UEA Research Could Revolutionise Genomic Sequencing of Drug-Resistant Bacteria

New nanopore DNA sequencing technology on a device the size of a USB stick could be used to diagnose infection - according to new research from the University of East Anglia and Public Health England.

Researchers tested the new technology with a complex problem – determining the cause of antibiotic resistance in a new multi-drug resistant strain of the bacterium that causes Typhoid.

The results, published in the journal Nature Biotechnology, reveal that the small, accessible and cost effective technology could revolutionise genomic sequencing.

Current technology for ‘long read’ detailed genomic sequencing can be performed using expensive instrumentation (around £500,000). It is complex to perform, and generally only available in specialist laboratories.

The research team tested a new device called MinION, produced by Oxford Nanopore Technologies Ltd. The machine produces long sequencing reads using a different methodology that does not require optical imaging – but at a small fraction of the instrument cost (expected to be around £650 per device). These long reads are important when trying to determine where resistance genes are.

Researchers proved its utility by successfully mapping the multi-drug resistance genes in a strain called Salmonella typhi haplotype H58 – which has recently emerged globally.

They successfully pinpointed the exact spot in the chromosomal structure that is home to the genes which makes it drug-resistant, known as an antibiotic resistance island. The MinION took just 18 hours to produce the results, with similar accuracy to current technologies.

Lead researcher Dr Justin O’Grady, from UEA’s Norwich Medical School, said: “This type of technology will revolutionise the way that we characterise the rapid spread of emerging antibiotic-resistant infectious diseases.

“This analysis would previously have taken months using traditional methods, due to extensive post-sequencing lab-based analysis. By the time the results are available, they might well be irrelevant for clinical diagnostics and guiding public health interventions.

“This is the first published research in the world to demonstrate the huge potential of MinION sequencing to solve important and complex biological problems.

“Public health and clinical laboratories could soon have easy access to this rapid, cheap technology which, in combination with short read sequencing, is capable of providing fully assembled bacterial genomes. Further improvements to the system are likely to remove the need for short read sequence data.

“MinION technology could potentially enable bacterial identification, diagnosis of infectious diseases and detection of drug-resistance at the point of clinical need.

“This type of technology makes next generation sequencing accessible to scientists everywhere.”

The research was funded by Norwich Medical School, UEA. The international team included researchers from UEA, Public Health England, the University of Sassari in Italy, and the National Reference Centre for Salmonellae in Germany.

‘MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island’ was published in the journal Nature Biotechnology on December 8, 2014.

Source: University of East Anglia

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World's Earliest Diagnostic Test for Ebola, Within 24 Hours of Infection

Marking a critical turning point in the fight against Ebola, a new Rapid Ebola Pre-Symptom Screening Test from IES Diagnostics, Inc. can accurately identify if someone is infected with the Ebola virus as early as 24 hours after they contract the virus. This is days to weeks before they are symptomatic and contagious. No other method can test for Ebola infection this early after the virus enters the body.

At this time, the Rapid Ebola Pre-Symptom Screening Test can detect the effects of virus infection within one day of exposure. Researchers anticipate that upcoming validation tests will show this test can detect Ebola effects even earlier--within a few hours of infection, or in less time than a transcontinental airplane trip.

Using a drop of blood, the Rapid Ebola Pre-Symptom Screening Test is designed to detect the unique combination of interferons (called a signature) produced immediately after cells are infected with the Ebola virus. Interferons are the body’s “first responder” proteins, produced only when cells are infected. The Rapid Ebola Pre-Symptom Screening Test can find the body’s interferon signature to Ebola even before the virus itself can be detected.

All other Ebola tests currently used or in development can only diagnose infection when the virus has replicated to levels high enough to be directly detected--typically 8-21 days after infection. At that stage, a person may already be contagious.

Delaying an early, accurate diagnosis can lead to lengthy--and often unnecessary—quarantine periods. Conversely, it can prevent quarantines where needed. For example, the limited sensitivity of current Ebola tests may provide “false negative” results. That may lead an infected person to forego quarantine. This action may unintentionally spread this deadly disease as well as delay an infected individual’s early treatment – significantly decreasing their overall chance of survival. The Rapid Ebola Pre-Symptom Screening Test is a vital tool for detecting Ebola infection earlier, limiting the spread of infection while allowing those infected to be treated earlier.

Key scientific facts about the new Rapid Ebola Pre-Symptom Screening Test:

• Once validation tests are completed, the Rapid Ebola Pre-Symptom Screening Test can be readily implemented in the field using existing methods. IES Diagnostics’ test uses PCR (polymerase chain reaction), the same test approach currently used to detect Ebola virus RNA. Also, clinical samples are collected, stored and transported the same way those from antibody Ebola tests are.
• This pivotal interferon signature screening tool is the only test in the world that can diagnose a person’s response to Ebola infection several days before viral RNA is measurably detected.
• Tests identifying Ebola antibodies through protein detection are less sensitive than IES Diagnostics’ interferon signature testing. Antibody detection tests also are limited in that they must be used when antibody levels are robust enough for detection—days to weeks following exposure. Additionally, antibody tests are surrogate markers of exposure, and do not necessarily indicate active infection.

“Interferon response is the ideal rapid diagnostic tool since it appears immediately after cells are infected with a virus,” explains Ronald Jubin, PhD, molecular biologist and Founder/President of IES Diagnostics, Inc. “Because each response is virus-specific, we can tell if someone has Ebola based on the interferon signature that appears.”

“Being able to test people long before they are contagious is a critical turning point in the fight against Ebola,” Jubin says. “Our ultimate target is to develop a handheld device capable of producing a result within minutes of infection.”

IES Diagnostics’ interferon signature-based technology was developed collaboratively with the US Food and Drug Administration (FDA) and licensed exclusively to IES Diagnostics, Inc. by the US National Institutes of Health (NIH).

IES Diagnostics, Inc. is a molecular diagnostics company in the United States working at the forefront of companion diagnostics and personalized medicine. Its proprietary, patented assay is the first and only test capable of detecting and quantifying all of the unique interferon signatures for any disease or virus with an interferon reaction. Interferon-associated diseases include viral infections, auto-immune diseases (such as lupus, rheumatoid arthritis and multiple sclerosis, among many others) and cancers. With IES Diagnostics’ interferon signature assay, science and medicine have a sensitive, accurate tool to detect and diagnose disease and infection earlier. These signatures also provide disease-specific knowledge that can lead to targeted, effective treatment options with fewer side effects.

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WHO: Rapid, Sensitive, Safe and Simple Ebola Diagnostic Tests Urgently Needed

The goal of interrupting chains of Ebola virus transmission depends heavily on laboratory support. This support is needed to confirm or discard suspected cases, guide triage and clinical decisions, aid contact tracing, and facilitate the early detection of cases in people with an exposure history. The WHO goal of aggressive case detection and isolation likewise depends on laboratory support.

Efforts to contain the Ebola outbreaks in West Africa are currently hampered by cumbersome, slow and complex diagnostic tests that impose a number of additional logistical challenges, including requirements for a high level of laboratory biosafety and staff expertise in using sophisticated machines.

The standard molecular assays currently used in mobile and other laboratories supporting the Ebola response include the reverse-transcriptase polymerase chain reaction, or RT-PCR test. The test, which involves a number of laborious procedures, provides very accurate results when performed by trained staff. Each test requires a full tube of blood, takes from 2 to 6 hours, and costs around $100. These requirements are difficult to meet in resource-constrained West African settings, thus severely limiting testing capacity.

Lost time – for patients and treatments

The time lost transporting patient samples over bad roads to West Africa’s limited number of laboratories means that anxious patients and their families may need to wait several days for test results.

Lost time means that infected people may remain in the community, with a severe risk of unknowingly transmitting the virus to others. Moreover, in the absence of rapid laboratory support, people with other common infectious diseases, such as malaria and dengue, that have similar early symptoms may be unjustifiably held in an Ebola “transit” centre as a precautionary measure. If they did not have Ebola when entering the centre, they may unfortunately get it there.

Apart from posing a severe risk to families and communities, undiagnosed and unmanaged patients contribute to the cyclical transmission pattern currently being seen, whereby cases begin to fall as control measures take effect, only to spike again as new chains of transmission are ignited.

Perhaps most importantly, a recent research study, based on the management of more than 700 Ebola patients in Monrovia, Liberia, strongly suggests that clinical decisions guided by results from rapid point-of-care diagnostic tests could significantly improve treatment outcomes.

Moreover, having such tests readily available could restore some order to West African health systems, which have been devastated by fear of contagion as well as by the demands of managing a deadly and dreaded disease.

Apart from expediting the immediate outbreak response, rapid diagnostic tests will have enduring value in countries where many other endemic diseases mimic the early symptoms of Ebola. Having such tests in hand will also leave health services better prepared for a possible recurrence of Ebola in West Africa and elsewhere.

WHO initiatives to stimulate diagnostic innovation and deployment of suitable tests

For all these reasons, WHO has launched two urgent initiatives to stimulate diagnostic innovation and expedite the delivery of better and faster tests to West African countries – compressed into months instead of years.

