TB 101 Disease, Clinical Assessment and Lab Testing

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1 TB 101 Disease, Clinical Assessment and Lab Testing Pacific Islands Tuberculosis Controllers Association Conference (PITCA) Clinical Laboratory Breakout

2 None Disclosure

3 Objectives Be able to list and explain the test methods available and turn-around-times for MTB testing. Understand and be able to explain the mycobacteriology testing algorithm for the following MTB test methods: AFB smear, culture, NAAT and drug susceptibility testing. Be able to explain possible reasons for uncommon and unexpected MTB test results. Be able to discuss the clinical impact of uncommon or unexpected MTB test interpretations.

4 M. tuberculosis Culture, Molecular and Clinical Laboratory Testing Nucleic acid testing -RNA, DNA -Viable and non-viable -MTB growth confirmed by direct nucleic acid probe detection -MTB nucleic acid amplification (NAAT) -GeneXpert MTB/RIF (PCR) Interferon Gamma Release Assay (IGRA) -QuantiFERON -TB Gold -ELISpot (T-SPOT.TB) Culture -Viability -Transport considerations -Collection to processing -Shipping and handling -Drug susceptibility testing (DST) -Limit of detection -MTB fitness in culture

5 Mycobacterial Testing Culture, Identification and DST Methods Microscopy & AFB stains Types Ziehl-Neelsen ( Hot ) Kinyoun ( Cold ) Auramine ( Fluorescent ) LOD (10K bacteria/ml) Direct AFB smear (ZN/K sensitivity 20 80%) Not M. tuberculosis specific Hours for processing and testing Experienced technologist

6 Mycobacterial Testing Culture, Identification and DST Methods Culture (Liquid/solid media) Solid (e.g. Middlebrook, Lowenstein- Jensen ), liquid (MGIT realtime) LOD (Single viable MTB) Culture identification Phenotypic and Biochemical tests (e.g. Niacin, Nitrate) Molecular is more common Nucleic acid probes» MTB, MAC, M. kansasii, M. gordonae (Hologic, Accuprobe) Sequencing (16S, Pyrosequencing) HPLC MALDI TOF MS (NTM) 2-8 weeks for final result Highly experienced technologists

7 Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis MMWR - January 16, 2009 / 58(01);7-10 AFB smear with the added value of NAA testing Greater positive predictive value (>95%) with AFB smear-positive specimens in settings in which nontuberculous mycobacteria are common Confirm rapidly the presence of M. tuberculosis in 50%--80% of AFB smear-negative, culture-positive specimens. Compared with culture, NAA tests can detect the presence of M. tuberculosis bacteria in a specimen weeks earlier than culture for 80%--90% of patients suspected to have pulmonary TB whose TB is ultimately confirmed by culture. Can impact patient care and TB control efforts Avoiding unnecessary contact investigations or respiratory isolation for patients whose AFB smear-positive specimens do not contain M. tuberculosis.

8 Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis MMWR - January 16, 2009 / 58(01);7-10 Culture remains the gold standard for laboratory confirmation of TB and is required for isolating bacteria for drug-susceptibility testing and genotyping. NAA testing should become standard practice for patients suspected to have TB. NAA testing for TB can shorten the time needed to diagnose TB from 1--2 weeks to 1--2 days. More rapid laboratory results should lead to earlier treatment initiation, improved patient outcomes, and increased opportunities to interrupt transmission. Rapid laboratory confirmation of TB also can help reduce inappropriate use of fluoroquinolones as empiric monotherapy of pneumonias (i.e. Can lead to development of fluoroquinolone-resistant M. tuberculosis).

9 Mycobacterial Testing Culture, Identification and DST Methods Drug susceptibility testing (DST) Phenotypic methods Culture and susceptibility testing 2-4 weeks (e.g. MGIT, agar proportion) Genotypic methods RealTime PCR Molecular Beacons (Selected labs)» 2-4 hours» Rifampin (rpob gene) INH (katg, inha, aphc, ndh) Heminested realtime PCR» Cepheid Xpert MTB/RIF Mtb direct and concentrated detection with rifampin resistance detection 2 hours Line Probe (Inno-Lipa) HAIN GTMD Sequencing and Rep-PCR 4-8 hours Next generation sequencing (NGS) 1. Inoculate broth wells containing bacterial inhbitiors, growth supplements, and antibiotic dilutions with 800 µl decontaminated sputum 2. Examine wells through an inverted microscope starting on day seven (7).

