Researchers Develop Rapid, Multiplexed High-Res Melt Assay for Microbial Species Differentiation

Differentiating species using real-time PCR is critical to clinical and forensic labs, but developing inexpensive and rapid workflows for high-throughput testing is not always straightforward.

Now, researchers at Towson University in Maryland have described a fast and low-cost method to develop species differentiation assays using high-resolution melt temperature and a saturation dye from BioMérieux subsidiary BioFire Defense. Published in Analytical Biochemistry, the method enables multiplexing with differentiation by melt temperature rather than by color.

Corresponding author Kelly Elkins is a professor at Towson and teaches in an undergraduate and masters program for forensics science and chemistry accredited by the Forensic Science Education Programs Accreditation Commission. Her team's multiplex molecular assay may have an application in biodefense and foodborne pathogen testing, as it detects and differentiates Salmonella enterica substrain typhimurium, Escherichia coli, and Shigella flexneri. These three bacteria are considered potential bioterror agents by the US Centers for Disease Control and Prevention.

However, it has other potential forensic utility as well, as the authors noted that it can be used to differentiate plant species like marijuana, morning glory, and jimson weed, which will be the subject of a subsequent report on differentiating so-called "legal high" species. That test has also been run with case-work samples provided by Maryland State Police, Elkins said.

The method can also be adapted for clinical and research biochemistry, Elkins said.

The workflow uses BLAST searching for primer design. It then uses a program called OligoCalc to estimate the melting temperature of the PCR products. The program has been around since the late 1990s and is freely available online from Northwestern University.

"Before we went and purchased the primers and tried out the assay in the lab for our multiplex and assay development, we tested right there at OligoCalc to determine if, from the DNA sequence, the length, and the GC content, it would produce an amplicon of a different melt temperature than the amplicons that we already had in the assay," Elkins said.

That computer work saved considerable time and money, and the results were within a few percent of the OligoCalc calculations, she said.

The group also used internal positive controls in the assay that were from commercial kits, and also designed its own IPC.

Initially, the food-borne pathogens assay was developed using SYBRgreen, but that dye "was not giving us the sensitivity with the melts that we needed," Elkins said.

She then read some reports that used a dye called LCGreen Plus from BioFire Defense. "Immediately when it came in, I went to the lab and tried it, and the first test got beautiful results," she said.

LCGreen Plus dye was originally developed for mutation scanning and SNP detection, Matt Scullion, vice president of sales and marketing at BioFire Defense told GenomeWeb. "This article presents an interesting application and demonstrates the excellent performance of the dye," he commented.

The dye was developed when BioFire was still an independent company and called Idaho Technologies. Other so-called saturating dyes, which do not inhibit PCR even if used at concentrations that give maximum fluorescence, were later identified, including EvaGreen and SYTO9.

These all give "much more sensitive melt curves" than other dyes, Elkins explained.

Previous work had shown HRM could be used to differentiate species, and LCGreen Plus has been used for a number of clinical applications. Studies from the lab of Carl Wittwer at the University of Utah in the mid-2000s showed the dye could be used to create multiplex assays to distinguish three mycobacteria species as well as four Aspergillus species using rapid-cycle PCR followed by high-resolution melt curve analysis.

The novelty of Elkins' study was that most other work used melt-curve difference plots and kinetics of the melt curves. "While the data are interesting, it appears that it is easier to automate the interpretation of high-resolution melt assays when you differentiate based on the melt temperature," Elkins said.

Additionally, one previous example differentiated by melt temperature, but only of a duplex assay. Elkins said she had not seen other assays differentiating multiple species in the same assay, specifically using melt temperature in this way.

"The idea of being able to see a triplex or a tetraplex is pretty exciting, and it is pushing the boundaries of HRM," Elkins said.

The method is easily extensible to other projects and interests. The LCGreen master mix costs about $1 per assay, and the method can be run on standard PCR instruments with HRM capabilities. Elkins' lab runs 72 samples at a time in about two hours, but she said the run time has not been optimized yet and will likely be shortened in the future. And, for bioterrorism, which might require sampling sick individuals and food from many sites, high throughput is a necessity.

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Texas Children's Hospital, Houston Methodist Hospital Release Country's First Rapid Test for Zika Virus

Collaboration between two Texas Medical Center institutions has resulted in today's release of the country's first hospital-based rapid tests for the Zika virus.

Pathologists and clinical laboratory scientists at Texas Children's Hospital and Houston Methodist Hospital developed the Zika direct test in a matter of weeks as part of the L.E. and Virginia Simmons Collaborative in Virus Detection and Surveillance. This sponsored program was designed to facilitate rapid development of tests for virus detection in a large metropolitan area. The tests are customized to each hospital's diagnostic laboratory and will provide results within several hours. They can be performed on blood, amniotic fluid, urine or spinal fluid, according to James Versalovic, M.D., Ph.D., pathologist-in-chief at Texas Children's and leader of the Texas Children's Zika test development team along with James Dunn, Ph.D., director of medical microbiology and virology at Texas Children's.

Zika virus, which is mostly transmitted through mosquitoes, is a flavivirus that contains RNA as its genetic material. The new diagnostic test identifies virus-specific RNA sequences to directly detect Zika virus.

"With travel-associated cases of the Zika virus becoming more prevalent in the United States, coupled with the looming increase in mosquito exposure during spring and summer months, we must be prepared for a surge of Zika testing demand," said Versalovic. "We must provide answers for anxious moms-to-be and families who may experience signs and symptoms or may simply have travel history to endemic areas."

Before this test was developed, physicians faced the possibility of long delays of testing in local and state public health laboratories and the Centers for Disease Control.

"Hospital-based testing that is state-of-the-art enables our physicians and patients to get very rapid diagnostic answers. If tests need to be repeated or if our treating doctors need to talk with our pathologists, we have the resources near patient care settings," said James M. Musser,M.D., Ph.D., Chair of the Department of Pathology and Genomic Medicine at Houston Methodist Hospital and leader of the Houston Methodist test development team along with Randall J. Olsen, M.D., Ph.D., director of the molecular diagnostics laboratory.

Musser said the test was designed to detect the genetic material of the virus, its RNA, so that virus is directly detected in pregnant women and any other adult or child with this virus infection. This test is specific and can distinguish Zika virus infection from Dengue, West Nile or Chikungunya virus infections. Every viral particle contains genes in its RNA and these RNA sequences are directly detected during pregnancy in amniotic fluid or anytime in blood, spinal fluid or urine.

