GR2011-12 Breath of TB - Dr Banaei

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Detection of Volatile Organic Compounds in the Breath of TB Patients : 

Detection of Volatile Organic Compounds in the Breath of TB Patients Niaz Banaei MD Assistant Professor of Pathology and Medicine Director of Clinical Microbiology Stanford University niazbanaei@stanford.edu

Slide 2: 

• TB Epidemiology • Lab diagnosis of TB • Development of breath test Overview

Slide 3: 

http://www.who.int/tb/publications/global_report/2011/ Estimated TB Incidence Rates, 2010

Slide 5: 

MDR-TB Among New TB Cases, 1994–2009 http://www.who.int/tb/publications/global_report/2011/

Slide 6: 

MDR-TB Among Treated TB Cases, 1994–2009 http://www.who.int/tb/publications/global_report/2011/

Slide 7: 

Prevalence of MDR-TB Among New and Previously Treated Cases WHO/IUATLD Fourth Global Report

Slide 8: 

Science 15 September 2006:Vol. 313. no. 5793, p. 1554

Slide 9: 

Primary Drugs Secondary Drugs Isoniazid Amikacin or Rifampin Capreomycin or Kanamycin + Quinolone MDR + = XDR MMWR Morb Mort Wkly Rep 2006 Anti-tuberculosis Drugs

Slide 10: 

Distribution of Countries (n=58) Reporting at Least One Case of XDR-TB as of January 2010 http://www.who.int/tb/publications/global_report/2011/

Slide 11: 

Prevalence of MDR-TB Among New and Previously Treated Cases 13% XDR 15% XDR WHO/IUATLD Fourth Global Report

Slide 12: 

MDR-TB Among New TB Cases, 1994–2009 http://www.who.int/tb/publications/global_report/2011/

Slide 13: 

MRSA MDR TB Laboratory Challenges of Mycobacteriology Day 1 Day 21

Slide 14: 

Specimen Processing Routine Bacteriology Mycobacteriology BSL2 BSL3 2 min 2 hrs (batch) Incubate 1-2 days Incubate up to 8 wks

Slide 15: 

Diagnosis by Microscopy MRSA MDR TB Ziehl-Neelsen Auramine Gram strain (min) Acid-Fast Stain (hrs) Overall Sensitivity of AFB smear: 22-80% Routine Bacteriology Mycobacteriology

Slide 16: 

Culture Detection and Identification Routine Bacteriology Mycobacteriology BSL2 BSL3

Slide 17: 

MGIT 960 320 tubes per unit Positive Automated Liquid Culture Systems Average TTD: 7days

Slide 18: 

Solid Media Absolute conc. Proportion method Resistance ratio Drug Susceptibility Testing Methods for M. tuberculosis MGIT 960 Average TAT: 8 wks 2-3 weeks

Slide 19: 

Look at wells through an inverted microscope starting on day 7 INH RIF Microscopic Observation Drug Susceptibility Assay (MODS) No abx No abx

Slide 20: 

INH RIF Microscopic Observation Drug Susceptibility Assay (MODS) No abx No abx Day 15 Day 7 Look at wells through an inverted microscope starting on day 7

Slide 21: 

Laboratory Challenges of TB Diagnosis Specimen processing BSL3 2 hr (batch) Solid Culture Broth Culture TAT: 4 wk TAT: 2 wk Ziehl-Neelsen Low sensitivity Microscopy

Slide 22: 

Laboratory Capacity is Lacking in Endemic Countries

Slide 23: 

Laboratory Training is Lacking in Endemic Countries

Slide 24: 

Contemporary diagnostics for MDR-TB are available for <1/2 of high MDR-TB burden countries http://www.who.int/tb/publications/global_report/2011/a

Slide 25: 

Patients Travel Long Distances to Seek Care

Slide 26: 

Patients Travel Long Distances to Seek Care

Slide 27: 

Novel Technologies Combine Sample Processing and Nucleic Acid Amplification Sputum treated with “buffer” for 20 min 120 minutes ID Sensitivity: 99% for smear pos; 70-90% for smear neg RIF Resistance sensitivity: 99% Boehme et al NEJM 2010

Slide 28: 

Tuberculosis Breath Test

Slide 29: 

Affordable Sensitive Specific User-friendly, no need for trained techs Robust, rapid Equipment minimal, no need for lab Deliverable to those who need them Tuberculosis Breath Test

Slide 30: 

Does TB Smell? Average daily sensitivities: 72% to 100% Average daily specificities: 91.9% to 99.3%

Slide 31: 

Does TB Smell? Average daily sensitivities: 72% to 100% Average daily specificities: 91.9% to 99.3%

Slide 32: 

Mass Spectrometery Detects TB-Specific VOCs Mass Spectra Breath Urine Phillips et al 2007 Banday et al 2011

Slide 33: 

Breathscanner: GC for separation /surface acoustic wave detector (SAW

Slide 34: 

TB “Point of Care” Diagnostics in Development

Colorimetric Sensor Array for Detection of Volatile Signatures : 

