Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing
syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such
assays can play an important role in outbreak investigations and environmental screening for intentional or accidental
release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens.
The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of
massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of
hundreds of infectious agents simultaneously.
The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and
Mercy Hospital Research Laboratory located in Bo.
We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a
suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was
observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the
study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a
possible pathogen.
The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak
investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases
when different agents are suspected. This strategy enables quick and cost effective detection of low probability events
such as outbreak of a rare disease or intentional release of a biothreat agent.
A previously developed resequencing microarray, "Tropical and Emerging Infections (RPM-TEI v.1.0 chip)", designed
to identify and discriminate between tropical diseases and other potential biothreat agents, their near-neighbor species,
and/or potential confounders, was used to characterize the microbes present in the silt/clay fraction of surface soils and
airborne dust collected from the Middle East. Local populations and U.S. military personnel deployed to the Middle
East are regularly subjected to high levels of airborne desert dust containing a significant fraction of inhalable particles
and some portion require clinical aid. Not all of the clinical symptoms can be directly attributed to the physical action of
material in the human respiratory tract. To better understand the potential health effects of the airborne dust, the
composition of the microbial communities associated with surface soil and/or airborne dust (air filter) samples from 19
different sites in Iraq and Kuwait was identified using RPM-TEI v.1.0. Results indicated that several microorganisms
including a class of rapidly growing Mycobacterium, Bacillus, Brucella, Clostridium and Coxiella burnetti, were present
in the samples. The presence of these organisms in the surface soils and the inhalable fraction of airborne dust analyzed
may pose a human health risk and warrants further investigation. Better understanding of the factors influencing the
composition of these microbial communities is important to address questions related to human health and is critical to
achieving Force Health Protection for the Warfighter operating in the Middle East, Afghanistan, North Africa and other
arid regions.
A comprehensive resequencing microarray "Tropical and Emerging Infections (TessArray RPM-TEI 1.0 array)" has
been developed to identify and distinguish between biothreat organisms of interest and genetically close related species.
This array has undergone validation using an innovative approach where synthetic DNA fragments are used for
organisms that it is not safe to work with outside a biosafety 3 facilities. The approach was confirmed from testing a
subset of target organisms, such as Ebola viruses and Lassa viruses, at USAMRIID. Most potential biothreat organisms
are actually endemic in some part of the world. Proper surveillance of biothreat agents will require some form of
monitoring the evolution of the indigenous organisms under their natural environment, so when changes in the organisms
occur, the diagnostic assays for these organisms can be reviewed to assure they still provide detection. Using the
resequencing microarray (RPM) for detection in locations such as the Africa can support indigenous monitoring as it
provides sequence information. An ongoing collaboration with Njala University aims to establish a broad-spectrum
pathogen surveillance capability in the Republic of Sierra Leone, West Africa using RPM technology combined with a
Geographic Information System. This has the potential to improve the public health efforts in an infected area as well as
provide monitoring of the changes occurring to a biothreat organism, i.e. Lassa viruses, in its natural location.
The increasing demand for portable devices to detect and identify pathogens represents an
interdisciplinary effort between engineering, materials science, and molecular biology. Automation
of both sample preparation and analysis is critical for performing multiplexed analyses on real world
samples. This paper selects two possible components for such automated portable analyzers:
modified silicon structures for use in the isolation of nucleic acids and a sheath flow system suitable
for automated microflow cytometry.
Any detection platform that relies on the genetic content (RNA and DNA) present in complex
matrices requires careful extraction and isolation of the nucleic acids in order to ensure their
integrity throughout the process. This sample pre-treatment step is commonly performed using
commercially available solid phases along with various molecular biology techniques that require
multiple manual steps and dedicated laboratory space. Regardless of the detection scheme, a major
challenge in the integration of total analysis systems is the development of platforms compatible
with current isolation techniques that will ensure the same quality of nucleic acids. Silicon is an
ideal candidate for solid phase separations since it can be tailored structurally and chemically to
mimic the conditions used in the laboratory.
For analytical purposes, we have developed passive structures that can be used to fully ensheath one
flow stream with another. As opposed to traditional flow focusing methods, our sheath flow profile
is truly two dimensional, making it an ideal candidate for integration into a microfluidic flow
cytometer. Such a microflow cytometer could be used to measure targets captured on either
antibody- or DNA-coated beads.
Conference Committee Involvement (6)
Advances in Global Health through Sensing Technologies 2015
20 April 2015 | Baltimore, MD, United States
Sensing Technologies for Global Health, Military Medicine, and Environmental Monitoring IV
5 May 2014 | Baltimore, MD, United States
Sensing Technologies for Global Health, Military Medicine, and Environmental Monitoring III
29 April 2013 | Baltimore, Maryland, United States
Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II
23 April 2012 | Baltimore, Maryland, United States
Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring
25 April 2011 | Orlando, Florida, United States
Sensors, and Command, Control, Communications, and Intelligence (C3I) Technologies for Homeland Security and Homeland Defense IX
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