8/28/2012. Rapid Detection of Mycobacteria. Overview. Acid Fast Bacteria. Mycobacterial Cell Wall Structure. Phylogenetic Tree of Bacteria

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1 Overview Rapid Detection of Mycobacteria Niaz Banaei MD Assistant Professor of Pathology and Medicine Director, Clinical Microbiology Laboratory Stanford University - Taxonomy/cell wall properties - Pathogenesis - Detection of active disease - Specimen processing - AFB microscopy - Culture and identification - Antibiotic susceptibility testing - Novel diagnostics - Detection of latent TB infection - Unknown cases Phylogenetic Tree of Bacteria Phylogenetic Tree of Mycobacteria Devulder et al Int J Syst Evol Microbiol 2005 Acid Fast Bacteria Mycobacterial Cell Wall Structure Acid Fast 61-70% GC Pink: mycolic acid Black: Peptidoglycan Red: Cell membrane Green: Outer membrane Extractable { Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan 1

2 M. tuberculosis Mycolic Acids Mycobacterial Cell Wall Structure Extractable { Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan Glickman et al Mol Cell 2000 Mycobacterial Ghost Cells in Gram Stain Mycobacterial Ghost Cells in Gram Stain 1000X 1000X Fuchsin stains: Ziehl-Neelsen and Kinyoun Fuchsin stains: Ziehl-Neelsen and Kinyoun Extractable { Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan Extractable { Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan Ziehl-Neelsen stain Kinyoun stain 2

3 Mycobacteria Stained with Kinyoun Fluorescent stains: Auramine and Auramine/Rhodamine 1000X 1000X 1000X Cording of M. tuberculosis Cording of M. chelonae in Blood Culture Auramine 400x Kinyoun,1000X M. tuberculosis Mycolic Acids Acid Fast Actinomycetes Order - Actinomycetales Suborder - Streptomycineae Suborder - Corynebacterineae Family - Corynebacteriaceae Genus - Corynebacterium Genus - Dietzia Genus - Turicella Family - Nocardiaceae Genus - Gordona Genus - Nocardia Genus - Rhodococcus Genus - Tsukamurella Family - Mycobacteriaceae Genus - Mycobacterium Family - Dietziaceae Suborder - Propionibacterineae Glickman et al Mol Cell

4 What distinguishes Acid Fast From Modified-Acid Fast? Acid Fast vs. Modified-Acid Fast Stain Ingredients of bacterial stains Gram AFB Modified AFB Stain Iodine Carbol fuchsin Carbol fuchsin Destain Acet-alcohol 3% HCl-alcohol 1% H 2 SO 4 -alcohol Counterstain Safranin Methylene blue Methylene blue Acid Fast vs. Modified-Acid Fast Bacteria Tsukamurella in AFB Stains Extractable { Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan Mycolic Acid Chain Mycobacteria:60-90 Nocardia: AFB (Kinyoun) Partial-AFB 1000X 1000X 1000X Phylogenetic Tree of Mycobacteria M. avium complex M. kansasii M. marinum M. tuberculosis complex Pathogenesis of Tuberculosis 1. Human pathogen 2. Opportunist M. fotuitum group M. chelonae abcessus group Devulder et al Int J Syst Evol Microbiol

5 Th1 Cell-Mediated Immunity Th1 Cell-Mediated Immunity APC APC Lymphoid Tissue IL-12 + Th 0 Lymphoid Tissue IL-12 + Th 0 Th 1 _ IFN- Th 2 Th 1 _ IFN- Th 2 Site of Infection Th 1 IFN- + TNF- M Site of Infection Th 1 IFN- + TNF- M Kaplan et al Infect Immun 2003 Th1 Cell-Mediated Immunity Pulmonary disease APC Lymphoid Tissue IL-12 + Th 0 Th 1 _ IFN- Th 2 Site of Infection Th 1 IFN- + TNF- M Blood Stream Infection Mycobacterial Ghost Cells in Blood Culture Mycobacterial Infection of the Joint Auramine 1000X Gram stain 1000x 5

6 Mycobacterial Infection of the Spine Mycobacterial Infection of the Soft Tissue AFB stain 600X M. marinum & ulcerans M. marinum - Disease in fish - Fish tank granuloma Clinical Significance of Rapid Growers Wound / bone infection - 60% M. fortuitum Pulmonary infection - 80% M. abcessus M. ulcerans - Buruli ulcer Disseminated cutaneous Disease - 75% M. chelonae - 20% M. abcessus Nosocomial - Post surgical wound infection - Catheter associate Winthrop et al NEJM 2002 Mycobacterial Isolates at Stanford in 2011 Mgord 10% Laboratory Detection and ID of Mycobacteria MAC 35% MTC 14% MCAG 29% n=165 unique isolates 6

7 Laboratory Detection and ID of Mycobacteria Specimen processing AFB Microscopy Culture (6-10 wk) Identification Susceptibility testing Specimen Processing for AFB Assays - Nonsterile samples - Decontaminated - Concentrated - Sterile samples - Fluids - Concentrated - Tissue - Homogenize In order to increase the sensitivity of culture for M. tuberculosis, the volume of a sputum specimen should exceed 5 ml Kent and Kubica JCM 1985 Processing Contaminated Specimens 1. Decontaminate specimen with NaOH. 3. Neutralize and dilute with buffer. AFB Specimen Processing in BSL3 2. Liquify with a mucolytic agent, e.g. N-acetyl-Lcysteine Specimen 4. Centrifuge at 3000 x g. Processing Contaminated Specimens Inoculation of Media and Microscopy MGIT liquid medium Specimen pellet Growth supplement PANTA 7H11/7H11S agar LJ slant Specimen sediment 5. Inoculate media & make a smear. Glass slide with 15mm specimen area 7