The first initiative aims to minimize the barriers faced by diagnostic companies to develop and deploy their tests by clearly defining the needs, by identifying channels to access early validation materials and clinical samples for research and development, and by preparing the deployment of these new tests in the affected countries. It is moving forward in close collaboration with manufacturers, academic researchers, staff from Médecins sans Frontières (MSF), and the non-profit organization Foundation for Innovative New Diagnostics, or FIND.

The “ideal” assay

To clearly define the needs and thus guide the development of tests, WHO issued a detailed profile of the “ideal” rapid, sensitive, safe and simple diagnostic test considered most likely to accelerate interruption of virus transmission in severely resource-constrained settings.

For example, the ideal test should be suited for use in peripheral health clinics with no laboratory infrastructure in place. Testing procedures should involve fewer than 3 steps, produce results in less than 30 minutes, and have no biosafety requirements beyond the wearing of personal protective equipment.

Additional operational specifications pertain to the easy storage and reconstitution of reagents and staff training that takes less than half a day. The ideal test and related portable equipment should need no power supply and require no maintenance.

In the absence of regulatory oversight of most commercial Ebola tests, WHO is also assessing the quality of these tests. To date, no rapid tests, either on the market or under development, have undergone full regulatory assessment, underscoring the need for an independent review of these products before they are used in the field. Some tests are marketed for research use only, and not for use with patients.

A new mechanism for emergency quality assessment

The second WHO initiative comes with the establishment of an emergency quality assessment mechanism. This is a rapid review process for assessing a diagnostic’s quality, safety and performance.

In early October, an invitation was sent to manufacturers, known to be working on diagnostic tests for Ebola virus, to submit documentation setting out the evidence they have compiled on the safety, quality and performance of the test. Information to be submitted includes the recommended specimen types; evidence of test performance, including sensitivity and specificity; suppliers of critical components or raw materials and services, and data on current inventory and manufacturing capacity and quality.

Sixteen sets of documentation were received by mid-October. These include conventional RT-PCR diagnostic kits, automated “desktop” PCR systems with integrated specimen processing, and new point-of-care tests that could – within minutes – detect Ebola virus infection with blood from a finger-prick instead of a full tube. The documentation is independently assessed for WHO by expert virologists; the performance of the most promising tests will be verified through a rapid laboratory evaluation using clinical specimens.

The emergency quality assessment mechanism is currently assessing the first 5 of the 16 documentation sets submitted to date. Australian, Belgian and Dutch reviewers are participating in the evaluation, together with staff from the WHO Prequalification of In Vitro Diagnostics Programme.

The overarching objective is to guide bulk procurement decisions by WHO and other partners that will get the best tests to West Africa within the next few months.

The good news

As WHO has noted, the development of rapid and simple to use assays is technically readily achievable. Their commercial price is expected to be less costly than conventional PCR tests. Biomarkers already exist. No significant technical hurdles stand in the way.

Further progress and outstanding needs will be discussed during a WHO expert consultation on innovative Ebola diagnostic tests to be held in Geneva in December.

Source: WHO

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The Next Generation Rapid Ebola Diagnostic Tests

Responsible for almost 5000 deaths, the Ebola epidemic has ravaged West Africa and acted as a catalyst for global health initiatives. More recently, it has surfaced in the United States, prompting blaring, dramatic headlines and mounting fears of the dangerous disease. And with cases of Ebola—not to mention panic—on the rise, researchers and IVD manufacturers are scrambling for better methods of early detection and tests that yield faster results in order to slow the spread of disease.

SP-IRIS Label-Free, On-Site Rapid Diagnostic Test

A cross-functional team of researchers at Boston University (BU) has developed a prototype of a label-free, chip-scale photonics device designed to provide rapid, accurate, on-site detection of Ebola and other infectious diseases. Dubbed the single-particle interferometric reflectance imaging sensor (SP-IRIS), the device has the potential to minimize or possibly eliminate sample preparation time and to reduce processing time from two or more hours down to approximately one hour, according to an article on the university's Web site. If commercialized, the device would also offer a low-cost, relatively simple-to-use alternative to current label-based lab equipment, according to the team.

"To detect and size nanoparticles, SP-IRIS shines light from visible LED sources on nanoparticles bound to the sensor surface, which consists of a silicon-dioxide layer on top of a silicon substrate," Selim Ünlü, a BU professor of biomedical engineering, electrical and computer engineering, and materials science and engineering, told MD+DI. "Interference of light reflected from the sensor surface is modified by the presence of particles producing a distinct signal that reveals the size of the particle."

"We have successfully detected low-index dielectric particles down to 60 nm and various viruses—H1N1, VSV, Ebola—with accurate size discrimination," Ünlü added. "Single nanoparticle detection with IRIS led to virus detection as well as  protein and DNA arrays with attomolar detection sensitivity in serum and unprocessed whole blood." The developers are currently working on further reducing the time the device takes to yield results—with an aim of 30 minutes—and anticipate that a field-ready version of the device may be available in five years.

Recombinant Diagnostic Tests

Backed by a three-year, $2.9-million NIH grant, Colorado-based diagnostic test kit company Corgenix Medical Corp., in collaboration with the Viral Hemorrhagic Fever Consortium, is tackling the challenge of developing rapid diagnostic tests for Ebola. "We have developed prototype lateral flow (LFI) and ELISA diagnostic tests for detecting Ebola," Corgenix CEO Douglass Simpson told MD+DI. "Our LFI prototypes are currently in testing—clinical testing in Africa and analytical testing in controlled lab settings here in the United States."

In the face of the escalating Ebola crisis, the company has ramped up its efforts; however, it has been quick to quell speculation and misguided expectations about when a rapid, point-of-care test capable of diagnosing or ruling out Ebola will be available. "I want to strongly emphasize that this is a new three-year project, building off work commenced in 2010,” Simpson said in an August statement. “We understand the critical nature of this virus and are doing everything possible to accelerate efforts to develop reliable and rapid result diagnostic kits in order to be ready for the next outbreak.”

The company announced in August that it had entered into an agreement to be acquired by German specialty diagnostics company Orgentec Diagnostika.

Firefly Dx

Leveraging real-time polymerase chain reaction (PCR) chemistry, Florida-based PositiveID Corp.'s handheld Firefly Dx system also aims to deliver faster test results, thereby expediting diagnosis, containment, and quarantine of infected patients. "Unlike currently available technologies that are labor intensive, very expensive, and often limited to laboratory or clinical settings—thus falling short of truly effective point-of-need or timely site-specific monitoring—Firefly Dx can be used at the point of need and derive an accurate test result in less than 20 minutes," according to Allison Tomek, senior vice president, corporate development, at PositiveID. "Currently used lab tests for Ebola can take hours or even days to provide a result, and require trained personnel with the appropriate lab equipment to process."

In contrast, the Firefly system does not require any specific training to use and, in turn, can be used in remote areas where trained personnel can be scarce. The system is also not limited to blood; it can yield a diagnosis using saliva, a cheek swab, and urine, among other sample types.

The company is making progress tailoring its current platform for Ebola. "The system is currently in bench-top prototype development, but the cartridge has already demonstrated the ability to detect and identify common pathogens and diseases using DNA and RNA analysis, which is how they currently test for pathogens ranging from influenza to Ebola," Tomek said. "Firefly Dx is built to test for pathogens using any assay, and the assay currently used in labs to detect Ebola will run on Firefly. We expect to have units to test in the field in late 2015."

N-Assay Rapid Test

Last month, Ohio-based biotech company NanoLogix Inc. announced that it, too, would enter the fray by exploring how to adapt its N-Assay Rapid diagnostic kits to detect Ebola. “The company has been aware of the virus detection capabilities of the technology since the N-Assay development. Viruses viewed as public health concerns prior to Ebola and Enterovirus were Influenza, the common cold, and HIV, none of which have an immediate or near-immediate threat of mortality," NanoLogix CEO Bret Barnhizer said in an October statement. "Until recently, NanoLogix has been focused solely on the use of their N-Assay kit for bacteria detection and identification. Now, with the recent outbreaks of both Ebola and Enterovirus, there is an immediate need for configuration of the N-Assay for viruses."

The roadblock for NanoLogix, however, is that its facilities are not equipped for virus testing. In light of this barrier, the company is seeking a corporate partner that has the necessary facilities to help bring the N-Assay for Ebola to market. If the company is able to find a suitable partner, it believes that the N-Assay ELISA could yield results for Ebola in 30 minutes to several hours and could be used in both developed and remote areas.

NESDEP IU

Earlier this year, F Cubed LLC was awarded the 2014 North America Frost & Sullivan Award for Technology Innovation Leadership for its portable NESDEP IU MRSA test kit, for which it is about to start clinical trials. Now, the Indiana-based molecular diagnostics company is examining how it can extend the technology and apply the system, fueled by the company's F3 microfluidic biochip, to Ebola detection.

"We know we can test for any molecular material," Les Ivey, president and CEO of F Cubed told WNDU last month. "Ebola is an RNA virus, so it has a strand of RNA and we can detect that. The job of our NESDEP device is to extract that RNA from the virus. If it matches with the Ebola then we know we had a successful detection."

