Saturday, June 29, 2013

Introduction to Rapid Microbiological Methods (RMM)

The implementation of rapid methods is increasing in many areas, including pharmaceutical manufacturing and QA/QC. Additionally, global regulatory authorities, such as the FDA and EMA, have embraced their use and stated that these new technologies will improve product quality and patient safety. 

A new, comprehensive 90-minute webinar provides an introduction to the world of rapid methods and their scientific principles, applications, validation strategies, return on investment, and how to navigate the current regulatory environment. This is an excellent overview that will provide attendees with the framework for implementing rapid methods at their own facilities.

DATE: July 15, 2013.

TIME: There are five (5) identical webinar sessions to accommodate most geographic locations.


  • Review the benefits of alternative and RMM technologies as compared with classical microbiological methods.
  • Opportunities for use and areas of application including microbial detection, quantification and identification.
  • Quality enablers and current regulatory perspectives.
  • Introduction to validation strategies.
  • Overview of how to develop a business plans based on return on investment calculations.
  • Describe the scientific basis for available RMM technologies.
  • Review published and online information and guidance related to RMMs.

Tuesday, June 25, 2013

Fighting Malaria Requires New Diagnostic Tools

Dr. Sanjeev Krishna, Professor of Molecular Parasitology and Medicine at St George's, University of London, has written an interesting article on the need for developing better diagnostic tools for fighting malaria. The following is an excerpt of his paper.

Malaria hits rural dwellers in poor countries the hardest. Those bitten by the wrong mosquito often do not know for many days that they have contracted malaria. Some have little or no access to doctors. There are times when, even if the tests confirm the disease soon enough, standard treatments may not help because they may be suffering from the drug-resistant strain of the parasite. 

The World Health Organisation recommends diagnosis of malaria before treatments are begun, because if drug-resistant varieties are on the rise. Despite support from big funding agencies, such as the Global Fund to fight AIDS, Tuberculosis and Malaria, such diagnoses are not happening often enough.

With more than 200 million cases and over 660,000 deaths reported annually, the need to develop a more reliable, cheap and effective tool to detect malaria without the need for trained personnel has never been higher.

Catch ‘em Quick

Rapid diagnostic tests (RDTs) developed for malaria in recent years have made it much easier to diagnose without using the conventional method of staining blood films and then examining them under a microscope.

RDTs still suffer from limitations, though. They need trained personnel to generate reliable results. Even though these test can be performed without the need for electricity, other field conditions such as high ambient temperatures encountered during transport and storage can interfere with the quality of results.

Access to trained personnel in rural places is difficult. Could we develop a method that does not require trained personnel? A recent large-scale experiment suggests we can. In the experiment, 1000 untrained individuals across 60 countries were able to diagnose malaria as accurately as experts.

There are a number of efforts to build better diagnostic tools. For example, a group of students at Makerere University in Uganda have developed a new needleless malaria-testing application that uses a light sensor connected to a tablet device to detect the infection. This could perhaps be used to screen large groups of patients quickly before suspected infections are confirmed.

Another tool comes from researchers at Michigan State University. They have identified a test that can determine which children with malaria are likely to develop cerebral malaria, a much more life-threatening form of the disease. Only about 1% of children with malaria develop the life-threatening form of the disease, yet thousands of African children die from it each year.

One More Solution

Researchers at St. George’s University of London where I work, along with researchers in Sweden and German, have been working on the Nanomal project to build another tool that could make malaria diagnosis cheap and more effective. The handheld device can diagnose malaria on-site in less than 15 minutes.

Successful diagnosis depends on reliable and detailed results based on a patient’s blood sample. Fortunately, each parasite has specific DNA markers that differentiates them. This device consists of a nanowire (billionths of a metre thick) that changes how much electricity it conducts based on which DNA marker it comes in contact with.

The diagnosis, which can be completed in a few minutes, is made by analysing a blood sample of a patient obtained by a small prick on the finger. Parasite’s DNA is then extracted from this sample and analysed by the nanowire.

If, say, the malaria is caused by Plasmodium falciparum, then it is likely that it is a drug-resistant variety. A doctor or nurse on site can then give more personalised prescription, helping increase chances of successful treatment while also reducing the chances of drug-resistance buildup.

The device will be tested in the field this year. If successful, the price of each device is expected to be about the same as a smartphone initially. A single-test cartridge will be around £10 to begin with.

In addition to improving immediate patient outcomes, the project will allow the researchers to build a better picture of levels of drug resistance in stricken areas. It will also give them information on population impacts of antimalarial interventions.

There are other challenges such as ensuring that the device can be quickly modified to include new DNA markers as more drug-resistant parasites are selected. And the price of each device and its cartridges need to be cut further, so that those in poor countries can afford it on a large scale.

