New Biosensor Catches 'Superbug' MRSA In Record Time

Scientists have developed a new test for antibiotic-resistant bacteria that could reduce the time for diagnosis from hours to minutes. This discovery will be invaluable to clinicians trying to treat patients and disinfect hospital facilities.

Antibiotic resistance is rapidly spreading in the world of microbes, which poses serious dangers to public health. Following a major outbreak this spring of the superbug Carbapenem-resistant Enterobacteriaceae (CRE), the director of the U.S. Centers for Disease Control (CDC) labelled emerging antibiotic resistance a "nightmare."

"Our strongest antibiotics don't work and patients are left with potentially untreatable infections," said CDC director Tom Frieden.

Until new treatments are developed, surveillance is the best method for stemming the spread of superbugs. However, current diagnostics for antibiotic resistance can take hours - sometimes days - to complete, increasing the likelihood that another person will catch the disease.

"Our [new] method can determine bacterial antibiotic resistance in 10-12 minutes, while other methods take hours," said senior author Dr. Vodyanoy, a physiologist at Auburn University. The project was a joint collaboration between Auburn and the Keesler Air Force Base with funding from the U.S. Air Force.

To create this method, the researchers relied on a natural enemy of these germs: bacteriophages.

A bacteriophage is a simple virus that can target and kill bacteria. It's name literally means "to eat bacteria."

Another common superbug - methicillin-resistant Staphylococcus aureus (MRSA) - was used to validate their system.

Tiny gold strips, the size and shape of sticks of gum, were placed in petri dishes and coated with modified bacteriophages. These tiny plates can ensnare staph bacteria from a biological sample - spit or blood.

Special markers for MRSA were then added to the dish to detect the presence of antibiotic-resistant strains.

While only samples of MRSA were used in this study, "it can also be applied to other antibiotic resistant bacteria," said co-author Iryna Sorokulova, Ph.D., professor of microbiology at Auburn University.

The study was published in the video journal JOVE, so that other scientists can learn these techniques.

"We felt it would be very beneficial and educational to publish [in JoVE]," said Dr. Vodyanoy. "We are interested in someone else reproducing our results; this technology can be used on a larger scale and for antibiotic resistance other than Staphylococcus."

Source: Medical Daily 

Student's Fast Start in Research Contributes to Life-Saving Rapid Test

As if the story of breakthrough research were not enough, there is the story of the researcher. The accomplishment, in this case, is a rapid test for detecting devastating bacterial infections. The researcher is undergraduate pre-med student Kristin Wiggins, finishing her junior year at Northern Arizona University while working as an intern at Translational Genomics Research Institute North in Flagstaff.

At the heart of the story is someone who wants to make a difference in the world by saving people's lives—a motivation rooted in instinct and a family tradition of public service and health care. 

"I have always been fascinated with biology and I've had a lot of exposure to people in the medical and science professions," said Wiggins, whose great-uncle was a pharmacogenomics researcher and father was a firefighter. "All of my experiences with them have affirmed my passion to help others with science and medicine." 

From the age of 16, Wiggins has applied her passion by getting into the lab and going to work. As a junior in high school, she received a Helios Internship that placed her in the Cancer and Cell Biology Division of TGen in Phoenix. 

"I worked in the Brain Tumor Unit on Medulloblastoma and I absolutely loved it," Wiggins said. The summer internship stoked her passion for research. After her freshman year at NAU, she contacted TGen North and was invited to interview, which resulted in an internship in the Center for Public Health and Clinical Pathogens. 

"Pathogen research is extremely different from cancer research," Wiggins said. "I was really excited to be offered the opportunity to participate in the revolutionary pathogen research that goes on every day at TGen North." 

Wiggins explained that her research group handles six different dangerous agents. "We work with pathogens that are locally relevant but have a global impact," she said. 

One of those pathogens, Klebsiella pneumonia, is emerging worldwide as a public health crisis, Wiggins said. The pathogen's resistance to antibiotics fuels its danger as a secondary infection in hospital environments. The ability to identify it quickly—in a process known as a rapid KPC test—can save lives. Still experimental, the rapid test can accomplish in 90 minutes what now takes physicians two–three days.

In the lab, Wiggins' role was to grow Klebsiella isolates and extract their DNA for validation purposes. The task involved extreme repetition in the pursuit of virtual perfection. "It is an amazing experience seeing the research process from beginning to end, and all the hard work in between," Wiggins said. "I am thrilled that as an undergraduate intern, I can play a role in every single one of these steps." 

It's a role that has gotten noticed. According to David Engelthaler, director of programs and operations at TGen North, "Kristin is the exact model of what we hope to see in our interns—someone who has immediate dedication to making herself a better and smarter researcher, so she can achieve her long term vision of making a difference in the world." 

Wiggins said the research group hopes to begin publishing papers on its findings soon. "I think that after some more validation and research, we can start translating this into the clinic so that doctors can start using it to diagnose their patients," she said. "Physicians could start using our assay to shorten detection time, reduce the risk of false positives and enable improved treatment." 

While continuing the internship and the research project, Wiggins plans to finish her biomedical science major, with minors in chemistry, philosophy and mathematics. Then she intends to become a full-time research associate at TGen North while pursuing a master's degree and possibly even a Ph.D. before turning to a medical career. 