10 Phenotypic Drug Susceptibility Testing (DST) M. tuberculosis Culture and detection of M. tuberculosis growth in the presence of anti-tb drugs Proportion, resistance ratio or absolute concentration methods on solid media (3 weeks) Broth-based semiautomated (4 12 days) BACTEC MGIT 960 TB system MGIT 960 system may be used directly on sputum samples, additional time and labor is needed for identification of M. tuberculosis. Other phenotypic tests Liquid media-based microscopic observation drug susceptibility (MODS) test Phage-based assays using mycobacteriophages

11 Countries reporting XDR-TB as of February 2008 (Based on information provided to WHO Stop TB Department) 1-5 Any drug-resistant TB describes TB with resistance to one of multiple drugs. Multidrug-resistant TB (MDR-TB) is defined as having resistance to the two most potent anti-tb drugs, isoniazid and rifampin. Extensively drug-resistant TB (XDR-TB) is newly defined as having multidrug resistance (resistance to isoniazid and rifampin) as well as resistance to at least two additional second-line medications, a fluoroquinolone and one or more injectable agents (amikacin, kanamycin, or capreomycin). 1. Zignol M, Hosseini MS, Wright A, et al. Global incidence of multidrug-resistant tuberculosis. J Infect Dis. 2006;194: Iseman MD. Treatment of multidrug-resistant tuberculosis. N Engl J Med. 1993;329(11): Weyer K, Lancaster J, Brand J, van der Walt M, Levin J. Survey of tuberculosis drug resistance in South Africa Paper presented at: Medical Research Council; 2004; Pretoria, South Africa. 4. Centers for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs worldwide, MMWR. 2006;55: Shah NS, Wright A, Bai GH, et al. Emergence of extensively drug resistant tuberculosis. Emerg Infect Dis. 2007;13(3):

12 M. TUBERCULOSIS, DRUG RESISTANCE GENES AND THE DEVELOPMENT OF RAPID METHODS FOR DRUG RESISTANCE DETECTION (I.E. MDR-TB/XDR-TB) S. Ahmad, et.al Res. Med. 103:1777

13 Rifampin Drug Resistance Rifampin (RIF), a lipophilic rifamycin derivative, binds to b- subunit of RNA polymerase (encoded by rpob) and inhibits RNA transcription ( protein synthesis) in Mtb 23 Mutations in rpob gene confer RIF resistance (drug binding) Monoresistance to RIF is rare except in HIV-co-infected patients RIF resistance is often a surrogate marker for MDR-TB 85-90% RIF resistant strains are also resistant to INH 90-95% of RIF-resistant strains carry missense mutations or small in-frame insertions/deletions within an 81-bp hotspot region (HSR) mainly involving rpob codons 531, 526 and 516 Resistance in 5-10% RIF-resistant isolates is due to mutations in N-terminal or other regions of the rpob gene or due to some unknown mechanism. Adapted from: S. Ahmad, et.al Res. Med. 103:1777

14 Boehme, et.al. NEJM 363:1005. Sept. 9,

15 Mycobacteriology Testing Scheme

16 Mycobacteriology Testing Scheme

17

18 Mycobacteriology Testing Scheme

19 Mycobacteriology Testing Scheme

20 Clinical Case #1

21 Laboratory Findings Non-tuberculosis mycobacteria (NTM) overgrowth What is the significance of NTM growth? Will M. tuberculosis recovery be possible with the co-isolation of NTM in culture? What are the alternatives for the approach to laboratory testing in the case of NTM overgrowth?

22 Clinical Case #2

23 Laboratory Findings Non-AFB overgrowth with specimen redigest What is the cause of non-afb overgrowth? Can this situation be prevented in any subsequent specimen recollections? This specimen has been treated 2X with NaOH, can we redigest a 3 rd time? If a third NaOH digest is performed, what is the effect on M. tuberculosis culture recovery?

24 Clinical Case #3

25 Laboratory Findings Gastric specimen with delayed specimen neutralization and no growth on culture Why is it important to list the specific source of the specimen for MTB AFB smear and culture? How is a gastric specimen different from a respiratory or other type of specimen? Are gastric specimens handled and transported differently from respiratory specimens?

26 Gastric Aspirate Collection and Processing Usually performed in young children Early morning collection (NPO required) Sodium carbonate neutralization to ph 7.0 Add 100 mg sodium carbonate/5-10 ml of gastric aspirate specimen following collection

27 Clinical Case #4

28 Laboratory Findings Direct USAPI AFB smear negative with DLS concentrated >+/- 1 grade and MTB growth Why is there a concern when the direct AFB smear is >+/- 1 grade from the concentrated smear? What is the possible consequence of this type of AFB smear discordance? What is the root cause of this type of discordance? Specimen? Technologist? Other causes?

29 Clinical Case #5

30 Laboratory Findings AFB smear positive and Xpert MTB/RIF Detected/Not Detected and NO MTB cultured How would this type of finding be interpreted to the ordering HCP? What if this finding was the result of a first time specimen collection on a new MTB, untreated patient? Specimen collection issue? Specimen handling and transport issue? Specimen processing issue? Specimen analytical or post-analytical phase issue?

31 MAJURO EBEYE

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