Current methods for microbial detection: a) Labor & time intensive cultivation-based approaches that can fail to detect or characterize all cells present. b) Requires collection of samples on orbit and transportation back to ground for analysis. Disadvantages to current detection methods: a) Unable to perform quick and reliable detection on orbit. b) Lengthy sampling intervals. c) No microbe identification

Birmele, Michele

Castro, Victoria

Jones, Kathy U.

Ott, Mark C.

Oubre, Cherie M.

Roman, Monserrate C.

Vaishampayan, Parag A.

Venkateswaran, Kasthuri J.

NASA Technical Reports Server

International Conference 
on Environmen al Systems 
Partnering To Engineer the Future 
Microbial Monitoring of Common Opportunistic 
Pathogens by Comparing Multiple Real-time 
PCR Platforms for Potential Space Applications 
Monserrate C. Roman and Kathy U. Jones 
Marshall Space Flight Center 
Cherie M. Oubre, Victoria Castro, and C. Mark Ott 
Johnson Space Center 
Michele N. Birmele 
Kennedy Space Center 
Kasthuri J. Venkateswaran and Parag A. Vaishampayan 
Jet Propulsion Laboratory 
43nd ICES, 14-18 July 2013, Vail, Colorado 
https://ntrs.nasa.gov/search.jsp?R=20140002375 2019-08-29T15:03:30+00:00Z
DEDICATION 
This presentation is dedicated 
In honor of 
Angela Johnston 
Marshall Space Flight Center 
2 
History 
• Current methods adequate for monitoring & safeguarding 
short-term spaceflight missions and ISS 
• Will not be sufficient for long term spaceflight missions 
- Keep air & water free of microbes 
- Keep crew healthy 
- Be autonomous & robust for long spacecraft missions 
• 2011 Workshop at JSC reviewed cutting edge technology 
- Environmental microbiology 
- Infectious diseases/Pathogens 
- Food Safety 
3 
History 
• JSC Conference determination 
- Should replace or supplement the current practices 
• Reviewed current methods :: ·-·-·-· 
..... ~~ 
- Real-time qPCR 
- ATP bioluminescence 
- Flow cytometry 
..... 
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1 2 J' '" t • t ••nuu,.,t,.u•m»7122JS)IIa"'n'aa10~»»J<I••,••• 
-
- Matrix assisted laser desorption/ionization (TOF) 
- Microscopy 
4 
Challenges 
• Challenges ahead for long-term spaceflight 
- No COTS units to fulfill the needs 
Recommendations for Instrument or Method 
• Easy to use High throughput 
• Effects of microgravity 
• Cost 
• Phylogenetic resolution 
• Live vs Dead 
• Quantitative 
• Easy to interpret data 
• Multipurpose 
• Real time information 
• Compact 
• Short time from sample to 
answer 
• Work with multiple samples 
5 
Introduction: 
• Current methods for microbial detection 
- Labor & time intensive cultivation-based approaches 
that can fail to detect or characterize all cells present 
- Requires collection of samples on orbit and 
transportation back to ground for analysis 
• Disadvantages to current detection methods 
- Unable to perform quick and reliable detection on orbit 
- Lengthy sampling intervals 
- No microbe identification 
6 
Background: 
• Molecular-based technology 
• Polymerase Chain Reaction (PCR) for real-time quantification and 
characterization 
• Identifies specific targets or total heterotrophic growth be and the 
current capabilities aboard ISS 
• Provide rapid assessments of environment 
• High reproducibility and accuracy 
• Low detection limits on culturable & unculturable m1cro es 
• Utilize commercial off the shelf (COTS) PCR units 
• Operational under microgravity conditions 
• Meet ISS interface and safety conditions 
7 
Goals: 
• Develop a rapid microbial identification system 
- Reduce crew time & expedite operational decisions 
- Provide an in-flight identification system 
- Increase monitoring of crew health 
- Monitor air, water and surfaces for potential pathogeAs 
- Reduce or eliminate reliance on ground support 
- Provide independent system for long-term space flight 
8 
Materials and Methods: Evaluate Commercial Off the 
._____Shelf Units (COTS) -~----------------' 
