phage based diagnostic technique for tuberculosis. Indian J. Tuberc. Jan. 2007; 54(1): Haldar, S., Chakravorty, S., Bhalla, M., De Majumdar, S., Tyagi, JS. Simplified detetion of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons. J. Med. Microbiol. 2007;56(Pt.10): Nahid, P., Pai, M., Hopewell, P. Advances in the diagnosis and treatment of tuberculosis. Proc. Am. Thorac. Soc. 2006; 3: , UCSF 300 Frank H. Ogawa Plaza, Suite 520 Oakland, CA; Office (510)

3 TB CASE MANAGEMENT AND CONTACT INVESTIGATION INTENSIVE NOVEMBER 1-4, 2016 Somoskovi, A., Dormandy, J., Mitsani, D., Rivenburg, J., Salfinger, M. Use of smearpositive samples to assess the PCR-based genotype MTBDR assay for rapid, direct detection of the Mycobacterium tubercuslosis complex as well as its resistance to isoniazid and rifampin. J. Clin. Microbiol. 2006;44(12): Lin, G., Probert, W., Lo, M., Desmond, E. Rapid detection of isoniazid and rifampin resistance mutations in Mycobacterium tuberculosis complex from cultures or smearpositive sputa by use of molecular beacons. J. Cli. Microbiol. 2004;42(5): Barnes, P., Cave, D. Molecular epidemiology of tuberculosis. NEJM. 2003;349(12): Review article. Dowdy, D.W., Maters, A., Parrish, N., Beyer, C., Dorman, S.E. Cost-effectiveness analysis of the gen-probe amplified mycobacterium tuberculosis direct test as used routinely on smear-positive respiratory specimens. J. Clin. Microbiol. 2003;41(3): Drobniewski, F.A., Caws, M., Gibson, A., Young, D. Modern laboratory diagnosis of tuberculosis. Lancet Infect. Dis. 2003;3(3): Van Der Zanden, A.G., Te Kopppele-Vije, E.M., Vijaya Bhanu, N., et al. Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis. J. Clin. Microbiol. 2003;41(3): Gillespie, S. Minireview. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob. Agents Chemother. 2002;46(2): Mostowy, S., Behr, M.A., Comparative genomics in the flight against tuberculosis: diagnostics, epidemiology and BCG vaccination. Am. J. Pharmacogenomics. 2002;2(3): Somoskovi, A., Mester, J., Hale, Y.M., Parsons, L.M., Salfinger, M. Laboratory diagnosis of nontuberculous mycobacteria. Clin. Chest Med. 2002;23(3): Breese, P.E., Burman, W.J., Hildred, M., et al. The effect of changes in laboratory practices on the rate of false-positive cultures for Mycobacterium tuberculosis. Arch Pathol. Lab Med. 2001;125(9): Somoskovi, A., Parsons, L.M., Salfinger, M. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir. Res. 2001;2(3): Hale, Y.M., Desmond, E.P., Jost, K.C., Jr., Salfinger, M. Access to newer laboratory procedures: a call for action. Int. J. Tuberc Lung Dis. 2000;4(12 suppl 2):S , UCSF 300 Frank H. Ogawa Plaza, Suite 520 Oakland, CA; Office (510)

4 LABORATORY METHODS: Mycobacterium tuberculosis Mark W. Pandori Alameda County Public Health Laboratory Oakland, CA Presentation Goals Discuss specimen collection Microscopy & Culture (growth): traditional identification Including susceptibility testing Nucleic Acid based detection (aka molecular testing 1

5 Slides: ACPHL, M.W.P. Dr. Ed Desmond, CDPHL The United States Agency for International Development (USAID) TB Response (TBCARE 1) Specimen collection and transport Specimens (sputum, bronchial washings, urine, etc.) should be collected in a laboratory-approved sterile, leak-proof, non-breakable container Containers must be labeled with patient s name and date collected Collect specimens prior to initiation of therapy 2

6 Specimen collection and transport (2) Sputum is the most common specimen Collect 5-10 mls of an early morning specimen, prior to eating Usually 3 specimens on 3 different days are recommended for diagnosis Specimen collection and transport (3) Contaminated specimens can be minimized by: Instructing the patient to rinse mouth with preferably sterile water before collecting the specimen Returning the specimen to the lab as soon as feasible after collection 3

7 o Specimen collection and transport (4) Indicate type of specimen on laboratory requisition form o Keep all specimens refrigerated and transport as soon as possible to the lab How many specimens to collect? The greater the number of specimens, the higher the probability of a positive Law of diminishing returns: 4 specimens doesn t give many more positives than 3, so 3 is usual guideline 4

8 Processing pulmonary specimens Digestion and decontamination Pulmonary specimens are exposed to a mucolytic agent to dissolve mucin and to liquefy the specimen N-acetyl-L-cysteine (NALC) is the most common mucolytic agent used Processing pulmonary specimens (2) o Digestion and decontamination Specimens are also treated with a liquid decontaminant, generally sodium hydroxide, a strong alkali which is more toxic to oral flora than AFB Material is concentrated by centrifugation 5