Colorimetric Sensor Array for Detection of Volatile Signatures

Colorimetric Sensor Array for Detection of Volatile Signatures : 

Colorimetric Sensor Array for Detection of Volatile Signatures

Nanoporous Pigment Sensor Arrays : 

Nanoporous Pigment Sensor Arrays Chemo-responsive Pigment Classes: Lewis Acid Dyes: metal ion containing dyes BrØnsted Acid/Base Dyes: pH indicators Dyes with Large Dipoles: solvatochromic Metal Nanodots: redox active pH Indicators: Methyl Red, Thymol Blue, Phenol Red, …

Origin of High Sensitivity : 

Our Sensor Array relies on strong interactions. metal-analyte bond: ~40 to ~200 kJ/mol. In soln., often Keq106 M-1. Raoult’s Law approx.,  sensitivity ~2 ppb vapor, as observed. Physical adsorption and H-bonding are weak: ~ 5 to 20 kJ/mol, ~ 1/10 of a metal bond.  intrinsically ~104 less sensitive.▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬ few ppb: amines, carboxylic acids, phosphines sub-ppm: thiols, aldehydes <100 ppm: alcohols, esters >100 ppm: arenes, alkenes, alkanes Origin of High Sensitivity

Difference Maps are Molecular Fingerprints : 

Difference Maps are Molecular Fingerprints Every volatile organic has a unique pattern! Suslick et al 2007

HCA of 100 VOCs: All Distinct and Identifiable : 

HCA of 100 VOCs: All Distinct and Identifiable // // // // // // // // //

Slide 41: 

Starbuck’s Columbian Maxwell House Original Decaf Starbuck’s Espresso Starbuck’s Sumatra Folgers Columbian Maxwell House Original 8 O'Clock Hazel Nut Folgers Grande Supreme Decaf 8 O'Clock Columbian Café Mai Traditional Example: 10 Arabica Coffees Easy to distinguish among different coffee brands.

Slide 42: 

Example: 18 Beers Separated by Class Zhang et al 2006

Slide 43: 

Can Colorimetric Sensor Array Detect TB Signature in Breath?

Colorimetric Array Setup for Detection of VOC Signatures In Vitro : 

Colorimetric Array Setup for Detection of VOC Signatures In Vitro Inoculum concentration: 50 million CFU for Mtb, 1 million for others.

Colorimetric Array Setup for Detection of VOC Signatures In Vitro : 

Colorimetric Array Setup for Detection of VOC Signatures In Vitro Incubated at 37C in 5% CO2 for 4 d and 20 hrs, respectively.

Slide 46: 

Representative Time Response Profiles

Slide 47: 

Species-specific decision tree classifier for 14 different bacterial species grown on blood agar plates 99% correct classification among 402 trials.

Slide 48: 

Representative time response profiles of pneumonia-causing bacteria and M. tuberculosis (6 trial per bug)

Slide 49: 

Species Separation of Pneumonia-Causing Bacteria Using a Decision Tree

Slide 50: 

Breath TB Breath Study at Highland Hospital Colorimetric Array Urine

IRB Approvals : 

Alamedia County Medical Center Approved on April 6, 2011 Stanford Approved on June 7, 2011 IRB Approvals

Slide 52: 

CSA for TB breath analysis instrument ΔR, ΔG, ΔB, ΔUVx,y,z iSense Breath Analysis Prototype Instrument (in evaluation at Cleveland Clinic for lung cancer)

Slide 53: 

iSense Breath Analysis Prototype Instrument (in in evaluation at Cleveland Clinic for lung cancer) Engineering features: - CO2 monitoring - Exhale against resistance

Slide 54: 

iSense Breath Analysis Prototype Instrument (in in evaluation at Cleveland Clinic for lung cancer) Safety features: Disposable mouth piece and tubing Biofilter mouth piece Culture monitoring (Highland study) Biofilter Disposable

Study Design : 

Study Design Inclusion criteria TB suspects in respiratory isolation for “rule out active TB” Stanford Study coordinator contacted Page (650-723-8222; ID 12217) Email generated by HIS

Study Coordinators : 

Samantha Mix Dr. Vasu Rangaswamy Study Coordinators

Slide 57: 

Breath tested Urine collected 5-7 min Study Design Study ID# Study ID#

Slide 58: 

Breath tested Urine collected 5-7 min Study Design Study ID# Study ID# Questionnaire Smoking habit Food consumed Immune status

Study Design : 

Study Design Train a classifier on an unblinded set of culture-positive TB patients and controls Validate the classifier using a blinded set of culture-positive TB patients and controls.

Translational Objectives : 

Translational Objectives Pilot Validation

Slide 61: 

Acknowledgements Stanford University Samantha Mix Vasundhara rangaswamy Indre Budvytiene Rajiv Gaur Alameda County Medical Center Herbert Schub Sang-ick Chang Alameda County Public Health Robert Benjamin iSense Paul Rhode Ray Martino Sung Lim Brian Taba Kenneth Suslick Financial Support Stanford SPARK/C-IDEA

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