Tuberculosis: Iden0fica0on and Suscep0bility Tes0ng. Andrew Hansen, MD Pathology Seminar Series 7/23/2012
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1 Tuberculosis: Iden0fica0on and Suscep0bility Tes0ng Andrew Hansen, MD Pathology Seminar Series 7/23/2012
2 James (Jim) Bowie April 10, 1796 March 6, 1836
3 Outline TB review Iden0fica0on Smears Cultures Molecular methods Others (we don t do here) Drug suscep0bility tes0ng
4 M. tuberculosis complex Clinical disease E0ologic agent: Rapidly growing nontuberculous mycobacteria (NTM) Tuberculosis (TB) Mycobacterium tuberculosis M. mucogenicum complex M. Avium (MTBC) including M. tuberculosis Slowly growing nontuberculous mycobacteria M. tuberculosis M. fortuitum complex Photochromogens M. bovis M. fortuitum M. kansasii M. africanum M. peregrinum M. marinum M. micro7 M. porcinum Scotochromogens M. cane8 M. chelonae M. gordonae M. abscessus M. scrofulaceum M. massiliense Nonchromogens M. smegma7s M. avium complex M. intracellulare M. terrae complex M. ulcerans M. xenopi M. simiae M. malmoense M. szulgai M. asia7cum (C/O Alan Brown, MD) M. haemophilum
5 Mycobacterial cell wall (lipoarabinomannan)
6 Robbins & Cotran Pathologic Basis of Disease. Elsevier, (LAM)
7 Who gets secondary TB? Immunocompetent person: 5-10% lifetime risk HIV+: 7-10% per year Robbins & Cotran Pathologic Basis of Disease. Elsevier, 2009.
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10 Jim Bowie Presents Cough, shortness of breath, fever, malaise, weight loss Clinician wants to rule out TB What sample should they send?? Please spit in this cup.
11 Henry's clinical diagnosis and management by laboratory methods. 22 nd ed. Elsevier, (or 8-24 hours apart with at least one early morning)
12 Mr. Bowie s sputum is received What do we do with it? Before anything else: Digest (i.e. demucify) n acetyl l cysteine (NALC) Decontaminate (kill non- mycobacteria) Sodium hydroxide (NaOH) Concentrate centrifuge
13 Sample digest/decontaminate AFB stain (fluorochrome) culture broth medium (MGIT tubes) culture agar plate (7H11) What s different about TB (and other mycobacteria)? E. coli doubling time: ~20 minutes In 24 hours, 1 bug 2 72 bugs M. tuberculosis doubling time: ~24 hours In 24 hours, 1 bug 2 bugs may take up to 6 weeks!
14 Gram stain Henry's clinical diagnosis and management by laboratory methods. 22 nd ed. Elsevier, 2011.
15 AFB stain (carbol fuchsin) Ziehl-Neelsen: requires heating Kinyoun: no heat, cold Henry's clinical diagnosis and management by laboratory methods. 22 nd ed. Elsevier, 2011.
16 Fluorochrome stain Auramine- rhodamine dye Can view low mag, faster Sensi0vity: 25-75% per stain Repeat 3 0mes Specificity: 99% What do we detect? mycobacteria
17 Sample digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT tubes) culture agar plate (7H11) Pt treated for NTM (MOTT) Molecular test for MTBC: TMA PCR + Pt treated for TB await cultures
18 Transcrip0on Mediated Amplifica0on Luminometer (TMA) Hybridization protection assay (HPA) Gen- Probe technology Principle: there s more rrna than DNA Hill. Gen Probe Transcription-Mediated Amplification: System Principles. Gen Probe Inc., 1996.
19 Transcrip0on Mediated Amplifica0on (TMA) What do we detect? M. tuberculosis complex (MTBC) RNA Advantages: Sensi0vity >91%, specificity >99.5% in smear- posi3ve (Dowdy, et al. 2003) >95% posi0ve predic0ve value (PPV) for MTBC Versus 50%- 80% PPV for acid- fast stain (CDC. 2009) Disadvantages: Hands- on assay: tech 0me >1 hour Cost: >$1,000 per kit (exp in 4 weeks) Not approved for bloody specimens
20 Polymerase Chain Reac0on (PCR) Bonus result! Boehme, et al. NEJM, 2010.
21 Polymerase Chain Reac0on (PCR) What do we detect? MTBC (DNA) Bonus!: rifampin resistance Sensi0vity (for MTBC detec0on in smear- posi0ve): 98.2% (Boehme, et al. 2010) Specificity: 99.2% Can be done on bloody specimens Tech 0me: 15 minutes Cost: $60 per cartridge Disadvantage: not FDA approved yet But strongly recommended by WHO
22 Sample digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT tubes) culture agar plate (7H11) Pt treated for NTM (MOTT) Molecular test for MTBC: TMA PCR + Pt treated for TB await cultures Other options for rapid detection of MTBC?