8 AFB Smears of Processed Specimens Reporting of Acid-fast Smears Carbol fuchin stained smear - Kinyoun or Ziehl-Neelsen - Read 300 high power fields - ~32-94% sensitive vs Cx Flurochrome stained smear - Auramine +/- Rhodamine - Read 30 low power fields - 52%-97%; average 10% more sensitive % sensitivity in HIV+ - ~60-70% sensitive Kinyoun stain (x1000) Auramine (x250) Steingart et al Lancet Infect 2006 Report Fuchin stain Fluorochrome stain (high power) (low power) Suspicious 1-2/300 F (3 sweep) 1-2/30 F (1 sweep) /100 F (1 sweep) 3-9/10 F /10 F 1-9/ F / F 10-90/ F 4+ >9/ F >90/ F Factors That Limit AFB Smear Sensitivity Overall Sensitivity 22-80% Processing Contaminated Specimens Bacterial load cfu/ml of specimen 60% positive smear Specimen volume Direct versus concentrated smears Specimen type: 20-55% for CSF Pretreatment Specimen sediment Staining method (Flurochrome>ZN>Kinyoun) Experience of reader Patient population Parsons et al Clin Mirobiol Rev 2011 Early Detection of M. tuberculosis in Respiratory Specimens Amplification Assays for Direct Detection of M. tuberculosis in Specimens 1. MDT Direct Test*, Gen-Probe Inc Method: TMA 2. COBAS AMPLICOR*, Roche Method: PCR ELISA 3. Cepheid GeneXpert Method: Real-Time PCR 4. Homebrewed PCR Method: Real-Time PCR, conventional * FDA approved 8

9 MMWR January 16, 2009 / 58(01);7-10 Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis Guidelines for the use of nucleic acid amplification (NAA) tests for the diagnosis of tuberculosis (TB) were published in 1996 (1) and updated in 2000 (2). Since then, NAA testing has become a routine procedure in many settings because NAA tests can reliably detect Mycobacterium tuberculosis bacteria in specimens 1 or more weeks earlier than culture (3). Earlier laboratory confirmation of TB can lead to earlier treatment initiation, improved patient outcomes, increased opportunities to interrupt transmission, and more effective public health interventions (4,5). Because of the increasing use of NAA tests and the potential impact on patient care and public health, in June 2008, CDC and the Association of Public Health Laboratories (APHL) convened a panel of clinicians, laboratorians, and TB control officials to assess existing guidelines (1,2) and make recommendations for using NAA tests for laboratory confirmation of TB. On the basis of the panel's report and consultations with the Advisory Council for the Elimination of TB (ACET),* CDC recommends that NAA testing be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities, such as contact investigations. These guidelines update the previously published guidelines (1,2). Performance of NATs for Detection of M. tuberculosis in Clinical Specimens Parsons et al Clin Mirobiol Rev 2011 DNA Extraction for Real-Time PCR Detection of TB in Respiratory Specimens by Real-time PCR Reaction 1. MTC target: 16S rrna gene Reaction 2. MTC target: IS6110 gene Concentrate DNA Extract Reaction 3: human b-actin Luo et al JCM 2010 IS6110 Copies in Clinical Strains Performance of Real-time PCR for Detection of TB in Respiratory Specimens Organism Sensitivity Specificity M. tb complex 92% 95% Gold standard: Culture positive M. tuberculosis complex Sreevatsan et al PNAS

10 Asian patient with suspected miliary TB 1+ AFB smear PCR ID: Mycobacterium tuberculosis complex Asian patient with suspected miliary TB 1+ AFB smear PCR ID: Mycobacterium tuberculosis complex Reaction 1 target: 16S rrna gene Reaction 2 target: IS6110 gene 7 y/o Indian boy with lung lesions and LAN Exposed to INH-R mom, previously treated with RIF Lymph node, AFB stain 60xa - Multicopy IS6110 target is 75% sensitive and 100% specific for detection of of M. tuberculosis complex - Sensitivity is independent of AFB stain but affected by age 7 y/o Indian boy with lung lesions and LAN Exposed to INH-R mom, treated with RIF Lymph node Novel Technologies Combine Sample Processing and Nucleic Acid Amplification Reaction 1: MTC, Genus Reaction 2: Genus, MTC Sputum treated with buffer for 15 min 120 minutes 10

11 Boehme et al NEJM 2010 LOD of GX MTB/RIF in Seeded Samples - No difference in sensitivity between tests on direct sputum and decontaminated pellet (P = 0.16) - GX test performed directly on sputum was more sensitive than Amplicor (94.6% vs. 86.8%, P<0.01) and similar to ProbeTec (83.7% vs. 83.9%, P = 0.96) performed on extracted DNA from sputum pellets -GX indeterminate rate of 3.7%, lower than the overall culture-contamination rate (5.5%) (P<0.001). One repeat test, dropped indeterminate to 1.2% (63 of 5190 tests). Boehme et al NEJM 2010 Taylor et al JCM 2012 Cycle Threshold of MTB/RIF Assay on CSF Samples Floated with Sucrose and NaCl Direct Genotypic Susceptibility Testing Drug Targets Sensitivity Rifampin INH PZA Ethambutol Streptomycin Quinolones rpob (katg), inha (pnca) emb rpsl & rrs gyra Taylor et al JCM 2012 WHO 2008 Lin et al JCM

12 Anti-tuberculosis Drugs What Defines MDR-TB? Primary Drugs Secondary Drugs Primary Drugs Secondary Drugs Isoniazid Rifampin Ethambutol Pyrazinamide Amikacin Capreomycin Ethinamide Ethambutol Kanamycin Linazolid Quinolone p-aminosalicylic acid Rifabutin Streptomycin Isoniazid Rifampin MDR Amikacin Capreomycin Ethinamide Ethambutol Kanamycin Linazolid Quinolone p-aminosalicylic acid Rifabutin Streptomycin What Defines XDR-TB? M. tuberculosis rpob Resistance Determining Region for Rifampin (98% Sensitivity) Primary Drugs Secondary Drugs Isoniazid Rifampin MDR + Amikacin or Capreomycin or Kanamycin + Quinolone = XDR Codons 507 through 533 of the M. tuberculosis rpob gene in 307 RIF-resistant isolates MMWR Morb Mort Wkly Rep 2006 Musser Clin Mic Rev 1995 M. tuberculosis rpob Resistance Determining Region for Rifampin (98% Sensitivity) Mechanism of INH Resistance InhA Codons 507 through 533 of the M. tuberculosis rpob gene in 307 RIF-resistant isolates Musser Clin Mic Rev