As its first step, the company is working on developing a test kit that identifies whether or not Ebola is present in objects or surfaces that a potentially infected person may have touched. However, the company hopes to then progress the test kit and navigate the more demanding regulatory hurdles involved with detecting and diagnosing Ebola in patients. The company expects that its technology could deliver test results in about 90 minutes. It's also portable—although not exactly lightweight at 50 lbs—and doesn't require highly skilled professionals to operate, according to the company. The device also carries a hefty price tag, WNDU reported, costing $50,000 while test kits range from $20 to $50 each.

Gene-RADAR

The emerging field of nanobiophysics—combining physics, nanotechnology, and biomedicine—has spawned development of the GENE-Radar device by Massachusetts-based Nanobiosym Diagnostics. Led by CEO and founder Anita Goel and featuring influential advisors such as MDEA Lifetime Achievement Award winner and MIT professor Robert Langer, Nanobiosym was established to "commercialize the Gene-RADAR technology platform to empower people worldwide with portable, rapid, and accurate information about their own health," according to the company's Web site. "Employing the latest advances in nanotechnology, Gene-RADAR is a fully portable, chip-based diagnostic that can recognize any disease with a genetic fingerprint from a single drop of blood or saliva without the need for lab infrastructure, trained health care personnel, electricity, or running water."

The technology has generated a great deal of buzz for its potential to rapidly and easily diagnose HIV and other diseases. But the company has also stated that it is investigating the possibility of adapting the platform to test for Ebola. If successful, the technology could diagnose the disease within an hour for a mere several dollars per test, according to the company.

BioThreat-E Test

While most rapid diagnostic tests for Ebola are still in development, the BioThreat-E test quickly became a technology to watch upon receiving FDA Emergency Use Authorization (EUA) for Ebola last month. Manufactured by Utah-based BioFire Diagnostics—which was acquired by bioMerieux earlier this year—the test is currently available to high and moderate complexity clinical laboratories in the United States, and can deliver results in one hour. The company's previously cleared BioFire FilmArray system, on which the fast-tracked EUA Ebola test runs, is a PCR molecular diagnostic instrument designed to identify infectious diseases.

“This EUA and FilmArray test for Ebola are the result of a long collaboration and close working relationship with U.S. Department of Defense. The FDA has been an excellent working partner, providing the feedback needed to ensure the efficacy of such an important diagnostic test,” Kirk Ririe, CEO of BioFire Defense, said in a statement last month. “It is our mission to help improve public health and the rapid turnaround and ease-of-use of FilmArray will certainly be a great help for the healthcare professionals in the context of the Ebola outbreak.”

Source: Medical Device and Diagnostic Industry Online

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Researchers Continue to Seek a Fast, Finger-Prick Ebola Test

Searching for a new way to attack Ebola, companies and academic researchers are now racing to develop faster and easier tests for determining whether someone has the disease.

Such tests might require only a few drops of blood rather than a test tube of it, and provide the answer on the spot, without having to send the sample to a laboratory.

The tests could be essential in West Africa, where it can take days for a sample to travel to one of the relatively few testing laboratories, leaving those suspected of having the disease in dangerous limbo.

Rapid tests might also be used to screen travelers at airports, providing a more definitive answer than taking their temperatures.

“There’s a great deal of interest in a technology that can screen large numbers of people from a finger prick in only a few minutes,” said Cary Gunn, chief executive of Genalyte, a company in San Diego that says its approach can do just that. “You can imagine testing an entire planeload of passengers and screening through them cost-effectively.”

The World Health Organization is encouraging development of rapid tests, as is the federal government. The Food and Drug Administration is giving emergency authorization for use of qualified Ebola tests. On Oct. 25, it gave such a clearance for a one-hour test developed by BioFire Defense, although that test requires more than a few drops of blood and is typically sent to a laboratory. “It would have taken years to get this product approved through the traditional process,” said Kirk Ririe, chief executive of the company, which is based Salt Lake City and owned by the French diagnostics firm BioMérieux.

Companies are hoping to get their tests into the field in Africa in the next few months, but it is not clear how many will be in time to make a difference in the outbreak. And some health specialists caution that while one company after another is announcing an Ebola test, there is little information about their accuracy.

“It’s a wild, wild West a little bit in development,” said Dr. Mark Perkins, chief scientific officer of FIND, or the Foundation for Innovative New Diagnostics, a nonprofit organization working with the World Health Organization to assess Ebola tests. “Eight Ebola products on the market with no one knowing how they work is not helpful.”

The need for better testing is most acute in West Africa, which has too few testing labs. Dr. Perkins said testing capacity needed to be more than doubled to about 12,000 tests a week in Guinea, Liberia and Sierra Leone and labs placed closer to where people with symptoms are coming for care.

Samples now sometimes have to be transported for hours or days over rutted roads to a laboratory. People suspected of having Ebola must wait in holding rooms until the results come back. If they do not have Ebola when they enter the room, they might have it by the time they leave.

“People are waiting four to five days,” for test results, said Dr. J. Daniel Kelly, who is working in Sierra Leone for the aid group Wellbody Alliance. “They are watching people around them die. Horrible experience. You are locked in there at night.”

Even in the United States, delays can occur, with samples being sent to the Centers for Disease Control and Prevention in Atlanta or some state public health laboratories.

It took about two days for the confirmation that Thomas Eric Duncan was infected after his second visit to a Dallas hospital.

With flu season coming, hospitals might benefit from having a quick way to rule out Ebola for certain patients as their emergency rooms fill with people with fevers and other symptoms that overlap with those of Ebola.

The standard testing technique for Ebola is known as reverse transcription polymerase chain reaction, or RT-PCR. It amplifies the genetic material of the virus, allowing even tiny quantities to be detected. It is very accurate if done correctly.

But PCR generally requires a tube of blood and is performed by trained personnel on a sophisticated machine. A test can take two to six hours or more and cost about $100.

BioFire’s FilmArray test, the one authorized for emergency use by the FDA, is a PCR test that can be done in one hour.

More than 300 hospitals already have one of BioFire’s $39,000 machines, which they have been using to test for other diseases. Now they can test for Ebola on site, though positive findings are supposed to be confirmed by the CDC.

A BioFire machine is in a special isolation ward used to treat Ebola patients at Emory University Hospital, and an instrument was delivered to Bellevue Hospital Center in New York after Dr. Craig Spencer was admitted for treatment.

Still, some say that in Africa, reducing PCR testing time by a couple of hours is not that important.

“The bottleneck is not the speed of the technology,” but the transport time and lack of testing capacity, said Rob Powell, director of research and development at Primerdesign, a British company. It is developing a PCR test using materials that do not require refrigeration, so that it can get “to more far-flung places,” he said.

What would be most valuable in Africa, some health specialists say, is a test that takes a few minutes, as the person awaits results, and costs a few dollars at most.

Various companies and organizations are working to develop such tests, including France’s Atomic Energy Commission, the Institut Pasteur, the German company Senova and the American companies Corgenix, NanoBioSym, Nanomix and others.

“We have been working since March, shoulders against the wheel, to be ready to roll these out in quantities that would make a difference in the outbreak,” said Dr. Robert Garry, a professor of microbiology at the Tulane University School of Medicine.

The test he is working on, which is being manufactured by Corgenix, is similar to a home pregnancy test. It uses antibodies that bind to one of the proteins of the Ebola virus. A finger prick blood sample is put on a test strip, which is then put into a tube containing a solution. After about 15 minutes, a line appears if the virus is present.

Because they do not amplify the viral genes as PCR does, such rapid tests are not expected to be as sensitive in detecting the virus. Using inaccurate tests would be a “perilous endeavor,” said Brian Bird, a veterinary medical officer at the CDC.

If a test falsely says someone has Ebola, that person would be put into an Ebola treatment center, where he or she might catch the disease. And missing an infection would allow a person to return to the community and possibly infect others.

Airport screening is also tricky. A fast test would presumably be more accurate, albeit slower, than thermometers, because body temperature can be raised for many reasons or suppressed by fever-relieving medicines.

But the main problem with screening is not that thermometers are inaccurate. Rather it is that people typically do not develop fever or other symptoms until six days or more after being infected. So they can pass through airport screening, as Mr. Duncan and Dr. Spencer did.

Yet health specialists say the rapid tests probably will not reliably detect the virus until the person has symptoms, because viral levels in the blood will be too low. Even the PCR test has trouble detecting infection much before symptoms appear.

Still, Dr. Garry of Tulane said even detecting the virus in people with mild symptoms might help. He said his test probably would have picked up the infection in Amber Vinson, the Dallas nurse who flew from Cleveland back to Dallas with a slight temperature and was later found to have Ebola.

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Synthetic Gene Networks Printed on Paper for Rapid and Inexpensive Detection of Ebola

In their new paper in the journal Cell, Keith Pardee, James J. Collins and colleagues from Collins’ lab at Harvard’s Wyss Institute for Biological Inspired Engineering talk about networks, printing circuits, programming, and even orthogonality.

They’re not talking about electronics, though. They’re describing how they developed “paper-based synthetic gene networks” into a practical, and potentially revolutionary, diagnostic tool for detecting a wide range of biomolecular targets such as glucose and viruses.