23 Serology? Tests are available Recent systema0c review and meta- analysis: Sensi0vity: 76% (smear- posi0ve) Specificity: 92% Conclusion: serology not accurate Steingart, et al. PLoS Medicine, 2011.
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25 Weetjens, et al. Int J Tuberc Lung Dis, 2009.
26 Weetjens, et al. Int J Tuberc Lung Dis, 2009.
27 Mr. Bowie Fluorochrome stain + TMA and PCR + for MTBC Clinicians start treatment with RIPE Rifampin Isoniazid Pyrazinamide Ethambutol 3 days later, another specimen is sent for AFB smear Is this appropriate?
28 Quiz The CDC recommends that pa0ents with suspected TB disease should remain under airborne precau0ons while they are hospitalized un0l they have a) had 3 consecu0ve nega0ve AFB smears, collected in hour intervals, with at least one being an early morning specimen b) received standard mul0drug an0tuberculosis treatment (minimum of 2 weeks) c) demonstrated clinical improvement d) all of the above Jensen, et al. CDC, 2005.
29 When Will Smears be Nega0ve? Time to sputum conversion in pts with smear- posi0ve TB: Mean: 33 days Median: 23 days Telzak, et al. Clin Infect Dis, I m calling you about Mr. Bowie. Please don t send us anymore specimens for a while. Thanks!
30 Sample digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT TM tubes) culture agar plate (7H11) Pt treated for NTM (MOTT) Molecular test for MTBC: TMA PCR + Pt treated for TB Specimen and antibiotic supplement added await cultures Silicone with O 2 -sensitive fluorescent compound
31 BD BACTEC TM MGIT TM 960
32 Broth- Based Culture (BD BACTEC TM MGIT TM 960) Sensi0vity >99% Time to detec0on: median 14 days At 21 days: 88.2% At 28 days: 100% for ini0al specimens 95% for follow- up Can we report the culture nega0ve before 6 weeks? Tyrrell, et al. CDC, 2012.
33 Sample digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT tubes) _ + culture agar plate (7H11) Pt treated for NTM (MOTT) Molecular test for MTBC: TMA PCR + Pt treated for TB await plate cultures DNA probe for MAC, others fluorochrome and gram stain subculture to 7H11 DNA probe for MTBC _ + - Pt treated for TB - send-out for susceptibility await cultures
34 Sample Middlebrook 7H11 Agar digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT tubes) _ + culture agar plate (7H11) + _ Molecular test for MTBC: TMA PCR + Pt treated for TB await plate cultures fluorochrome and gram stain subculture to 7H11 Henry's clinical diagnosis and management by laboratory methods. 22 nd ed. Elsevier, Pt treated for NTM (MOTT) DNA probe for MTBC _ DNA probe for MAC, others + fluorochrome and gram stain - Pt treated for TB - send-out for susceptibility finalize as negative await cultures Alternative solid medium: Löwenstein Jensen (egg-based)
35 Sample digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT tubes) _ + culture agar plate (7H11) + _ Pt treated for NTM (MOTT) Molecular test for MTBC: TMA PCR + Pt treated for TB await plate cultures fluorochrome and gram stain subculture to 7H11 DNA probe for MTBC _ DNA probe for MAC, others + fluorochrome and gram stain - Pt treated for TB - send-out for susceptibility finalize as negative await cultures
36 TB Drugs Henry's clinical diagnosis and management by laboratory methods. 22 nd ed. Elsevier, Mul0- drug resistant (MDR) TB: Resistant to at least 2 primary drugs (ojen isoniazid and rifampin)
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39 TB Resistance Mechanisms Spontaneous chromosomal muta0ons: Not via mobile gene0c elements (e.g. plasmids) ~ 1 bacillus per bacilli lung cavity of 2.5 cm has 10 8 bacilli Need to treat with mul0ple drugs! Pt needs to be compliant! Nachega, et al. Clin Infect Dis, 2003
40 Rifampin resistance: marker for MDR: usually resistant to at least one other agent codons 526, 531 confer high-level resistance codons 511, 516, 519, 522 confer low-level resistance
41 Sample digest/decontaminate AFB stain (fluorochrome) + culture broth medium (MGIT tubes) _ + culture agar plate (7H11) + _ Pt treated for NTM (MOTT) Molecular test for MTBC: TMA PCR + Pt treated for TB await plate cultures fluorochrome and gram stain subculture to 7H11 DNA probe for MTBC _ DNA probe for MAC, others + fluorochrome and gram stain - Pt treated for TB - send-out for susceptibility finalize as negative await cultures
42 Sensitivity: 97.6% Specificity: 98.1% Boehme, et al. NEJM, 2010.
43 Drug Suscep0bility Phenotypic: Gold standard: plate- based Resistance: > 1% resistance to single concentra0on of drug 4- week turnaround 0me Available at CDC
44 Drug Suscep0bility Phenotypic: Broth- based Resistance: O 2 - sensi0ve fluorescence detected in tube with drug Results in 5-8 days Poten0al to bring in- house
45 Recent case MTB/RIF PCR detected rifampin resistance Phenotypic (plate) tes0ng revealed sensi0vity to rifampin codons 526, 531 confer high- level resistance codons 511, 516, 519, 522 confer low- level resistance Is it a low- level resistance muta0on? How could we find out?