"This is a significant development as health authorities are recommending all pregnant women who have traveled to a place with a Zika virus outbreak get tested," Musser said.

At the current time, only registered patients at Texas Children's or Houston Methodist hospitals can receive the test but the labs will consider referral testing from other hospitals and clinics in the future.

The test will be initially offered to patients with a positive travel history and symptoms consistent with acute Zika virus infection such as a rash, arthralgias or fever or asymptomatic pregnant women with a positive travel history to any of the affected countries. The World Health Organization is now advising pregnant women to consult their doctors before traveling to places with Zika virus outbreaks and consider delaying travel. The CDC issued similar guidelines to American women last month.

The collaboration between Texas Children's and Houston Methodist Hospital was made possible by philanthropists, Virginia "Ginny" and L.E. Simmons who created the program after the 2014 Ebola virus scare highlighted the need for more focus on these infectious diseases.

"It is so great to see the progress these teams have made in such a short time. The work they are doing has such an impact on so many lives," Simmons said. "I am so grateful to know that the funds we donated are being used to make these types of advances in the Texas Medical Center."

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George Wash. Univ. Tests New Method for Rapid Detection of Infection in Wounds

A new method for detection of infection in wounds could take physicians less than a minute to complete, rather than the current 24 hours it takes to plate bacteria and leave it to incubate overnight, according to research by the George Washington University's (GW) Victoria Shanmugam, M.D.

Shanmugam and co-authors Edgar Goluch, Ph.D., DiPietro Assistant Professor of Chemical Engineering at Northeastern University College of Engineering, and Agnes Chan, Ph.D., assistant professor at the J. Craig Venter Institute, published this new method in the journal Wound Repair and Regeneration (see below). The method uses an electrochemical detection strategy to identify molecules produced by the bacteria Pseudomonas, which commonly infects chronic wounds.

The team tested the use of an inexpensive, disposable electrochemical sensor that immediately reveals bacteria based on the detection of pyocyanin, a bacterial quorum sensing molecule produced by Pseudomonas. The probe correctly identified the presence of the bacterium 71 percent of the time and correctly identified absence of the bacterium 57 percent of the time.

"Being able to detect Pseudomonas and other infectious organisms at the time of the clinic visit will greatly enhance our ability to take care of patients," said Shanmugam, director of the Division of Rheumatology at the GW School of Medicine and Health Sciences and principal investigator of the WE-HEAL study, a National Institutes of Health-funded study investigating the interplay of the host immune response and wound bed microbiome in patients with chronic wounds. "We would not have to wait for culture results before making a decision about antibiotics, and this would allow us to better tailor therapies for our patients."

After further enhancement and testing, probes harnessing this methodology could potentially provide a way for physicians to detect wound infections at the bedside, allowing physicians to switch from broad-spectrum antibiotics to specific directed therapies sooner, lowering health care costs, minimizing drug resistance, and improving patient care outcomes.

"Infections are a major challenge in medicine, and by using this probe, we were able to harness one of the unique molecules produced by bacteria to detect infection," said Shanmugam. "Through this ongoing collaboration with Dr. Goluch's team of engineers, we plan to continue to refine this testing method and hope to scale it up for detection of other bacteria and to optimize it for clinical use."

Link to Paper: Electrochemical detection of Pseudomonas in wound exudate samples from patients with chronic wounds.

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Australia's Atomo Secures $6M to Deploy its Rapid HIV Self-Diagnostic in Emerging Markets

The Global Health Investment Fund (GHIF) announced it has provided a $6 million loan to Atomo Diagnostics (Atomo) to support the scale-up of production operations and the expansion of commercial activities related to the award-winning AtomoRapid™ rapid diagnostic test (RDT) platform. Funds will also be used to commercialize a self-test solution for HIV and other infectious diseases.

Structured by JPMorgan Chase & Co. and the Bill & Melinda Gates Foundation, with founding support from Grand Challenges Canada, KfW and SIDA, GHIF focuses its investments in companies addressing key global health challenges. Curt LaBelle, MD, president of GHIF, said, "We are committed to financing initiatives and technologies that can significantly benefit global health. We believe the company's unique and innovative rapid test solutions support point-of-care testing across a variety of settings and have many exciting applications in both the developing world and developed markets."

As part of the collaborative funding agreement, GHIF and Atomo have made global access commitments to support and facilitate the launch and commercialization of Atomo's professional use and self-test products in low and middle-income countries to ensure that they achieve the greatest possible global impact.

Rapid testing in near patient settings is well established and enables quick diagnosis of many types of diseases and conditions using only a small drop of capillary blood from the fingertip. However, current generation lateral flow rapid test kits require multiple test components to perform a test, making them unsuited for use outside of the laboratory. User errors are common with these kits, even among clinicians, and they are generally considered unsuitable for self-testing. Atomo's RDT solutions replace these multi-component kits with a single integrated user-friendly device, designed specifically to simplify rapid testing and support consumer self-testing.

John Kelly, chief executive officer of Atomo, said, "Our mission has always been to provide the market with products that meet the needs of users.  We set out to develop a solution to obsolete the current generation 'bits in a box' test kit format, responsible for hundreds of thousands of errors and incorrect diagnoses globally each year. The investment from GHIF not only enables us to scale up our global health activities, it is also a clear endorsement of our innovative AtomoRapid™ technology as a preferred solution for rapid diagnostic testing."

AtomoRapid™ HIV for professional use was launched in early 2014 and has demonstrated high rates of user preference over existing RDT kits in the field. It has also won a number of prestigious international awards, including 'Best in Show' at the 2014 Medical Design Excellence Awards.

Dr. Linda-Gail Bekker, executive director of the Desmond Tutu HIV Foundation and president of the International AIDS Society, said, "Young South Africans surveyed at the Foundation's mobile and youth clinics preferred self-testing with the Atomo product over traditional HIV test kits.  We urgently need innovation that will facilitate the UNAIDS ambitious goal of 90% of all HIV positive people knowing their status by 2020. GHIF's investment in Atomo has laid a foundation towards that 90% goal through HIV self-testing."

As well as supporting tests for HIV, malaria and Ebola, Atomo is finalizing development of an AtomoRapid™ Hepatitis C test on the platform. The company is currently also refining a combination test platform that can diagnose multiple diseases at once, as well as solutions with digital readers and e-health capabilities.