It took them less than a day to produce a slip of paper that can detect the Ebola virus. Armed only with that slip and smartphone camera, a healthcare worker in the field could know within two hours—and sometimes in as little as 20 minutes—whether a patient is infected or not. And the doctor, nurse, or volunteer could do this without advanced skills, extensive sample preparation, expensive reagents, laboratory instruments, or even refrigeration.

“Our paper-based system could not only make tools currently only available in laboratory readily fieldable, but also improve the development of new tools and the accessibility of these molecular tools to educational programs for the next generation of practitioners,” wrote Collins.

To produce a synthetic gene network, selected chunks of DNA, RNA, proteins, organelles (including, importantly, the ribosomes that read messenger RNA, or mRNA, and translate it into proteins), and other biomolecules are freed from their cellular casings and isolated into a complete but non-living physiological pathway.

The Wyss researchers engineered their synthetic network, painted the stew onto paper (or cloth, or any other porous medium), and freeze-dried it into an inert dot. Add water and a bit of a triggering analyte—DNA from a suspicious virus, say—and the synthetic network goes to work, activating a cascade of reactions that causes the printed dot to change color. The approach could be used for detecting not just viruses, but a staggering variety of other targets.

A “toehold hairpin RNA” sensor is a key to the process. If a single strand of RNA includes complementary sequences at separated stretches along its length, it can fold back upon itself to form a hairpin. The Wyss researchers engineer an RNA sequence so that it includes: a stretch of detector RNA that will bind to messenger RNA produced by the target (a transcript Ebola virus produces to build coat proteins for new viruses, for example); a ribosome binding site sequence, which will prompt the ribosome to grab the molecule and start reading its instructions to make protein; a “closure” sequence that binds to the detector RNA, hiding the ribosome binding site in the loop of the hairpin; and mRNA instructions for an enzyme (such as beta-galactosidase) that will alter the structure of a reporter molecule (such as a yellow form of galactose) to change its color (say, to a purple). See the following video:

The design leaves the toehold, a short strip of detector RNA, dangling free at the bottom of the hairpin. The target RNA latches onto the toehold and starts zipping up along the rest of the detector sequence—and unzipping the closure sequence. This opens up the ribosome binding site in the hairpin. The unfolded mRNA then passes through the ribosome, and the ribosome produces the enzyme. The enzyme reacts with the reporter and, voila, the color changes.

The color changes can be seen with the naked eye or digitally quantified. Conventional laboratory plate readers will certainly do the job. But along the way, the Wyss team also developed algorithms that allow most digital color cameras, including those available in cellphones, to quantify color changes in the gene-network dots.

Pardee, Collins, and their colleagues report that paper-based synthetic gene networks offer a number of advantages, including cost, speed, and rapid development, over conventional diagnostic approaches.

Cost. Paper-based diagnostics could cost as little as US $0.02 to $0.04 per sensor, they say. This is dramatically lower than the $0.45 to $1.40 for familiar antibody-based rapid diagnostics tests (RDTs) like home pregnancy and glucose kits, and the $1.50 to $4.00 cost of the reagents used in a PCR (polymerase chain reaction) DNA assay.

Speed. The paper-based synthetic gene network diagnostics the Harvard team produced are about as fast as antibody-based home tests, a little faster than PCR, and much faster than the bacterial and viral culture methods that have been a diagnostic mainstay. The Wyss group’s paper diagnostics produce detectable color changes in 20 to 40 minutes (or perhaps longer, depending on the assay and the concentration of the target molecule). Antibody-based RDTs show results in about 20 minutes. And PCR assays require at least 60 minutes…and a well-equipped laboratory.

Rapid Development. As an exercise, the Wyss researchers gave themselves a day to develop an assay capable of detecting the Ebola virus, and of distinguishing between its Sudan and Zaire strains. They produced 24 Ebola sensors in less than 12 hours.

“Taken together,” the Wyss researchers conclude, “and considering the projected cost, reaction time, ease of use, and no requirement for laboratory infrastructure, we envision paper-based synthetic gene networks significantly expanding the role of synthetic biology in the clinic, global health, industry, research, and education.”

Source: IEEE Spectrum

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Novel Biosensor Technology Could Allow Rapid Detection of Ebola Virus

In 2010, Ahmet Ali Yanik published his first paper on the rapid detection of Ebola virus using new biosensor technology he and colleagues at Boston University had invented. But he found there was little interest at the time in developing the technology further.

"People told me that there wasn't any profit in it because this disease only affects people in the developing world," Yanik said.

Now, however, Ebola hemorrhagic fever has captured the attention of first world countries in a big way. The current outbreak in West Africa began spreading out of control just as Yanik was setting up his lab as a new faculty member at UC Santa Cruz, where he is an assistant professor of electrical engineering. Yanik plans to resume his work on virus detection in addition to ongoing projects involving biosensors for other biomedical applications. The current Ebola crisis may subside before his technology can be perfected, since there are still many challenges to overcome, but the need will remain for simple and inexpensive virus detection techniques, he said.

"The truth is that Lassa virus, which is related to Ebola and also causes hemorrhagic fever, infects nearly half a million people every year in Africa and kills more people than Ebola, but it doesn't make the news. So there has been an ongoing crisis with hemorrhagic fever viruses, and now it's finally getting some serious attention," Yanik said.

His goal is to create a low-cost biosensor that can be used to detect specific viruses without the need for skilled operators or expensive equipment. "We need a platform for virus detection that is like the pregnancy tests you can use at home," Yanik said. "The initial symptoms of hemorrhagic fever are similar to the flu, and you just cannot treat every person with flu symptoms as a potential Ebola-infected patient. It needs to be simple and cheap."

Nanotechnology

Nanotechnology may provide a solution. Advances in nanotechnology have enabled researchers to fabricate novel materials with precise structures on the scale of nanometers (a nanometer is one billionth of a meter). A "plasmonic nanohole," for example, is a tiny hole a few hundred nanometers across. Yanik's 2010 paper described a biosensor based on arrays of nanoholes in a metallic surface that interact with light in predictable ways. Using antibodies on the sensor surface to bind specific viruses, the researchers showed that binding of the virus caused a detectable change in the color of light transmitted by the nanohole arrays.

Detecting the color change, however, required the use of a spectrometer. Yanik later figured out how to make a sensor that could be read with the naked eye, without any need for electronic instruments. "You can use sunlight as the light source and the human eye as the detector," he said.

He published that technique in a 2011 paper in the Proceedings of the National Academy of Sciences. The sensor technology involves realms of physics far more complex than the relatively straightforward enzymatic reactions involved in a pregnancy test. Light transmission through nanohole arrays occurs through a phenomenon known as "surface plasmon resonances," which involves the oscillations of free electrons in a metallic surface. Yanik's 2011 paper showed that light transmission could be greatly enhanced by exploiting a phenomenon called Fano resonances (named for Italian physicist Ugo Fano).

The transmission of light through plasmonic nanohole arrays changes with the binding of specific proteins or virus particles, as shown in the illustration above.

Label-free detection

"This effect causes a huge difference in light transmission that you can see with the naked eye," Yanik said. Unlike conventional laboratory tests such as PCR and ELISA (currently the standard tests for Ebola infection), Yanik's approach does not rely on labeling with fluorescent tags or other markers to see the results. "The results can be read immediately after the pathogen binds to the sensor," he said.

Further work is needed, however, to improve the sensitivity of the sensor to the point where it could be effective for virus detection in routine "point-of-care" clinical evaluations. Yanik plans to work with Jin Zhang, professor of chemistry and biochemistry at UC Santa Cruz, to develop a new version of the sensor. Zhang's research group works on advanced nanomaterials for a wide range of applications.

"He has the exact technology I need to make the biosensor more sensitive for virus detection, so we are working on a proposal to combine his approach with ours," Yanik said.

Another major focus of Yanik's research is the detection and isolation of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs can spread cancer to other parts of the body (metastasis), and detecting them in blood samples can have important prognostic and therapeutic implications. Yanik is working on methods for capturing CTCs with high efficiency and isolating them for molecular analysis. He began this work as a research associate at Harvard University Medical School and Massachusetts General Hospital before coming to UC Santa Cruz. At UCSC, he is working with Zhu Wang, an assistant professor of molecular, cell, and developmental biology, who studies genetic alterations in prostate cancer.

"It was a natural match," Yanik said. "We met during an orientation meeting for new faculty, and our collaboration spun off from there."

Both plasmonic biosensors and CTC technology are crowded fields with lots of competition, but Yanik has confidence in the methods he has developed. "There are a lot of different approaches to these problems, but we have achieved some unprecedented results with our technology," he said. "I am pretty optimistic that we can make a difference in people's lives."

Source: University of California Santa Cruz

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FDA Guidelines Restrict Use of Ebola Scanning Device at Hospitals

It took two days for the US Centers for Disease Control and the Texas Department of State Health Services to confirm that Thomas Eric Duncan, the first patient to be diagnosed with Ebola in the US during the current outbreak, had in fact tested positive for the hemorrhagic fever. For a virus that claimed the 42-year-old Liberian national's life in less than two weeks, two days might have made all the difference — and it appears that a device in Texas Health Presbyterian Hospital's arsenal could have turned around the results in less than an hour.

