Saturday, December 19, 2015

Electrically Induced Arrangement of Bacteriophages Improves Bacteria Biosensors


Viruses that attack bacteria - bacteriophages - can be fussy: they only inject their genetic material into the bacteria that suit them. The fussiness of bacteriophages can be exploited in order to detect specific species of bacteria. Scientists from Warsaw have just demonstrated that bacteriophage-based biosensors will be much more efficient if prior to the deposition on the surface of the bacteriophage sensor their orientation is ordered in electric field.

In the future, an effective method of detecting a particular species of bacteria will be a bacteriophage-based biosensor. The sensitivity of current sensors coated with bacteriophages, that is, viruses attacking bacteria, is far from ideal. In the journal Sensors and Actuators B: Chemical, researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw, Poland, have presented a method for creating layers of bacteriophages which significantly increases the efficiency of detection. This achievement, funded by the Polish National Centre for Science within SONATA and MAESTRO grants, paves the way for the production of low-cost biosensors, capable of rapidly and reliably detecting specific species of bacteria.

The late detection and identification of bacteria have been - and, unfortunately, still are - the causes of many a tragedy. The lack of reliable and rapid medical tests that, even these days, doctors only find out after several hours which bacterial species is wreaking havoc in the body of the patient. As a result, instead of administering the optimal antibiotic at an early stage of the disease, they have to guess - and often get it wrong, with disastrous consequences for the patient.

"Hospital-acquired infections, to which 100 thousand patients in the United States alone succumb each year, are just some of the problems arising from the lack of good methods for the detection of undesirable bacteria. Industrial contamination is no less important. Nobody wants to sell - much less buy - for example, carrot juice with the addition of dangerous bacteria causing typhoid fever or sepsis. However, such cases continue to occur," says Dr. Jan Paczesny (IPC PAS).

Attempts have been under way for some time to construct sensors to detect bacteria in which the key role is played by bacteriophages. A single phage, with a length of about 200 nanometers, consists of a head (capsid) containing DNA or RNA and a tail through which genetic material is injected into the interior of the bacteria. The mouth of the tail is surrounded by fibrils. They perform a very important function: they are receptors detecting the presence of bacteria and recognizing their species. The bacteriophage cannot take any risks: its genetic material must reach the interior of only those bacteria that have suitably matching genetic machinery. If the phage were to make a mistake and inject its genetic code into the wrong bacteria, then rather than duplicating itself, it would self-destruct.

The specific structure of bacteriophages means that when they are deposited on the surface they are arranged at random, and most of them cannot effectively penetrate the space around them with their receptors in search of bacteria. As a result, only a few bacteriophages in the detection layer of current biosensors can fulfill their role and the equipment's sensitivity is greatly reduced.

"Phage heads are electrically negatively charged, whereas the filaments penetrating the surroundings are positive. The bacteriophage is therefore an electrically polarized entity. This gave us the idea of 'ordering' the bacteriophages using an electric field," says PhD student Kinga Matula (IPC PAS).

The idea was simple, but its implementation proved to be far from trivial.

"There is a high pressure of up to 50 atm in phage heads. This is what enables the bacteriophage to inject its genetic material. That's fine, only that this means that bacteriophages like highly saline solutions, because then the pressure difference between the head and the environment is reduced. Such solutions are highly conductive, and therefore the electric field inside them is present only in a thin layer at the surface, further on it drops to zero. And there is a problem. Fortunately, we have managed to solve it," explains PhD student Lukasz Richter (IPC PAS).

During their experiments, the Warsaw-based scientists, led by Prof. Robert Holyst, used an appropriately selected constant electric field. Bacteriophages were deposited on a carefully constructed glass substrate, coated first with titanium and then with gold. The titanium served as the glue binding the gold with the glass, while the gold was the main 'bait' to which the bacteriophages bound. Unfortunately, not only bacteriophages like gold, so do bacteria. To prevent the binding of random bacteria with the gold layer, the empty spaces between the deposited bacteriophages were covered with a neutral protein (casein).

T4 bacteriophages that attack Escherichia coli bacteria were used to construct the new detection layer at the IPC PAS. The phages for the studies were prepared by the team of Prof. Marcin Los from the Department of Biology, University of Gdansk.

"Virtually all of the bacteriophages in our detection layers stand on the substrate's surface, so they can easily spread out their receptors. The situation is somewhat similar to what is seen at a rock concert, where fans often raise their hands high above their heads in unison and wave them cheerfully in all directions. We have the impression that our phages are even happier, because we try not to place them too close to each other. After all, the neighbours' receptors should not interfere with each other," says Prof. Holyst with a smile.

Meticulous laboratory tests have established that the bacteriophage layers produced using the method developed at the IPC PAS trap up to four times more bacteria than existing layers. As a result, their sensitivity is close to that of the best biosensors that use other, more time consuming and expensive, methods for the detection of bacteria.