13 Mechanism of INH Resistance Novel Technologies Combine Sample Processing and Nucleic Acid Amplification Sputum treated with buffer for 15 min KatG S315T InhA 120 minutes Direct Genotypic Susceptibility Testing Drug Targets Sensitivity Rifampin rpob 98% INH (katg), inha 85% PZA (pnca) Ethambutol emb Streptomycin rpsl & rrs Quinolones gyra Boehme et al NEJM 2010 WHO 2008 Lin et al JCM 2004 Accuracy of PyroSequencing DST results by MGIT 960 (N=166) HAIN GenoType MTBDRplus MDR TB from sample and culture INH 0.1 µg/ml RIF 1 µg/ml OFX 2 µg/ml AMK 1 µg/ml CAP 2.5 µg/ml KAN 2.5 µg/ml Overall agreement 97% 97.6% 97% 91.6% 92.8% 80.1% Sensitivity 96.5% 89% CA 97.1% 95.5% 84.6% 89.2% 70% Specificity 100% 100%* 100% 100% 96.4% 100% * With the knowledge of the association of a mutation and RIF MIC, mutations not conferring resistance were NOT interpreted as associated with resistance. 77 Lin et al unpublished 13

14 HAIN GenoType MTBDRsl XDR TB from sample and culture Direct Mycobacterial Identification Cx Time after culture (days) Leukemic girl from Portland with diffuse rash. Skin biopsy, H&E Leukemic girl from Portland with diffuse rash. Skin biopsy, AFB stain, 600X Culture: Negative AFB: 60x Direct Bacterial ID by 16S rrna Sequencing Mycobacterial 16S rrna Sequence Identification focuses on the signature sequences in the 16S rrna gene hypervariable regions A and B, which correspond to the Escherichia coli positions around 129 to 267 and 430 to 500. CLSI MM18-A Kirschner et al JCM

15 Bacterial ID by 16S rrna Sequencing CLSI Cut-Off Values for Percent Identity Scores (Mycobacterium) GENBANK: egablast&page_type=blastsearch&show_defaults=on&link_loc=blasth ome# LEBIBI: RIDOM CLSI MM18-A CLSI MM18-A DNA Sequencing: Sanger Method M. haemophilum100% Identity Laboratory Detection and ID of Mycobacteria Specimen processing AFB Microscopy Culture (6-10 wk) Identificaiton Susceptibility testing CDC recommends the use of broth culture and solid media Liquid Media - More rapid - Automated Solid Media - Mixed morphologies visible - Contamination recoverable FDA Approved Methods for AFB Culture Method BACTEC BacT/ALERT MGIT Marker 14 CO 2 CO 2 O 2 BACTEC 460 BacT/ALERT 3D MGIT

16 MGIT 960 MGIT Culture System 320 tubes per unit Inoculation of Media and Microscopy MGIT liquid medium Specimen pellet Growth supplement PANTA 7H11/7H11S agar LJ slant 5. Inoculate media & make a smear. Positive Glass slide with 15mm specimen area Solid Media Used to Culture Mycobacteria Solid Media Used to Culture Mycobacteria - Egg-based 1. Lowenstein-Jensen (LJ slant) - Agar-based 2. Middlebrook (7H11 plates) - Egg-based 1. Lowenstein-Jensen (LJ slant) - Agar-based 2. Middlebrook (7H11 plates) 7H11/7H11S biplate incubated at 37 C With hemin paper discs. Incubated at 30 C. The CHUM Group Incubation of Mycobacterial Cultures M. chelonae (some varieties) M. haemophilum M. marinum M. ulcerans, agent of Buruli ulcer Media Incubation - MGIT cultures 37 C 6-10 wk - Middlebrook 7H11 agar CO 2, 30/37 C 6-10 wks 16

17 J Clin Microbiol Jul 25. Probability of Negative Mycobacterium tuberculosis Complex Cultures Based on Time-to-Detection of Positive Cultures: A Multi-Center Evaluation of Commercial Broth-based Culture Systems. Tyrrell FC, Budnick GE, Elliott T, Gillim-Ross L, Hildred MV, Mahlmeister P, Parrish N, Pentella M, Vanneste J, Wang YF, Starks AM. Mycobacterial Identification U.S. Centers for Disease Control and Prevention, Atlanta, Georgia. Abstract We conducted a multicenter study to determine whether Mycobacterium tuberculosis complex (MTBC) cultures in automated broth-based systems could reliably be considered as negative sooner than six weeks. Laboratory sites used MGIT or BacT/ALERT and tracked results of time to detection of mycobacteria (TTD-all, n = 1547) and MTBC (TTD-MTBC, n = 466) over six-month periods from primarily (93%) respiratory specimens. Cumulative percentages by day detected and median TTD of initial and follow-up specimens were analyzed. The median TTD-MTBC for MGIT (n = 6 sites) was 14 days. For laboratories using standard processing procedures, 100% of MTBC were detected from initial and follow-up specimens in 28 and 35 days, respectively, and no yield of MTBC on solid or MGIT liquid media was observed after five weeks. The median TTD-MTBC for BacT/ALERT (n = 3 sites) was 18 days, with 95% and 100% detected within 37 and 42 days respectively. Analysis of TTD of positive MTBC cultures in broth can predict the probability of culture negativity at defined time points. Receipt of interim negative reports earlier than six weeks could assist clinicians in considering alternative diagnoses, and could alter timing and prioritization of public health interventions. Laboratories should analyze their own TTD data to inform protocol decisions. Laboratories using MGIT could issue no growth of MTBC reports on initial specimens as early as four weeks and on patients undergoing treatment as early as five weeks post-inoculation. 0 Cx Time after culture (days) What happens when the broth turns positive? Positive MGIT broth Gram stain Sheep blood agar Mycobacterial Culture Identification 1. Biochemical Tests 2. Nucleic Acid Tests - Gen-Probe AccuProbe - Line Probe - PCR - Sequencing 3. HPLC Auramine stain Commercial Identification Methods Gen-Probe AccuProbe AccuProbe (Gen-Probe) Target Mycobacterial ID Sensitivity Specificity Mycobacterium tuberculosis complex 99.2% 99.0% Mycobacterium avium complex 99.9% 100% Mycobacterium avium 99.3% 100% Mycobacterium intracellulare 100% 100% Mycobacterium gordonae 98.8% 99.7% Mycobacerium kansasii 92.8% 100% 17