The device is called the FilmArray, a sleek diagnostic scanner that can identify more than a dozen different viruses and bacteria. With the right kit, these capabilities include testing for Ebola. In fact, healthcare workers at Emory University Hospital in Atlanta used the device to diagnose US aid workers Dr. Kent Brantley and Nancy Writebol after they contracted the disease in Liberia.

Another proponent of the device is the US military, which has funded the company behind the machine, BioFire, to tailor the device for testing diseases like Ebola — an investment that has proven worthwhile considering the military is utilizing the FilmArray as apart of its Ebola response efforts in West Africa.

BioFire confirmed to DefenseOne that the Dallas hospital does in fact have a FilmArray in its arsenal, begging the question as to why it wasn't used to test Duncan during his emergency room visit, instead of sending his samples to a CDC-sanctioned lab in Texas and the agency's Atlanta headquarters.

First and foremost, the hospital would have needed a special Ebola panel for the device — a panel that most hospitals probably don't have, Dr. Stephen Morse, an epidemiology professor at Columbia University, told VICE News. But perhaps the biggest obstacle is the fact that, despite military backing and high profile cases of use, the device is not technically approved by the Food and Drug Administration for diagnosing Ebola.

Currently, FilmArray is approved for diagnosing gastrointestinal and respiratory problems, but when it comes to Ebola the scanner must get special approval by the FDA, an allowance which falls under the agency's "research use only" guidelines. This means that even if the FDA gave a hospital the green light to use the Ebola panel for the FilmArray, it would have to be strictly for research, not simply for determining whether a patient has the disease.

According to Morse, once the hospital is given permission to use a device under the research guidelines, it would be up to clinicians to determine what that means — leaving a definite gray area in which testing could be done.

In the case of a potential Ebola patient, it may seem reasonable for healthcare workers to cite an emergency situation and use the diagnostic test. But Dr. Peter Jacobson, a health law and policy professor at the University of Michigan, told VICE News that using the test could open a hospital up to liability issues.

"If you're a hospital, before you use a device, you ask the FDA for a waiver because if you don't and you get it wrong, you have a liability issue," he said. "In an emergency situation, you've got to give healthcare administrators leeway, but you have to be careful and justify why it's an emergency."

Jacobson said there is a strong defense for allowing emergency use of FilmArray for Ebola diagnosis.

"Unchecked Ebola has obviously dire consequences," he said, explaining that if you can argue that something would work to prevent the spread of Ebola or cure someone, you would have a strong defense. "Particularly with this device, one argument could be that the only way to actually find out if it works is to use it during an outbreak." Jacobson noted an important caveat: FilmArray should not be used exclusively — its results should be backed up by other testing methods.

The FDA said it has been reaching out to commercial companies in order to encourage them to develop rapid diagnostic tests for Ebola, in addition to acknowledging the importance of such technologies. As Morse points out, diagnostic tests are especially important for tackling the outbreak at its source West Africa. He explained that rapid diagnostic capabilities are important on the ground in order to differentiate between Ebola and diseases with similar symptoms, especially Malaria, which is very common in the region.

Early diagnostics also help to jumpstart contact tracing efforts, ensuring that resources are not used to follow the contacts of someone who doesn't actually have the virus.

Regardless of the fervor surrounding the outbreak, the FDA sticks by its current use guidelines for FilmArray. The agency said it works to quickly decide on these cases once they receive a request.

"The FDA may not authorize the use of a diagnostic test before reviewing data about its performance in detecting Ebola virus in human specimens and determining that the standard for authorization is met. Doing so would also be irresponsible and potentially unsafe," the FDA said in a statement.

According to Jacobson, the FDA is constantly under attack for being to slow to respond or to slow to approve pharmaceutical and medical devices.

"But there's a reason why we have the FDA," he said. "FDA approval needs to be systematically thorough and robust so we don't have devices on the market that don't work and/or that cause more harm."

Jacobson said the debate brings up larger issues that the government will have to resolve going forward.

"We know that we need some type of policy to deal with this kind of outbreak," he said. "The way Ebola spreads and the fear it induces suggests we need to think differently about devices and pharmaceuticals in an outbreak."

Source: news.vice.com

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US Federal Government Sits on a Rapid Ebola Test While the Private Sector Ramps Up Its Efforts

Researchers at a government lab have developed a minimally invasive test for Ebola that could cut the time it takes to diagnose cases of the lethal virus from days and hours to minutes or even seconds, International Business Times has learned. The Department of Energy, which invented the procedure at its Oak Ridge National Laboratory in Knoxville, Tennessee, is now scrambling to find a partner to commercialize the technology.

The development comes amid fears that the Ebola virus may spread in the U.S. after the first cases appeared in Dallas in the past week. On Friday, President Obama named former vice presidential chief of staff Ron Klain as the nation’s first “Ebola czar.”

In a solicitation-for-contractors document, DOE describes its test as a “rapid, portable viral diagnostic for RNA viruses,” including, specifically, Ebola hemorrhagic fever. RNA viruses are made from genetic material comprising ribonucleic acid. In addition to Ebola, the DOE said the test can quickly detect Hanta, Dengue, West Nile and several other exotic viruses.

DOE posted its solicitation late Wednesday to a federal contracting database. A public records search showed that as of Friday one contractor had expressed interest: Healtheon Inc., of New Orleans, which manufacturers a range of diagnostic tools. Healtheon president Jasmeet Walia did not immediately respond to a request for more information.

A DOE spokesperson said the agency has been directed to refer all calls related to Ebola to the National Security Council. NSC officials did not immediately return phone calls.

In its solicitation, the DOE said rapid diagnostics “are critical elements of an effective response to viral outbreaks, but are limited by both available technology and implementation. ORNL researchers have developed a diagnostic for active, acute viral infections using a highly fieldable, and nearly reagentless system.”

A reagentless system would not require blood samples or other bodily fluids from suspected Ebola sufferers to be transported to a lab to be mixed with other chemicals.

An expert who viewed DOE patent documents on behalf of International Business Times said the technology appears to be a legitimate breakthrough. Dr. Amar Safdar, director for Transplant Infectious Diseases at New York University’s Langone Medical Center, said it could significantly reduce the time and cost of diagnosing new Ebola cases. “It’s cutting-edge,” said Safdar.

Health care facilities currently test for Ebola using a method known as polymerase chain reaction (PCR). The method requires several steps in a lab to isolate and then amplify the virus’ RNA.

DOE’s technology is “a major improvement on the existing, time-consuming PCR technology,” said Safdar, adding it could be used to conduct on-the-spot screenings at airports and other points of entry. Subjects could be asked to provide a drop of blood or nasal swab, which could then be checked using test strips that change color. “If there is a good, rapid test, then that is extremely desirable,” said Safdar.

Beyond speed, the technology could promote more widespread Ebola testing in developing nations in western Africa, where the current outbreak originated. PCR testing requires access to sophisticated and expensive lab equipment, which is not widely available in the region. Health care workers deployed to the area are in some cases shipping blood samples to Europe for testing.

Safdar cautioned, however, that rapid virus testing may not be as accurate as PCR. Rapid testing for human immunodeficiency virus (HIV), which has been available for several years, is known to deliver a very small percentage of false positives, though no false negatives. “This may be better at ruling people out than ruling them in,” said Safdar.

Positive results detected through rapid testing should be confirmed in a lab, Safdar added. He said that the DOE’s technology, as is the case with PCR testing, would only work on individuals who are symptomatic. Humans can harbor the Ebola virus for as long as three weeks before showing signs such as severe headaches, diarrhea and vomiting.

In the meantime, a number of private contractors are working on their own versions of the technology.

Colorado-based Corgenix has partnered with Tulane University to develop a rapid diagnostic kit. A production-ready version is several months in the offing, according to reports. Another vendor, Nanōmix, is also participating. It offers a device that can check for multiple infections at once. “We did do some field testing, but we need to do more,” Tulane scientist Dr. Robert Garry told The New Republic.

The U.S. military is also using commercially produced hardware in the fight to contain Ebola. Troops deployed to western Africa are screening suspected patients with a device called FilmArray, from BioFire Diagnostics of Salt Lake City. The box scans for the genetic markers of Ebola and a number of other viruses. “It will take the Ebola cells, break them open, expose the [ribonucleic acid] in the Ebola and match those with a target we’ve identified,” BioFire reps said in a statement to DefenseOne.

The New York Post reported on Thursday that startup Nanobiosym has “an iPhone-sized device” that can detect Ebola and other diseases in less than an hour. Numerous other companies are working on technologies that can be used to support Ebola containment efforts to the point at which investors are now tracking the market.

The 2014 Ebola outbreak has to date claimed about 4,500 lives, according to the Centers for Disease Control and Prevention in Atlanta. There has been one death in the U.S. Thomas Eric Duncan passed away after traveling from Liberia to a hospital in Dallas. Two nurses who treated Duncan were infected and are receiving treatment.

Source: International Business Times

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This New Ebola Test Is As Easy As a Pregnancy Test, So Why Aren’t We Using It?

The best weapon against the outbreak may be one we’re not using: Scientists at Tulane University are sitting on millions of rapid diagnostic kits capable of spotting the virus instantly.