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Abbott to Acquire Alere, Becoming Leader in Point of Care Testing and Significantly Advancing Global Diagnostics Presence

Abbott and Alere Inc. announced a definitive agreement for Abbott to acquire Alere, significantly advancing Abbott's global diagnostics presence and leadership. Under the terms of the agreement, Abbott will pay $56 per common share at a total expected equity value of $5.8 billion. Once the transaction is completed, Abbott will become the leading diagnostics provider of point of care testing. Abbott's total diagnostics sales will exceed $7 billion after the close.

Abbott will be able to provide new, flexible, cost-effective, high-quality products to help health systems meet growing demand in both in-patient and out-patient settings. Alere's complementary portfolio of products will provide Abbott access to new channels and geographies, including entry into fast-growing outlets, such as doctors' offices, clinics, pharmacies and at-home testing.

"The combination of Alere and Abbott will create the world's premier point of care testing business and significantly strengthen and grow Abbott's diagnostics presence," said Miles D. White, chairman and chief executive officer, Abbott. "We want to offer our customers the best and broadest diagnostics solutions. Alere helps us do that."

Upon completion of the transaction, the combined business will offer the broadest point of care menu of infectious disease, molecular, cardiometabolic and toxicology testing, expanding Abbott's platforms to include benchtop and rapid strip tests. Abbott will be able to better serve an expansive customer base around the world while also accelerating innovation in point of care diagnostics. More than half of Alere's $2.5 billion in sales are in the U.S. Alere also has a growing presence in key international markets, where Abbott's capabilities and infrastructure will drive accelerated growth of Alere's portfolio.

"Today's announcement marks an exciting and transformative milestone for Alere and one that provides an immediate benefit for our stockholders," said Namal Nawana, president and chief executive officer, Alere. "Our leading platforms and global presence in point-of-care diagnostics, combined with Abbott's broad portfolio of market-leading products, will accelerate our shared goal of improving patient care. I'd like to thank our global workforce of nearly 10,000 employees whose hard work and dedication has enabled Alere to contribute to improved patient outcomes throughout the world."

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ZeptoMetrixto Release NATtrol Inactivated Zika Virus for the Development of Molecular Diagnostic Assays and Ongoing Clinical Laboratory Quality Control

ZeptoMetrix™ Corporation (ZMC), announced today the release of several new products to aid in the rapid development and ongoing quality control of molecular assays targeting the Zika Virus.

"Our level of concern with the Zika Virus outbreak is extremely high," Dr. Gregory R. Chiklis, President and CEO of ZeptoMetrix, relayed to his Executive Team. "We need to support the molecular diagnostic industry with accurate, reliable and non-infectious tools to alleviate the level of uncertainty that exists. These molecular based assays are without a doubt the most sensitive and specific ways to identify and monitor a Zika Virus infection."

To complement its current Tropical Disease microorganism offerings, ZeptoMetrix has added 3 strains of Zika Virus for the development and verification of molecular diagnostic assays. These strains can be used to optimize test sensitivity. Other arboviruses from the ZeptoMetrix collection, such as Dengue Virus, Yellow Fever, and Chikungunya can be used to test that an assay is specific for the Zika Virus.

For clinical laboratory use, ZeptoMetrix has treated Zika Virus with its proprietary NATtrol™ inactivation process to manufacture non-infectious, molecular testing controls and an assay range validation panel. Together, these quality controls products can be utilized to verify test performance, track variations in results, follow lot-to-lot test kit reproducibility, monitor individual operator performance and determine aberrant long-term trends in laboratory results.

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CosmosID Announces Partnership with QIAGEN for Microbial Analysis

CosmosID, a leading genomic big data company, today announced a partnership with QIAGEN to launch its metagenomics analysis platform in QIAGEN's CLC Genomics Workbench. This will complete the comprehensive metagenomics application offered in QIAGEN's microbial genomics solution.  CosmosID will enable QIAGEN's customers to employ shotgun metagenomics and microbiome analysis for fields such as infectious disease, animal health, agriculture, environmental, and food safety.

By adding CosmosID's platform to the CLC Genomics workbench, researchers will be able to rapidly profile microbial communities from whole genome shotgun data, and determine their relative abundance. CosmosID's platform identifies to strain level by placement on a phylogenetic tree and allows comparative analyses of datasets employing heat maps and principal component analysis.

"Researchers running metagenomic, microbial and other applications often need to rely on complicated data analysis tools from different sources", says Laura Furmanski, Senior Vice President of QIAGEN Bioinformatics business area. "The addition of CosmosID expands the range of metagenomics applications that QIAGEN customers can access in our single, user-friendly NGS data interpretation platform."

Chris Mason, Associate Professor in Genetics from Weil Cornell Medical School said, "I have explored the microbiome of everything from the NY subway to outer space and the software analysis component is critically important. CosmosID provides a robust tool for the quantification and detection of microorganisms and antimicrobial resistance markers and we are actively using it for onsite in situ diagnostics."

In the agricultural space, CosmosID's microbiome analysis can be used for increased crop production and better plant performance in harsh or changing environmental conditions. For animal health applications, the technology can support disease diagnostics and outbreak surveillance. Additionally, food safety can be improved through rapid and accurate detection of both common and novel food borne pathogens. With a targeted database for hospital acquired infections (HAIs), hospitals can better monitor, prevent and predict HAI outbreaks.

Rita Colwell, Founder and Chair, says, "As a pioneer in the field of microbial ecology, I am very familiar with the need to understand microbial community structure and function. CosmosID developed an accurate, reproducible system that provides actionable identification of bacteria, viruses, fungi, and parasites, literally within minutes, using DNA sequencing."

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Startup Develops Quick Breath Test for TB Based on UNM Tech

Doctors could soon determine if patients have tuberculosis in less than 10 minutes with a simple breath test being developed by a local startup company with technology from the University of New Mexico.

Avisa Pharma Inc., which licensed the testing process from UNM in 2010, and has been working to develop and market it, conducted its first field test on patients in South Africa last fall. The trial, which included nearly three dozen people in a region where co-infection with HIV and tuberculosis is common, produced rapid results that in some cases were even more accurate than standard sputum smear tests, said Avisa President and CEO David Joseph.

