Thursday, April 18, 2013

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.

Thursday, April 11, 2013

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 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.

Tuesday, April 9, 2013

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.”