"One of the things that has surprised me the most is how much of an impact we have," Wiggins said. "We do not do research for the sake of research. Our mission is to advance public health and clinical medicine, and we strive for this every single day."

Source: Northern Arizona University

U of Alberta Researchers Seek Ways to Rapidly Diagnose Diseases in Developing World

A pair of University of Alberta researchers have each received a $100,000 grant to develop new tests that can be used easily and cheaply to detect diseases in developing nations.

The grants come from the federally funded Grand Challenges Canada, which provides money to develop what it calls “bold new global health ideas.”

The two U of A recipients, John Davis and Darryl Adamko, are from different fields, yet their projects revolve around the same idea of using metabolites — small molecules associated with a biological process in the body — as a method of detection for diseases.

“There’s a lot of literature and more coming out every day that says various metabolites are associated with various diseases, sometimes in blood, sometimes in urine and sometimes in breath,” said Davis, a physicist.

Davis’s project is to create a breath test that would be delivered through a hand-held device similar to a breathalyzer used by police. Patients would blow into the box, which would have the ability to sense a number of different metabolites indicating the presence of diabetes, various cancers, tuberculosis, cholera and other illnesses.

“We have been working on these tiny little vibrating nanomechanical devices that are really sensitive to their external environment and their intrinsic properties,” he said.

Davis uses the analogy of guitar string to describe how the devices work. A guitar string’s sound can be changed several ways, such as by increasing its thickness or tension, or by doing more extreme things like plunging it under water. In the same way, the nanomechanical devices can be altered to become sensitive to various types of molecules, he said.

“If a cop pulls you over and gives you a breathalyzer, he is testing for one thing, ethanol, but what we would like to have is array of devices that are functionalized differently so each string is sensitive to a different thing,” Davis said.

“One of things we still have to do is make these things chemically functional so they respond to particular molecules, because right now they just kind of respond to everything. And we’re trying to make it all miniaturized to be hand held. Right now it’s big, laboratory scale of apparatus.”

Once the development is further along, Davis’s team plans to connect with a researcher in Bangladesh to test the device.

He said for diagnostic technology to work in the developing world, it needs to be portable, rapid and rugged. Many tests currently are done by taking swabs and then growing cultures, which is slow. This is problematic as it often requires patients to travel long distances to a clinic and endure lengthy waits to get results when they need to be at home or work.

A hand-held device could provide results right away, and could even be taken into remote villages rather than having patients come to the clinic.

“Just like the breath analysis a cop does, he knows right away, he doesn’t take a sample, culture it for a few days and then tell you if you can leave the crime scene,” Davis said. “If we can develop this rapid test, even if it were not 100-per-cent accurate, it would tell someone right away whether they needed to do a secondary test or if people were free to go home.”

As for Adamko, a children’s lung disease expert, his project involves using a simple urine test to look for respiratory diseases such as asthma, bronchitis, tuberculosis and pneumonia.

There are currently few effective measures of airway diseases that can be done in a family doctor’s office, so the idea is to create something that can be done easily and non-invasively, he said. That’s where metabolite science is handy, because each type of breathing disorder involves a different kind of that can be seen the fluids patients excrete.

“As a pediatrician, when I heard they were doing metabolic analysis in adults using sputum, I said that isn’t going to work for kids because kids aren’t going to spit, but what about urine tests?”

The idea is that children visiting a doctor’s office would provide a urine sample, which would then be sent to a lab. The lab would then send back a metabolic profile of the patient using a roadmap Adamko is developing. His information would tell the doctor that the presence of a particular molecule or set of molecules indicates a particular disease.

“It’s the pattern of metabolites,” he said. “Based on a urine test, you should be able to get an idea of whether they are sick or not sick, what type of breathing disease they have and also whether it’s getting better or worse.”

Adamko, who recently moved back to the University of Saskatchewan, started a company called Respirlyte and maintains a lab at the U of A to further develop the concept. Eventually the plan is to test the project in India.

“I would like to do a clinical trial with a lot of people and going to a place like India would be awesome,” he said.

Davis and Adamko were among 102 researchers selected to receive a Grand Challenges grant out of 436 applications.


Source: Edmonton Journal 

Portable Nanodevice Provides Rapid, Accurate Diagnosis of TB and Other Pathogens

A handheld diagnostic device that Massachusetts General Hospital (MGH) investigators first developed to diagnose cancer has been adapted to rapidly diagnose tuberculosis (TB) and other important infectious bacteria. Two papers appearing in the journals Nature Communications and Nature Nanotechnology describe portable devices that combine microfluidic technology with nuclear magnetic resonance (NMR) to not only diagnose these important infections but also determine the presence of antibiotic-resistant bacterial strains.

"Rapidly identifying the pathogen responsible for an infection and testing for the presence of resistance are critical not only for diagnosis but also for deciding which antibiotics to give a patient," says Ralph Weissleder, MD, PhD, director of the MGH Center for Systems Biology (CSB) and co-senior author of both papers. "These described methods allow us to do this in two to three hours, a vast improvement over standard culturing practice, which can take as much as two weeks to provide a diagnosis."