When all these tools are put together, there is great hope that they will help cut down the number of those who die from malaria every year. The sooner this happens the better.

Source: The Conversation

Palm-Sized Microarray Grows 1,200 Individual Cultures of Microorganisms

A new palm-sized microarray that holds 1,200 individual cultures of fungi or bacteria could enable faster, more efficient drug discovery, according to a study published in mBio, the online open-access journal of the American Society for Microbiology.

Scientists at the University of Texas at San Antonio and the U.S. Army Institute of Surgical Research at Fort Sam Houston have developed a microarray platform for culturing fungal biofilms, and validated one potential application of the technology to identify new drugs effective against Candida albicans biofilms. The nano-scale platform technology could one day be used for rapid drug discovery for treatment of any number of fungal or bacterial infections, according to the authors, or even as a rapid clinical test to identify antibiotic drugs that will be effective against a particular infection.

"Even though we have used the antifungal concept for development, it is a universal tool," says co-author Jose Lopez-Ribot of the University of Texas at San Antonio. "It opens a lot of possibilities as a new platform for microbial culture. Any time you need large numbers of cultures, this has a big advantage over other methods."

"The possibility exists to use this same technology for pretty much any other organism," he says.

Microbiology and medicine have become increasingly reliant on micro- and nano-scale technologies because of the increased speed and efficiency they can offer, but until now the cultivation of microorganisms has mostly been conducted on larger scales, in flasks and in trays called micro-titer plates. The microarray technology enables the user to rapidly compare hundreds or thousands of individual cultures of bacteria or fungi, a big benefit in the search for new drugs to treat infections. And like many nano-scale techniques, the nano-culture approach described in the mBio® study is also automated, a feature that saves time, improves reproducibility, and prevents some types of user error.

To test the technique, the authors embedded cells of the opportunistic pathogen C. albicans in each of the 1,200 tiny dots of alginate on the surface of the microarray. Under the microscope, these nano-biofilms of C. albicans, each of which was only 30 nanoliters, exhibited the same growth habits and other outward characteristics as conventional, macroscopic biofilms, and achieved maximum metabolic activity within 12 hours. The tiny cultures were then treated with a wide range of candidate drugs from the National Cancer Institute library, or with different FDA-approved, off-patent antifungal drugs in combination with FK506, an immunosuppressant, for identifying individual or synergistic combinations of compounds effective against biofilm infections. Co-author Anand Ramasubramanian of the University of Texas at San Antonio says that the tests prove the utility of the technology in screening combinations of drugs.

"The antifungal screening results were similar to results in larger macroscale techniques. That gives us confidence that it could be used as a tool to replace existing techniques," says Ramasubramanian.

Going forward, Ramasubramanian says he and his colleagues are testing the microarrays with polymicrobial cultures - mixtures of fungi and bacteria - to see whether the technology can be used to explore treatments for mixed infections. They are also exploring clinical applications for the technique, testing patient samples against an array of drugs or combinations of drugs to develop tailored therapies.

Lopez-Ribot says their microarray technique is just the latest development in a decades-long trend toward the tiny in science. "Things are moving toward smaller scale, more powerful techniques. You don't need millions of cells for these assays like we used to - maybe a few cells will do."


Friday, June 21, 2013

NPL Trials Identify Improved Bioaerosol Monitoring Technology

Trials conducted by the National Physical Laboratory (NPL) have identified improved methodologies for sampling and measuring bioaerosols at composting facilities. Commissioned by Defra, the first project began in 2008 and the results of a larger series of trials will be published later this summer.

As the UK seeks to reduce the quantity of waste going to landfill, there has been a growth in demand for composting, particularly to accommodate ‘green bin’ waste. In addition there has been an increase in the variety of wastes that are being composted, so it is important to be able to understand the emissions from these processes in order to minimise any impact on the environment and human health.

Micro-organisms are necessary for the composting process, so they will always be present in large quantities within the bulk material. Any handling process, such as moving, sorting or turning, is likely to create airborne dust that will contain micro-organisms, and studies have shown that exposure to the pathogenic fungus Aspergillus fumigatus can trigger asthma, bronchitis and allergic responses, so workers and residents near composting sites are potentially at risk.

Traditional bioaerosol sampling techniques rely on the impaction of particles on a solid agar medium. However, these methods can be time-consuming and are limited by low flow rates and unreliable impaction. They are also restricted to particles that can be cultivated. In contrast, the wet walled cyclonic technology employed by the Coriolis instruments, rapidly collects biological particles in liquid at a high flow rate with validated efficiency, and the liquid containing the particles is compatible with a number of rapid microbiological analysis methods, including qPCR (quantitative polymerase chain reaction), which enables the quantification and qualification of most targets.