The story of Thomas Eric Duncan’s experience with Ebola is one that has played out thousands of times in West Africa. The patient fell ill with a fever. Doctors misdiagnosed him. A vial of his blood was shipped to a high-tech laboratory to be tested. By the time virologists confirmed it was Ebola—in this case, four days later—isolation was long overdue.

It’s a sequence of events that captures one of the biggest, and often overlooked obstacles to curbing the spread of the disease in West Africa: inefficient testing.

There is only one approved, working test that can detect whether or not Ebola is present in the blood. One. The powerful technology required is slow, complicated, and requires both a laboratory and equipment. It is not that more-efficient tests do not exist; it’s that we don’t yet have the permission needed to use them.

As Ebola cases in West Africa surpassed 6,000 last week, Dr. Bob Garry, a scientist at Tulane University, is working harder than ever to get one possible solution—a rapid diagnostic test—approved. Until that happens do, testing will remain the epidemic’s secret ally.

The vial of blood taken from Duncan in Dallas was sent to the Centers for Disease Control and Prevention’s Atlanta lab, where a single test was performed. It’s the same one used by doctors in West Africa, or anywhere in the world. The test, called polymerase chain reaction (PCR), uses a method to amplify the DNA to a point where the virus can actually be detected. But the technology, while powerful, is cumbersome and takes anywhere from 12 hours to four days to yield a result.

This isn’t the best test for this epidemic, but it’s the only one.

Garry says the biggest problem with this test is in its high-tech, time-consuming method.

For example, a vial of the patient’s blood is transported to one of the few laboratories in West Africa with the suitcase-size PCR equipment needed to test for the virus. These facilities, among other more sophisticated equipment, require central electricity. In many parts of West Africa, this is hard to find. The test can take anywhere from one day to four. Factoring in travel, an answer to a sick patient’s question could be as much as a week away.

At the outset of this process, the patient is told to remain at the treatment center until a diagnosis can be made. But by the time the test results come back, the vast majority are gone. Some have fled the hospital in fear. Some have gone into hiding. Some have gone home to die. All of those who are positive have now needlessly spread the virus to countless others.

It’s the breakdown in care caused by delay that Garry and his team are hoping to fix. The scientists began working in West Africa roughly 10 years ago on another fever called Lassa. Over the course of a few years, they developed a rapid diagnostic test that allowed doctors to give patients a diagnosis on the spot. When the first cases of Ebola began popping up in West Africa, Garry and his team began “fortifying” existing labs they had in the area. In the creation of the Lassa fever test strips, they had also made a similar, but separate, Ebola test. But without any presence of Ebola in the region until this year, they were unable to test them until now.

The value of the rapid diagnostic test lies in its simplicity. It consists of a small white lancet, which requires just a small drop of blood. In 15 minutes or less, a positive or negative line will appear on the test, indicating Ebola positive or negative. "They work like pregnancy tests except its blood," says Garry.

“What our tests would permit one to do is to basically see if a person has Ebola on the spot,” Garry tells me. “They are not perhaps as sensitive as a PCR. That’s a very sophisticated test, but they don’t really have to be. What we’re most interested in doing is coming out with a test that could detect when someone is infectious, immediately.”

Taken into a clinical setting in West Africa, the test would allow physicians to immediately determine the condition of their patient, improving both the health of that individual and the safety of those around them. By immediately isolating patients that test positive, Liberia, Guinea, and Sierra Leone could prevent infected patients from going back to their village and spreading the virus further.

Dr. Alan Wu, a chemistry lab director at San Francisco General and professor at lab medicine at University of California SF, said he believes the rapid diagnostic testing is necessary in the U.S. as well. At his hospital in San Francisco, he’s received CDC training in preparation for an outbreak. But if a case comes in that he suspects is Ebola, he won’t be able to test it himself. Wu has been instructed to send the vial to the CDC’s headquarters in Atlanta—one of 12 labs in the nation (according to the CDC) capable of performing the test. “Not everyone can do molecular tests, whereas anyone can do a finger stick,” he says. “This is not going to go away. This is not a handful of patients. It’s going to have an impact on Americans as other people interact with people from that part of Africa, so we need products and we need a nimble system that will allow products to be available in advance.”

So if the test is so promising, why aren’t we already using it? “We have to prove that it works,” says Garry. “The timing is what’s holding us up.” In July at the American Association for Clinical Chemistry (AACC)’s Annual Meeting and Clinical Lab Expo, Corgenix (the company behind the rapid test) and the Viral Hemorrhagic Fever Consortium were awarded $2.9 million in grant money by the National Institutes of Health to continue work on the development of the Ebola test kit.  [See our post on the Corgenix-Tulane project for more information.]

Three months later, Garry and his team are prepped and ready to have hundreds of thousands, “even millions,” of the rapid tests ready to send to West Africa. But without 100 percent proof that it works, they’re at a standstill. With the news of an American being diagnosed with Ebola on U.S. soil, Garry hopes the process may be sped along. Not only so the test can be used in the States—but on planes.

With Garry and Corgenix’s previous rapid tests for Lassa used extensively—and successfully—in Sierra Leone and elsewhere, the evidence is even more compelling that their Ebola test will get approval. In the eyes of Wu, the test should already be out in the field, despite a lack of 100 percent proof. “If you have a positive, even if it’s a false positive, you’re erring on the side of caution. That is good.”

But not all physicians see rapid diagnostic tests as the answer. Kent Sepkowitz, an infectious disease specialist from New York (and contributor to The Daily Beast) said he thinks the technology is simply coming too late. “It will be great when the Ebola outbreak starts in 2016, or whenever the next one is. Would have nipped that in the bud.” says Sepkowitz. “But I think they need gowns and hospital rooms much more than rapid tests.”

While the rapid diagnostic test could help this epidemic, it’s by no means a longterm solution West Africa, where thousands die of other viruses each year. "We will get to a point where Ebola is not the only thing people are thinking about,” says Doug Simpson, President and CEO of Corgenix. In effort to address the larger issue, Simpson’s company has teamed up with Nanomix, a a leading nanotechnology company focused on the development of next generation Point of Care diagnostic tests. The joint venture entails Corgenix migrating the rapid diagnostic tests into a handheld device designed by Nanomix. In a matter of minutes, using multiplex detection on a drop of blood, the handheld device can distinguish between at least five different common disease ranging from Malaria to Lassa Fever. "The longterm answer is you have to be able to differentiate between the diseases,” says Simpson. “I think that’s the answer."

As the epidemic rages on, Garry is working tirelessly to get the test through the U.S.’s clunky approval process. The delay, he says, is not for lack of backing. “At the very highest level of government they know about it,” says Garry. “We’re trying to work as quickly as we can.” While he stresses that the test isn’t scientifically smarter than the existing PCR, he’s aware of its power to potentially change the tide of the epidemic.  “If you could quarantine people on the spot… it would make a big difference in trying to shut this thing down.”

The response to the Ebola epidemic in West Africa has surged in the past few weeks, delivering more money, supplies, and doctors. As the region is flooded with goods, Garry and his team are stripping the virus testing process bare. It’s not another Ebola test that the world needs, but a simpler one.

Source: The Daily Beast and rapidmicromethods.com

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Detecting Ebola with Nanotechnology

By late January, 1.4 million people in Liberia and Sierra Leone could be infected with the Ebola virus. That’s the worst-case scenario of the Ebola epidemic in West Africa recently offered by scientists at the US Centers for Disease Control and Prevention. The CDC warns that  those countries could now have 21,000 cases of the virus, which kills 70 percent of people infected.

One of the big problems hindering containment of Ebola is the cost and difficulty of diagnosing the disease when a patient is first seen. Conventional fluorescent label-based virus detection methods require expensive lab equipment, significant sample preparation, transport and processing times, and extensive training to use. One potential solution may come from researchers at the College of Engineering and the School of Medicine, who have spent the past five years advancing a rapid, label-free, chip-scale photonic device that can provide affordable, simple, and accurate on-site detection. The device could be used to diagnose Ebola and other hemorrhagic fever diseases in resource-limited countries.

The first demonstration of the concept, described in the American Chemical Society journal Nano Letters in 2010 and developed by an ENG research group led by Selim Ünlü, a professor of biomedical engineering, electrical and computer engineering, and materials science and engineering, in collaboration with Bennett Goldberg, a College of Arts & Sciences professor of physics, showed the ability to pinpoint and size single H1N1 virus particles. Now, after four years of refining the instrumentation with the collaboration of John Connor, a School of Medicine associate professor of microbiology, and other hemorrhagic fever disease researchers at the University of Texas Medical Branch, the team has demonstrated the simultaneous detection of multiple viruses in blood serum samples—including viruses genetically modified to mimic the behavior of Ebola and the Marburg virus.

Mentioned in Forbes magazine as a potentially game-changing technology for the containment of Ebola, the device identifies individual viruses based on size variations resulting from distinct genome lengths and other factors. Funded by the National Institutes of Health, the research appears in the May 2014 ACS Nano.

