Lawrence Livermore National Laboratory developed a novel method for transporting pathogens at ambient temperatures using amoeba. This system enhances pathogen viability and allows for the detection of various bacterial and viral pathogens without loss of virulence during transport. A prototype device for field deployment is being built to address sample contamination and improve recovery rates in clinical settings.

1. LLNL-PRES-658976
This work was performed under the auspices of the
U.S. Department of Energy by Lawrence Livermore
National Laboratory under contract DE-AC52-07NA27344.
Lawrence Livermore National Security, LLC
Sahar El-Etr, Biomedical Scientist

2.  Develop better methods for transport of
pathogens from the field to the laboratory at room
temperature without loss of viability to enable live
pathogen characterization.
 Existing transport devices are modification of a
swab with a transport medium. Storage containers
are usually cryogenic vials frozen at -80oC. Sample
clean up and enrichment currently occurs using
pathogen-specific transport/culture media.
Lawrence Livermore National Laboratory LLNL-PRES-658976
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3.  Environmental amoeba exist as trophozoites (active feeding form)
under favorable conditions and encyst (make cysts) under stress.
 Technology takes advantage of the fact that many microbial
pathogens are known to survive in amoeba cysts for many years
and to emerge after the amoeba excyst.
 Decided to make use of mother nature and developed first living
system for the transport and storage of pathogens.
Lawrence Livermore National Laboratory LLNL-PRES-658976
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Trophozoites Cysts

4. Add nutrient
rich media
cysts
transport to
lab
encystment
replication
survival
in cysts
: Pathogen of Interest
: Skin/ Environmental
Flora
excystment
trophozoites
excystment
Lab: pure pathogen
recovery and ID
Field
Inoculate
Device
Swab wound
Sample
Decontamination
and Pathogen
Enrichment in
Amoeba
Lawrence Livermore National Laboratory LLNL-PRES-658976 4

5.  We have demonstrated the ability of the system to detect as few as
10 bacterial colony forming units (CFUs) after 6-weeks of
inoculation at temperatures ranging from 4-42oC with no loss of
viability.
 We can detect Gram-negative (e.g. A. baumannii), Gram-positive
(e.g. MRSA) fastidious bacterial pathogens, select agents and
gastroenteritis causing viruses.
 Emerging microbes do not show enhanced resistance to antibiotics
or biocides and do not show mutations due to growth in amoeba.
 May not work for anaerobic bacteria or enveloped viruses.
 We are currently building a prototype device for field deployment.
Lawrence Livermore National Laboratory LLNL-PRES-658976
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6.  System can be kept at ambient temperatures with no requirement
of freezing or cold chain.
 Bacterial pathogens maintain their clinical phenotype with no loss
of virulence, since they demonstrate similar rates of survival in
mammalian cells as macrophage grown strains.
 Since Amoeba naturally feed on bacteria, samples contaminated
with patient or environmental flora are “cleaned up” during
transport.
 System enriches for the pathogen of interest.
Lawrence Livermore National Laboratory LLNL-PRES-658976
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7. Application
Description
Setting Target Customers Current Practice
#1 Sample
collection device
Field and hospital
deployment
Military Medical Service
personnel
Lawrence Livermore National Laboratory LLNL-PRES-658976
Use of traditional media-containing swabs at ambient
temperatures, major problem of sample
contamination or loss of genetic mobile elements
#2 Sample
collection device
Clinic/hospital
deployment
Civilian physicians and
nurses
Use of traditional media-containing swabs at ambient
temperatures, major problem of sample
contamination or loss of genetic mobile elements
#3 Sample cleanup
and enrichment
device
Diagnostic Military
and civilian
laboratories
Diagnostic laboratory
personnel
Attempting to culture samples and isolate pathogen
of interest
#4 Storage Device Research
Laboratories
Research Personnel Long-term storage at -80 oC. Requires freezing and
does not protect sample from contamination and loss
of virulence
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8. Kevin Christopher
christopher6@llnl.gov
Lawrence Livermore National Laboratory LLNL-PRES-658976 8