The objective of the initial work was to improve the accuracy and speed of traditional measurement techniques, and one of the conclusions of the project was that the wet walled cyclonic technology employed by the Coriolis, gave the best performance for quantifying biological species such as fungi and bacteria, when used in conjunction with qPCR. Some of the experimental work was carried out at the Health Protection Agency (HPA) to quantify the efficiency of sampling and analysis methods for the measurement of airborne Aspergillus fumigatus spores. This work demonstrated good correlation between Coriolis/qPCR and the HPA’s ‘standard’ method for these measurements.

As a result of the initial work, NPL now offers an Aspergillus fumigatus bioaerosol monitoring service to quantify airborne spore levels at composting sites using a rapid qPCR technique. The key advantages of this monitoring service over traditional microbiological methods are: Short sampling times; Rapid analysis; High sensitivity and broad detection range; Species specific; Detects total spore count (viable and non-viable), which overcomes any issue of emission underestimation as a result of damage to the spores during collection; Aids differentiation between background spore levels and site specific emission.

A full report in the early work has now been published on the Defra website, and further studies have been commissioned. The most recent studies have involved bioaerosol sampling with the Coriolis sampler at four different sites, every quarter during 2012. NPL’s David Butterfield says “The objective of the latest trial was to assess the sampling and monitoring technologies in greater detail, under differing weather conditions and with different sources.”

At the same time, a working group at CEN, the European Committee for Standardisation, is working on a new bioaerosol monitoring standard that is likely to accommodate the latest technology and will necessitate demonstration of equivalence.

Looking forward, Jim Mills from Air Monitors (UK), the company which launched the Coriolis in the UK, says “It will take some time before this new technology becomes standard practice, but in the meantime, with the benefit of the work that has been conducted by NPL and others, there is no reason why Coriolis should not be utilised widely to improve the efficiency and effectiveness of bioaerosol sampling at composting sites, and in many other applications such as hospitals, legionella investigations, cooling towers, animal housing and pharmaceutical manufacture.”

Source: Environmental Technology Online 

Monday, June 17, 2013

Last Week to Register for the FREE Myths of Rapid Methods Webinar

Please CLICK HERE to register for this FREE webinar, which will be held on MONDAY, JUNE 24. Four (4) sessions will be held to accommodate most geographic locations. But these are filling up fast, so please register quickly to secure your preferred time slot.

The webinar will debunk the myths and misconceptions that RMMs cannot be validated, that they are too costly to implement, and that the regulatory authorities either do not understand or accept RMMs as the future solution for pharmaceutical microbiology. 

Friday, June 14, 2013

CDC Issues Guidelines For Avian Influenza H7N9 Testing, Specimen Collection And Rapid Testing

To date, there has been a total of 132 laboratory-confirmed cases of human infection with avian influenza A(H7N9) virus reported to the World Health Organization (WHO) since the outbreak began–131 cases in China and one in Taiwan.

Human infection appears to be related to exposure to live poultry or contaminated environments. However,much remains unknown about this virus, including the animal reservoir(s) in which it is circulating, the main exposures and routes of transmission, and the scope of the spread of this virus among people and animals. Investigations are ongoing.

Although there has been no cases of human infection with the virus in the United States, the Centers for Disease Control and Prevention (CDC) has provided interim guidance for clinicians and public health professionals on appropriate specimen collection, storage, processing, and testing for patients who may be infected with avian influenza A (H7N9) virus.

The CDC says since there currently is no data describing prolonged shedding of infected individuals with this virus, the estimated duration of viral shedding is based upon seasonal influenza virus infection.

Specimen collection for avian influenza A (H7N9) virus testing should be as soon as possible after illness onset, ideally within 7 days of illness onset.

The specimens of choice include a nasopharyngeal swab, or a nasal aspirate or wash, or  two swabs combined into one viral transport media vial (e.g., combined nasal swab with oropharyngeal swab or combined nasopharyngeal swab with oropharyngeal swab).

An acceptable alternative would be a single nasal swab or single oropharyngeal swab.

If the case is a lower respiratory infection, an endotracheal aspirate or bronchoalveolar lavage is preferred.

In addition, the CDC has specific requirements on types of swabs used and shipping requirements.

Concerning diagnostic testing, the CDC notes that the performance of current Food and Drug Administration (FDA) cleared diagnostic tests for influenza has been demonstrated for seasonal human influenza viruses as described by the manufacturer package insert. Performance has not been demonstrated with novel influenza A viruses.

Molecular assays may detect novel influenza A viruses, but will not differentiate novel influenza A viruses from seasonal influenza A viruses. For these assays a novel influenza A virus may give an influenza A “unsubtypable” result.