• Market survey of available platforms 
• Evaluate technologies & initial proof of concept 
- Flight feasibility 
• Determine LLOD for each platform 
- Using identical cultures prepared at KSC 
• Capability to monitor ISS potable water system 
9 
Materials and Methods : Market Survey 
• Platform overview including size, weight, ease of operation 
• Number of reactions/samples that can be processed 
simultaneously 
• Reagents required for sample to answer 
• Platform and hardware components 
• Power, data, refrigeration requirements 
See ppr 
appendix B 
10 
Materials and Methods: Proof of Concept on 3 PCR-based 
.___instruments ______________ _ 
• iCubate, iCubate 2.0 system, 
Huntsville, AL- JSC 
• BioFire, RAZOR EX and Film 
Array, Salt Lake City, UT-
KSC 
• Cepheid Smartcycler, 
Sunnyvale, CA- JPL 
_/ 
11 
Materials and Methods: Attributes of PCR-based platforms 
• iCubate, 2.0 System 
- Multiplex, semi-quantitative system 
- Sample to answer 
- Self-contained cassette pre-loaded with all PCR 
reagents 
- Evaluate up to 30 microorganisms simultaneously 
- Ability to customize reactions for additional 
I 
organ1sms 
12 
Materials and Methods: RAZOR EX 
• BioFire RAZOR EX 
- Field-portable, real-time PCR unit 
- Semi-quantitative 
- Uses raw or prepared samples 
- Pouch system contains optimized freeze dried 
reagents 
- Customizable designs for additional microbes 
- Sample to answer in less than 1 hour 
13 
Materials and Methods: Film Array 
• BioFire Film Array 
- Multi-plex PCR all-·in-one integrated system 
- Windows-based instrument 
- Automated analyses 
- Freeze-dried reagent format 
- Sample to answer in less than 1-hour 
14 
Materials and Methods: Cepheid Smartcycler 
• Cepheid Smartcycler 
• Modular real-time PCR instrument 
• Barcode scanners 
• Solid-state optical system 
• Smart-tube sample processing 
• Software capable of real-time analysis 
15 
Materials & Methods: Other platforms reviewed 
• LOCAD 
- Lab-on-a-chip Application Development 
- Biomarkers for bacteria or fungi 
• WET LAB 2 - NASA Ames Research Center 
- Considered 9 platforms for in-flight 
- Smartcycler selected for deployment 
• MIDASS- European Commission & ESA 
- Microbial detection in air system for space 
- PCR based detection system for air & surfaces 
16 
Proof of Concept: LLOD Determination 
• Tested three of the PCR-based platforms 
• Single target in vendor's reagent assay kit 
- Challenge organism - Salmonella enterica (Arcc 14028) 
- Functional negative control- Pseudomonas aeruginosa 
(A TCC 700888) 
• 1 x 105 to 1 x 102 CFU/ml serial dilutions 
• LLOD determined for each platform 
• Mixed culture of both organisms 
• Varied based on LLOD 
17 
Materials and Methods: Proof of Concept Testing 
• All testing completed under identical environmental 
conditions 
- Ambient room temperature 
- Test organisms cultured at one location and shipped to 
each test site 
- DNA extracted from Salmonella at JPL, evaluated on 
Nanodrop 1000 and tested on each platform 
18 
Results: Market Survey 
Instrument iCubate 2.0 RAZOR EX Film Array Smartcycler Attribute 
No. of samples 4 X 12 12 102 16 
Volume 40 JJI 100 JJI 100 J.JI 1 ~I 
Size (in) 14 X 15 X 14 & 17 in3 25.4 x 11.4x19 10 X 15.5 X 6.5 12x12x10 
Weight (lb) 177 11 20 22 
Power Standard 24V 4A power supply & battery Standard Standard 
Reagents Pre-loaded cassettes Pre-loaded pouches Pre-loaded pouches Sealed, preloaded SmartTube 
Time to answer 6-8 h 30m 30m Labor intensive 
Sample Type Raw or DNA Raw or DNA Raw or DNA DNA only 
19 
Results: Proof of Concept 
Instrument iCubate 2.0 RAZOR EX Smartcycler 
Salmonella 
1 X 104 1 X 104 1 X 103 
LLOD 
Combined culture 
1 X 105 1 X 105 1 X 104 
LLOD 
Minimum cells needed 
400 50 94 
per reaction 
20 
Discussion & Conclusions: 
• Three platforms had capability to detect s 400 
cells Salmonella enterica 
• Two platforms considered for further testing 
- iCubate 2.0 system & -RAZOR EX 
- SmartCycler removed from future testing 