“Our success rate was very high,” Joseph said. “Three of the patients had negative smear tests, but our tests confirmed that those same patients did have tuberculosis. That shows that this test can detect disease with even more sensitivity than today’s standard tests.”

A rapid breath test for tuberculosis could provide an immense boost in global efforts to combat the disease, particularly in Africa and other developing regions where tuberculosis is rampant and access to modern medical facilities is limited.

A report from the World Health Organization released last fall called tuberculosis the world’s leading infectious disease killer, alongside HIV/AIDS, with about 1.5 million deaths in 2014. The organization said a new, rapid diagnostic system is urgently needed to help stem the disease, which currently infects nearly 10 million people globally.

The problem is lack of a reliable testing method for early detection of tuberculosis and to rapidly monitor patient progress on antibiotics to determine if treatments are working, Joseph said. Today’s sputum smear tests generally take days or weeks to confirm disease. The results can be erroneous because the sputum samples don’t provide a full examination of the lungs. And children or patients sick with AIDS and other diseases often can’t produce enough sputum for an effective test.

“We can detect tuberculosis in less than 10 minutes and it doesn’t require sputum,” Joseph said. “Since the test takes just minutes, doctors can also monitor antibiotic treatments to see if they’re working or need to change.”

The breath test was originally developed by Graham Timmins, a UNM professor of medical chemistry and toxicology, and Vojo Deretic, chair of the Molecular Genetics and Microbiology Department.

They created a urea-based drug that turns to carbon dioxide when it comes in contact with bacteria. That happens because a lot of bacteria in the lungs contain an enzyme that breaks down urea to absorb the nitrogen and other elements in it, leaving carbon dioxide behind.

The drug acts as a biomarker for the enzyme. Patients breathe the compound in and, when they exhale, the carbon dioxide levels are measured.

“The breath test shows the presence of the enzyme in the lungs, which shouldn’t be there, and the system says something is wrong and you need to probe further,” Timmins said. “It’s almost like a bacterial thermometer for the lungs.”

The compound can detect the presence of bacteria anywhere in the lungs, making the breath test more accurate than culture-based sputum samples, which examine only limited regions of the respiratory tract, Joseph said.

Since 2010, Avisa has built a portable device to measure carbon dioxide levels as patients breathe in and out. The laptop-sized machine is fairly simple to use, making it ideal for remote, rural locations, said David Karshmer, vice president for technology development.

“It’s a small, lightweight, battery-powered sampling system with a simple user interface,” Karshmer said. “We went into the field to clinics and hospitals to come up with a simple, easy-to-use design that you can put in a backpack and carry anywhere.”

The company now has a prototype, which it used in the South Africa trials, and, by midyear, Joseph expects to send the design to a contract manufacturer in Texas.

Avisa is now preparing for a large, clinical trial for approval by the U.S. Food and Drug Administration and regulatory agencies in other countries. That trial, which would likely take place in summer of 2017, would include up to 1,000 people in the U.S., Europe, China and South Africa.

Once that trial concludes, it could take up to a year to receive FDA approval, potentially paving a path to market by 2018, Joseph said. At that point, Avisa would initiate more trials to apply the technology to detection of other lung diseases, such as cystic fibrosis and chronic obstructive pulmonary disease.

That diversity in potential market applications has helped attract venture investment in the company.

Avisa has raised about $8 million in private equity to date, including a $4 million round of venture investment that closed last year. It’s now raising a later-stage, $10 million round of investment to carry the company through to its clinical trials.

Santa Fe-based Sun Mountain Capital – which manages the State Investment Council’s $150 million co-investment fund for direct investments in startup companies – has contributed several million dollars to Avisa Pharma, said Sun Mountain managing partner Brian Birk.

“We originally got interested in the technology because it has a number of different applications, not just tuberculosis,” Birk said. “And the technology itself is relatively simple, giving it a shorter path to commercial development with a reasonably low amount of capital compared with other pharmaceutical startups. Avisa is one of those local companies that’s been flying under the radar, but really doing some great stuff.”

The company now employs seven people at a 2,000-square-foot office in Santa Fe. It expects to ramp up its workforce over the next year in preparation for the clinical trial.

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UC San Diego Researchers Develop Novel Detection Method for Antibiotics Resistance

A recent article by UC San Diego researchers, published in the journal EBioMedicine, reported the development of a novel, rapid and efficient technique able to determine whether bacteria are resistant to antibiotics in only a few hours.

Pathogenic bacteria are a potential cause of human infections or aggravation of diseases including a variety of lung illnesses such as chronic obstructive pulmonary disease (COPD), pneumoconiosis, bronchiectasis, tuberculosis, and cystic fibrosis. Most of these infections are treated through the use of antibiotics, but bacteria are developing resistance to antibiotics much faster than the development of new anti-resistance therapies.

This results in a substantial number of infections, estimated to cause 2 million illnesses annually, according to the Centers for Disease Control and Prevention (CDC).

From a diagnostic viewpoint, because these infections could progress quickly, it is always critical for physicians to rapidly differentiate between drug resistant and drug sensitive strains, in order to prescribe the appropriate therapy. Furthermore, with the current methods, it often takes days for a physician to receive results related to bacterial resistance. This requires doctors to treat patients based on the assumptions that the infection is drug-resistant.

The new method developed at UC San Diego has two key advantages: It is fast, taking only a few hours for typical culture-based assays, and it does not require any detailed understanding of the bacterium that causes the infection or the genes responsible for the resistance.

The method is based on a previously developed technique in the lab of Kit Pogliano, Ph.D. a biology professor at UC San Diego who headed the research team. “Previously we developed a microscopy-based method that performs an autopsy on bacterial cells that allows us to determine how each cell died, and we have shown that this method can identify new antibiotics and help understand how these antibiotics work,” Pogliano said in a UC San Diego news release.

Researchers tested whether the method could be used to determine antibiotic susceptibility in Staphylococcus aureus strains, bacteria responsible for roughly 60 percent of the hospital-acquired infections, which can cause pneumonia.

“We tested to see if this method could be applied to antibiotic susceptibility testing,” Pogliano said. “Surprisingly, we not only found that our method was able to accurately differentiate sensitive S. aureus strains from resistant MRSA [methicillin-resistant S. aureus] strains, but that we were able to identify two subgroups of MRSA strains, one of which is susceptible to combinations of antibiotics that could be used in the hospital.