Investigators at the MGH CSB previously developed portable devices capable of detecting cancer biomarkers in the blood or in very small tissue samples. Target cells or molecules are first labeled with magnetic nanoparticles, and the sample is then passed through a micro NMR system capable of detecting and quantifying levels of the target. But initial efforts to adapt the system to bacterial diagnosis had trouble finding antibodies – the detection method used in the earlier studies – that would accurately detect the specific bacteria. Instead the team switched to targeting specific nucleic acid sequences.

The system described in the Nature Communications paper, published on April 23, detects DNA from the tuberculosis bacteria in small sputum samples. After DNA is extracted from the sample, any of the target sequence that is present is amplified using a standard procedure, then captured by polymer beads containing complementary nucleic acid sequences and labeled with magnetic nanoparticles with sequences that bind to other portions of the target DNA. The miniature NMR coil incorporated into the device – which is about the size of a standard laboratory slide – detects any TB bacterial DNA present in the sample.

On the 2.5- by 7.5-cm cartridge (see above picture), DNA extracted from sputum samples is amplified in the chambers on the left. TB-specific sequences are magnetically labeled in the microfluidic mixing channels in the center and detected by passage through the micro-NMR coil on the right. Credit: Center for Systems Biology, Massachusetts General Hospital

Tests of the device on samples from patients known to have TB and from healthy controls identified all positive samples with no false positives in less than three hours. Existing diagnostic procedures can take weeks to provide results and can miss up to 40 percent of infected patients. Results were even stronger for patients infected with both TB and HIV – probably because infection with both pathogens leads to high levels of the TB bacteria – and specialized nucleic acid probes developed by the research team were able to distinguish treatment-resistant bacterial strains.

The Nature Nanotechnology paper, being issued online today, describes a similar system using ribosomal RNA (rRNA) – already in use as a bacterial biomarker – as a target for nanoparticle labeling. The investigators developed both a universal nucleic acid probe that detects an rRNA region common to many bacterial species and a set of probes that target sequences specific to 13 clinically important pathogens, including Streptococcus pneumoniae, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

The device was sensitive enough to detect as few as one or two bacteria in a 10 ml blood sample and to accurately estimate bacterial load. Testing the system on blood samples from patients with known infections accurately identified the particular bacterial species in less than two hours and also detected two species that had not been identified with standard culture techniques.

While both systems require further development to incorporate all steps into sealed, stand-alone devices, reducing the risk of contamination, Weissleder notes that the small size and ease of use of these devices make them ideal for use in developing countries. "The magnetic interactions that pathogen detection is based on are very reliable, regardless of the quality of the sample, meaning that extensive purification – which would be difficult in resource-limited setting – is not necessary. The ability to diagnose TB in a matter of hours could allow testing and treatment decisions within the same clinic visit, which can be crucial to controlling the spread of TB in developing countries."

Hakho Lee, PhD, of the MGH Center for Systems Biology. co-senior author of both papers, notes that the system will alsohave important applications in developed countries. "The capacity of the system not only to identify bacterial species but also to differentiate factors such as antibiotic resistance will help clinicians treat patients with the 'right' drugs from the start, which also helps reduce the emergence of treatment-resistant strains. The fact that this device requires only a tiny drop of the sample to be tested will be helpful in instances when specimens can be hard to obtain, such as treating children or seniors."

Source: http://phys.org

Beach Bacteria: DNA Tests Offer Quicker Results

Just in time for swimsuit season, federal researchers are touting a faster, more accurate water-quality test to keep beaches open and people healthy.

But it’s expensive, and most of the nation’s cash-strapped cities and counties can afford it.

Local officials traditionally check for bacteria in ocean and lake water with tests that take about 24 hours to complete. Now the U.S. Environmental Protection Agency is recommending testing at the molecular level – tagging DNA and counting bacteria – which provides results within hours.

“Water quality can change significantly in 24 hours. This way we’re identifying threats to human health almost immediately,” said Meredith Nevers, a research ecologist with the U.S. Geological Survey who is studying the EPA’s new DNA test.

An estimated 3.5 million people get sick every year after a trip to the beach because of E. coli or other pathogens from sewage overflows, spills and polluted runoff, according to the EPA. Exposure can cause gastrointestinal illness, skin rashes and infections.

About 43 percent of beaches along the East and West coasts and the Great Lakes had at least one water-quality advisory in 2011, according to EPA data.

Counties and cities test beaches routinely – often weekly but depending on the location and season. Beaches also are tested after spills to determine when they can be reopened.

Slow Results

Traditional tests involve waiting 24 hours to allow E. coli, coliform or enterococci to grow in a water sample, then counting the colonies. That means decisions to close down beaches are based on samples collected the day before.

The new method speeds up DNA replication. Researchers then use fluorescent probes to see how many bacteria are present in a water sample by counting the DNA copies that fragmented. Other same-day tests are in various stages of research, but all generally use two steps: capturing microbes and tagging them so they can be counted.

Comparing the new method to the culture-based tests at Lake Michigan beaches, Nevers and colleagues concluded that beach closures and illness rates “could be minimized.” They couldn’t predict how many unnecessary closings or illnesses it would prevent, but it would be “significant,” Nevers said.