18 Line Probe Assay (LiPA) - Nitrocellulose strip - Specific probes attached as parallel lines - Requires a PCR amplification step - Visual detection or automated reader Line Probe Assay (LiPA) - Innogenetics Infectious Diseases Mycobacteria - INNO-LiPA Rif.TB - INNO-LiPA MYCOBACTERIA v2 Mycobacterial Culture Identification with Line Probe Assay HAIN GenoType Mycobacterium CM/AS Identification of MTC and up to 40 NTM from culture INNO-LiPA MYCOBACTERIA v2 Mycobacterial Culture Identification Commercial Identification Methods 1. Biochemical Tests 2. Nucleic Acid Tests - Gen-Probe AccuProbe - Line Probe - PCR - Sequencing 3. HPLC HPLC { Extractable Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan 18

19 Commercial Identification Methods HPLC Lacks Resolution for Species ID HPLC M. avium complex chromatogram M. tuberculosis M. bovis HPLC Lacks Resolution for Species ID M. chelonae abscessus group M. fortuitum group Mycobacterial Culture Identification 1. Biochemical Tests 2. Nucleic Acid Tests - Gen-Probe AccuProbe - Line Probe - PCR - Sequencing 3. HPLC Mycobacterial Identification Mycobacterial Isolates at Stanford in 2011 Mgord 10% PCR Cx+ MAC 35% Time after culture (days) MTC 14% MCAG 29% n=165 unique isolates 19

20 DNA Extraction for Real-Time PCR Solid Media added to water MGIT960 BD concentrated in water AFB + Culture Multiplex, Real-time PCR Design for Identification of AFB Isolates Rxn 1 Rxn 2 1. M. tb complex 2. M. avium complex 3. AFB genus 1. M. chelonae/abscessus 2. M. fortuitum group 3. AFB genus Mycobacterial rrna Sequences Targeted AFB + Culture Multiplex, Real-time PCR Design for Identification of AFB Isolates Target Length Tm Rxn 1 1. M. tb complex ITS M. avium complex 16S AFB genus 16S Conventional PCR Rxn 2 1. M. chelon/abscess ITS M. fortuitum group 16S AFB genus 16S Kirschner et al JCM 1993 Multiplex, Real-time PCR for Speciation of M. tb complex and M. avium complex Multiplex, Real-time PCR for Speciation of M. fortuitum group and M. chelonae-abscessus Reaction 1 M. tuberculosis Reaction 1 M. avium MTC 77 C MAC 86 C Reaction 2 M. fortuitum Reaction 2 M. chelonae MFG 86 C MCAG 77.5 C Reaction 1 M. fortuitum Reaction 1 M. mucogenicum Reaction 2 M. tuberculosis 20

21 Performance of Multiplex, Real-time PCR Multiplex, Real-time PCR Paradigm for Identification of AFB Isolates Organism Sensitivity (n) Specificity (n) M. tb complex 100% (96) 100% (215) M. avium complex 100% (95) 99.5% (216) M. chel-absc group 100% (68) 100% (238) M. fortuitum group 95% (19) 100% (287) M. mucogenicum 100% (9) 99.7% (297) AFB + Culture Rxn 1 Rxn 2 1. M. tb complex 2. M. avium complex 3. AFB genus 1. M. chelonae/abscessus 2. M. fortuitum group 3. AFB genus Richardson et al JCM 2009 Heart transplant patient with new lung lesion: BAL sent for AFB culture. Heart transplant patient with new lung lesion: BAL sent for AFB culture. Put in respiratory isolation and treated for TB (4 abx), and MAC (1abx) TB or NTM? BAL Cx AFB - BAL Cx AFB - Auramine (x1000) Cx+ 3/26 4/2 4/9 4/16 Time post-culture (days) 3/26 4/2 4/9 4/16 Time post-culture (days) Heart transplant patient with new lung lesion: BAL sent for AFB culture. Put in respiratory isolation and treated for TB (4 abx), and MAC (1abx) Reaction 1: MTC, Genus, MAC Reaction 2: : MCAG, Genus, MFG Heart transplant patient with new lung lesion: BAL sent for AFB culture. Put in respiratory isolation x and treated for TB (4 abx), and MAC (1abx) x Reaction 1: MTC, Genus, MAC Reaction 2: MCAG, Genus, MFG Genus 81 C MAC 86 C Genus 81 C Genus 81 C MAC 86 C Genus 81 C BAL Cx AFB - PCR Cx+ BAL Cx AFB - PCR Cx+ 3/26 4/2 4/9 4/16 Time post-culture (days) 3/26 4/2 4/9 4/16 Time post-culture (days) 21

22 2 y/o Indian boy with preauricular lymphadenitis: FNA positive for AFB. On therapy for TB (4 abx) and NTM (3 abx) 2 y/o Indian boy with preauricular lymphadenitis: FNA positive for AFB. On therapy for TB (4 abx) and NTM (3 abx) TB or NTM? TB or NTM? FNA AFB+ FNA AFB+ Auramine (x1000) Solid Cx+ 2/22 3/14 4/4 4/25 Time post-culture (days) 2/22 3/14 4/4 4/25 Time post-culture (days) 2 y/o Indian boy with preauricular lymphadenitis: FNA positive for AFB. On therapy for TB (4 abx) and NTM (3 abx) Reaction 1: MTC, Genus, MAC Reaction 2: MCAG, Genus, MFG 2 y/o Indian boy with preauricular lymphadenitis: FNA positive for AFB. On therapy for TB x (4 abx) and NTM (3 abx) Genus 81 C Genus 81 C TB x or NTM? FNA AFB+ Solid Cx+ PCR FNA AFB+ Solid Cx+ PCR 2/22 3/14 4/4 4/25 Time post-culture (days) 2/22 3/14 4/4 4/25 Time post-culture (days) Multiplex, Real-time PCR Design for Speciation of M. tuberculosis Complex AFB + Culture Rxn 1 Rxn 2 M. tb complex M. tuberculosis M. bovis M. bovis BCG M. africanum M. mongi M. microti M. pinnipedii M. canettii M. caprae Mangoose: M. mongi Vole: M. microti M. tuberculosis M. canettii, BCG M. africanum Goat: M. caprae Cow: M. bovis Seal: M. pinnipedii Badger & Antelope: M. bovis 22