Rapid influenza diagnostic tests (RIDTs) and immunofluorescence tests also have unknown sensitivity and specificity to detect human infection with avian influenza A (H7N9) virus in clinical specimens.

The federal health agency says specimens to be tested for avian influenza A (H7N9) virus should be sent first to public health laboratories.

All state public health laboratories should use the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel to screen specimens for InfA, InfB, and RP.

Health departments should test all InfA-positive specimens with the CDC Influenza A Subtyping kit using all primer/probe sets: H1, H3, pdmInfA, and pdmH1. Avian influenza A (H7N9) viruses will be positive for the influenza A target, but negative for the seasonal influenza A (H3) target, negative for the seasonal influenza A (H1) target, negative for the pandemic 2009 (pdmH1) target, and negative for the nucleoprotein (NP) gene target (pdmInfA) using the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. Public Health officials should contact CDC immediately if they obtain unsubtypable results when testing an influenza specimen.

Thursday, June 13, 2013

Does the 5-Second Rule Apply Aboard the International Space Station?

A crew member "drops" his or her sandwich aboard the International Space Station (ISS), and it hits a surface. Quick! Grab it within five seconds or it is spoiled?

If this rule really did apply, and the sandwich was picked up few seconds too late, not much bacteria would be found, according to the latest published results of the Lab-on-a-Chip Application Development -- Portable Test System (LOCAD-PTS).

The paper, titled Rapid Culture-Independent Microbial Analysis Aboard the International Space Station (ISS) Stage Two: Quantifying Three Microbial Biomarkers, provided molecular data on the distribution of microbial molecules -- parts of microbes that are single-cell organisms -- in every livable space of the station.

"This means that the LOCAD-PTS analyzed samples collected from surfaces in every habitable module that was part of the ISS from 2007 to 2009," said Heather Morris, LOCAD-PTS scientist at NASA's Marshall Space Flight Center in Huntsville, Ala. "The molecules that it can detect are cell wall molecules of bacteria and fungi."

The handheld LOCAD-PTS device rapidly detects biological and chemical substances on surfaces aboard the station. Astronauts swab surfaces within the cabin, mix swabbed material with a liquid before adding it to the LOCAD-PTS and obtain results within 15 minutes on a display screen.

The lightweight system has three different types of cartridges for detecting endotoxin, a marker of gram-negative bacteria; glucan, a type of fungi; and lipoteichoic acid, a marker of gram-positive bacteria. The category of gram-positive bacteria includes multiple pathogens, specifically the well-known Streptococcus and Staphylococcus, making the identification of these bacteria important for crew health.

The study showed a rapid indication of biological cleanliness to help the crew monitor microorganisms in the space station environment. Likewise, this technology can provide a quick screen for bacterial/fungal contamination in a hospital or other clinical setting, particularly after cleaning, to assess the sanitization of the surface.

"The major benefits of the LOCAD-PTS are the low, relative cost and minimal crew time to operate with rapid results," said Norm Wainwright, Ph.D., the principal investigator for the system from Charles River Laboratories in Charleston, S.C. "It can be used right at the place where contamination may have occurred, only requiring a small sample, and performs the analysis in a contained environment with no growth of organisms required."

In the paper, investigators reported that most surfaces used by the station crew are relatively free of microbial molecules. However, the number of microbial molecules were elevated at sites frequently contacted by crew members, including the workout bike in the U.S. Laboratory module; the Japanese Experiment Module (JEM) airlock handle; and foot rests, drawers and the waste and hygiene compartments in the Zvezda and Tranquility modules.

The first molecular test of surfaces in the newly docked JEM were performed using the cartridges designed to detect gram-positive bacteria, including the microorganisms that can cause staph infections and strep throat. The results revealed relatively clean surfaces.

"By and large, we found that the station is a clean environment as the crew does have a stringent cleaning protocol," said Lisa Monaco, Ph.D., scientist for the LOCAD-PTS at Marshall. "Our in-orbit testing revealed some areas of hardware/software/testing modifications that need to occur, and we've begun working on all of them, but overall we're very satisfied with the results."

Simple panels on walls generally returned low readings of microbial molecules, but sometimes sleep stations would give the astronauts both high and low results.

"We wondered if that was due to the roughness of the surfaces and our swab," said Wainwright. "Sometimes it would collect more, perhaps because of the way and the angle the swab was used...[P]anels are flat, smooth surfaces, so if a molecule or a cell is on them, it's going to be a little easier to pick up. But if it's on a rough, corrugated surface, they might be buried down into something. So we'd see variations mostly with fabrics."