46 Suscep0bility Tes0ng by Gene Sequencing Available at CDC (Atlanta, GA)
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48 Key Points MTBC mycobacteria grow very slowly! Special methods for rapid iden0fica0on and suscep0bility tes0ng are crucial Laboratory directors and staff must: understand and streamline algorithmic tes0ng for MTBC maintain open and effec0ve communica0on with clinical services create efficient working rela0onships with reference laboratories
49 References 1. Ryan, Kenneth J., C. George Ray, and John C. Sherris. Sherris medical microbiology: an introduc7on to infec7ous diseases. 5th ed. New York: McGraw- Hill, Kumar, Vinay, Abul K. Abbas, Nelson Fausto, Stanley L. Robbins, and Ramzi S. Cotran. Robbins and Cotran pathologic basis of disease. 8th ed. Philadelphia: Elsevier Saunders, CDC Surveillance Slides (Tuberculosis). Accessed 07/2012 via default.htm. 4. McPherson, Richard A., Marhew R. Pincus, and John Bernard Henry. Henry's clinical diagnosis and management by laboratory methods. 22nd ed. Philadelphia: Saunders Elsevier, Hill, C. Gen- Probe Transcrip0on- Mediated Amplifica0on: System Principles. San Diego: Gen- Probe Incorporated, Accessed via probe.com. 6. Dowdy DW, Maters A, Parrish N, Beyrer C, Dorman SE. Cost- effec0veness analysis of the gen- probe amplified mycobacterium tuberculosis direct test as used rou0nely on smear- posi0ve respiratory specimens. J Clin Microbiol Mar;41(3): CDC. Updated Guidelines for the Use of Nucleic Acid Amplifica0on Tests in the Diagnosis of Tuberculosis. MMWR 2009 Jan;58(1): Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, Allen J, Tahirli R, Blakemore R, Rustomjee R, Milovic A, Jones M, O'Brien SM, Persing DH, Ruesch- Gerdes S, Gotuzzo E, Rodrigues C, Alland D, Perkins MD. Rapid molecular detec0on of tuberculosis and rifampin resistance. N Engl J Med Sep 9;363(11): Steingart KR, Flores LL, Dendukuri N, Schiller I, Laal S, Ramsay A, Hopewell PC, Pai M. Commercial serological tests for the diagnosis of ac0ve pulmonary and extrapulmonary tuberculosis: an updated systema0c review and meta- analysis. PLoS Med Aug;8(8):e Weetjens BJ, Mgode GF, Machang'u RS, Kazwala R, Mfinanga G, Lwilla F, Cox C, Jubitana M, Kanyagha H, Mtandu R, Kahwa A, Mwessongo J, Makingi G, Mfaume S, Van Steenberge J, Beyene NW, Billet M, Verhagen R. African pouched rats for the detec0on of pulmonary tuberculosis in sputum samples. Int J Tuberc Lung Dis Jun;13(6):
50 References, con0nued 11. Jensen P, Lambert L, Iademarco, MRidzon, R. Guidelines for Preven0ng the Transmission of Mycobacterium tuberculosis in Health- Care Sexngs, CDC, Recommenda0ons and Reports Dec;54(RR17); Telzak EE, Fazal BA, Pollard CL, Turer GS, Justman JE, Blum S. Factors influencing 0me to sputum conversion among pa0ents with smear- posi0ve pulmonary tuberculosis. Clin Infect Dis Sep;25(3): Tyrrell F, Starks A. Mul0- Center Evalua0on of Time to Nega0ve Mycobacterium tuberculosis Complex Cultures in Commercial Broth Systems: Can Culture Nega0vity be Determined Sooner? CDC conference, Nachega JB, Chaisson RE. Tuberculosis drug resistance: a global threat. Clin Infect Dis Jan 15;36(Suppl 1):S Laurenzo D, Mousa SA. Mechanisms of drug resistance in Mycobacterium tuberculosis and current status of rapid molecular diagnos0c tes0ng. Acta Trop Jul;119(1):5-10. Epub 2011 Apr CDC. Tuberculosis (TB): Molecular Detec0on of Drug Resistance (MDDR). Accessed 07/2012 via hrp:// Special thanks to: Dr. Jorgensen Mildred Dr. Dallas
51 Quiz What is the purpose of trea0ng sample sputum with NaOH prior to tes0ng for AFB? a) Increase ph for increased dye affinity during staining b) Increase ph for op0mal mycobacterial growth condi0ons c) Kill contamina0ng normal flora d) Decrease sample viscosity by diges0ng mucin
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