9 MTB are Acid Fast Bacilli (AFB) Once they are stained (with any of a variety of stains) -they resist de-colorization by acid-alcohol treatment -they have waxy, tough outer membranes and walls Staining concentrated smear Fluorescent stains Fluorochrome stained smears require a fluorescent microscope Auramine-rhodamine is an example of such a stain where the AFB appear yellow against a black background 6

10 Staining concentrated smear (2) Carbol fuchsin-based stains Utilize a regular light microscope Must be read at a higher magnification Two types: Ziehl-Neelsen and Kinyoun. (ZN is more sensitive) Smear is then decolorized with acid-(hci) alcohol and counter-stained with methylene blue 7

11 From CDC Lab Manual REPORTING AFB SMEAR RESULTS* Number of AFB found: Report: 0 Negative 1-2 / 300 fields 1-9 / 100 fields / 10 fields / field 3+ >9 / field 4+ *CDC System (WHO system goes up to 3+ only) 8

12 Growth media for culture: 2 kinds of media Solid media Liquid Media Two types most commonly used are: Essentially one: 7H9 Lowenstein- Jensen (eggbased) Middlebrook 7H10 or 7H11 The MGIT broth It is: Liquid 7h10 minus the Vit. B6 and malachite green Growth media for culture: Advantages of solid media: Organisms (colonies) can be seen on the surface of the medium; morphology is visible If there is mixed growth or contamination, picking individual colonies can allow you to obtain a pure culture 9

13 Solid culture media 7H10 7H11 Solid culture media: Lowenstein-Jensen 10

14 Inoculating growth media for culture Liquid media Liquid or broth medium has the advantage of allowing detection of AFB more quickly Drug susceptibility testing using growth in liquid media leads to more rapid reporting of results Examples of liquid media are Trek and MGIT systems 11

15 MGIT Incubator 12

16 Accuracy problems in the TB lab False positive results, due to: o Cross-contamination during specimen processing o Specimen mix-up or mislabeling Inadequate primary culture media (some labs use only solid media) Identification of acid-fast bacilli (AFB) Growth characteristics (preliminary ID) Preliminary indication of M. tb can be made from physical parameters (in addition to microscopic observation) Rate of growth; temperature Colonial morphology Pigmentation 13

17 Rate of growth Rapid-grower: isolated in less than a week not TB Slow-grower: usually 3 weeks, up to 6 weeks could be TB Growth temperature Incubation: 36 ± 1 C M. tuberculosis does not grow at lower or higher temperatures. 14

18 Pigment production Non-chromogen TB Chromogens non-tb Colonial Morphology Smooth Rough 15

19 Identification of acid-fast bacilli (AFB) Biochemical tests There is a battery of 8-12 biochemical tests used to differentiate within the mycobacterium genus Nitrate reduction and niacin accumulation are definitive for M. tb Biochemical tests Niacin production Nitrate reduction Catalase negative at 68 C Always use pure cultures, otherwise they will yield false results. Test should be performed in the BSC aerosols are produced. 16

20 Identification of acid-fast bacilli (AFB) Nucleic acid probe tests (non-amplified) Requires a pure colony of organism DNA probe tests are species-specific Require less time than biochemical tests for identification Commercial probes are available for M. tb complex, MAC, M. kansasii and M. gordonae Identification of acid-fast bacilli (AFB) (2) High performance liquid chromatography (HPLC) HPLC uses a chromatography method to identify mycobacteria based on their mycolic acid profiles (cell wall composition) Instrument is expensive/usually reserved for larger laboratories MALDI-TOF aka Mass Spec. (matrix assisted laser desorption ionization-time of flight) is now being validated Like HPLC, expensive instrument, but quicker 17

21 Susceptibility testing of M. tuberculosis When to test: All primary M. tb isolates from patients should be tested isolates from relapse or re-treatment cases when drug resistance is suspected Susceptibility testing of M. tuberculosis (2) Methods for susceptibility testing Agar proportion method compares growth on agar media with and without one of the four primary drugs Broth based (MGIT, Trek) Requires inoculation of the strain in broth with each of the (5) primary drugs, plus control vial Growth of the strain in a vial with a drug indicates resistance to that drug 18

22 MGIT System (Becton Dickinson) 19

23 Nucleic Acid Amplification Tests AKA Molecular Tests Molecular Tests -- Allow for direct detection of MTB in a clinical specimen -- No culture required; they amplify DNA/RNA -- Highly specific -- Very fast 20

24 Molecular Tests (2) CDC guidelines recommend as standard of practice Will allow quicker diagnosis in some smear neg patients Only if patients are true TB suspects Only for untreated patients test smear negatives when clinical suspicion of TB is moderate or high More recommendations to use Molecular/ NAAT Pascopella (2004) J. Clin. Microbiol 42:4209 For smear neg patients, health care providers often/typically don t start Rx until culture is + Results in a delay in initiation of Rx, typically ~3 weeks NAA would detect many of these patients, earlier initiation of Rx MMWR guidelines: Jan. 16, (1):