“We are excited by the accuracy and speed of this test, as well as by its unanticipated ability to identify these two types of MRSA infections, which would have been missed by other tests,” she said.

Joe Pogliano, Ph.D., also a professor of biology at UC San Diego, said, “Regardless of the type of bacterium, a healthy and growing bacterium looks different from a dead bacterium, so whenever we detect a difference in how the cells look, we know that the bacterium is sensitive to the antibiotic we have applied. When we combine careful culture conditions, cutting edge imaging methods and a detailed quantitative analysis, we can turn this simple approach into a reliable test.”

“Rapid and precise identification of antibiotic sensitivity patterns allows the most potent and effective drug to be administered,” said Victor Nizet, M.D., a professor of pediatrics and pharmacy. “Equally important, more specific antibiotic therapy can help preserve the normal bacteria living in our gut microbiome that play an important role in our health and immune system function.”

The researchers believe the novel method could be applied to various types of bacteria.

“Our new method worked surprising well at rapidly detecting antibiotic resistant strains of S. aureus,” said Diana Quach, a graduate student and lead author of the study. “We are now optimizing it to provide a more accurate test for other types of antibiotic resistant bacteria, such as Pseudomonas aeruginosa.”

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Obama Calls for Rapid Zika Research and Development of Diagnostic Tests as Virus Seen Spreading

President Barack Obama on Tuesday called for the rapid development of tests, vaccines and treatments to fight the mosquito-transmitted Zika virus, which has been linked to birth defects and could spread to the United States in warmer months.

U.S. health officials are stepping up efforts to study the link between Zika virus infections and birth defects, citing a recent study estimating the virus could reach regions where 60 percent of the U.S. population lives.

Obama was briefed on the potential spread of the virus by his top health and national security officials in January.

"The president emphasized the need to accelerate research efforts to make available better diagnostic tests, to develop vaccines and therapeutics, and to ensure that all Americans have information about the Zika virus and steps they can take to better protect themselves from infection," the White House said in a statement.

The virus has been linked to brain damage in thousands of babies in Brazil. There is no vaccine or treatment for Zika, a close cousin of dengue and chikungunya, which causes mild fever and rash. An estimated 80 percent of people infected have no symptoms, making it difficult for pregnant women to know whether they have been infected.

On Monday, the World Health Organization predicted the virus would spread to all countries across the Americas except for Canada and Chile.

In a blog post, National Institutes of Health Director Dr. Francis Collins cited a Lancet study published Jan. 14 in which researchers predicted the Zika virus could be spread in areas along the East and West Coasts of the United States and much of the Midwest during warmer months, where about 200 million people live.

The study also showed that 22.7 million more people live in humid parts of the country where mosquitoes carrying the virus could live year round.

Given the threat, Collins said "it is now critically important to confirm, through careful epidemiological and animal studies, whether or not a causal link exists between Zika virus infections in pregnant women and microcephaly in their newborn babies." Microcephaly results in babies being born with abnormally small heads.

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Sample6 Awarded New Patent for Phage Engineering Detection Technology

Food safety innovator Sample6 announced that it has earned a new patent for its phage-based bacterial detection assay.

The company was awarded its first patent by the U.S. Patent and Trademark Office (USPTO) for its phage engineering technology on January 10, 2016. The new patent, U.S. Patent No. 13/627,060, entitled "Recombinant Phage and Methods" contributes to Sample6's unique process for phage engineering. Phage engineering is at the core of the company's continued food safety innovations, which drive faster, more accurate safety measures.

"As an industry, we need to set a new bar for food safety programs -- widespread illness outbreaks and massive recalls should not happen in this day and age. Contaminated food should be found and stopped before it ever enters the food chain," noted Sample6 CEO Tim Curran. "We are proud that our team of brilliant scientists continues to earn recognition for driving the innovations that will set new standards for food safety programs in the years ahead."

Approved by the USDA, AOAC and plants across the country, Sample6 DETECT/L is the first in-plant, in-shift pathogen detection for listeria. In fact, the company now helps ensure food safety at more than 30 plants in the United States and Canada, including leading ice cream, soy product and consumer packaged meats suppliers.

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Fingerprint to Identify Foodborne Pathogens Moves Closer to Production

A Purdue University innovation that creates a "fingerprint-like pattern" to identify foodborne pathogens without using reagents has been licensed by Hettich Lab Technology.

Hettich Lab Technology designs, engineers and commercializes software and automated incubation systems for identifying pathogens using elastic light scatter techniques that fire lasers at a pathogen colony to create a light-scatter field that gives the pathogen a pattern or fingerprint.

"The use of elastic light-scatter technology could change the way foodborne pathogens are identified," said Klaus-Günter Eberle, Hettich's CEO and general manager. "We are excited about the potential of the technology to advance the process of protecting society from foodborne pathogens."

Foodborne pathogens continue to be an international health and safety concern. In the United States, the Centers for Disease Control and Prevention estimates that one in six Americans, or 48 million people, become ill from foodborne illnesses with 128,000 becoming hospitalized and nearly 3,000 deaths. In addition, an estimated cost of $152 billion in medical expenses, lost productivity and business, lawsuits and compromised branding is attributed to foodborne illnesses.

"Improving our ability to quickly and accurately detect foodborne pathogens is a top priority for the USDA's Agricultural Research Service," said George Paoli, research microbiologist and lead scientist at the USDA-ARS in Wyndmoor, Pennsylvania. "ARS has supported the development of the BARDOT technology through funding and collaboration with Purdue's Center for Food Safety Engineering. The potential applications of BARDOT (Bacteria Rapid Detection using Optical Scattering Technology) for bacterial classification and identification are intriguing, particularly for the facile, rapid and low-cost detection of bacterial foodborne pathogens, because foodborne pathogen identification often takes days to complete using conventional microbiological detection methods."

The USDA-ARS, National Science Foundation and National Institutes of Health all provided funding to the Purdue colleges of Agriculture and Veterinary Medicine to develop the technology, which uses an optical sensor in the detection and identification of foodborne pathogens and other bacteria of interest.

"The development of this innovation is a prime example of how research funding from public and private sources can positively lead to innovations to help our global society," said Dan Hasler, president of the Purdue Research Foundation, which is the licenser of the technology. "We are eager to see this technology come to fruition and move to the public."