The crew compared results from the LOCAD-PTS with those obtained by the current standard protocol. This method requires pads of gelatinous, agar-based media called contact slides to be held flat to a surface for one to two seconds to capture any microbes. After a sample is taken, the astronaut incubates them for five days and then photographs the slides to capture any colony growth.

"While the data from LOCAD-PTS did not directly correlate to the colonies that we observed growing on the contact slides that had sampled sites close to the ones that we sampled, both tests did provide similar results some of the time," said Morris.

"And there are explanations for the differences. Microbial density varying across a surface or differences in sampling -- contact slide versus swab tool -- could account for some of the discrepancies observed."

By having cartridges that could detect all three classes of microorganisms, this study greatly expands the usefulness of the LOCAD-PTS, which had previously only been able to detect gram-negative and fungal contamination.

Currently, the technology is being used to assess fluids used in pharmaceutical processing. It also will provide environmental testing capabilities that may serve homeland security. Additionally, future iterations of the technology could provide rapid medical diagnostics in clinical applications.

The LOCAD-PTS team continues to seek out additional hardware capability that can discriminate live cells versus dead ones. New rapid microbial testing methods are needed to keep the crew and equipment healthy whether in low Earth orbit or on long-duration space missions.

Source: Space Daily

U of Penn Researchers Attach Lyme Disease Antibodies to Nanotubes

Early diagnosis is critical in treating Lyme disease. However, nearly one quarter of Lyme disease patients are initially misdiagnosed because currently available serological tests have poor sensitivity and specificity during the early stages of infection. Misdiagnosed patients may go untreated and thus progress to late-stage Lyme disease, where they face longer and more invasive treatments, as well as persistent symptoms.

Existing tests assess the presence of antibodies against bacterial proteins, which take weeks to form after the initial infection and persist after the infection is gone. Now, a nanotechnology-inspired technique developed by researchers at the University of Pennsylvania may lead to diagnostics that can detect the organism itself.

The study was led by professor A. T. Charlie Johnson of the Department of Physics and Astronomy in Penn’s School of Arts and Sciences along with graduate student Mitchell Lerner, undergraduate researcher Jennifer Dailey and postdoctoral fellow Brett R. Goldsmith, all of Physics. They collaborated with Dustin Brisson, an assistant professor of biology who provided the team with expertise on the bacterium.

Their research was published in the journal Biosensors and Bioelectronics.

“When you’re initially infected with the Lyme disease bacterium, you don’t develop antibodies for many days to a few weeks,” Johnson said. “Many people see their physician before antibodies develop, leading to negative serological test results. And after an initial infection, you’re still going to have these antibodies, so using these serological diagnostics won’t make it clear if you’re still infected or not after you’ve been treated with antibiotics.”

The research team’s idea was to flip the process around, using laboratory-produced antibodies to detect the presence of proteins from the organism.  This is an extension of previous work Johnson’s lab has done connecting other biological structures, such as olfactory receptors and DNA, to carbon nanotube-based devices.

Click on the picture (above) to enlarge the illustration of a Lyme antibody attached to a carbon nanotube.

Carbon nanotubes, rolled-up lattices of carbon atoms, are highly conductive and sensitive to electrical charge, making them promising components of nanoscale electronic devices. By attaching different biological structures to the exteriors of the nanotubes, they can function as highly specific biosensors. When the attached structure binds to a molecule, that molecule’s charge can affect the electrical conduction of the nanotube, which can be part of an electrical circuit like a wire. Such a device can therefore provide an electronic read-out of the presence, or even concentration, of a particular molecule.

To get the electrical signal out of these nanotubes, the team first turned them into transistor devices.

“We first grow these nanotubes on what amounts to a large chip using a vapor deposition method, then make electrical connections essentially at random,” Johnson said. “We then break up the chip and test all of the individual nanotube transistors to see which work the best.”

 In their recent experiment, Johnson’s team attached antibodies that naturally develop in most animals that are infected with the Lyme disease bacterium to these nanotube transistors. These antibodies naturally bind to an antigen, in this case, a protein in the Lyme bacterium, as part of the body’s immune response.

“We have a chemical process that lets us connect any protein to carbon nanotubes. Nanotubes are very stable, so we have a very reactive compound that binds to the nanotube and also has a carboxylic acid group on the other end. For biochemists, getting any kind of protein to bind to a carboxylic acid group is just child’s play at this point, and we have worked with them to learn how to perform this chemistry on the side wall of nanotubes. “

After using atomic-force microscopy to show that antibodies had indeed bound to the exteriors of their nanotube transistors, the researchers tested them electrically to get a baseline reading. They then put the nanotubes in solutions that contained different concentrations of the target Lyme bacteria protein.