“Rapid testing is great – the faster the results, the better,” said Shannon Briggs, a toxicologist at the Michigan Department of Environmental Quality, which works with communities throughout the state on beach testing. “The goal is to keep beaches open all the time and keep people safe and this will help.”

But it’s not cheap.

DNA testing requires new labs and newly trained staff – a significant hurdle for cities and counties. Nevers estimated the new tests would cost about twice as much as the old ones.

Universities throughout Michigan are partnering with some coastal communities to help get the ball rolling.

Only Racine

The only community using the method right now to make beach safety decisions is Racine, Wis. The city received clearance last year from the EPA to use the DNA test on its two beaches that frequently exceed safe bacteria counts after years of testing it, said Julie Kinzelman, a research scientist at the Racine Health Department.

“We used to have to say, ‘well, you swam yesterday and now we know the water wasn’t clean. Sorry we couldn’t tell you in time,’” Kinzelman said.

Racine still uses culture tests to check the reliability of the DNA testing. Kinzelman said over the years the two different tests have been at about 90 percent agreement.

Racine’s lab was purchased with grant money, and it has expanded with grant and city money. Kinzelman had to be trained and now uses college students to help run the tests. She credits the city’s beach pride for being on the cutting edge of beach testing.

“Beaches are our identity,” she said. “We’re Racine on the lake.”

Too Costly for Chicago, California

But in Chicago, Cathy Breitenbach, director of lakefront operations for the city’s Park District, said it’s not economical.

“We have 24 beaches, we’d have to move up and down 26 miles of coastline, take samples and get them to a lab,” Breitenbach said. “We’d have to start at 2 in the morning” in order to take advantage of the same-day benefits.

No one is using the rapid method in California, said John Griffith, a marine microbiologist at the Southern California Coastal Water Research Project, a research institute that studies coastal pollution.

“We barely have money to do our regular culture testing,” he said.

The EPA should subsidize the DNA test method for states, said Steve Fleischli, water program director at the Natural Resources Defense Council, an environmental advocacy group.

“People want to know what the water is like when they’re in it, not what it was like days beforehand,” Fleischli said.

But Griffith said rapid testing only makes sense at certain beaches. “If a beach never has a problem, or if you have a chronically contaminated beach, you don’t need a rapid answer and it’d be hard to justify this cost,” Griffith said.

Griffith and colleagues are working on different rapid testing methods with communities along Southern California’s coast. One method, still in the testing phase, is a mobile DNA sampler that sends results to the lab directly from the field.

Predicting Water Quality

Some beach managers are pairing testing with predictive software, which projects water quality based on weather and water conditions. Briggs said the software is about 90 percent accurate in most Michigan locations.

“Before it was always a guess. Now we’re getting accurate information, and getting it faster,” Briggs said.

And it seems to be working. Michigan beaches were open 97 percent of the time in 2011, according to the EPA. Chicago is focusing on predictive modeling at 15 beaches and the decisions were more accurate than those made by the culture tests, Breitenbach said.

Predictive modeling, however, isn’t used in many ocean communities, where larger waves and less summer rainfall make it much less useful than in the Great Lakes, Griffith said.

The DNA test is included in a set of voluntary recommendations the EPA released last November when it updated water-quality criteria. The EPA’s guidelines recommended that states test beach water and notify the public more quickly.

But the EPA’s new proposed 2014 budget would eliminate federal funding for beach monitoring completely – a $9.9 million takeaway.

States and others that used the funding “now have the ability and knowledge to run their own programs without federal support,” according to the budget proposal.

Source: Environmental Health News

NUS Team Develops World's First Microfluidic Device for Rapid Separation and Detection of Non-spherical Bioparticles

A bioengineering research team from the National University of Singapore (NUS) team led by Associate Professor Zhang Yong has developed a novel microfluidic device for efficient, rapid separation and detection of non-spherical bioparticles. Microfluidic devices deal with the behavior, precise control and manipulation of fluids that are geometrically constrained to sub-millimeter scale. This new device, which separates and detects non-spherical bioparticles such as pathogenic bacteria and malaria infected red blood cells, can potentially be used for rapid medical diagnostics and treatment.

Bioparticles such as bacteria and red blood cells (RBC) are non-spherical. Many are also deformable - for example, our blood cells may change shape when affected by different pathogens in our body. Hence, the team's shape-sensitive technique is a significant discovery. Currently, separation techniques are mostly designed for spherical particles.

Though the team is focusing mainly on the rapid separation and detection of bacteria from pathological samples at the moment, their device has potential as a rapid diagnostic tool as well. Their new technique can potentially replace an age-old method of detection based on bacterial culture.

Explained Assoc Prof Zhang, "The old method was developed about 100 years ago, but it is still being used today as the mainstream technique because no new technique is available for effective separation of bacteria from pathological samples like blood. Many of the pathogenic bacteria are non-spherical but most of microfluidic devices today are for separating spherical cells. Our method uses a special I-shape pillar array which is capable of separating non-spherical or irregularly-shaped bioparticles."

The method developed by the NUS team can complete the diagnosis process in less than an hour compared to 24-48 hours required for bacterial detection by using conventional methods. Their device is also efficient in separating red blood cells (RBCs) from blood samples as RBCs are non-spherical. This enables rapid detection of diagnostic biomarkers which reside in blood sample.

