TB Laboratory for Nurses

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1 TB Laboratory for Nurses Shea Rabley, RN, MN Consultant Mayo Clinic Center for Tuberculosis 2014 MFMER slide-1

2 Disclosures None 2014 MFMER slide-2

3 Objectives Participants will be able to: 1. Name 2 safety procedures utilized in a TB Laboratory. 2. Name 2 types of tests conducted in a TB Laboratory MFMER slide-3

4 BioSafety Laboratory Level 3 Since mycobacterium tuberculosis is an airborne disease, laboratories that routinely deal with Mtb are required to be BioSafety Level 3 (BSL3). All procedures involving the manipulation of infectious materials are conducted within a negative air flow laboratory room using biological safety cabinets, specially designed hoods, or other physical containment devices, has a handwashing area, a shower area and maintains limited access. These labs require the use of personal protective equipment (gowns, gloves, safety glasses and respirators) while working with the specimens MFMER slide-4

5 TB Laboratory Tests Sputum Specimen arrives at Lab Decontamination and Concentration of Specimen Smear Liquid or Broth Culture Solid Media Culture Genotyping NAAT/ NAA TB Probe Drug Susceptibility Tests

6 Sputum specimens arriving at the TB Lab and ready to begin the decontamination and concentration process 2014 MFMER slide-6

7 Decontamination & Concentration Process Most respiratory specimens contain microorganisms other than mycobacteria. Therefore, the specimens collected for culture and susceptibility testing of M. tuberculosis should be refrigerated if a transportation delay is expected or if the temperature would encourage growth. Before culturing in the laboratory, specimens from nonsterile body sites (sputum) must have a pretreatment which involves digestion, homogenization, decontamination, and concentration. This will kill the more rapidly growing organisms such as normal flora, other bacteria and fungi, while not seriously affecting the viability of the mycobacteria. If specimens are not handled correctly and overgrowth occurs, the specimen can not be used MFMER slide-7

8 SMEAR Microscopy Smear microscopy is conducted on all specimens. A specimen is smeared directly onto the glass slide (direct smear), stained, washed in an acid solution, and then placed under the microscope for visual examination. This test is used to detect acid-fast bacilli on a smear, but will not differentiate between species of mycobacteria. Individuals with sputum smear-positive for AFB are considered to have TB until proven otherwise and are considered more infectious than those with smear-negative sputum MFMER slide-8

9 AFB Smear AFB (shown in red) are tubercle bacilli Red Snappers 2014 MFMER slide-9

10 Nucleic Acid Amplification Tests (NAA/NAAT) NAATs can rapidly identify Mtb directly in clinical samples by amplification of the DNA and RNA of the mycobacterium in the specimen. At least 1 NAAT should be performed on the first specimen of each pulmonary TB suspect. A single negative NAAT does not exclude TB MFMER slide-10

11 Nucleic Acid Amplification Tests (NAA/NAAT) Two NAATs are approved for use in the United States by the FDA: The Enhanced Amplified Mycobacterium Tuberculosis Direct Test (E-MTD, Gen-Probe, San Diego, California) is approved for detection of M. tuberculosis complex bacteria in acid-fast bacilli (AFB) smear-positive and smear-negative respiratory specimens. The Amplicor Mycobacterium Tuberculosis Test (Amplicor) (Roche Diagnostic Systems, Inc., Branchburg, New Jersey) is approved for detection of M. tuberculosis complex bacteria in acid-fast bacilli (AFB) smear-positive respiratory specimens MFMER slide-11

12 Nucleic Acid Amplification (NAA) Test MTD Amplicor 2014 MFMER slide-12

13 Culture The CDC gold standard for the detection and confirmation of TB is the culture. To achieve this standard, the recommendation is to inoculate at least one tube each of solid and liquid media, even if the smear or NAAT is negative. Organisms are grown on media so that they can be identified; a positive culture for M. tuberculosis contains tubercle bacilli, whereas a negative culture contains no detectable tubercle bacilli. Results are usually available within 4 14 days when liquid medium systems are used. It can take up to 8 weeks for solid media results MFMER slide-13

14 Liquid or Broth Culture All types of clinical specimens, pulmonary as well as extrapulmonary (except blood and urine), can be processed for primary isolation of Mtb by culture methods. VersaTREK system (Trek Diagnostic Systems) is an automated system that detects mycobacterial growth by automatically monitoring the rate of oxygen consumption within the headspace of the culture bottle. MGIT, BacTec MGIT : Mycobacterium Growth Indicator Tube, Becton Dickinson and Co. Is a commercial nonradiometric broth-based mycobacterial culture system MFMER slide-14

15 VersaTREK System BacTec MGIT System 2014 MFMER slide-15

16 Solid Media Culture Lowenstein-Jensen (LJ): Refers to conventional culture method that uses solid media to grow mycobacteria. LJ slant is an old and very dependable method, but can take from 3 to 8 weeks because mycobacteria grow slowly. Regardless, solid media cultures are considered to be the gold standard for TB diagnosis MFMER slide-16

17 Solid Media Culture Plates can also be utilized for culturing Mtb 2014 MFMER slide-17

18 Colonies of M. tuberculosis Growing on Media Described as rough buff 2014 MFMER slide-18

19 MTB Probe AccuProbe (Gen-Probe Inc., San Diego, CA) is a nucleic acid hybridization kit that can detect Mtb complex in cultures. It can be used with both solid culture mediums and liquid mediums. The probe assay does not include an amplification step so it is not sensitive enough to be used directly on a clinical specimen but is usually capable of identifying Mtb complex in contaminated liquid cultures (depending on the extent of the contamination). M. Tb complex consists of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, and M. canetti MFMER slide-19