“When we wash away the solution and test the nanotube transistors again, the change in what we measure tells us that how much of the antigen has bound,” Johnson said. “And we see the relationship we expect to see, in that the more antigen there was in the solution, the bigger the change in the signal.”

The smallest concentration the nanotube devices could detect was four nanograms of protein per milliliter of solution.

“This sensitivity is more than sufficient to detect the Lyme disease bacterium in the blood of recently-infected patients and may be sufficient to detect the bacterium in fluids of patients that have received inadequate treatment,” Brisson said.

“We really want the protein we are looking to detect to bind as close to the nanotube as possible, as that is what increases the strength of the electrical signal,” Johnson said. “Developing a smaller, minimal version of the antibody  — what we call a single chain variable fragment — would be a next step.

“Based on our previous work with single chain variable fragments of other antibodies, this would probably make such a device about a thousand times more sensitive.”

The researchers suggested that, given the flexibility of their technique for attaching different biological structure, eventual diagnostic tools could incorporate multiple antibodies, each detecting a different protein from the Lyme bacterium. Such a setup would improve accuracy and cut down on the possibility of false-positive diagnoses.

“If we were to do this type of test on a person’s blood now, however, we would say the person has the disease,” Johnson said. “The first thought is that if you detect any protein coming from the Lyme organism in your blood, you are infected and should get treatment right away.”

This research was supported by the Department of Defense U.S. Army Medical Research and Materiel Command, the National Institutes of Health, Penn’s Nano/Bio Interface Center, the National Science Foundation and Penn’s Laboratory for Research on the Structure of Matter.

Source: University of Pennsylvania News

Monday, June 10, 2013

FREE Rapid Methods Webinar: Myths and Misconceptions

Although the momentum for implementing rapid microbiological methods (RMMs) has improved over the last few years, the industry still remains hesitant in validating and routinely using RMMs. Fortunately, a web seminar has been developed that will debunk the myths and misconceptions that RMMs cannot be validated, that they are too costly to implement, and that the regulatory authorities either do not understand or accept RMMs as the future solution for pharmaceutical microbiology. 

Taught by one of the industry's leading experts on rapid micro methods, this FREE webinar will immerse the participants in a thought-provoking discussion on how to get past the myths and successfully implement RMMs. Following the webinar, attendees will be able to ask questions about the presentation or any other topic associated with the implementation of RMMs.

The webinar will take place on MONDAY, JUNE 24, 2013, and is being offered FOUR different times to accommodate all geographic regions. Each session is LIMITED TO 100 attendees, so please register soon, as it is expected that all sessions will fill up very quickly. 

CLICK HERE or type in to register for this FREE web seminar. 

Friday, June 7, 2013

New Single Virus Detection Techniques for Faster Disease Diagnosis

To test the severity of a viral infection, clinicians try to gauge how many viruses are packed into a certain volume of blood or other bodily fluid. This measurement, called viral load, helps doctors diagnose or monitor chronic viral diseases such as HIV/AIDS and hepatitis. However, the standard methods used for these tests are only able to estimate the number of viruses in a given volume of fluid. 

Now two independent teams have developed new optics-based methods for determining the exact viral load of a sample by counting individual virus particles. These new methods are faster and cheaper than standard tests and they offer the potential to conduct the measurements in a medical office or hospital instead of a laboratory. The teams will present their latest results at the Conference on Lasers and Electro-Optics (CLEO: 2013), to be held June 9-14, in San Jose, Calif. 

One research group, led by electrical engineer and bioengineer Aydogan Ozcan of UCLA, is working to directly image single virus particles using holographic microscopy. The other, led by electrical engineer Holger Schmidt of the University of California, Santa Cruz (UCSC), is detecting single particles tagged with fluorescent labels on a microfluidic chip. Both teams expect to use their work to develop commercial instruments useful for on-site diagnosis and monitoring with rapid results and fast turnaround. 

Ozcan’s UCLA team has demonstrated the ability to capture optical images of single viruses and nanoparticles over a comparatively large field of view – about the size of a postage stamp – using nanolenses that self-assemble around the virus particles like little magnifying glasses. 

“Because viruses are very small--less than 100 billionths of a meter--compared to the wavelength of light, conventional light microscopy has difficulty producing an image due to weak scattering of sub-wavelength particles,” Ozcan says. When lighted, the team’s new nanolens-nanoparticle assembly projects a hologram that can be recorded using a CMOS imager chip (a type of semiconductor-based light detector) and digitally reconstructed to form an optical image of the particle. “The resulting image improves the field-of-view of a conventional optical microscope by two orders of magnitude,” says Ozcan. 

This wide field of view allows the device to form images of many nanoparticles in a single photograph and provides a high-throughput platform for a direct and accurate viral load count. The instrument can be made sufficiently compact and lightweight for field applications and, attached to a cell phone, could become useful even in remote locations. 

