Science 1663

• Los Alamos Science and Technology
  Magazine
  JANUARY 2007
• About 1663     |     Past issues

Foiling the Flu Bug

Cold, flu, or something worse? Los Alamos researchers are working on a home-use kit that would give the answer QUICKLY.

Dr. David Fox, shown with his healthy daughter, is keenly aware of how influenza can spread through simple social interactions. He is part of the Los Alamos team that is developing a rapid, home-use influenza detector.

It's a device straight out of Star Trek—a hand-held cartridge that can tell you in about an hour if you've come down with the flu. For an individual, that simple piece of information could be the key to a fast recovery. For a community, it could help contain a deadly influenza outbreak.

To Hong Cai, Xiaoyun Lu, David Fox, and their collaborators in the Bioscience Division of Los Alamos National Laboratory, early detection is the key to both fast recovery and the prevention of an epidemic. The team is developing the cartridge—a self-contained biochemistry lab about the size of a deck of cards—to be an inexpensive, portable device that can be used by nearly anyone, whether they are first responders, point-of-care health providers, or homemakers, to detect harmful viruses.

"We call it the dipstick," says Cai, "because it's as easy to use as a dipstick-style home pregnancy test. But it's a really sophisticated little detector."

Most people simply recover from influenza on their own, albeit after a few miserable days of fever, chills, and muscle aches. But influenza can be exceptionally deadly.

The World Health Organization considers influenza to be one of the foremost biothreats facing the planet. The organization's web site includes references to the influenza outbreak of 1918-1919—the "Spanish" flu. A common influenza virus (there are many influenza viruses) had mutated into a variant to which the human immune system had never been exposed. Once the virus entered the human population, the result was a pandemic, a viral blitzkrieg that killed more than 40 million people worldwide.

A similar situation exists today. The H5N1 avian influenza virus, or bird flu, is infecting and killing birds around the world. As with the Spanish flu virus, our immune system has no experience with H5N1. If the bird flu virus mutates and becomes easily transmissible between humans, the world would likely face another devastating pandemic.

The flu is easily spread by sneezing.
Photo by Andrew Davidhazy, School of Photo Arts and Sciences/RIT.

The prototype dipstick will detect not only the H5N1 influenza virus but also others that produce flu-like symptoms, such as respiratory syncytial virus (RSV), the SARS (severe acute respiratory syndrome) virus, and the common cold virus. However, its greatest advantage over current detectors is speed. Pandemics are fueled, in part, by a lack of timely information; the virus spreads before infected persons can be identified and properly quarantined. In today's highly mobile society, early detection is an imperative.

"Eventually, we hope to be able to go from sample collection to results in less than an hour, even in patients who are not fully symptomatic," says David Fox, an Agnew National Security Postdoctoral Fellow on the dipstick team. "That would give people an incredible head start for initiating response strategies."

The Laboratory is funding the project through an Exploratory Research Grant. And though the bulk of Los Alamos research is not geared towards family health care, the dipstick is aligned with the Lab's long-standing efforts to combat biothreats.

For example, the Laboratory developed methods to detect Bacillus anthracis—the bacterium that produces anthrax—and has designed and run computer simulations that predict the course of an epidemic. Los Alamos is also responsible for databases that gather and organize vast amounts of genomic information about AIDS, hepatitis C, and influenza, information that is used by researchers around the world to identify pathogens and design strategies to thwart them.

"All of this information is being compiled about pathogens," says Bioscience Division Leader Gary Resnick. "We needed a way for first responders and health-care providers to apply that information on a daily basis. The dipstick will do that."

Sensitivity and Simplicity

To be effective for home or field use, the dipstick needs to be both sensitive and specific when targeting a small number of virus particles. In Cai's mind, that requirement limited her choices to DNA-based detection methods.

Dr. Xiaoyun Lu, a member of the Los Alamos dipstick team, developed the dipstick detection method.

Broadly speaking, there are two ways to detect harmful organisms: protein-based tests or DNA-based tests. Protein-based tests are immunoassays that detect the proteins (antigens or antibodies) that signal the presence of an invading organism. The invader may be friendly (a developing baby—the home pregnancy test is an immunoassay) or unfriendly (a pathogen such as a virus or bacterium). These tests are simple and inexpensive because the proteins are floating freely in samples of urine or blood serum and can be accessed with no special preparation. However, immunoassays lack sensitivity and often fail when there is not enough protein in the sample.