“Others have developed different label-free systems, but none have been nearly as successful in detecting nanoscale viral particles in complex media,” says Ünlü, who is also ENG associate dean for research and graduate programs, referring to typical biological samples that may have a mix of viruses, bacteria, and proteins. “Leveraging expertise in optical biosensors and hemorrhagic fever diseases, our collaborative research effort has produced a highly sensitive device with the potential to perform rapid diagnostics in clinical settings.”

Whereas conventional methods can require up to an hour for sample preparation and two hours or more for processing, the current BU prototype requires little to no sample preparation time and delivers answers in about an hour.

“By minimizing sample preparation and handling, our system can reduce potential exposure to health care workers,” says Connor, a researcher at the University’s National Emerging Infectious Diseases Laboratories (NEIDL). “And by looking for multiple viruses at the same time, patients can be diagnosed much more effectively.”

The shoebox-sized battery-operated prototype diagnostic device, known as the single particle interferometric reflectance imaging sensor (SP-IRIS), detects pathogens by shining light from multicolor LED sources on viral nanoparticles bound to the sensor surface by a coating of virus-specific antibodies. Interference of light reflected from the surface is modified by the presence of the particles, producing a distinct signal that reveals the size and shape of each particle. The sensor surface is very large and can capture the telltale responses of up to a million nanoparticles.

In collaboration with BD Technologies and NexGen Arrays, a start-up based at the Photonics Center and run by longtime SP-IRIS developers David Freedman (ENG’10) and postdoctoral fellow George Daaboul (ENG’13), the research team is now working on making SP-IRIS more robust, field-ready, and fast—ideally delivering answers within 30 minutes—through further technology development and preclinical trials.

SP-IRIS devices are now being tested in several labs, including a Biosafety Level-4 (BSL-4) lab at the University of Texas Medical Branch that’s equipped to work with hemorrhagic viruses. Other tests will be conducted at BU’s NEIDL once the facility is approved for BSL-4 research. Based on the team’s current rate of progress, a field-ready instrument could be ready to enter the medical marketplace in five years.

Source: BU Today

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Ph. Eur. Revises Bacterial Endotoxin Chapter - Adds Recombinant Factor C as an Alternative Method

The European Directorate for the Quality of Medicines & HealthCare  (EDQM) has recently proposed a significant revision to European Pharmacopoeia Chapter 5.1.10, Guidelines for Using the Test for Bacterial Endotoxins.

The proposed revision includes the use of alternative reagents to the Limulus amoebocyte lysate (LAL), such as recombinant factor C: this practice avoids the use of endangered animal species and can be considered in the context of the use of an alternative method as described in the General Notices.

The proposed revision includes the following additions:

• Replacement of the rabbit pyrogen test required in a pharmacopoeial monograph by an amoebocyte lysate test, or by other methods such as the monocyte-activation test or a test using recombinant factor C as a replacement for the amoebocyte lysate, constitutes the use of an alternative method of analysis and hence requires demonstration that the method is appropriate for the given substance or product and gives a result consistent with that obtained with the prescribed method as described in the General Notices.
• Replacement of a rabbit pyrogen test or a bacterial endotoxin test prescribed in a monograph by a test using recombinant factor C or any other reagent as a replacement of the amoebocyte lysate is to be regarded as the use of an alternative method in the replacement of a pharmacopoeial test, as described in the General Notices.
• The test and assays described are the official methods upon which the standards of the Pharmacopoeia are based. With the agreement of the competent authority, alternative methods of analysis may be used for control purposes, provided that the methods used enable an unequivocal decision to be made as to whether compliance with the standards of the monographs would be achieved if the official methods were used. In the event of doubt or dispute, the methods of analysis of the Pharmacopoeia are alone authoritative.

The deadline to submit comments on the proposed revision is December 31, 2014.

The revision is found in Pharmeuropa, Issue 26.4. The online version can be accessed at http://pharmeuropa.edqm.eu/home/. Access to the online version requires registration, but this is a free service.

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Presenting Our Latest Rapid Methods Webinar Series

In response to the positive feedback received from last year's rapid microbiological methods webinar series, we have developed five new seminars that will be presented in October and November.  Visit our Calendar of Events Page for agendas and registration information. Sessions will include an introductory-level webinar on applications, scientific principles and implementation, as well as advanced online courses on validation strategies, RMM technologies, global regulatory expectations and how to develop a business case and return on investment model.

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ASM Statement on National Strategy to Combat Antibiotic Resistant Bacteria

The American Society for Microbiology (ASM) congratulates the Administration for its September 18 announced White House National Strategy for Combating Antibiotic Resistant Bacteria (CARB).   The strategy outlines bold steps to slow the public health threat of antibiotic resistant bacteria, including efforts to stimulate innovative research.   Importantly, the Strategy will establish a new Task Force for Combatting Antibiotic Resistant Bacteria which is directed to submit an action plan to the President by February 2015. This elevated attention at the highest level of government is needed because in the United States alone, antibiotic resistant bacteria cause 2 million infections a year and 23,000 deaths.

Innovative research is needed to discover new, effective antibiotics and to ensure existing antibiotics are properly targeted. Research will lead to innovative diagnostics to improve detection and tracking of pathogens, new vaccines targeted to drug resistant organisms and new antibiotics in partnership with private industry. Cutting edge genetic sequencing technologies used at point of care can enhance surveillance of antimicrobial resistance, enabling rapid tracking of genetic signatures and ensure rapid, accurate diagnosis and appropriate use of antibiotics saving lives and reducing resistance resulting from inappropriate treatment.  Because an estimated half of antibiotic prescriptions are inappropriate, encouraging the development of rapid, point of care tests is critical to identifying and tailoring treatment of resistant bacteria and minimizing the use of broad spectrum antibiotics.  

The National Institutes of Health (NIH), the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA) will play leading roles in the national response. The collaborative efforts of the these agencies will be extremely important to advance development and use of rapid diagnostic tests for identifying drug resistant infections. Enhanced regulatory processes and reduction in approval cycle time will be key. Reimbursement of new diagnostic tests will also be a major incentive for development of new diagnostics by the private sector. The recommended expansion of DNA sequencing capacity and collection of microbial genetic sequences in a centralized National Database of Resistant Pathogens will allow comparison of outbreak stains with the database collection, improving their control.

The emphasis on tracking resistance in humans, animals and food and promoting antibiotic stewardship across the food chain is vitally important, as well as minimizing antibiotic use for non-health purposes. The Executive Order calls for work internationally, recognizing that efforts must be global to reduce the burden of antimicrobial resistance and its spread.

The National Strategy articulates national goals, priorities and specific objectives that provide an overarching framework for federal investments to combat antimicrobial disease. It will be extremely important that new and adequate funding is provided to accomplish this comprehensive agenda. The ASM appreciates the new initiatives and is committed to working with federal agencies and Congress as this ambitious agenda to address the threat of antimicrobial resistance gets underway.

Source: American Society for Microbiology.

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FDA Food Safety Challenge to Spur New Technologies for Fighting Foodborne Illness

The U.S. Food and Drug Administration is asking for potential breakthrough ideas on how to find disease-causing organisms in food – especially Salmonella in fresh produce.

The 2014 FDA Food Safety Challenge was developed under the America COMPETES Reauthorization Act of 2010, which grants all federal agencies broad authority to conduct prize competitions to spur innovation, solve tough problems, and advance their core missions. This challenge offers a total prize pool of $500,000.

Concepts must be able specifically to address the detection of Salmonella in minimally processed fresh produce, but the ability of a solution to address testing for other microbial pathogens and in other foods is encouraged.

Food safety experts such as scientists, academics, entrepreneurs, and innovators, as well as those new to the field, are encouraged to participate in the challenge, which launches September 23. A panel of food safety and pathogen detection experts from the FDA, the Centers for Disease Control and Prevention, and the U.S. Department of Agriculture will judge the submissions, determine finalists, and select a winner or winners.

“We are thrilled to announce the FDA’s first incentive prize competition under the America COMPETES Act,” said Michael Taylor, the FDA’s deputy commissioner for foods and veterinary medicine. “This is an exciting opportunity for the federal government to collaborate with outside experts to bring forth breakthrough ideas and technologies that can help ensure quicker detection of problems in our food supply and help prevent foodborne illnesses.”

While the American food supply is among the safest in the world, an estimated 1 in 6 Americans is sickened by foodborne illness annually, resulting in about 3,000 deaths, according to the CDC.  Salmonella is the leading cause of deaths and of hospitalizations related to foodborne illness, estimated to cause 380 deaths and 19,000 hospitalizations in the United States each year.

Those interested in participating should submit concepts to the FDA by Nov. 9, 2014. Up to five submitters will be selected to advance as finalists. Finalists will be awarded $20,000 and will have the opportunity to be coached by FDA subject matter experts who will help them mature their ideas before they present their refined concepts to the judges.

For a complete list of challenge rules and to submit a concept, visit http://www.foodsafetychallenge.com.

The FDA, an agency within the U.S. Department of Health and Human Services, protects the public health by assuring the safety, effectiveness, and security of human and veterinary drugs, vaccines and other biological products for human use, and medical devices. The agency also is responsible for the safety and security of our nation’s food supply, cosmetics, dietary supplements, products that give off electronic radiation, and for regulating tobacco products.