25 2 FDA Approved NAA Tests Cepheid GeneXpert and Gen-Probe MTD test the only FDA-approved options MTDis labor intensive and time consuming MTDmay be the most sensitive method GeneXpert easy to do; provides drug susceptibility data Homebrew / RUO Tests All are PCR -based Homebrew: rather inexpensive Performance can be excellent (See Halse & Musser JCM 2010, NYS lab) But: They can require much more initial set-up / quality control 22

26 NAAT methods: challenges Culture still rules: higher sensitivity -higher specimen volume is tested by culture than by NAAT (MTD & GeneXpert may be exceptions) -TB is a bit tougher than other organisms -sputum often doesn t contain many MTB organisms (compared to viral specimens (herpes, flu etc..) for example) Gene Xpert (Cepheid) Single use cartridges Extraction and amplification: in the cartridge Fully Automated 23

27 Using the Cepheid Gene Xpert: Clinical Specimen Treat with NALC- NaOH and make concentrate Gene Xpert, results Nested PCR: rpob gene Take product of PCR 1, use as target in reaction 2 1. Increase specificity by having two sets of primers needed for amplification 2. Increase sensitivity by amplifying target prior to second PCR 24

28 Target DNA sequence: rpob gene The target of rifampin: RNA polymerase subunit B PCR amplifies a small region relevant for rifampin resistance; uses 5 probes to assess for mutations probes Cepheid MTB: positive result five probes -assay has an Internal PCR Control (for inhibition assessment) Test gives semi-quantitative results: high, medium, low, very low and negative 25

29 Cepheid MTB: negative result Summary of GXPsensitivities described in the literature: smear positive smear-negative Moure et al (2011), JCM ND 75.30% Boehme et al (2010) NEJM 98.20% 72.50% Marlowe et al (2011) JCM 98% 72% Helb et al (2010) JCM 98.40% 71.70% Armand et al (2011) JCM 100% 48% 26

30 Genotyping methods Spoligotyping (spacer oligonucleotide typing) MIRU / VNTR (mycobacterial interspersed repetitive units/ variable number of tandem repeats) RFLP fingerprinting (restriction fragment length polymorphism) Spoligotyping summary Not too powerful at discriminating different strains. Sometimes strains that are not part of the same outbreak will have the same spoligotype e.g., Manila strain & Beijing strain Is now performed at CDC, using DNA sequencer 27

31 MIRU summary A PCR-based method, like spoligotyping Like spoligotyping, the result is a number (24 digits) Uses a DNA sequencer instrument to analyze the PCR products Like spoligotyping, MIRU sometimes doesn t discriminate between unrelated strains RFLP typing of TB strains Involves DNA electrophoresis, and requires a lot of DNA Must have a pea-sized lump of TB bacteria to start A complicated procedure that takes ~ a week Result is a visual pattern easy to compare by eye, but difficult to make a database 28

32 IS6110-RFLP fingerprinting Genotyping: Uses 1. Cross contamination studies 2. Outbreak investigation 3. TB Control needs, such as identifying settings where transmission occurs 29

33 Molecular Detection of Drug Resistance: PCR DNA Sequencing Principles of Molecular Detection of Drug Resistance methods PCR Sequencing Fast More time than PCR Uses probes to look for precharacterized mutations; GeneXpert can only assess Rif susceptibility Can be integrated directly into testing Reads entire DNA code Detects any and all mutations Cannot be fooled Can be fooled by silent mutations Either can guide treatment until culture DST is completed 30

34 Suggestion for requesting molecular detection of drug resistance: Acid-fast smear-positive specimen Some of the specimen sediment is available for sending to reference lab (Public Health Lab or CDC) Drug resistance is suspected, or A susceptible population has been exposed, or The culture is mixed or non-viable, so regular drug suscept. testing can t be done CDC also has Molecular Detection of Drug Resistance (MDDR) program: tests for mutations associated with resistance to additional drugs ethambutol, pyrazinamide Notice: Next Generation Sequencing The ability to generate massive amounts of DNA sequence data very quickly Can be used to Deep Sequence which means to sequence everything within a given specimen Can be used to perform Whole Genome Sequencing Platforms becoming affordable: ~$100 per sample Can multiplex samples Available now at our Laboratory New York State PH Lab uses this for all Drug susceptibility testing 31

35 Whole Genome Sequencing: Perhaps achievable ~24 hours Performed on an isolate (and possibly a specimen) You get: Whole Genome Sequencing: Perhaps achievable ~24 hours Performed on an isolate (and possibly a specimen) You get: E V E R Y T H I N G 32

36 Whole Genome Sequencing: Perhaps achievable ~24 hours Performed on an isolate (and possibly a specimen) You get: E V E R Y T H I N G That means: -species -genotyping; strain -virulence factors -Drug Susceptibility Thank you 33