The device, called the Bacteria Rapid Detection using Optical Scattering Technology, or BARDOT, has shown great promise in identifying dangerous pathogens such as listeria, staphylococcus, salmonella, vibrio, and E. coli. Since the technology does not require a reagent, it reduces the cost of the pathogen identification. The technology can be used to test any food source for contamination, changing the model for rapid and definitive identification of pathogens.

"The technology can transmit a pathogenic organism fingerprint across the country instantly without the danger of physically transporting the pathogenic organism. This can be achieved without any reagents or assay requirements, which makes this a unique feature for this technology," said J. Paul Robinson, the SVM Professor of Cytomics in the Purdue Department of Basic Medical Sciences, and member of the Purdue Center for Food Safety Engineering who helped develop the technology. "Another attribute is that the technology evaluates every colony on a Petri-dish, so it eliminates or significantly minimizes the sampling bias, and as a result dramatically lowers dramatically the rate of false negatives - something that no other technology in organism identification can claim."

Other innovators of this technology are Dan Hirleman, Purdue's chief corporate and global partnerships officer; Arun Bhunia, professor of food science; Bartlomiej Rajwa, research associate in the Department of Basic Medical Sciences; and Euiwon Bae, senior research scientist in the Department of Basic Medical Sciences. The Purdue Center for Food Safety Engineering, under the leadership of director Lisa Mauer, also contributed to the development of the technology.

"The Purdue Center for Food Safety Engineering, established as a cooperative agreement with USDA-ARS, has a valuable history of combining engineering and microbiological expertise to develop pathogen detection technologies," Mauer said. "The technologies developed by the center translate science and engineering into practical tools for improving the detection of foodborne hazards."

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Warning That UK Lacks Capability to Respond to a Domestic Ebola Outbreak

Britain's response to the Ebola outbreak was marked by “systemic delay” and it is “naive” to think that deadly epidemics that can cross national borders are a thing of the past, a cross-party group of MPs warns in a hard-hitting report published in january.

The committee is concerned that in the “unlikely but possible event of a domestic outbreak” the UK “lacks the capability to go further and manufacture enough vaccines to vaccinate UK citizens in an emergency”.

The MPs state: “Existing facilities are degraded and new plant will take years to build, leaving the UK in a vulnerable position.”

They want the UK to act now and negotiate with vaccine manufacturers to “establish pre-agreed access to manufacturing capabilities that can be called upon quickly in an emergency.

he Westminster Science and Technology committee warns that “delays were evident at every stage” of the UK Government’s response.

The MPs state: “The biggest lesson that must be learnt from this outbreak of Ebola is that even minor delays in responding cost lives. Rapid reaction is essential for any hope of success in containing an outbreak.”

Warning that how the Government responds to global health crises can have a “profound” impact on the lives of people in the UK, they write in today’s report: “While we hope that the world will never experience an Ebola outbreak of this magnitude again, it would be naïve to assume that epidemics with the potential to cause death and devastation, and cross national borders, can be consigned to the past. Our global health policy will have a profound impact on the lives of people in the UK and beyond.

“It is therefore vital that the Government clearly sets out what would trigger an in-country response to a disease emergency and what capability the UK should be able to deploy readily overseas.”

The outbreak, the MPs argue, “highlighted structural weaknesses in the UK’s capacity to absorb and withstand shocks to the system arising from emergencies”.

They want leading experts to publish an “emerging infectious disease strategy” and were “dismayed” to learn that a rapid diagnostic test for Ebola developed by the Defence Science and Technology Laboratory was not released for general use.

The committee wants the Government to “clarify urgently why the rapid diagnostic test for Ebola was not released for use.”

The MPs warn that the UK and other countries were “not ‘research ready’ when the outbreak began” and the response was “uncoordinated”.

They state: “The failure to conduct therapeutic trials earlier in the outbreak was a serious missed opportunity that will not only have cost lives in this epidemic but will impact our ability to respond to similar events in the future.”

The committee members are “not convinced” the UK Government has “looked ahead and considered how a more timely, coordinated and robust response could be achieved when the next epidemic emerges.”

There were “inadequate” systems to share advice and information between the UK and Sierra Leone and this had “harmful repercussions”

On a positive note, the committee states: “The willingness of Government agencies, third sector organisations, health and aid workers, universities, and pharmaceutical companies to go above and beyond to help tackle the outbreak was phenomenal. The swift pace at which clinical trials were approved and conducted particularly stood out.”

A Welsh Government spokesman said: “All four UK countries have developed a collaborative approach to preparing for and responding to emerging infectious diseases, such as Ebola as well as a pandemic. Our NHS has proven experience of dealing with such infectious disease emergencies.

“It is very important to learn lessons from the Ebola crisis, to help inform responses to future crises, both on a national and global level. We will consider this report with our UK partners.”

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New Test Could Detect Elusive Pathogens in Patients at High Infection Risk

Researchers from the University of Pennsylvania have created a test that they say has the potential to quickly detect ambiguous pathogens among patients with compromised immune systems, for whom certain infections can be life-threatening.

In the journal Cancer, Biology & Therapy, study leader Erle Robertson, PhD, professor and vice chair of otorhinolaryngology at the university's Perelman School of Medicine, and colleagues applied the test - called PathoChip - to tissue samples of a patient with relapsed acute myelogenous leukemia (AML).

The patient - a middle-aged man - had undergone chemotherapy for the cancer, a treatment that is well known to weaken the immune system, increasing susceptibility to infection. As a result, he developed an unknown fungal infection.

Such incidents are not uncommon; according to the Centers for Disease Control and Prevention (CDC), each year, around 60,000 cancer patients in the US are hospitalized as result of low white blood cell counts, and around 1 in 14 of these patients die.

It goes without saying that rapid identification of the cause of infection is key to fast and effective treatment for these patients. But this is where a major problem lies; there are certain pathogens for which, at present, doctors lack efficient identification techniques.

One of these pathogens is a rare fungus known as Rhizomucor, which is a cause of the hard-to-treat fungal infection zygomycosis in humans. Such fungal species can take a very long time to culture in the lab, and some cannot be cultured at all, making patient diagnosis challenging.

However, in their study, Robertson and colleagues reveal how they used PathoChip to identify a species of Rhizomucor as the cause of their study subject's "unknown" fungal infection.

PathoChip detected cause of fungal infection in little over 24 hours

The researchers describe the PathoChip as a microarray technology that is capable of testing human tissue for the possible presence of thousands of pathogens.