The method of preparing layers of ordered bacteriophages developed in Warsaw has numerous advantages. The creation of an external electric field, which is necessary to put the bacteriophages in order, is not very costly. The field acts through space and therefore direct contact of the electrodes with the solution is not required. The presence of an external electric field also means there is significantly less physicochemical interference than in the situation where current is passed through the solution. At the same time, the method is fast and universal: it can be used not only for bacteriophages but also for other electrically polarized molecules.

Department of Homeland Security Bioterror Detection Program Unreliable, GAO Says

Although the US has made significant strides towards improving preparedness for a bioterrorist attack, the nation’s biosurveillance capabilities are a far cry from adequate, according to a recent report by
the Government Accountability Office (GAO), which found he nation’s billion dollar biosurveillance detection system can’t be counted on to actually work.

In April, the House Committee on Homeland Security’s Subcommittee on Emergency Preparedness, Response and Communications said a biological terrorist attack on the US is an "urgent and serious threat."

The US began to recognize a bioterrorist attack as a serious and urgent threat just days after the terrorist attacks of Sept. 11, 2001 when anonymous letters laced with deadly anthrax spores were sent through the mail, sickening 17 people and killing 5 others. The anthrax attacks awakened the nation to the catastrophic impact of a bioterrorist attack in the US.

Shortly thereafter, the Department of Homeland Security (DHS) quickly rolled out a biosurveillance program known as BioWatch to provide early warning of a biological weapon attack in the US. Deployed in more than 30 metropolitan areas throughout the country, the system uses aerosol collectors to detect the intentional release of select aerosolized biological agents.

However, GAO determined the rapid deployment of the program in 2003 did not allow for sufficient testing and evaluation of the system’s capabilities. The report said that without sufficient testing, DHS could not support the claim that the program could meets its operational objective to detect catastrophic attacks, which they define as attacks large enough to cause 10,000 casualties.

“DHS officials told us that in the 12 years since BioWatch’s initial deployment, they have not developed technical performance requirements against which to measure the system’s ability to meet its objective,” GAO stated.

Over the years, numerous false alarms have plagued the program, exasperating local and state officials where the detection system is deployed. From 2003 through 2014, BioWatch generated 149 mistaken detections — all of which have been termed false positives by scientists at the US Centers for Disease Control and Prevention and other experts GAO consulted.

False positives are more than just a mere annoyance. They can lead to the shutdown of major transportation and economic facilities, such as airports and shopping centers, as well as the unnecessary medication of an uninfected public.

“I am supportive of efforts in early detection and mitigation of a biological attack against our homeland,” said Senate Committee on Homeland Security and Governmental Affairs Chairman Ron Johnson (R-Wis.). “However, GAO raises serious questions about the uncertainty in the capabilities of the current BioWatch system. We may be missing opportunities to properly support our biodefense infrastructure.”

The current BioWatch system in use is referred to as Gen-2, which expanded deployment of the system to additional jurisdictions and included the addition of indoor monitoring capabilities in three high-threat jurisdictions. While DHS has taken steps to mitigate the limitations associated with not testing the Gen-2 system in an operational environment with live biothreat agents, GAO determined DHS did not systematically test the Gen-2 system under the most realistic possible conditions.

“Because it is not possible to test the BioWatch system directly by releasing live biothreat agents into the air in operational environments, DHS relied on chamber testing and the use of simulated biothreat agents, which limit the applicability of the results,” GAO’s audit report report stated. “These limitations underscore the need for a full accounting of statistical and other uncertainties, without which decision makers lack a full understanding of the Gen-2 system’s capability to detect attacks of defined types and sizes and cannot make informed decisions about the value of proposed upgrades.”

As soon a DHS deployed the BioWatch program in 2003, they began working on an autonomous detection capability known as Gen-3 in order to reduce operational costs, as well as the time required to detect biothreat agents. DHS envisioned that the system would automatically collect air samples, conduct analysis to detect the presence of biothreat agents every 4 to 6 hours, and communicate results to public health officials via an electronic network without manual intervention.

However, after a GAO audit recommended DHS examine the acquisition process, DHS subsequently commissioned an analysis of alternatives, which was interpreted by DHS as showing that any advantages of an autonomous system over the current manual system were insufficient to justify the cost of a full technology switch.

DHS cancelled the Gen-3 acquisition in April 2014, and made Gen-2 the official program of record for aerosol biological threat detection. In the next year, some Gen-2 equipment will reach the end of its lifecycle; consequently, DHS will need to make decisions about reinvesting in the program.

DHS is considering autonomous detection as an upgrade to Gen-2, with possible benefits including reduction in casualties and clean-up costs. But, GAO said, “The extent of these benefits is uncertain because of several assumptions, such as the speed of response after a detection, that are largely outside of DHS’s control.”