One of the most challenging aspects for the team was designing and fabricating a device that is capable of detecting even the smallest dimension of bioparticles and still provide reasonably good throughput (amount which can be processed through the system in a given time).

How it works and moving forward 

Scientists have tried to address the problem of separating non-spherical bioparticles by using techniques such as restricting the flow of particles but these have not shown to be as effective. However, the NUS Bioengineering team's I-shape pillar array device has proven to be successful.

The I-shape pillar array induces rotational movements of the non-spherical particles which in turn increases the effective hydrodynamic size of the bioparticles flowing in the device, allowing for efficient separation. Their design is able to provide 100 percent separation of RBCs from blood samples, outperforming conventional cylindrical pillar array designs.

The device can also potentially separate bioparticles with diverse shapes and sizes. The team has tested their device successfully on rod-shaped bacteria such as Escherichia coli (common bacteria which can cause food poisoning). So far, this has been difficult to achieve using conventional microfluidic chips.

The team's findings were published in the reputed journal Nature Communications on 27 March 2013, in a manuscript titled "Rotational separation of non-spherical bioparticles using I-shaped pillar arrays in a microfluidic device".

Said Assoc Prof Zhang, "With our current findings, we hope to move on to separate other non-spherical bioparticles like fungi, with higher throughput and efficiency, circumventing the spherical size dependency of current techniques."

Source: National University of Singapore

European Union Funding 5-Year Safe Drinking Water Project

The European Union is funding a new research initiative to improve the safety of drinking water and reduce outbreaks of waterborne diseases.

The five-year, 9 million-euro ($11.8 million) Aquavalens project will be led by Paul Hunter of the University of East Anglia’s Norwich Medical School in eastern England, the school said today in a statement. It will seek to develop more rapid methods of detecting viruses, bacteria and parasites in water before they make people sick, according to the statement.

Globally, about 2 million deaths a year are attributed to unsafe water, sanitation and hygiene and almost 1 billion people lack access to safe drinking water, according to the World Health Organization.

“With the technologies we currently have it can take two or more days to identify infectious risks in drinking water and by then the affected water is likely to have been consumed,” Hunter said. “This project will develop more rapid methods so that problems can be identified earlier.”

The project hopes to find low-cost testing technologies that can monitor water supplies year-round to identify unacceptable levels of viruses or bacteria instead of weekly or in some cases even less frequent spot checks, Hunter said.

Gene probes, nano-technologies and bio-sensors will be explored, he said.

“Although the focus is on Europe, hopefully the technologies that will be developed will have applicability that is far wider than just Europe,” Hunter said. “The main activity will be development of water technologies that could be used globally.”

The European Union’s Framework Programme 7, which supports scientific and technological research in the region, is providing funding for the project. Academic groups and biotechnology companies in 13 European countries will be participating in the project, according to the statement.

ID Deadly Pathogens Without Growing Bacteria

Metagenomics has allowed researchers to reconstruct the genome sequence of a deadly Shiga-toxigenic E. coli outbreak without having to grow bacteria in the lab.

“The outbreak of Shiga-toxigenic Escherichia coli illustrated the effects of a bacterial epidemic on a wealthy, modern, industrialized society, with more than 3,000 cases and more than 50 deaths reported in Germany between May and June of 2011,” says Mark Pallen, professor of microbial genomics at Warwick Medical School.

“During an outbreak such as this, rapid and accurate pathogen identification and characterization is essential for the management of individual cases and the outbreak as a whole.

“Traditionally, clinical bacteriology has relied primarily on laboratory isolation of bacteria in pure culture to identify and characterize an outbreak strain.”

The team of researchers was able to reconstruct the genome sequence through the direct sequencing of DNA extracted from microbiologically complex samples.

The study, published in a genomics-themed issue of JAMA, highlights the potential of this approach to identify and characterize bacterial pathogens directly from clinical specimens.

Metagenomics has been used previously in a clinical diagnostic setting to identify the cause of outbreaks of viral infection, but this is its first reported use in an outbreak of bacterial infection.

Often, the process of laboratory culture proves slow and the recognition of an outbreak strain can be difficult if it belongs to an unknown variety or species for which specific laboratory tests and diagnostic criteria don’t already exist.

“There are numerous drawbacks to the use of nineteenth-century approaches such as microscopy and culture when it comes to classification,” says Pallen.

“Our results illustrate the potential of metagenomics as an open-ended, culture-independent approach for the identification and characterization of bacterial pathogens during an outbreak. There are challenges, of course, including speeding up and simplifying workflows, reducing costs, and improving diagnostic sensitivity.

“However, given the dizzying pace of progress in high-throughput sequencing, these are not likely to remain problems for very long.”
Researchers from the University of Birmingham, the University of Glasgow, the University Medical Centre Hamburg-Eppendorf in Germany and the sequencing company Illumina contributed to the findings.