23 Phylogeny of M. tuberculosis Complex Phylogeny of M. tuberculosis Complex RD1 BCG Ernst et al J Clin Invest 2007 Pinsky and Banaei JCM 2008 Multiplex, Real-time PCR Design for Speciation of M. tuberculosis Complex Multiplex, Real-time PCR Design for Speciation of M. tuberculosis Complex Pinsky and Banaei JCM 2008 Pinsky and Banaei JCM y/o Mexican girl with culture-positive peritoneal TB. On therapy for M. bovis (RIF, INH, EMB, PZA). 4 y/o Mexican girl with culture-positive peritoneal TB. On therapy for M. bovis (RIF, INH, EMB, PZA). M. tuberculosis or M. bovis? Reaction 1 Reaction 2 Genus control RD4 deleted 23

24 Multiplex, Real-time PCR Design for Speciation of M. chelonae-abscessus group Multiplex, Real-time PCR Design for Speciation of M. chelonae-abscessus group AFB + Culture Rxn 1 AFB genus M. chelonae M. abscessus 76.5 C 81.5 C Rxn 2 M. chelonae/abscessus 1. M. chelonae 2. M. abscessus 3. M. immunogenum M. immunogenum 81.5 C 79.4 C Guarin et al 2010 Accuracy of Multiplex, Real-time PCR Design for Speciation of M. chelonae-abscessus group Mycobacterial Isolates at Stanford in 2011 Guarin et al 2010 n=350 isolates Multiplex, Real-time PCR Paradigm for Identification of AFB Isolates AFB + Culture Rxn 1 1. M. tb complex 2. M. avium complex 3. AFB genus Mycobacterial Isolates at Stanford in % Mgord 10% MAC 35% Rxn 2 Rxn 3 1. M. chelonae/abscessus 2. M. fortuitum group 3. AFB genus 1. M. gordonae MTC 14% MCAG 29% NTM n=165 unique isolates 24

25 Bacterial ID by 16S rrna Sequencing Mycobacterial 16S rrna Sequence Identification focuses on the signature sequences in the 16S rrna gene hypervariable regions A and B, which correspond to the Escherichia coli positions around 129 to 267 and 430 to 500. CLSI MM18-A Kirschner et al JCM 1993 Bacterial ID by 16S rrna Sequencing CLSI Cut-Off Values for Percent Identity Scores (Mycobacterium) GENBANK: egablast&page_type=blastsearch&show_defaults=on&link_loc=blasth ome# LEBIBI: RIDOM CLSI MM18-A CLSI MM18-A Laboratory Detection and ID of Mycobacteria CLSI Document M24-A Specimen processing AFB Microscopy Culture (6-10 wk) Identificaiton Susceptibility testing 25

26 Drug Susceptibility Testing for M. tuberculosis Anti-tuberculosis Drugs MTC 14% Mgord 10% NTM: MAC Environmental 35% MCAG 29% - First isolate - Retest if fail to respond - Retest if culture pos after 3 months Primary Drugs Isoniazid Rifampin Ethambutol Pyrazinamide Secondary Drugs Amikacin Capreomycin Ethinamide Ethambutol Kanamycin Linazolid Quinolone p-aminosalicylic acid Rifabutin Streptomycin FDA Approved Drug Susceptibility Testing Methods for M. tuberculosis MGIT960 Drug Susceptibility Assay Solid Media BACTEC 460 MGIT 960 Control Resistant Sensitive 1. Absolute conc. 2. Proportion method 3. Resistance ratio No Antibiotic TAT: 4-13 days <4 or >13 days: No result SM INH RMP EMB MGIT960 AST Set Report Growth Unit Status Conc. Drug Name 400 C Growth Control 50 S 1 g/ml Streptomycin 110 R 0.1 g/ml Isoniazid 0 S 1 g/ml Rifampin 0 S 5 g/ml Ethambutol 400 units in 4-13d. >400 in <4 d is invalid; <400 in 13 d is invalid Growth Unit Status Conc. Drug Name 400 C Growth Control 60 S 100 g/ml Pyrazinamide Microscopic Observation Drug Susceptibility Assay (MODS) No abx No abx INH RIF Look at wells through an inverted microscope starting on day 7 26

27 Microscopic Observation Drug Susceptibility Assay (MODS) No abx No abx INH RIF Look at wells through an inverted microscope starting on day 7 Day 7 Day 15 Indirect Genotypic Susceptibility Testing Drug Targets Sensitivity Rifampin rpob INH (katg), inha PZA (pnca) Ethambutol emb Streptomycin rpsl & rrs Quinolones gyra WHO 2008 Lin et al JCM 2004 Overall agreement Accuracy of PyroSequencing DST results by MGIT 960 (N=166) INH 0.1 µg/ml RIF 1 µg/ml OFX 2 µg/ml AMK 1 µg/ml CAP 2.5 µg/ml KAN 2.5 µg/ml 97% 97.6% 97% 91.6% 92.8% 80.1% Line Probe Assay (LiPA) - Nitrocellulose strip - Specific probes attached as parallel lines - Visual detection or automated reader Sensitivity 96.5% 89% CA 97.1% 95.5% 84.6% 89.2% 70% Specificity 100% 100%* 100% 100% 96.4% 100% * With the knowledge of the association of a mutation and RIF MIC, mutations not conferring resistance were NOT interpreted as associated with resistance. 159 Lin et al unpublished Line Probe Assay (LiPA) Innogenetics INNO-LiPA Rif. TB - Innogenetics Infectious Diseases Mycobacteria - INNO-LiPA Rif.TB - INNO-LiPA MYCOBACTERIA v2 INNO-LiPA Rif.TB

28 HAIN GenoType MTBDRplus MDR TB from sample and culture HAIN GenoType MTBDRsl XDR TB from sample and culture Indicators to Monitor the Quality of AFB Culture Drug Susceptibility Testing for NTM Monitor date collected and date received in the laboratory. Determine the percentage of AFB-positive smears reported and the percentage of smear-positive and culture positive as well as smear-negative and culture-positive specimens. Determine the percentage of specimens reported as being contaminated. Contamination rates of 3 to 5% for solid media and less than 10% for liquid media are generally considered acceptable. Determine the turnaround time for smear, culture, and DST. MTC 14% Mgord 10% NTM: MAC Environmental 35% MCAG 29% - Methods not standardized - First isolate macrolides vs. MAC - Test if treatment failure Overview - Taxonomy/cell wall properties Tuberculosis Urine Test - Pathogenesis - Detection of active disease - Specimen processing - AFB microscopy - Culture and identification - Antibiotic susceptibility testing - Novel diagnostics - Detection of latent TB infection CSA for TB breath analysis instrument - Affordable - Sensitive - Specific - User-friendly, no need for trained techs - Robust, rapid - Equipment minimal, no need for lab - Deliverable to those who need them - Unknown cases 28