GAO added, “As a result, the effectiveness of the response—and the number of lives that could be saved—is uncertain.”

Consequently, GAO recommended DHS not pursue upgrades or enhancements for Gen-2 until it reliably establishes the system’s current capabilities. Additionally, DHS should incorporate best practices for testing in conducting any system upgrades.

DHS generally concurred with GAO’s recommendations.

“The findings by the GAO bring into focus shortcomings in the BioWatch program at a time when concerns about the threat of a bioterrorism event are elevated,” said House Homeland Security Committee Chairman Michael McCaul (R-Texas), ranking member Bennie G. Thompson (D-Miss.), Emergency Preparedness, Response and Communications Subcommittee Chairman Martha McSally (R-AZ), and Emergency Preparedness, Response and Communications Subcommittee ranking member Donald Payne, Jr. (D-NJ) in a joint statement.

“Earlier this month, the co-chairs of the Blue Ribbon Study Panel on Biodefense testified before our Committee on the threat posed by bioterrorism,” McCaul, Thompson, and McSally added. “They made it clear that that we must act aggressively and deliberately to bolster our ability to detect and rapidly respond to a bioterror event. We also know terrorist groups, like ISIS, aspire to conduct attacks using biological agents. These facts make the GAO’s findings about BioWatch all the more concerning.”

Just weeks ago, Homeland Security Today reported that the bipartisan Blue Ribbon Panel on Biodefense strongly encouraged renewed focus on the need for rapid diagnostics and prioritization of the development of a fully functional environmental detection system to replace BioWatch.

“The entire BioWatch system is dying for lack of innovation,” the report stated. “DHS attempted and failed to acquire next-generation BioWatch technology (Generation 3) that could have reduced time to-detection to as few as six hours. Even if the acquisition had been successful, the system would still have been flawed: like the current system, it would have addressed only a small number of biological agents, inactivated them, and relied on non-random air currents.”

“To date, no fully automated, tested, and evaluated autonomous detection system has been deployed that adequately addresses the airborne biological threat or sufficiently provides operational response information,” the report added.


Bioterrorist attack: hype or reality?

GAO’s report emerged amid a time of heightened concern over the nation's vulnerability to biological terrorism. Moldovan police working alongside the Federal Bureau of Investigation recently uncovered multiple attempts by gangs with suspected Russian connections to sell radioactive material to Islamic State militants.

Homeland Security Today recently reported that the possibility of a bioterrorist event is not simply hype—it is a reality. Of particular concern is the threat of terrorist use of a “dirty bomb,” a type of radiological dispersal device (RDD) that combines conventional explosives such as dynamite with radioactive material like Cobalt 60.

Although terrorists have yet to launch a successful RDD attack on US soil, the threat is real and terrorists have shown an interest in RDDs. The material needed to make a dirty bomb is almost everywhere and the technical sophistication required to create such a device is minimal. Moreover, the intent to create and use such a device is certainly there.

“There can be absolutely no doubt as to the aspirations of terrorist groups, particularly Islamic organizations like ISIS, to acquire and use any and all weapons of mass destruction they can,” said former CIA WMD counterterrorism official Charles Faddis told Homeland Security Today. “ISIS holds an apocalyptic worldview. It is not simply in a battle with the West -- it is in the final battle. They believe the world is literally coming to an end, and any and all means necessary must be employed to ensure they emerge victorious. There is no such thing as too far or too horrific.”

Just last month, the House Committee on Transportation and Infrastructure Subcommittee on Coast Guard and Maritime Transportation recently held a hearing to discuss the vulnerability of US ports to terrorist attacks using a dirty bomb.

During the hearing, Dr. Stephen Flynn, director of the Center for Resilience Studies at Northeastern University, testified that if a dirty bomb ends up in the wrong hands, our country is at grave risk. Currently, there is a real and present danger that containers will be used as modern-day Trojan horses, since the reality is no one really knows what is inside a container except those who are there when the container is packed.

“Should a dirty bomb that originated overseas be set off in a US port, it would represent a major security breech in the global supply system that will result in US port closures,” Flynn said. “This, in turn, will place the intermodal transportation system at risk of widespread economic disruption generating tens of billions of dollars in losses, and potentially endangering lives as the shipments of critical time-sensitive goods such as medical supplies and defense-related materials are interrupted.”

Furthermore, earlier this year, Homeland Security Today conducted an exclusive interview with Brig. Gen. JB Burton, the commanding general of the United States Army 20th Chemical, Biological, Radiological, Nuclear and Explosives Command (CBRNE), in which he stated both ISIS and Al Qaeda’s leaders and their determined affiliates around the world have made it exceedingly clear they seek weapons of mass destruction – especially chemical, biological, radiological and nuclear – to use in attacks on the West – in particular, the United States.