Source: University of Warwick

The JAMA article may be reviewed at http://jama.jamanetwork.com/article.aspx?articleid=1677374. Below is the abstract:

A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4. Nicholas J. Loman, MBBS, PhD; Chrystala Constantinidou, PhD; Martin Christner, MD; Holger Rohde, MD; Jacqueline Z.-M. Chan, PhD; Joshua Quick, BSc; Jacqueline C. Weir, MSci; Christopher Quince, PhD; Geoffrey P. Smith, PhD; Jason R. Betley, PhD; Martin Aepfelbacher, MD; Mark J. Pallen, MA, MD, PhD

Importance  Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain.

Objective  To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture.

Design, Setting, and Patients  In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain.

Main Outcomes and Measures  The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples.

Results  During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered.

Conclusions and Relevance  These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

Rapid Mobile Bio-Threat Detector Developed By Sandia Lab

In the event of a mass casualty bio-attack, hospital emergency rooms will quickly be overwhelmed. Hospitals nationwide have limited surge capacity to handle a sudden influx of patients.

A pathogenic attack that’s unable to be identified quickly and early will compound the problem as increasing numbers of infected people flood hospitals’ ERs and mobile triage facilities that will have to be erected or established to accept the growing number of victims. The situation will be exacerbated if the pathogen is highly transmissible between humans.

But now scientists at Sandia National Laboratories (SNL) are on the verge of developing a technology that can be used in ERs and by first responders to rapidly identify a pathogenic WMD attack that’s potentially contagious. SNL researchers have made groundbreaking strides in developing a portable device that is able to quickly detect biological pathogens that terrorists might use in a weapon of mass destruction (WMD) attack, including anthrax, ricin, botulinum, shiga and SEB toxin.

But the technology, called SpinDx, also has much wider applicability to the homeland security community.

“Not only can the device screen clinical samples, we also expect it to be very useful to analyze water, aerosol effluent, milk, juice and other liquefied food to look for biothreat agents,” said Anup Singh, senior manager for Sandia’s biological science and technology group, in an e-mail response to questions by Homeland Security Today. "SpinDx does not require any off-device sample prep and hence, is compatible with many different types of samples. It can also be useful to screen cattle in an event of natural or man-made agricultural terrorism incident."

Once developed and approved by the Food and Drug Administration and commercialized, the technology not only would be used in emergency rooms in the event of a bioterrorism incident, but it also can be used by first responders.

“This is an unmet need for the nation’s biodefense program. A point-of-care device does not exist,” said Singh.

“In case of an alleged bioterror event, it is expected that emergency rooms will be overwhelmed by people who are infected or who think that may be infected,” said Singh. “Our ERs are not capable of screening large numbers of people quickly. One reason is that they do not have access to technology that can be used to screen people rapidly,” he said.

Sandia’s SpinDx device features centrifugal microfluidics, or “lab-on-a-disk” technology, which uses centrifugal forces to manipulate samples and reagents through microfluidic channels implanted on disks that are of the same size as a standard CD or DVD.

“We expect SpinDx to be a potential solution as it is fast, portable and simple to use,” said Singh. “An ER doctor or nurse can take a pin-prick blood sample from a person, insert into a disk, load the disk into SpinDx and run SpinDx. After about 15 min, SpinDx will display the relative amount of toxin present on an LCD panel on the device or on a computer linked to the device. It is possible to run samples from multiple persons simultaneously as well as look for multiple agents simultaneously.”

Sandia’s work is funded by a four year, $4 million grant from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (NIH), which has funded a number of recent projects at Sandia.

“This will take things to the next level,” said Singh. In addition to the broader suite of toxins and bacterial agents that the device would test for, the project includes comprehensive testing with animal (mouse) samples.

This is an important step, according to Singh, who explained that toxins may behave differently in live animals and humans than in laboratory blood samples.

“We are getting closer and closer to translational elements of research, which involves testing in animal and clinical facilities,” said Singh. “This is part of the maturation of our bioresearch activities at Sandia.”

The project also will increase what SpinDx can do, Singh said.

“When you look for bacterial agents, you don’t want to rely solely on proteins because you won’t get the detection sensitivity you need,” said Singh. “So we are also using other methods that may lead to better detection limits and additional confirmation.”

SNL said the new NIH project includes collaborators with expertise in animal modeling as well as device manufacturing.

The University of Texas Medical Branch and the US Department of Agriculture’s Western Regional Research Center in Albany, Calif., are providing Sandia with expert insight into toxins and diseases at animal lab facilities.

Bio-Rad, a manufacturer and distributor of a variety of devices and laboratory technologies, also is serving as a consultant on the project to evaluate plans for product development, assisting with manufacturers’ criteria on the device that is finally developed and providing important feedback when a prototype is built.

“You’ve got to keep innovating and coming up with the next thing,” said Singh. “Every technology has its lifecycle. As good as SpinDx is, we know there will be other technologies, better technologies that come along in the next few years. We have to continue to innovate to meet the needs of our customers, understand what other competing technologies are being designed to solve the problems and develop technologies that provide an improvement.”

Singh pointed out that the need for diagnostic devices for biodefense is not going away because there are always new diseases emerging for which there are inadequate diagnostic assays.

“Plus, we want dual-use devices that combat both man-made and nature-made problems,” said Singh. “We’re not just going to wait for the next anthrax letter incident to happen for our devices to be used and tested; we want them to be useful for other things as well, like infectious diseases.”