29 Mycobacterial Lipoarabinomannan (LAM) Extractable { Lipids (wax) Mycolic acids Arabinogalactan Peptidoglycan - LAM is a structurally essential kD heat-stable glycolipid - Abundant: 15% of the total weight - Immunogenic - Virulence factor - Released from metabolically active or degrading bacteria during TB infection. Performance Estimates of Urine LAM Assay in Culture Positive Cases Performance Estimates of Urine LAM Assay in Culture Positive Cases Range, 13 93% Range, 87 99% Minion et al Eur Respir J 2011 Minion et al Eur Respir J 2011 Tuberculosis Urine Test Does TB Smell? CSA for TB breath analysis instrument Average daily sensitivities: 72% to 100% Average daily specificities: 91.9% to 99.3% 29

30 Tuberculosis, 91:327-28, 2011 Breathscanner: GC for separation /surface acoustic wave detector (SAW Mass Spectrometery Detects TB-Specific VOCs Colorimetric Sensor Array for Detection of Volatile Signatures Breath Urine Mass Spectra Phillips et al 2007 Banday et al 2011 TB Breath Study at Highland Hospital Breath tested 5-7 min Colorimetric Array for Detection of Volatile Signatures Printed array of chemically responsive dyes. Digitally image before & after exposure & subtract. Difference Map is a molecular fingerprint : a unique 108-dimensional vector (36 ΔR, ΔG, ΔB). Urine collected Study ID# Before Exposure After Exposure Difference Map 9 mm (center avg. 300 pixels) Study ID# Biggest color changes are boxed in gray. Suslick et al Quim. Nova 2007 ammonia R after - R before, G after - G before, B after - B before 30

31 Difference Maps are Molecular Fingerprints Example: 10 Arabica Coffees decylamine sec-bu 2 amine aniline 2-picoline acetic acid Café Mai Traditional 8 O'Clock Columbian 8 O'Clock Hazel Nut Folgers Columbian Folgers Grande Supreme Decaf hexanethiol benzylthiol ethanol pentanol CF 3 CO 2 H PhMe 2 P Bu 3 P hexanal benzaldehyde Every volatile organic has a unique pattern! 1-octene Maxwell House Original Maxwell House Original Decaf Starbuck s Columbian Starbuck s Espresso Easy to distinguish among different coffee brands Starbuck s Sumatra Colorimetric Array Setup for Detection of VOC Signatures In Vitro Colorimetric Array Setup for Detection of VOC Signatures (a) incubator (b) scanner Inverted Petri dish Sensor array Scanner Inoculum concentration: 50 million CFU for Mtb, 1 million for others. Inoculum concentration: 50 million CFU for Mtb, 1 million for others. Incubated at 37 C in 5% CO 2 for 4 d and 20 hrs, respectively. Representative Time Response Profiles Species-specific decision tree classifier for 15 different bacterial species grown on blood agar plates 99% correct classification among 402 trials. 31

32 Representative time response profiles of pneumoniacausing bacteria and M. tuberculosis (6 trial per bug) Species Separation of Pneumonia-Causing Bacteria Using a Decision Tree TB Breath Study at Highland Hospital isense Breath Analysis Prototype Instrument Breath tested 5-7 min Urine collected Study ID# CSA for TB breath analysis instrument ΔR, ΔG, ΔB, ΔUV x,y,z,w Study ID# Laboratory Diagnosis of Tuberculosis Estimated TB Incidence Rates, 2010 Active TB - Symptomatic - Productive cough Latent TB - Asymptomatic 32

33 Model of Tuberculosis Transmission Model of Tuberculosis Transmission Active TB Active TB Latent TB Latent TB Model of Tuberculosis Transmission Laboratory Diagnosis of Tuberculosis Active TB Active TB - Symptomatic - Productive cough Latent TB Latent TB - Asymptomatic Diagnosis of Latent Tuberculosis - Tuberculin Skin Test Diagnosis of Latent Tuberculosis - Tuberculin Skin Test Disadvantages - Subjective - In vivo - Adverse effects - Boosting - Affected by BCG vaccination - Requires two visits 33

34 Diagnosis of Latent Tuberculosis Quantiferon Gold Intube Assay - Tuberculin Skin Test CD 4 IFN- + APC CD 4 Intube Elispot IFN- + APC CD 4 APC CD 4 IFN- + TNF- APC ESAT6 CFP-10 TB7.7 Diagnosis of Latent Tuberculosis Region of Difference 1 (RD1) - Interferon- Release Assays Quantiferon Gold In-tube (Cellestis Inc.) - FDA approved T-SPOT.TB (Oxford Immunotec Inc.) - FDA approval TB7.7 Origin of M. tuberculosis M. szulgai M. kansasii M. marinum M. flavescens TB7.7 (Rv2654; encoded by RD11) is another immunodominant antigen for diagnosis RD1 BCG Ernst et al J Clin Invest 2007 Devulder et al Int J Syst Evol Microbiol

35 Quantiferon Gold Intube Assay Sample Processing Quantiferon Gold Intube Assay Sample Processing - Draw 1 ml of blood - Shake until frothing - Transport to incubator immediately ( 16h) - Incubate 37 C h - Store rm temp x3 days - Perform ELISA for IFN Quantiferon Gold Intube Assay DSX: Automated ELISA Instrument Interpretation Criteria for QFT Gold-Intube Benefits & Disadvantage of IGRA Over TST Benefits - Single visit - Ex vivo - Less adverse effects - Eliminate boosting - Unaffected by BCG vaccination Disadvantage - Costs - Reagent - Capital - Laboratory - Blood draw Performance of IGRA for Detection of Latent TB Given Lack of Gold Standard Estimated sensitivity - In active TB patients - In high-risk individuals - Concordance with TST Estimated specificity - In low-risk individuals Reproducibility - Repeat testing 35