The technology contains 60,000 probes that simultaneously test for all known viruses, as well as a variety of bacteria, fungi, helminths (parasitic worms) and protozoa.

"We've run many tests to see if we could identify pathogens in the lab, just to see if the PathoChip has efficacy in identifying a variety of organisms, and we were able to identify all infectious agents tested," Robertson said. "But this was the first time we actually looked directly at a patient sample to identify a pathogenic agent."

By using the PathoChip to test preserved tissue samples from the AML patient, the researchers were able to identify one of the two species of Rhizomucor as the cause of the patient's fungal infection in just over 24 hours.

Commenting on the results, the authors say:

"This report highlights the value of PathoChip as a diagnostic tool to identify microorganisms to the species level, especially for those difficult to identify in most clinical laboratories.

It will also help clinicians to obtain a critical snapshot of the infection profile of a patient to plan treatment strategies."

PathoChip 'complementary to next-generation sequencing'

The researchers note that there are other methods - such as next-generation sequencing - that are capable of identifying unknown pathogens, but there are limitations of such technologies.

For example, Robertson says that in the case of next-generation sequencing, there needs to be a high level of nucleic acids in tissue samples in order for pathogens to be identified, and analyzing tissue using such a method is more time-consuming.

"We think this technology [the PathoChip] is complementary to next-generation sequencing in some ways, and even more finely tuned, because we have a much higher sensitivity in detecting agents or individual organisms present in any kind of sample, whether it's abiotic or biotic," says Robertson. "We can identify agents in soil, for example, in plant tissue, in animal tissue, or human tissue."

While the PathoChip holds promise for clinical use, the team says many more studies are required before it can reach that stage. "This will take a great deal more research to eventually lead to approval for frontline clinical use in hospitals," adds Robertson.

Last September, a study reported by Medical News Today revealed the development of a new test called ViroCap, which researchers say can detect almost any virus.

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Rapid Diagnostic Tests Detect Malaria in Pregnant Women When Used Early

Rapid diagnostic tests may be an effective tool to detect malaria among pregnant women in endemic regions, according to data from a recently published trial.

Due to waning sensitivity upon subsequent visits to antenatal care facilities, however, researchers proposed limiting the use of these diagnostics to initial visits to maintain cost efficiency.

New approaches needed as preventive treatments lose strength

For pregnant women living in malaria-endemic African countries, WHO guidance recommends preventive treatments of sulfadoxine-pyrimethamine at each antenatal clinic visit after the first trimester of pregnancy. According to WHO, these treatments have been shown to prevent peripheral parasitemia, maternal anemia and clinical malaria during pregnancy, and are also capable of reducing overall neonatal mortality.

However, Plasmodium falciparum resistant to sulfadoxine-pyrimethamine are becoming more prevalent in many of these affected regions, the researchers wrote, increasing the need for new strategies to limit malaria during pregnancy.

“Intermittent screening and treatment provides a possible approach to the management of malaria in pregnancy in certain epidemiological situations,” the researchers wrote. “There are, however, concerns that infections present at too low a density to be detected with a [rapid diagnostic test (RDT)] could have an adverse effect on the outcome of pregnancy.”

To gauge the sensitivity of Histidine-Rich Protein 2/parasite lactate dehydrogenase (HRP2/pLDH) RDT screening, researchers examined samples and data obtained from a large multicenter trial comparing preventive sulfadoxine-pyrimethamine treatment to screening and treatment. Participating primigravidae and secundigravidae from four West African countries were screened with the HRP2/pLDH RDT during multiple antenatal clinic visits, and treated with artemether-lumefantrine if positive. The researchers re-examined finger prick blood samples collected during each visit for malaria infection using microscopy and PCR assay, and compared these findings to RDT results reported during the primary study. Researchers also reviewed birth outcomes to identify any association between missed infections and consequences including low birth weight, hemoglobin concentration and active malaria infection of the placenta.

RDTs demonstrate improved specificity upon first clinic visit

Malaria prevalence at enrollment as determined by microscopy or PCR was 70% in Burkina Faso, 58.6% in Ghana, 35.9% in Mali and 15.1% in The Gambia, the researchers wrote. The overall sensitivity of the RDT was 87.4% (95% CI, 85.3%-89.4%), and 55.6% of all RDT detections occurred during the first visit to an antenatal clinic. Sensitivity ranged from 90.9% in Burkina Faso to 59.3% in The Gambia, which the researchers wrote may suggest a potential relationship between parasite density and RDT specificity. Among Ghanaian women alone, P. falciparum prevalence was highest at enrollment and RDT sensitivity was 88.8% at the first antenatal clinic, 83.7% at the second, 77.4% at the third and 48.6% at delivery.

The researchers saw no association between RDT results and any of the measured birth outcomes. After adjusting for covariates, the only identifiable risk factor of missed infection was pregnancy with a woman’s second child as opposed to her first (OR = 1.85; 95% CI, 1-3.43).

Taken together, these results led researchers to suggest RDT screening and treatment upon a pregnant woman’s first clinic visit as a cost-effective means to detect and treat many infections with no serious repercussions for the child. However, the diminished test accuracy in less affected regions is a major shortcoming in need of more investigation.

“The lowest sensitivity of the RDT seen in this study was at the site with the lowest transmission; it is not clear if this is a causal association due to lower average parasite densities but this is a possible explanation for this observation,” the researchers wrote. “It is also unclear if sensitivity would decline further over the course of pregnancy in lower transmission settings and this needs to be investigated. As the incidence of malaria falls in many previously highly endemic areas, new approaches to the control of malaria in pregnancy need to be developed and evaluated.”

RDT screening curbs overprescription

Previous data have also shown the potential of RDTs to reduce costs in other areas of malaria care.

In another recently published trial, researchers provided 10 geographic clusters of registered Ugandan drug shops with RDTs instruction to only provide artemisinin-based combination therapy (ACT) upon positive results. The control arm, which also consisted of 10 clusters, treated febrile patients with ACT presumptively. In total, 15,517 eligible patients with fever sought treatment from these 59 enrolled drug shops and were included in the study.

Nearly 60% of patients who purchased an RDT had positive test results, which were verified by the researchers to be 95% accurate. All but 1.5% of patients with negative RDT results adhered to the results and did not receive ACT.

When compared with controls, ACT prescriptions were greatly reduced within the intervention arm (99.8% vs. 62.5%) and resulted in a 72.6% reduction in overprescription.