Expanding into those areas will keep Sandia’s bioresearch efforts engaged for years to come, said Singh.

“That’s where the value of the national labs really comes in,” Singh said. “Our capabilities and culture are a very good fit for tackling long-term problems that require a sustained effort.”

Source: HSToday.us

Revolutionary Medical Dressing Uses Nanotechnology to Detect and Fight Infection

Researchers are using nanotechnology to develop a medical dressing which will detect and treat infection in wounds.

Scientists at the University of Bath and the burns team at the Southwest UK Paediatric Burns Centre at Frenchay Hospital in Bristol are working together with teams across Europe and Australia to create an advanced wound dressing.

The dressing will work by releasing antibiotics from nanocapsules triggered by the presence of disease-causing pathogenic bacteria, which will target treatment before the infection takes hold.

The dressing will also change colour when the antibiotic is released, alerting healthcare professionals that there is infection in the wound.

This is an important step in treating burns patients, particularly children, where infections can lead to toxic shock syndrome, a potentially fatal condition.

The €4.5 million European Commission funded project is a collaboration of 11 partners across Europe and Australia coordinated by Dr Renate Förch, at the Max-Planck-Institute for Polymer Research (Germany), which will develop the prototype dressing over four years.

The Bacteriosafe project includes chemists, cell biologists, clinicians and engineers. They will not only develop the dressing, but will also work with industry on a pre-pilot scale manufacturing process, so they could be available on the market within a few years after completion of the project.

University of Bath project leader, Dr Toby Jenkins said: “Your skin is normally home to billions of ‘friendly’ bacteria, which it needs to stay healthy.

“The dressing is only triggered by disease-causing bacteria, which produce toxins that break open capsules containing the antibiotics and dye.

“This means that antibiotics are only released when needed, which reduces the risk of the evolution of new antibiotic-resistant super-bugs such as MRSA.”

Dr Amber Young, a paediatric burn specialist at the South West UK Paediatric Burn Centre, based at Bristol’s Frenchay Hospital, will be the clinical consultant on the project.

She said: “We’re really excited about this project – every day we see young children who are seriously ill from burns who would hugely benefit from this research.

“Many people don’t realise that a burn from a cup of tea can be deadly if it becomes infected.

“Conventional dressings have to be removed if the skin becomes infected, which slows healing and can be distressing for the child.

“This advanced dressing will speed up treatment because it is automatically triggered to release antibiotics only when the wound becomes infected, meaning that the dressing will not need to be removed, thereby increasing the chances of the wound healing without scarring.

“The colour change acts as an early warning system that infection is present, meaning we can treat it much faster, reducing the trauma to the child and cutting the time they have to spend in hospital.”

The dressing could also be used for other types of wound, such as ulcers or by the military on the battlefield.

The researchers have already tested fabric coated with the nanocapsules, which are just one millionth of a millimetre in size. They have been shown to react specifically to harmful bacteria. Over the next four years the European team will be working on integrating the colour change technology into a suitable dressing and looking at cost effective routes for industrial production.

The following video describes the research:



Source: University of Bath

Plan to Fight Deadly TB Strain Advances in India, But Slowly

WHO has endorsed all of the discounted diagnostics and they are widely used globally. But Indian TB officials say they haven't validated some of them, and TB remains the leading cause of death by infectious disease in the country's adults.

An international health initiative has brokered a landmark accord that aims to halve the price patients pay for advanced tests for a strain of tuberculosis that is resistant to standard drugs.

The deal is between the makers of diagnostic equipment that can detect multi-drug-resistant TB and private Indian clinics that test patients. Together, they could fill a big hole in international efforts to combat more-virulent forms of the disease by creating a matchable model.

India is fighting a TB epidemic. In the past year, a Mumbai hospital has been reporting an increasing number of patients—now numbering 22, who are resistant to virtually all of the dozen treatments commonly used against the disease.

Under the accord—coordinated by the nonprofit Clinton Health Access Initiative and a McGill University professor—several diagnostics makers will give private Indian laboratories the same steep discounts offered to India and other poor governments. In return, the laboratories agreed to a top price for the tests that is about half of the current market price in India.

"We convinced the manufacturers that if they made the best diagnostics more affordable, a much larger numbers of patients would use them. That's good for the manufacturers and good for TB control," said Madhukar Pai, an associate professor at McGill University in Canada and one of the world's top TB experts, who helped organize the initiative.

The Indian government, supported by the World Health Organization, runs a national program to test and treat TB free of charge. But about half of TB patients don't use the government program because many believe they will receive poor medical care in the public-run sector.

Many TB victims instead seek care from private medical providers, who often offer cheap, inaccurate tests and inadequate treatments, fueling the spread of drug-resistant forms of the disease.

"Either a patient with no disease is put wrongly on TB treatment or a patient with disease is not treated," said Navin Dang, a New Delhi laboratory owner and an organizer of the initiative. "The poor TB patient in India suffers terribly as a result."

The potential impact of the new initiative remains uncertain due to the reluctance of India's Central TB Division to endorse it. New Delhi's support is crucial to persuade private physicians and patients to use their tests rather than the poor diagnostics, the initiative's organizers say.