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RT-PCR Used to Detect MERS Coronavirus in the Air of a Saudi Arabian Camel Barn

Saudi Arabian researchers have detected genetic fragments of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in the air of a barn holding a camel infected with the virus. The work, published this week in mBio®, the online open-access journal of the American Society for Microbiology, indicates that further studies are needed to see if the disease can be transmitted through the air.

MERS, a serious viral respiratory illness caused by the coronavirus, has been identified in 699 people as of June 11, according to the World Health Organization; 209 people have died from the condition. An additional 113 cases occurring between 2012 and 2014 were reported by the Saudi Arabian Ministry of Health on June 3.

For the study, researchers on three consecutive days last November collected three air samples from a camel barn owned by a 43-year-old male MERS patient who lived south of the town of Jeddah, who later died from the condition. Four of the man's nine camels had shown signs of nasal discharge the week before the patient became ill; he had applied a topical medicine in the nose of one of the ill camels seven days before experiencing symptoms.

Using a laboratory technique called reverse transcription polymerase chain reaction (RT-PCR) to detect gene expression, they found that the first air sample, collected on November 7, contained genetic fragments of MERS-CoV. This was the same day that one of the patient's camels tested positive for the disease. The other samples did not test positive for MERS-CoV, suggesting short or intermittent shedding of the virus into the air surrounding the camels, said lead study author Esam Azhar, PhD, head of the Special Infectious Agents Unit at King Fahd Medical Research Center and associate professor of medical virology at King Abdulaziz University in Jeddah.

Additional experiments confirmed the presence of MERS-CoV-specific genetic sequences in the first air sample and found that these fragments were exactly identical to fragments detected in the camel and its sick owner.

"The clear message here is that detection of airborne MERS-CoV molecules, which were 100% identical with the viral genomic sequence detected from a camel actively shedding the virus in the same barn on the same day, warrants further investigations and measures to prevent possible airborne transmission of this deadly virus," Azhar said.

"This study also underscores the importance of obtaining a detailed clinical history with particular emphasis on any animal exposure for any MERS-CoV case, especially because recent reports suggest higher risk of MERS-CoV infections among people working with camels," he added.

Meanwhile, he said, mounting evidence for camel-to-human transmission of MERS-CoV warrants taking precautionary measures: People who care for camels or who work for slaughterhouses should wear face masks, gloves and protective clothing, and wash their hands frequently. It is also important to avoid contact with animals that are sick or have tested positive for MERS-CoV. Those who visit camel barns, farms or markets should wash hands before and after contact with animals. In addition, pasteurization of camel milk and proper cooking of camel meat are strongly recommended.

The study was supported by King Abdulaziz University.

Source: The American Society for Microbiology

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Nanopore Technique Rapidly Decodes Long DNA Strands

A low-cost technology may make it possible to read long sequences of DNA far more quickly than current techniques.

The research advances a technology, called nanopore DNA sequencing. If perfected it could someday be used to create handheld devices capable of quickly identifying DNA sequences from tissue samples and the environment, the University of Washington researchers who developed and tested the approach said.

“One reason why people are so excited about nanopore DNA sequencing is that the technology could possibly be used to create ‘tricorder’-like devices for detecting pathogens or diagnosing genetic disorders rapidly and on-the-spot,” said Andrew Laszlo, lead author and a graduate student in the laboratory of Jen Gundlach, a UW professor of physics who led the project.

The paper “Decoding long nanopore sequencing reads of natural DNA”  describes the new technique. It appears June 25 in the advanced online edition of the journal Nature Biotechnology.

Most of the current gene sequencing technologies require working with short snippets of DNA, typically 50 to 100 nucleotides long. These must be processed by large sequencers in a laboratory. The cumbersome process can take days to weeks to complete.

Nanopore technology takes advantage of the small, tunnel-like structures found in bacterial membranes. In nature, such pores allow bacteria to control the flow of nutrients across their membranes.

UW researcher used the nanopore Mycobacterium smegmatis porin A (MspA). This bacterial pore has been genetically altered so that the narrowest part of the channel has a diameter of about a nanometer, or 1 billionth of a meter. This is large enough for a single strand of DNA to pass through. The modified nanopore is then inserted into a membrane separating two salt solutions to create a channel connecting the two solutions.

To read a sequence of DNA with this system, a small voltage is applied across the membrane to make the ions of the salt solution flow through the nanopore. The ion flow creates a measurable current. If a strand of DNA is added to the solution on one side of the membrane and then enters a pore, the bulky DNA molecules will impede the flow of the much smaller ion and thereby alter the current. How much the current changes depends on which nucleotides — the individual molecules adenine, guanine, cytosine and thymine that make up the DNA chain — are inside the pore. Detecting changes in current can reveal which nucleotides are passing through the nanopore’s channel at any given instant.

Since the technique was first proposed in the 1990s, researchers hoped that nanopore DNA sequencing would offer a cheap, fast alternative to current gene sequencing. But their attempts have been frustrated by several challenges. It is difficult to identify each nucleotide one-by-one as they pass through the nanopore. Instead, researchers have to work with changes in current associated with four nucleotides at a time. In addition, some nucleotides may be missed or read more than once. Consequently, current nanopore sequencing technology yields an imprecise readout of a DNA sequence.

The UW researchers describe how they bypassed these problems. The researchers first identified the electronic signatures of all the nucleotide combinations possible with the four nucleotides that make up DNA — a total of 256 combinations in all (4 x 4 x 4 x 4).

They then created computer algorithms to match the current changes generated when a segment of DNA passes through the pore with current changes expected  from DNA sequences of known genes and genomes stored in a computer database. A match would show that the sequence of the DNA passing through the pore was identical or close to the DNA sequence stored in the database. The whole process would take minutes to a few hours, instead of weeks.

To test this approach, the researchers used their nanopore system to read the sequence of bacteriophage Phi X 174, a virus that infects bacteria and that is commonly used to evaluate new genome sequencing technologies. They found that the approach reliably read the bacteriophage’s DNA sequences and could  read sequences as long as 4,500 nucleotides.

“This is the first time anyone has shown that nanopores can be used to generate interpretable signatures corresponding to very long DNA sequences from real-world genomes,” said co-author Jay Shendure, a UW associate professor of genome sciences whose lab develops applications of genome sequencing technologies.  “It’s a major step forward.”

Because the technique relies on matching readings to databases of previously sequenced genes and genomes, it cannot yet be used to sequence a newly discovered gene or genome, the researchers said, but with some  refinements, they added, it should  be possible to improve performance in this area. To accelerate research on this new technology, the scientists are making their methods, data and computer algorithms fully available to all.

“Despite the remaining hurdles, our demonstration that a low-cost device can reliably read the sequences of naturally occurring DNA and can interpret DNA segments as long as 4,500 nucleotides in length represents a major advance in nanopore DNA sequencing,” Gundlach said.

This work was supported by the National Institutes of Health, National Human Genome Research Institutes $1,000 Genome Program Grants R01HG005115, R01HG006321 and R01HG006283  and a graduate research fellowship from the National Science Foundation DGE-0718124.

Source: University of Washington

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USP 1223 Now Available for Public Review and Comment

The USP Expert Microbiology Committee has recently published it's draft of USP 1223, Validation of Alternative Microbiological Methods.  Interested parties should visit the USP Pharmacopeial Forum (PF) website to review the draft.  The web address is http://www.usp.org/usp-nf/pharmacopeial-forum.  You are required to register to gain access to the USP PF online, but this is a free service to the pharmaceutical community. The public can provide comments on the draft chapter 1223 until September 30, 2014.  

This proposed revision provides guidance on the selection and implementation of assay methodologies to serve as alternatives to compendial microbiological methods. The revised chapter describes important steps that should be taken to evaluate candidate alternative methods, to select the analytical technology, and ultimately to qualify the method with actual product. These steps include, but are not limited to, identification of a potentially suitable alternative methodology, demonstration that the method is equivalent and applicable as a replacement for a standard compendial method, development of user specifications for equipment selection, and qualification of the method in the laboratory. In addition, this chapter outlines four distinct options for demonstrating equivalence.

A separate draft chapter, 1223.1, Validation of Alternative Methods to Antibiotic Microbial Assays, is also available for review and comment.

Microbiological assay methods are used to quantify the potency, or antimicrobial activity, of antibiotics. These microbial assays provide a direct measure of the effectiveness of the antibiotic against a reference microorganism. However, microbial assays have limited selectivity and are not appropriate for evaluating organic impurities. Also, the specific skill sets required for performing microbiological antibiotic assays, their unique equipment requirements, and their comparative complexity deter many stakeholders from using these methods. In contrast, physicochemical analytical procedures, such as high-performance liquid chromatography (HPLC), allow for simpler preparation and analysis of samples and rapid data acquisition with improved precision, accuracy, selectivity, and specificity. Alternative methods can be used effectively for both potency assignment and organic impurity testing. This proposed general chapter presents points to consider for stakeholders who wish to use physicochemical methods such as HPLC as alternatives to microbial assay methods described in general chapter Antibiotics—Microbial Assays 81.

The public can also provide comments on the draft 1223.1 until September 30, 2014. 

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