“Our findings show that it is feasible to collaborate with the private health sector and introduce malaria rapid diagnostic tests in drug shops,” Anthony K. Mbonye, PhD, commissioner of health services at the Ugandan Ministry of Health, said in a press release. “The next step is to refine the strategy and understand the cost implications of scaling it up in Uganda. Our long-term aim is to provide evidence to help [WHO] develop guidance to improve malaria treatment in the private sector.”

Source: Healio Infectious Disease News

References:

Mbonye AK, et al. PLoS One. 2015;doi:10.1371/journal.pone.0129545.

WHO. WHO Evidence Review Group: Intermittent Preventive Treatment of malaria in pregnancy with Sulfadoxine-Pyrimethamine. 2012. www.who.int/malaria/mpac/sep2012/iptp_sp_erg_meeting_report_july2012.pdf.

Williams JE, et al. Clin Infect Dis. 2015;doi:10.1093/cid/civ1198.

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Micro Imaging Technology Adds Staph aureus to its Catalog of Identifiers

Micro Imaging Technology, Inc. announced that its MIT 1000 System can now identify Staphylococcus aureus ("S. aureus"), a bacterial pathogen which can cause skin infections and commonly leads to abscess formation. S. aureus sometimes can also lead to pneumonia, endocarditis, and osteomyelitis. It is also a common food contaminant. "This is a significant step forward for the MIT 1000 technology," said Dr. David Haavig, Micro Imaging Technology's Chief Scientist. "The completion of this S. aureus Identifier, which gives the MIT 1000 System the ability to identify S. aureus, continues to demonstrate the sensitivity of this non-biological bacterial identification technology and adds to MIT's growing catalog of Identifiers. Adding S. aureus to our catalog of Identifier's, gets MIT one step closer to creating an Identifier for the superbug, Methicillin Resistant S. aureus also known as MRSA."

Completing this S. aureus Identifier is a significant milestone in the previously announced collaboration with the Northern Michigan University (NMU) Department of Biology. The goal of this collaboration is to rapidly and cost-effectively identify and differentiate the healthcare threats, S. aureus and MRSA, using the MIT 1000 System.

The MIT 1000 is a rapid, bacterial cell-based detection and identification system that can identify pathogenic bacteria, now including Staph aureus. In addition to this new Identifier, the MIT 1000 can also identify Listeria genus, Staphylococcus genus, Salmonella enterica serotype Choleraesuis (S. Choleraesuis) and Enterococcus faecalis. All MIT 1000 System bacterial identification tests consist of a simple, chemical-free, very low-cost, one-minute sample preparation procedure and a two-minute average hands-off sample measurement.

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Cepheid Announces Diagnostic Collaboration With MedImmune And COMBACTE To Facilitate Clinical Trials Of New Monoclonal Antibodies To Prevent Serious Infectious Diseases

Cepheid announced a collaboration with MedImmune, the global biologics research and development arm of AstraZeneca, and COMBACTE, a European public/private partnership set up to promote the development of new drugs in the anti-infectives field, to develop a series of rapid diagnostic tests to identify Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) in respiratory secretions of mechanically ventilated patients.  These tests will be used to help identify patients for MedImmune's MEDI4893 and MEDI3902 clinical programs, which are being conducted within the COMBACTE consortium to explore the use of biologics in preventing ventilator associated pneumonia (VAP) infections in intensive-care-unit (ICU) patients.

MEDI4893 is a novel monoclonal antibody that targets alpha toxin produced by S. aureus and is currently being investigated by MedImmune and COMBACTE for the prevention of nosocomial pneumonia caused by S. aureus.  MEDI3902 is a bispecific antibody under investigation for the prevention of nosocomial pneumonia caused by P. aeruginosa, a highly drug-resistant bacterium.   The Xpert® tests are expected to help identify patients colonized with S. aureus or P. aeruginosa before they have clinical signs of pneumonia, so that these patients can be enrolled in the respective MEDI4893 or MEDI3902 clinical trials.

"Utilizing rapid diagnostics is a key component in effectively targeting serious healthcare-associated pathogens in our MEDI4893 and MEDI3902 clinical trials," said Steve Projan, head of Infectious Diseases and Vaccines, Innovative Medicines unit at MedImmune.  "We believe that the combination of rapid diagnostics and pathogen-specific antibodies will help physicians identify patients at risk and prevent serious and life-threatening infections in a way that is not possible today.  By developing diagnostic tests through this collaboration with Cepheid, we can ensure that novel life-saving antibodies are delivered to patients who need them in a rapid and efficient manner."

As part of the collaboration, Cepheid has adapted its existing Xpert MRSA/SA skin and soft tissue infection (SSTI) test cartridge for use in respiratory sample types for detection of patients with respiratory colonization with S. aureus and MRSA.  This test is being used in ongoing clinical trials for MEDI4893.  In addition, Cepheid has developed a new Xpert test cartridge to support the rapid identification of patients colonized with P. aeruginosa for patient enrollment in clinical trials for MEDI3902 starting early in 2016.

"Cepheid is pleased to be working with MedImmune and COMBACTE to address the critical challenges posed by serious bacterial infections. We believe this can be achieved through the use of molecular diagnostic tests to precisely target colonized patients, improve clinical trial efficiency, and accelerate these much needed therapeutics to market," said John Bishop, Cepheid's Chairman and Chief Executive Officer.  "These two initial projects give Cepheid an opportunity to demonstrate its technology leadership, while also building an installed base of GeneXpert systems that could be leveraged for future COMBACTE or broader Innovative Medicines Initiative programs."

Dr. Herman Goossens, the head of COMBACTE's laboratory network who is participating in both the MEDI4893 and MEDI3902 trials and evaluating the new assays added, "We are pleased that COMBACTE is supporting the implementation, training and validation of both the S. aureus and P. aeruginosa screening tests.  Our ability to engage the laboratories in the network will ensure timely implementation and standardization of the platform while allowing us to determine the utility of identifying colonized patients before they show clinical signs of pneumonia."

According to the Centers for Disease Control and Prevention, S. aureus and P. aeruginosa together account for >40% of VAP infections in the United States (Sievert DM, et al. 2013).  The length of stay in the ICU is extended on average 17 days after pneumonia onset when either S. aureus or P. aeruginosa are present, and the attributable mortality can reach 30% despite the use of currently available antibiotics.

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