The UCSC researchers will present the results of a collaborative effort between UCSC, Liquilume Diagnostics Inc., and the groups of infectious disease clinician and virologist Charles Chiu at University of California, San Francisco, and engineer Aaron Hawkins at Brigham Young. While Ozcan's group visually counts individual viruses, Schmidt’s counts them by detecting their nucleic acids--the genetic makeup of the viruses. The nucleic acids are labeled with a fluorescent dye, and light from the fluorescence is detected as they pass through a channel in a microfluidic chip about the size of a thumbnail. 

Current tests for determining viral load generally rely on a technique called polymerase chain reaction (PCR), which amplifies a small sample of nucleic acid, such as DNA, and makes it easier to detect. “The gold standard for viral load detection is PCR, due to its sensitivity and specificity,” Schmidt says, but PCR is limited to merely estimating the number of viruses. In contrast, the new method counts real particles as they pass through the fluorescence detector on the chip. “We have demonstrated actual virus counts of specific nucleic acids in less than 30 minutes with minimal sample workup,” Schmidt says. So far, the group has collected reliable data on samples diluted to a point well within the range required for clinical detection. 

Unlike direct visualization techniques, Schmidt's chip-based method requires that the targeted virus particles be labeled. The labeling technique would allow clinicians to target specific viruses while ignoring unlabeled background material. This makes the process potentially useful in situations where clinicians already know what they are looking for – often the case for viral load tests. 

The chip is currently housed in an instrument about one foot square, making the device portable. Along with rapid analysis turnaround, this portability should make the technique useful for point-of-treatment tests. In addition to detecting viruses, the device may also find uses as a sensor for cancer biomarkers, for environmental analyses of chemicals, and even in industrial production monitoring. 

CLEO: 2013 presentation AW1I.6. “High-throughput Imaging of Single Viruses using Self-assembled Nano-lenses and On-Chip Holography” by Aydogan Ozcan will take place Wednesday, June 12 at 12:15 p.m. in the San Jose Convention Center. 

CLEO: 2013 presentation CM1M.7. “Clinical Detection of Viral Infection on an Optofluidic Chip” by Philip Measor will take place Monday, June 10 at 9:45 a.m. in the San Jose Convention Center. 

Source: ScienceSelect

Thursday, June 6, 2013

JHU Postdoc Recognized for Work on Microfluidics and Bacterial DNA Identification

A Johns Hopkins research fellow who is developing novel approaches to quickly identify bacterial DNA and human microRNA has won the prestigious $500,000 Burroughs Wellcome Fund (BWF) Career Award at the Scientific Interfaces. The prize, distributed over the next five years, helps transition newly minted PhDs from postdoctoral work into their first faculty positions.

Stephanie Fraley is a postdoctoral fellow working with Samuel Yang, MD, in Emergency Medicine/Infectious Disease at the Johns Hopkins School of Medicine and Jeff Wang, PhD, in Biomedical Engineering with appointments in the Whiting School of Engineering and the medical school. The goal of her work is to develop engineering technologies that can diagnose and guide treatment of sepsis, a leading cause of death worldwide, while simultaneously leading to improved understanding of how human cells and bacterial cells interact.

“Sepsis is an out of control immune response to infection,” Fraley said. “We are developing tools that are single molecule sensitive and can rapidly sort and detect bacterial and host response markers associated with sepsis. However, our devices are universal in that they can be applied to many other diseases.”

Fraley is using lab-on-chip technology, also known as microfluidics, to overcome the challenges of identifying the specific genetic material of bacteria and immune cells. Her technology aims to sort the genetic material down to the level of individual sequences so that each can be quantified with single molecule sensitivity.

“Bacterial DNA is on everything and contamination is everywhere, so trying to find the ones associated with sepsis is like the proverbial search for the needle in the haystack,” Fraley said. “With microfluidics, we can separate out all the bacterial DNA, so instead of a needle in a haystack, we have just the needles.”

Another advantage to Fraley’s novel technology is that it will assess all the diverse bacterial DNA present in a sample, without presuming which genetic material is important. “Bacteria are constantly evolving and becoming drug resistant,” she said. “With this technology, we can see all the bacterial DNA that is present individually and not just the strains we THINK we need to look for.”

Fraley’s award will follow her wherever her career takes her. The first two years of the prize fund postdoctoral training and that last three years help launch her professional career in academia. During the application process, she had to make a short presentation on her proposal to BWF’s panel of experts. “It was like the television show ‘Shark Tank’ but for scientists,” she laughs. “ The panelists gave me many helpful suggestions on my idea.”