36 IGRAs for Screening of HCWs Sensitivity of TST & IGRAs in patients with active TB TST QFT-IT T-SPOT.TB Diel et al Chest 2010 Sensitivity of QFT-IT in patients with active TB Developed Countries Developing Countries Overall in HIV+ and -, T-SPOT sensitivity was higher but not significantly different from QFT-GIT sensitivity (sensitivity difference, 19%; 95% CI, 217% to 56%; P 5.3) (Table 2). Results were similar when restricted to HIV-infected individuals. Diel et al Chest 2010 Metcalfe et al JID 2011:204 (Suppl 4) Sensitivity of QFT-G-IT and T-SPOT.TB in HIV infected with confirmed active TB in low- and middle-income countries Sensitivity of QFT-G-IT and T-SPOT.TB in HIV uninfected with confirmed active TB in low- and middle-income countries Pooled sensitivity estimates: T-SPOT: 68%; 95% CI, 56% 80%; 5 studies) QFT-GIT: 65%; 95% CI, 52% 77%; 7 studies Metcalfe et al JID 2011:204 (Suppl 4) Pooled sensitivity estimates: T-SPOT: 88%; 95% CI, 81% 95% QFT-GIT: 84%; 95% CI, 78% 91% Metcalfe et al JID 2011:204 (Suppl 4) 36

37 Sensitivity difference between IGRA and TST results: No consistent evidence that either IGRA was more sensitive than the TST for active tuberculosis diagnosis Sensitivity of IGRA in Children? Sensitivity difference with TST: T-SPOT: 23%; 95% CI, 0% 45%; P 5.05 QFT-GIT: 7%; 95% CI, 29% to 23%; P 5.37 Metcalfe et al JID 2011:204 (Suppl 4) FIGURE 1. Meta-analysis of QFT sensitivity for diagnosing active TB disease (culture positive) in children. QFT sensitivity: pooled proportion 75% (95% CI, 63% 85%); heterogeneity tests: Cochran Q (df 4) FIGURE 2. Meta-analysis of QFT sensitivity for diagnosing active TB disease (all cases; clinical diagnosis) in children. QFT sensitivity: pooled proportion 66% (95% CI, 53 78) (Clinical + Lab Diagnosis) 86%(19/22) study of years old children with symptoms of TB (28 confirmed, 136 probable and 131 unlikely TB), 335 children in contact with adults with pulmonary TB and 156 community controls in Southern Ethiopia. The Tuberculin Skin Test (TST) and Quantiferon-In-Tube (QFT-IT) were performed. 37

38 Who is right? Menzies et al 2007 Sensitivity in patients with latent TB using progression to active TB as gold standard Specificity of IGRAs in Healthy Individuals QFT-IT T-SPOT.TB Herrera et al CID 2011 Diel et al Chest 2010 Reproducibility of QFT-IT Reproducibility of QFT-IT in HCW Coversions 2% to 15% Reversions 20 to 40% Pai and Elwood Can Respir J

39 Reproducibility of QFT-IT in Stanford HCW QFT-GIT Assay Standardization Reversions Standardized - Pre-analytical - Blood collection* - 37 ºC incubation - Plasma separation Not Standardized Conversions - Analytical - ELISA - Interpretation QFT-GIT Assay Standardization Quantiferon Gold Intube Assay Sample Processing Standardized - Pre-analytical - Blood collection* - 37 ºC incubation - Plasma separation - Analytical - ELISA - Interpretation Not Standardized - Pre-analytical - Skin preparation - Incubation delay* - Incubation duration* - Time of day - Day of month - Season - Diet - Infection - Antibiotics - Transport to incubator ( 16h) Effect of incubation delay on QFT-IT results Incubation delay increases indeterminate results Herrera et al JCM 2010 Herrera et al JCM

40 Mitogen Results Following Incubation Delay Mitogen Results Following Incubation Delay Herrera et al JCM 2010 Doberne et al JCM 2011 Indeterminate Results in Studies Using QFT-IT Effect of Incubation Delay on the Accuracy of QFT-IT Results Range: 0-41% Implementation of the immediate incubation at Stanford yielded an indeterminate result rate of 0.36% in 14,830 HCW tested during the first 12 months 128 study participants 3 QFT-GIT sets collected Incubation Delay 0 h Low risk & - TST&orQFT High risk & + TST&orQFT 6 h 12 h Diel et al Chest 2010 Doberne et al JCM 2011 TB Ag results following immediate and delayed incubation TB-Ag Results for Subjects with Discordant Results Reversion rate: 19% (5/26) with 6 h delay 22% (5/23) with 12 h delay Doberne et al JCM 2011 Doberne et al JCM

41 Risk factors for latent TB infection in volunteers with discrepant results Reproducibility of QFT-IT in Stanford HCW Reversions 0.35 Conversions 0.35 Doberne et al JCM 2011 Reproducibility of QFT-IT in Stanford HCW Reproducibility of QFT-IT in Stanford HCW Reversions Reversions Conversions Conversions An algorithm for LTBI screening with IGRAs Overview - Taxonomy/cell wall properties - Pathogenesis - Detection of active disease - Specimen processing - AFB microscopy - Culture and identification - Antibiotic susceptibility testing - Novel diagnostics - Detection of latent TB infection - Unknown cases 41

42 Case 1 A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive right neck mass. 7/14/12 ED visit HPI: Pt was living in India until 2 weeks prior to admission. Approximately 1.5 months before, she noted mild R neck swelling. She was evaluated in India where an US was done 4/27/12 and demonstrated multiple moderately enlarged discrete lymph nodes in her R neck with the largest measuring 3.2x1.2 cm. She then underwent a FNA of the mass on 4/30/12 with pathology read as caseous tuberculosis. She was started on Ethambutol, Pyrazinimide, Rifampin, and INH. A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive R neck mass. Host She then moved here 2 weeks prior to admission. 5-6 days before admission, she noticed the mass was enlarging. It was also more erythematous, mildly pruritic, and mildly tender. She denied any fevers, weight loss, cough, chest pain, difficulty breathing, difficulty swallowing. She called her PCP who recommended that she come to the ED for further evaluation. She was admitted for further evaluation and treatment. Bugs? Exposure A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive R neck mass. Host Immunocompetent Neck abscess Auramine stain, 1000x 2+ Acid-fast-bacilli Bugs? Exposure Children: M.tb & NTM Adults: M. tb Zoonotic: Bartonella, Rickettsia Sputum smears and culture 7/15/12 Fluorescent smear NEGATIVE for AFB; 7/15/12 Fluorescent smear NEGATIVE for AFB; 7/15/12 Fluorescent smear NEGATIVE for AFB; What else? Sputum smears and culture 7/15/12 Fluorescent smear NEGATIVE for AFB; Culture positive for M. abscessus 7/15/12 Fluorescent smear NEGATIVE for AFB; Culture positive for M. abscessus 7/15/12 Fluorescent smear NEGATIVE for AFB; Culture negative What else? 42