• Wetlab2 Project 
• Further requirements developed for technologies 
to be used in competitive proposal process 
21 
Further Research: Microbial Monitoring System 
• Platforms will be simultaneously analyzed 
- Quantification AND Identification abilities · 
- 20 targeted microbe populations in water samples 
- Culture independent technology 
• Quantitative & qualitative matrix developed 
- Science 
- Engineering 
- Functionality 
22 
Further Studies: Quantitative & Qualitative Matrix 
voc CCR Description Criteria (N) 
Safety: ensure safety of flight 
S: amount of potential hazards 
crew, ground personnel, public, Number of hazards 11 
flight vehicles, and environment 
produced by the system 
Pl: ability of system to accurately 
identify problematic microbes in a Number of microbes 
31 
Performance: system can identify sample when present above identified; Time to results 
target microbes within a sample detection limit 
P2: system uses molecular Number of microbes 
methods independent of culturing identified; Time to results 
16 
Operability: crew is able to 
01: ability of system to operate in 
Number of environmental 
ambient conditions both on the 8 
operate system in ambient conditions met 
conditions both on the ground 
ground and in the spacecraft 
and in the spacecraft 02: ease of use for operator 
Number of steps operator 
19 performs 
Fl: abil ity of system to function Number of functional 
26 
Functionality: system is physically with min imal resources requirements met 
capable of performing required F2: abil ity of system to store and 
functions transmit data to crew and ground 
Number of software 
13 
personnel 
requirements met 
Manufacturability: system can be M: abil ity of manufacturer to meet Number of requirements 
9 
modified for space flight requirements met 
23 
Further studies: Quantitative & Qualitative Matrix 
Milestone Task Status Outcome 
Phase 1 Define top-level Complete 1.Safety 
VOCs 2.Performance 
3.0perability 
4.Functionality 
5.Manufacturability 
Phase 2 Prioritize VOCs Complete VOCs weighted 
based on 
customer input 
(ISS Office) 
Phase 3 Define Critical Complete 8 CCRs defined and weighted 
Customer 
Requirements 
(CCRs) 
Phase 4 Data collection In-work Pending (collecting data for 133 
total criteria) 
Phase 5 Analysis using Awaiting data Data will be transformed into bins 
VOC software based on weights from MMS 
team; scores generated by Pugh 24 
Matrix method 
Acknowledgements 
The authors would like to thank 
Adesh Singhal - Marshall Space Flight Center, Huntsville, AL 
Angela Johnston - Marshall Space Flight Center, Huntsville, AL 
Tamra Ozbolt- Emerald City Initiatives, Inc., Grant, AL 
Jerry Owens - Marshall Space Flight Center, Huntsville, AL 
Daniel Jett- Teledyne Brown Engineering, Huntsville, AL 
Darrell Jan- Jet Propulsion Lab & Ames Research Center, CA 
Michael Roberts- Consolidated Safety Services, Inc, Dynamac, KSC, FL 
Gerard Newsham- ESC-Team QNA, KSC, FL 
Megan Morford - NASA, NE-Surface Systems, KSC, FL 
Janicce Care- ESC-Team QNA, KSC, FL 
Mary Hummerick- ESC-Team QNA, KSC, FL 
Airan Yoets - Enterprise Advisory Services, Inc. (Easi), JSC, Houston, Tx 
25 
Research Support Acknowledgements 
Research support was provided by 
• KSC Research & Technology Review Board CTC Grant for In-
flight Monitor. 
• Bioastronautics Contract Number NAS9-02078 (JSC). 
• Part of the research described in this study was carried out at the 
Jet Propulsion Laboratory, California Institute of Technology, 
under contract with the National Aeronautics and Space 
Administration. 
26 
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Microbial Monitoring of Common Opportunistic Pathogens by Comparing Multiple Real-time PCR Platforms for Potential Space Applications

Microbial Monitoring of Common Opportunistic Pathogens by Comparing Multiple Real-time PCR Platforms for Potential Space Applications

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