20 Drug-Susceptibility Testing Drug susceptibility testing (DSTs) refers to the laboratory testing done on mycobacterial cultures to determine susceptibility of the organisms to specific anti-tb drugs. Drug resistance: Inability of anti-tb medications to kill Mtb organisms. Drug susceptibility: Ability of anti-tb medications to kill Mtb organisms MFMER slide-20

21 Drug Susceptibility Testing Drug-susceptibility testing should always be conducted on the initial Mtb isolate, to determine the drugs to which the organism is susceptable. First-Line DSTs covers 5 drugs: INH, Rifampin, Ethambutol, PZA and Streptomycin. Second-line drugs include the fluoroquinolones, ethionamide, amikacin, kanamycin, capreolycin, cycloserine & para-aminosalicylic acid. Second-line DSTs are not recommended when cultures are susceptible to first-line drugs. Only laboratories with experience and competency in should perform second-line DSTs MFMER slide-21

22 Drug Susceptibility Testing The MGIT 960 system is currently the most validated and broadly used system for drug susceptibility testing of M. tuberculosis MFMER slide-22

23 Drug Susceptibility Testing The VersaTREK system is an automated, validated methodology for drug susceptibility testing of M. tuberculosis 2014 MFMER slide-23

24 Molecular Detection of Drug Resistance Drug resistance is caused by mutations in specific M. tb genes. Several molecular assays and tests can detect mutations. Generally conducted for INH and Rifampin mutations but can also be conducted to identify mutations associated with resistance to second-line drugs such as fluroquinolones, amikacin, kanamycin, and capreomycin MFMER slide-24

25 Molecular Detection of Drug Resistance GeneXpert MTB/RIF, Cepheid, Inc. is a commercial molecular assay for the direct detection of Mtb and mutations associated with rifampin resistance in clinical specimens. It received FDA approval in July 2013 for use in the United States. There are other tests available that can conduct this testing as well (some are not FDA approved), but molecular detection testing can be completed at several expert labs in the US MFMER slide-25

26 Therapeutic Drug Monitoring Therapeutic drug monitoring is recommended when a person is experiencing a slower than expected response to TB treatment and other reasons have been ruled out (not taking meds, adverse drug reactions, etc). These are not recommended as a routine part of monitoring. Blood is drawn 2 hours post medication administration (peak) and can be drawn 6 hours post medication administration (trough). When lower than expected drug levels are found, adjustment to the medications can be made to bring the levels within therapeutic range MFMER slide-26

27 Genotyping Laboratory-based approach that analyzes the genetic material of patient isolates 2014 MFMER slide-27

28 Genotyping Different strains of Mtb have different genotypic patterns that repeat information at different intervals. Genotyping reports include a spilogotype, MIRU 1, MIRU 2 and PCR. Funded by and available at CDC-designated labs in the US, specimens are automatically sent by state TB labs. Main purpose of genotyping: add to the understanding of TB transmission in the community 2014 MFMER slide-28

29 CDC Guidelines for Turn Around Times in the TB Laboratory When evaluating TATs, must remember all the steps in the process: Smears: < 24 hours NAAT: < 48 hours Identification for M. tuberculosis: < 21 days Drug Susceptibility: < 28 days 2014 MFMER slide-29

30 Nontuberculous mycobacteria (NTM): Also known as atypical mycobacteria or MOTT (mycobacteria other than tuberculosis). Members of the mycobacteria family other than M. tuberculosis. Some of the more prominent members are M.aviumintercellulare, M.kansasii, M.fortuitum, etc. Nontuberculous mycobacteria (NTM) is the preferred terminology MFMER slide-30

31 Other Laboratory Tests used in the TB Program 2014 MFMER slide-31

32 Screening for TB Infection: QuantiFERON-TB Gold In-Tube QFT-GIT requires 3 tubes provided by the lab GRP - each designed to allow only 1ml of blood to enter. Gray Negative Control or Nil background noise Red Antigen Response of the test Purple Positive Control or Mitogen shows immune status and correct handling & incubation of the tubes Will receive a numeric as well as a positive, negative or indeterminate result. The result is based on IFN-g concentration Positive - > 0.35 Negative - < 0.35 Indeterminate - < 0.35 or > 0.35 if Nil > 8.0 & any result in Mitogen tube OR any result in antigen & mitogen with > 8.0 in Nil 2014 MFMER slide-32

33 Screening for TB Infection T-Spot.TB T-Spot uses 1 standard green top tube, but requires differing amounts of blood: 6 ml: Adults & children over 9 years old 4 ml: Children 2 to 9 years old 2 ml: Children up to 2 years old Interpretation of Results: Interferon-gamma is captured and presented as spots from T cells sensitized to Mycobacterium tuberculosis antigens. There are 4 panels used. Results are interpreted by subtracting the spot count in the negative (NIL) control from the spot count in Panels A and B (response panels). The 4 th panel is the anitgen panel or positive control. Results are reported as positive (>8), negative (<4), borderline (5-7) and indeterminate (low mitigen or high nil) MFMER slide-33

34 Other Laboratory Tests Patient Type Recommended Test All TB Suspects/Cases LFTs (AST, ALT), bilirubin, alkaline phosphatase, and serum creatinine and a platelet count (CBC) Patients at risk for hepatitis B or C (e.g., injection drug user, born in Asia or, or HIV infected) Conduct serologic tests HIV-infected patients Obtain CD4+ lymphocyte count 2014 MFMER slide-34

35 Questions? 2014 MFMER slide-35

36 References MFMER slide-36

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