Rapid Concentration of Pathogens in Liquid Samples Presentation by A. Packingham

Transcription

1 1 Rapid Concentration of Pathogens in Liquid Samples Presentation by A. Packingham National Environmental Monitoring Conference August 2013 San Antonio, TX

2 2 InnovaPrep Overview To be covered Development of the wet foam elution process for rapid concentration of pathogens in liquid samples Environmental monitoring - testing history with 3 devices: HSC-40 Bench-top Research Concentrator HCI-40 Integratable Unit The Concentrating Pipette Capabilities and challenges Results: Enterococcus spiked ocean water samples Current R&D Pipeline

3 3 has developed a suite of sampling and sample prep equipment that rapidly collects particles / biological pathogens from Air, Surfaces and Liquids InnovaPrep and rapidly delivers the captured particles /pathogens in very small liquid volumes for rapid identification improves the capabilities of any analytical method

4 Aerosol Research Midwest Research Institute to AlburtyLab Inc. Dave Alburty CEO Andy Page President & CTO Pam Murrowchick Director of Laboratory Operations

5 AlburtyLab, Inc. Aerosol Research & Testing Aerosol Wind Tunnel Engineering & Research Laboratory Custom Aerosol Generators Microbiology Lab & BSL-2 Field Station Computational Fluid Dynamics /Flow modeling

6 R&D Motivation - Biodefense Aerosol Samplers collect into 2-15 ml samples Identifiers accept µl From 90% to 99.97% of the sample is not analyzed!

7 7 Rapid identification requires rapid sample prep! Problem Very small unconcentrated biological sample volumes = likely false negative results. The entire sample needs to be analyzed Concentration via centrifugation or enrichment + incubation are too slow and tedious Affinity capture only works for a target organism Filtration works but particle recovery from filters is difficult

8 8 R&D - recovering particles from filters Membrane Hollow Fiber Depth filters

9 Core Technology: Wet Foam Elution Volume Expansion Elution fluid expands to 6X its liquid volume Increased Viscosity The higher viscosity of foam prevents channeling and creates a more uniform flow across the filter surface. Bubble Dynamics The microbubbles in the foam behave as deformable solids. As they travel across the surface of the filter they move as a ridged body with a narrow lubricating layer; effectively squeegeeing the particles off of the surface. Exfoliating Action The turbulence and energy produced helps to lift particles that are adhering to the membrane. For rapid sample recovery 4

10 InnovaPrep Concentration Instruments HSC-40 Bench-top Research Unit 2010 HCI-40 Integratable 2012 Concentrating Pipette

11 11 HSC-40 Hollow fiber dead-end filtration wet foam elution Feed Permeate Retentate

12 12 HCI-40 Integratable The HCI-40 serves as a macro to micro interface between large sample volumes collected and the tiny volume needed for analysis

13 13 Autonomous BioAerosol Monitoring Air deposited into ~10 ml liquid Concentration Rapid ID (qpcr) Biowatch Gen 3 Department of Homeland Security project System runs autonomously for up to 30 days Detects bacteria, viruses, and toxins Provides results in as little as two hours Results reported via a secure wireless network in real time

14 14 Biological Concentration Plated sample of bacteria before and after 73x concentration with the HSC-40 and Separation Feed 6 CFU/mL Concentrate 440 CFU/mL Particle Mixture Bacterial Sized Particles Viral Sized Particles

15 Air to liquid concentration 15 validation testing Outdoor air samples collected at a rate of 60 CFM for periods of 48 to 72 hours. Particulate recovered from the filters by rinses with 0.05% Triton X- 100/TBS. The total air to liquid volume ratio was the same as that of the biodefense system. (2 hours at 400 Lpm concentrated into 10 ml

16 16 Hollow fiber concentration cell longevity testing

17 17 Concentrating Pipette Sample is contained within the disposable tip No cross contamination between samples No decontamination between samples needed Simplified user interface Canned elution fluid

18 18 Single-Use Tips Particle Size Capabilities: Tip specific current tips are 0.1 µm and 0.4 µm for bacteria, parasites, mold, spores and whole cells Speed: up to 100+ ml /min* Intake Volume: >1 Liter * Final Volume: user selectable from µL *dependent on matrix & tip type

19 19 Canned Elution Fluid Provides up to 50 elutions Does not inhibit detection Organisms remain viable 2 fluid types offered PBS recommended for classical analytical methods Tris recommended for rapid analytical methods Canned maintenance fluids for system decontamination

20 20 Concentrating Pipette video

21 Representative Results Relatively clean matrices such as drinking water ml spiked PBS samples concentrated down to 250 µl average run time 2 minutes.

22 Honey - pollen Concentrating difficult matrices Matrix Pretreatment required Vol. time 5 ml diluted to 100 ml / no prefilter 100 ml* ~ 2 min. Vegetable oil No pretreatment/ no prefilter 56 ml ~30 min. Hydrofracturing flow-back (frack fluid) Prefilter (330 µm mesh filter sleeve) 100mL ~ 3 min. Blood Pretreatment - enzymes 5 ml ~ 3 min. Urine (UTI) 3 ml diluted to 10 ml Prefilterd 10 ml* 1:30 min. Apple juice No pretreatment/ no prefilter 100 ml 19:15 min. Milk/dairy Pulverized spinach Environmental samples - high humic content Pretreatment enzymes Prefilter required Pretreatment, specially coated membranes 22 Ongoing method development

23 23 Pipette tip prefilters 330 µm mesh filter sleeve Common syringe filter with Luer connector

24 24 Ocean water samples Ocean Beach and Mission Bay samples - San Diego, CA

25 25 Testing method Sample type Mission Bay Ocean Beach Starting volume Enterococcus faecalis BioBall TM spike 100 ml each 0.9 CFU/mL 1.05 CFU/mL Pretreatment Autoclave 20 min none Pipette tip pre-filter none 10 µm syringe filter Filter tip type Control 0.4 Polycarbonate Track Etch (PCTE) PBS spike 1.2 CFU/mL # runs 3 each Analysis by plating, incubation, and enumeration

26 26 Results Mission Bay Ocean Beach PBS Time 1:11 1:10 1:04 Average final 295 µl 282 µl 189 µl concentrate volume Average % Efficiency 58.1% 63.8% 54.9% Concentration factor 203X 221X 287X Extract #2 Average % Efficiency 6.3% 7.9% 10% Average total efficiency Extract 1&2 64.4% 71.7% 65%

27 Additional runs with Tween 20 added to samples 27

28 28 Current R&D Single-use Hollow fiber packed Concentrating Pipette tips ~10X increased surface area to process larger volumes and more difficult sample matrices

29 Current R&D Sample Fractionation Cartridge (under phase 2 DHS funding) Remove environmental debris (including humics) Separates up to ~ 20 ml Sample into 5 fractions 1. Whole bacteria 2. DNA from bacteria 3. Proteins from bacteria 4. Free DNA and Viruses 5. Free Proteins Concentrates all fractions at once into separate 100 µl samples In less than 15 minutes 29

30 30 AutoElutor - for rapid concentration of cryptosporidium & giardia from large water volumes Pipeline Disposable zero-power handheld concentrator for use in austere environments

31 31 Funding Agencies

32 32 THANK YOU Ann Packingham Acknowledgments: R&D: Andy Page 1, Dave Alburty 1&2, Zach Packingham 1, Pam Murowchick 2 Water testing: Mike Hornback, PhD 1, Pam Murowchick 2, Rebecca Schmitt InnovaPrep, Drexel, MO, 2 - AlburtyLab, Inc, Drexel, MO,