DNA-based tests are very sensitive as well as very specific to targeted organisms. But unlike immunoassays, they require significant sample preparation and processing to obtain the required amount of genetic material. Still, the entire testing process, or protocol, boils down to just four steps. First, researchers isolate the organism from the rest of the sample (mucous obtained from a nasal swipe, for example), then they extract the organism's genetic material (DNA, or for most viruses, RNA). Next, they amplify, or copy repeatedly, a small region of that genetic material (a gene segment) in order to produce enough material to allow the final step, detection.

Influenza viruses. Photo courtesy of Cynthia Goldsmith.

Executing those steps has often required both sophisticated (and expensive) equipment and highly trained personnel. Those two factors relegated DNA identification to either research facilities or well-equipped clinical laboratories.

Determined to change all that, the Los Alamos dipstick team established collaborations with other scientists from the Bioscience Division to develop and/or adopt novel approaches to each of the protocol's four steps. From Laboratory scientist John Dunbar's work, the team developed a way to extract genetic material from complex samples. Jian Song and Murray Wolinsky, two bioinformatics experts, helped identify genetic similarities between viruses, which enabled the dipstick team to find gene segments that were unique to the viruses of interest. Amplifying only those segments, instead of those from other organisms, allowed them to separate the wheat from the chaff, so to speak.

Interestingly, the dipstick eliminates the polymerase chain reaction (PCR)—the biologist's standard method of amplifying gene segments—from the protocol. PCR typically relies on a "thermal cycler," a costly piece of equipment that takes the DNA through many time-consuming temperature cycles. Instead, the dipstick uses a cellular enzyme called a helicase to obtain a billion-fold amplification of gene segments. This strategy obviates the need for a thermal cycler since the helicase operates at a single temperature.

"Researchers had developed numerous isothermal amplification methods to amplify gene segments," notes Lu. "The one we chose and refined just matched up nicely with our detection scheme."

Larger view

How It Works: (1) The sample is inserted into the dipstick through the use of a cotton swab. Viruses fall from the swab and stick to protein-coated, magnetic beads, which are then collected by a magnet. The remainder of the sample is washed away. (2) The beads are resuspended in a solution that breaks the viruses open, releasing the genetic material. (3) A novel DNA-amplification scheme creates billions of copies of a targeted gene segment from, for example, virus B (orange). (4) Only those amplified segments can mate to both the label probes (red), which are attached to blue-colored marker beads, and the capture probes (green), which are secured at the virus B detection site. Thus, if a blue spot appears at that site, virus B was present in the sample.

That scheme is perhaps the most innovative part of the protocol (see part 4 of illustration). Once amplified, the gene-segment copies are mixed with blue-colored, microscopic beads that are coated with "label probes," which are short, virus-specific pieces of DNA. If one end of the gene segment matches the label probe, the two will bind and the segment will become labeled with a bead. The labeled gene segments then migrate over a detection site that is uniquely coated with millions of short "capture probes." If the free end of the gene segment matches the capture probe, then millions of labeled gene segments become anchored to the site, and a blue spot becomes visible to the eye. If the virus is not in the sample to begin with, no gene segments are amplified and no blue spot appears.

Laboratory tests confirmed that the protocol effectively detects targeted viruses and gives a positive result when as few as 100 virus particles are present.

Next, the dipstick needed to be brought out of the laboratory and transformed into an affordable device. Los Alamos is adept at that kind of technology transfer because it has an interdisciplinary approach to problem solving. The dipstick team called on Torsten Staab of the Applied Engineering Technologies Division, and he was able to engineer the world's first hand-held, disposable, DNA-based influenza detector.

Even so, team members were not satisfied to identify viruses alone. They designed the dipstick to test for bacteria as well. The only change to the protocol is in the first step, the one that isolates the organism.

Unfortunately, finding an isolation procedure for bacteria is difficult. "The problem is one of specificity," explains Lu. "Although we can isolate most viruses from the rest of the sample, we can't do the same for all types of bacteria." So the group is focusing on Bacillus anthracis, largely to address bioterrorism concerns.

The dipstick has caught the attention of an industrial manufacturer, who hopes to mass-produce the device at a projected retail cost of about $10 per dipstick. If that happens, we can all start thinking about clearing a little shelf space in our medicine cabinets.