43 A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive R neck mass. 7/14/12 ED visit HPI: Pt was living in India until 2 weeks prior to admission. Approximately 1.5 months before, she noted mild R neck swelling. She was evaluated in India where an US was done 4/27/12 and demonstrated multiple moderately enlarged discrete lymph nodes in her R neck with the largest measuring 3.2x1.2 cm. She then underwent a FNA of the mass on 4/30/12 with pathology read as caseous tuberculosis. She was started on Ethambutol, Pyrazinimide, Rifampin, and INH. She then moved here 2 weeks prior to admission. 5-6 days before admission, she noticed the mass was enlarging. It was also more erythematous, mildly pruritic, and mildly tender. She denied any fevers, weight loss, cough, chest pain, difficulty breathing, difficulty swallowing. She called her PCP who recommended that she come to the ED for further evaluation. She was admitted for further evaluation and treatment. A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive R neck mass. 7/30/12 ED visit HPI: Patient is a 29 F who is s/p I& D of a neck mass/abscess on 7/15/12 for presumed TB abscess. She was diagnosed with TB in India 6 weeks ago and started on RIPE therapy. She had an enlarging R neck mass despite the therapy and thus had I&D here nearly 2 weeks ago. Initial path was suggestive of TB, but cultures just grew out Mycobacterium abscessus as of 7/27/12. Highly likely to have M. abscessus lymphadenitis as well. She comes in to the ED today, with increasing neck pain at the I&D site which still has a penrose drain in place. She also has neck stiffness and decrease ROM limited by pain. But with negative sputum AFB cultures X3, suggesting TB lymphadenitis but no active pulmonary TB A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive R neck mass. Neck 1+ AFB smear PCR ID: Mycobacterium tuberculosis complex A 29 Y female recently started on RIPE therapy in India, who presented with AFB-positive R neck mass. Host Immunocompetent Reaction 1 target: 16S rrna gene Reaction 2 target: IS6110 gene M.tb Exposure Children: M.tb & NTM Adults: M. tb Zoonotic: Bartonella, Borrelia 20% of all TB cases More likely in immunocompromised patients. Case#2 4 y/o Mexican girl with peritonitis. On therapy for presumptive M. bovis Surgical pathology showed granulomas Positive AFB Culture PCR ID: Mycobacterium tuberculosis complex Reaction 1: MTC Reaction 2: Genus What additional testing should be done? 43

44 4 y/o Mexican girl with peritonitis. On therapy for presumptive M. bovis Surgical pathology showed granulomas Positive AFB Culture PCR ID: Mycobacterium bovis Case 3 2 y/o boy with travel to rural Korea presents with pneumonia, meningitis, partial stroke, and disseminated gastrointestinal lesions Colon biopsy, H&E 100X Reaction: 1 Mtb, Genus, BCG Reaction 2: M. africanum, M. bovis Genus control RD4 deleted 2 y/o boy with travel to rural Korea presents with pneumonia, meningitis, partial stroke, and disseminated gastrointestinal lesions Colon biopsy, AFB stain 400X 2 y/o boy with travel to rural Korea presents with pneumonia, meningitis, partial stroke, and disseminated gastrointestinal lesions PCR ID: Mycobacterium tuberculosis complex Reaction 1: MTC, Genus Reaction 2: Genus, MTC 2 y/o boy with travel to rural Korea presents with pneumonia, meningitis, partial stroke, and disseminated gastrointestinal lesions PCR ID: Mycobacterium tuberculosis Case 4 47yo h/o lymphoma s/p stem cell transplant AFBs found on recent biopsy of painful calcaneus Gram stain, 1000X Reaction: 1 Mtb, Genus, BCG Ghost cells Ghost cells 44

45 47yo h/o lymphoma s/p stem cell transplant AFBs found on recent biopsy of painful calcaneus H&E, 100X 47yo h/o lymphoma s/p stem cell transplant AFBs found on recent biopsy of painful calcaneus H&E, 400X 47yo h/o lymphoma s/p stem cell transplant AFBs found on recent biopsy of painful calcaneus AFB, 600X Bacterial ID by 16S rrna Sequencing Identification focuses on the signature sequences in the 16S rrna gene hypervariable regions A and B, which correspond to the Escherichia coli positions around 129 to 267 and 430 to 500. AFB: 60x CLSI MM18-A DNA Sequencing: Sanger Method Lymph node culture of M. haemophilum M. haemophilum100% Identity 45

46 TB Response (IU/mL) 8/28/2012 Case 5 A 44 year old health care worker presents for her annual TB exposure exam. US born Negative TST in the past No TB contacts Psychiatry nurse No travel history Quantiferon Gold In-tube Serial Results Axis Title 3/30/09 7/12/09 7/29/09 5/27/10 A 44 year old health care worker presents for her annual TB exposure exam. An algorithm for LTBI screening with IGRAs US born Negative TST in the past No TB contacts Psychiatry nurse No travel history Should she be treated for latent TB infection? Reproducibility of QFT-IT in Stanford HCW Acknowledgements 0.35 Reversions 0.35 Conversions Stanford University Pathology/Clinical Lab Samantha Mix Indre Budvytiene Rajiv Gaur David Doberne Mady Slater Nora Guarin Ben Pinsky Tom Richardson Divinia Samson Chemistry Eric Cool Alameda County Medical Center Herbert Schub Alameda County Public Health Robert Benjamin Ca Dept. of Health Services Ed Desmond Grace Lin Cepheid Ellen Jo Baron David Persing isense Paul Rhode Ray Martino Sung Lim Brian Taba Financial Support Stanford Pathology Stanford SPARK/ Global Health isense NIH 46