Dr. Dang said the WHO has endorsed all of the discounted diagnostics and they are widely used globally. But the Indian TB officials say they haven't validated some of them. In particular, after about a year of pilot tests, the TB division hasn't adopted GeneXpert—a rapid test to detect TB and drug resistance within two hours—and the pilot studies continue.

Last week, Dr. Dang said he and other organizers visited the TB division and invited officials to endorse their efforts to bring good discounted diagnostics to private laboratories.

"I was quite disgusted" by the reaction of the officials, Dr. Dang said. "They refused to come on board. They said these are not validated tests. How can they not be validated when the WHO themselves endorsed them? They've been widely reported in publications internationally as accurate tests."

Ashok Kumar, the head of the TB division, didn't return phone calls seeking comment.

"The government welcomes all initiatives that support the fight against tuberculosis," Anshu Prakash, joint secretary of health for India, who oversees TB, told The Wall Street Journal on Friday. He said he wasn't told why the TB division didn't endorse the effort but said it might have been because a written proposal wasn't submitted. He added that he also needed to review a proposal in writing before endorsing the initiative.

Dr. Pai and Dr. Dang said they canceled plans to announce the initiative with fanfare in New Delhi on Friday because of the TB division's refusal to participate. Both men said the initiative would still go forward, but it would be less likely to be widely adopted without the government's stamp of approval. An official with the Clinton group declined to comment.

"For the uptake to be huge, we need to be able to get the word out to private sector doctors and to patients that these are the tests they should be using and that we are discounting them," Dr. Pai said. "For that, the government's help is important."

Dr. Pai said even at the discounted price of 1,700 rupees, or $35, the GeneXpert and other good TB diagnostics would still be unaffordable to many Indians. "We acknowledge that we are still a long, long way from a test that will be affordable to the masses. There needs to be a 100-rupee test. But this is a critical first step."

Under the Indian government's TB program, TB deaths have declined. But TB remains the leading cause of death by infectious disease in the country's adults.

The airborne disease, usually affecting the lungs and spread through coughing and sneezing, can be cured with a treatment of four medicines taken for six months. But patients often go undiagnosed for months, spreading the disease to others. Inadequate treatment allows the TB bacteria to mutate into a stronger form, fueling drug resistance.

India is home to the largest number of TB patients—2.2 million of the world's 8.7 million, according to the WHO's latest estimates.

Despite the decline in regular TB, drug resistant strains of the disease have worsened and spread here and globally. There hasn't been a nationwide survey of drug resistance, but India and the WHO say 2% to 3% of India's TB patients are drug resistant. However, pilot tests of a new rapid diagnostic suggest a rate of 6.7% at 18 sites around the country. The same preliminary analysis showed rates were far higher for Mumbai, where 28% of patients tested at one clinic were resistant to the most powerful TB medicine.

Mumbai made international headlines last year when a physician researcher reported several patients whose disease didn't respond to 12 of the most commonly used TB medicines.

The Clinton group and Dr. Pai began a year ago visiting manufacturers of the GeneXpert and several other diagnostics, said Dr. Pai, convincing them to extend their public sector discounts to the private sector in India because so many poor people sought care there.

"The reality in India is poor people are getting treatment in the private sector. It's not just rich people. So if you want to improve TB care, you have you make sure accurate testing is available in the private sector," Dr. Pai said. "It also makes good economic sense for the manufacturers to try to increase volumes by lower the price."

Because the best diagnostics were unaffordable to most Indians, inaccurate tests have proliferated in India, TB expert say. The Indian government last year banned one of the inaccurate diagnostics, although many are still performed by small laboratories because few other cheap alternatives are on the market.

"I realized it wasn't enough to ban bad diagnostics. You had to make sure consumers had access to affordable good diagnostics," Dr. Pai said.

Dr. Pai said he and the Clinton group canvassed the Indian laboratory industry, convincing 27 of the 400 laboratories in the country to agree to a price ceiling on the diagnostics they purchased at the steep discount.

Dr. Dang, who owns Dr. Dang's Lab Private Ltd., said he signed up for the initiative in the national interest. The agreement requires the participating labs also not to use certain inaccurate tests and to register all patients with the Indian government.

"So much manpower is wasted because of TB," he said. "India cannot advance economically if we let more than two million people a year suffer from TB."

India's own strategic plan for TB calls for improving medical care in the private sector, in addition to scaling up testing and treatment of drug resistance nationwide.

India's director general of health this year said India would improve TB treatment in the private sector by soon extending the free drug program in the public sector also to patients being treated in the private sector—provided they were under the care of qualified doctors who registered them in a government database. The government last year required all private doctors to register TB patients, but few are complying.

Source: The Wall Street Journal

Clinical Microbiology Rapid Method Research Discussed in Our Latest Newsletter

The clinical sector has seen a tremendous increase in the use of rapid methods for the detection and identification of pathogenic microorganisms, and the pharmaceutical industry can learn from these experiences. In our latest Newsletter, we reviewed select abstracts from the current edition of the Journal of Clinical Microbiology, where almost one-third of the bacteriology-related papers focused on rapid microbiological and molecular methods.

To read our review, please sign up for our Newsletter at: http://rapidmicromethods.com/newsletter/.