Fraley earned her bachelor’s degree in chemical engineering from the University of Tennessee at Chattanooga and her doctorate in chemical and biomolecular engineering with Denis Wirtz, professor and director of Johns Hopkins Physical Sciences-Oncology Center. Wirtz is associate director for the Institute for NanoBioTechnology and Yang and Wang also are INBT affiliated faculty members.

BWF’s Career Awards at the Scientific Interface provides funding to bridge advanced postdoctoral training and the first three years of faculty service. These awards are intended to foster the early career development of researchers who have transitioned or are transitioning from undergraduate and/or graduate work in the physical/mathematical/computational sciences or engineering into postdoctoral work in the biological sciences, and who are dedicated to pursuing a career in academic research. These awards are open to U.S. and Canadian citizens or permanent residents as well as to U.S. temporary residents.

Source: Johns Hopkins University Institute for NanoBioTechnology

Saturday, June 1, 2013

iPhone Biosensor Detects Toxins, Proteins, Bacteria and Viruses

Researchers and physicians in the field could soon run on-the-spot tests for environmental toxins, medical diagnostics, food safety and more with their smartphones.

University of Illinois at Urbana-Champaign researchers have developed a cradle and app for the iPhone that uses the phone’s built-in camera and processing power as a biosensor to detect toxins, proteins, bacteria, viruses and other molecules.

Having such sensitive biosensing capabilities in the field could enable on-the-spot tracking of groundwater contamination, combine the phone’s GPS data with biosensing data to map the spread of pathogens, or provide immediate and inexpensive medical diagnostic tests in field clinics or contaminant checks in the food processing and distribution chain.

“We’re interested in biodetection that needs to be performed outside of the laboratory,” said team leader Brian T. Cunningham, a professor of electrical and computer engineering and of bioengineering at the U. of I. “Smartphones are making a big impact on our society – the way we get our information, the way we communicate. And they have really powerful computing capability and imaging. A lot of medical conditions might be monitored very inexpensively and non-invasively using mobile platforms like phones. They can detect molecular things, like pathogens, disease biomarkers or DNA, things that are currently only done in big diagnostic labs with lots of expense and large volumes of blood.”

The wedge-shaped cradle contains a series of optical components – lenses and filters – found in much larger and more expensive laboratory devices. The cradle holds the phone’s camera in alignment with the optical components.

At the heart of the biosensor is a photonic crystal. A photonic crystal is like a mirror that only reflects one wavelength of light while the rest of the spectrum passes through.  When anything biological attaches to the photonic crystal – such as protein, cells, pathogens or DNA – the reflected color will shift from a shorter wavelength to a longer wavelength.

For the handheld iPhone biosensor, a normal microscope slide is coated with the photonic material. The slide is primed to react to a specific target molecule. The photonic crystal slide is inserted into a slot on the cradle and the spectrum measured. Its reflecting wavelength shows up as a black gap in the spectrum. After exposure to the test sample, the spectrum is re-measured. The degree of shift in the reflected wavelength tells the app how much of the target molecule is in the sample.

The entire test takes only a few minutes; the app walks the user through the process step by step. Although the cradle holds only about $200 of optical components, it performs as accurately as a large $50,000 spectrophotometer in the laboratory. So now, the device is not only portable, but also affordable for fieldwork in developing nations.

In a paper published in the journal Lab on a Chip, the team demonstrated sensing of an immune system protein, but the slide could be primed for any type of biological molecule or cell type. The researchers are working to improve the manufacturing process for the iPhone cradle and are working on a cradle for Android phones as well. They hope to begin making the cradles available next year.

Cunningham’s group is now collaborating with other groups across campus at the U. of I. to explore applications for the iPhone biosensor. The group recently received a grant from the National Science Foundation to expand the range of biological experiments that can be performed with the phone, in collaboration with Steven Lumetta, a professor of electrical and computer engineering and of computer science at the U. of I. They are also are also working with food science and human nutrition professor Juan Andrade to develop a fast biosensor test for iron deficiency and vitamin A deficiency in expectant mothers and children.

In addition, Cunningham’s team is working on biosensing tests that could be performed in the field to detect toxins in harvested corn and soybeans, and to detect pathogens in food and water.

“It’s our goal to expand the range of biological experiments that can be performed with a phone and its camera being used as a spectrometer,” Cunningham said. “In our first paper, we showed the ability to use a photonic crystal biosensor, but in our NSF grant, we’re creating a multi-mode biosensor. We’ll use the phone and one cradle to perform four of the most widely used biosensing assays that are available.”

Cunningham also is affiliated with the Institute for Genomic Biology, the Beckman Institute for Advanced Science and Technology, and the Micro and Nanotechnology Laboratory, all at the U. of I.

Source: University of Illinois News Bureau