1. Point-of-Care Pulmonary Colonization Detection Protocol based upon an Ultra-Fast Gas Chromatograph Edward J. Staples, Ph.D., Q&A Solutions, Inc. and Chris Landon, M.D., Director of Pediatrics, Ventura County Medical Center Technology Description Screening for chemical biomarkers In-vitro Culture Results POC Analysis Summary

2. Cystic Fibrosis Cystic fibrosis is one of the most common fatal genetic disorders in the United States, affecting about 30,000 individuals. As a result of a genetic disorder, there is the presence of a thick mucus secretion which clogs the bronchial tubes in the lungs. Mucus blocks the airway passages and results in a predisposition toward chronic bacterial infections. The most common bacterium to infect the CF lung is Pseudomonas aeruginosa, a gram-negative microorganism with a propensity to live in warm, wet environments. Objective : To improve diagnosis speed and more effective antibiotics to improve lung function in CF patients

3. Approach: Odors associated with Bacteria Some bacteria have a very distinctive odor. Pseudomonas aeurginosa has a "grape-like" fruity odor. Many think that Proteus mirabilis smells like chocolate cake. sulphur-like smell may well be Salmonella typhi. Many anaerobes, bacteria that grow without oxygen, have very distinctive odors as well.

5. Point-of-Care Breath Analysis Screening for Cystic Fibrosis in Children Breath Sample Collection Chemical Biomarker Analysis with GC/SAW (zNose ® )

6. Point-of-Care Analysis using GC/SAW ( Ultra-Fast and Portable Gas Chromatography ) Portable (5 hour battery life) Disposable He =>350 runs 10-20 Second Chromatograms Universal Specificity C4-C24 Electronically Variable Sensitivity Blue-tooth connectivity Disposable He tank And battery GC/SAW Sensor Head

7. Chemical Odor Libraries Using Kovats Indices IndexOdorChemical Name 8 Seconds

8. Protocol: Breath Sample Collection aided by External Thairapy ® Vest

9. POC Analysis Protocol Pre-concentrator (Tenax Absorber) 100:1 enrichment  femto-gram sensitivity

10. Calibration (Tuning) with n-alkane standard (e.g. 125 ppm in ethanol) untuned tuned 20 Seconds Indices 400-3000 Virtual Chemical Sensors “magic perfume” …Allows electronic nose researchers to collaborate and compare their findings C 6 ….C 22

11. Zechman etal 1985 1 butanol Isopentanol Toluene 1-undecene 2-butanone 2-heptanone 2-nonanone 2-undecanone 2-aminoacetophenone Labows etal 1980 2-nonanone 2-undecanone 2-aminoacetophenone Dimethyldisulfide Dimethyltrisulfide Butanol 2-butanone 1-undecene Isopentanol Biomarkers from P. Aeruginosa

12. 2-nonanone Standard Response 10 sec sample 60 deg detector Graphic regions define virtual chemical sensor arrays Index Amount

13. Culture medium (TSB) Aroma Response mcarthy pa no 3 30 sec 20 deg det 165 valve, 200 inlet, 10ps-2a1b TSB

14. Virtual Chemical Sensor Array based upon Indexed compounds in a PA Culture Aroma TSB

15. Cultures from CF patients show repeatable PA Characteristic pattern (Grape odor) A. Duarte No. 1 C. Lazenby No. 3 A. Duarte No. 2 A. Duarte No. 3 M Parez No. 3 C. lazenby No. 1 C. Lazenby No. 2

16. Comparing direct breath sample (black) with sputum culture headspace (Patient: M. Perez) Breath Ketones Hospital room Background (citronellal) …sputum vs culture vs breath sample

17. Virtual Chemical Sensor Arrays + Pattern Recognition Amino acetophenone nonanone

18. Naphthalene 1180-1205 (db5) Methylcyclododecane 1380 (squalane) Cyclohexane, 1,4-dimethyl-, trans- 776-785 (db5) Cyclohexane, 1,4-dimethyl-, cis- 805 Heptane 700 Benzene, pentamethyl- 1290 (HP-5) Cyclohexane, 1-methyl-4-(1- methylethyl)-, trans- 980-997 (SE-30) Cyclohexane, 1-methyl-4-(1- methylethyl)-, cis- 990-1000 3-Heptanone, 5-methyl- 939-954 (dB5) 923 (HP-1) 3-Heptanone, 2-methyl- 920 (HP- 1) 3-Heptanone, 4-methyl- 3-Heptanone, 6-methyl- Biomarkers from Tuberculosis

19. Research in Progress…. TB Biomarker Virtual Sensor Array TB Virtual Sensor Array

20. Summary Ultra-fast chromatography provides a portable and sensitive POC technology for odor analysis Standards empower collaboration and comparison of researcher findings Diagnosis of Pulmonary infections such as CF and TB can be detected and quantitatively monitored using UF/GC Specific Virtual Chemical Sensor Arrays using biomarker chemicals enable more accurate recognition of pulmonary infections Breath, Sputum, and Culture Analysis can lead to early detection and diagnosis of pulmonary infections