CBRNE and security experts are all saying the same thing: terrorists are intent on using a biological weapon to attack the United States. The failure of US bio-preparedness efforts like BioWatch is not only wasting taxpayer dollars, it is putting the safety of the American people at risk.

“Now more than ever we need reassurances that our efforts to combat and prevent bioterrorism are successful and trusted. It is clear that BioWatch has not lived up to the job it set out to do, and we must put our efforts toward finding a program that will be successful in detecting and preventing these catastrophic attacks,” said full committee chairman Fred Upton (R-Mich.), Oversight and Investigations Subcommittee Chairman Tim Murphy (R-Penn.), and Oversight and Investigations ranking member Diana DeGette (D-Co.) in a statement.

Source: Homeland Security Today.US

Researchers Propose Rapid Ebola Test Using Nanotechnology

Just as Ebola was finally fading from the headlines, it came back in the news with shocking reports: a Scottish nurse rehospitalized nine months after beating Ebola is now suffering from meningitis caused by the virus. A recent study also confirms the virus can live in semen for up to nine months, maybe more.

These stories, added to the continuing trickle of new cases out of Guinea, remind us of the persistence of the deadly virus and the need for quick diagnosis and treatment.

“Recent Ebola virus infection has taken more than 11,000 lives globally and there is still no way to target the virus and kill the disease,” says Ajeet Kaushik, assistant professor in the Center for Personalized Nanomedicine in the Institute of Neuroimmune Pharmacology at the Herbert Wertheim College of Medicine (HWCOM).

Kaushik decided to undertake an extensive review of the Ebola literature to see where and how there may be an improvement in the diagnosis and or treatment of the disease. Ebola Virus Disease (EVD) is lethal. When a patient gets infected with Ebola, the right diagnosis is not, unfortunately, a guarantee that the person will be saved. But recognizing the virus early greatly increases the chance the person will live, and catching it at later stages skyrockets the likelihood they will die.

Kaushik was aided in the review by colleagues Sneham Tiwari, Rahul Dev Jayant, and Center director Madhavan Nair, as well as by Dr. Aileen Marty. The paper,  titled “Toward detection and diagnosis of Ebola virus disease at point-of-care,” was recently published in the journal Biosensors and Bioelectronics: 75, 2016, 254–272.

Marty, a professor of Infectious Diseases in the Department of Medicine, Family Medicine and Community Health at the HWCOM, specializes in tropical medicine, infectious disease pathology and disaster medicine. She took two trips to Africa in 2014 and 2015 to offer aid and expertise in containing and treating the virus and offered a front-line perspective to the review.

The team’s conclusions after summarizing these articles: Early detection of the disease would be a critical tool in helping control the virus and save lives.

“Yes, recent vaccine trials have demonstrated effectiveness in preventing clinical disease, but only with fast detection and equally fast distribution of the vaccine to all those exposed, and the vaccines being tested only exist in limited supplies and have not yet been licensed,” Marty says.

It currently takes six to eight hours to perform the diagnostic testing that will prove or disprove that a patient has Ebola. This must be performed in a laboratory. In the meantime, the caregivers are facing shortages of resources, such as IV fluids and hospital beds, to help the sick while they await their fate.

“Conventional methods of detection are good but slow and should be performed at Biosafety Standard Level 3 laboratories which are expensive to maintain and require skilled personnel,” Kaushik says.

Marty, who was recently appointed to the Presidential Advisory Council on Combating Antibiotic-Resistant Bacteria, also noted that the lack of appropriate safety, work stations and effective selective rapid diagnostics methodologies will continue to be significant challenges in the fight against EBOV. But a cost-effective, rapid, sensitive and selective sensor that can detect Ebola at point-of-care could literally be lifesaving.

“It is important to devise more rapid, more sensitive, but also more specific and appropriate diagnostic assays for Ebola because there are grave social, economic and medical – even lethal consequences –to a misdiagnosis,” Marty says. “This requires that only diagnostics tests that have undergone independent, comprehensive assessment of quality, safety and performance be used in confirming the diagnosis of infection with Ebola virus. While this new nano-technology is extremely promising, we must be very careful to assure that a definitive diagnosis is given only if the specificity is extremely high – a criteria that becomes even more important as the incidence and prevalence of Ebola disease continues to decrease. This nanotechnology is currently likely to be a good screening test but it should still be backed up with nucleic acid testing using technologies such as polymerase chain reaction (PCR).”


Kaushik’s lab has been working on miniaturized sensing technology and integrating that into devices that are capable of detecting virus levels for quite some time. Combining the collaborative strengths of a biologist, nanotechnologist and engineers to develop smart compartments to integrate point-of-care (POC) Ebola sensing devices in a BSL-4 environment should be the aim of future research approach in this area.

“We believe that if we explore this option, the length of time it will take to detect the Ebola virus will be reduced to 40 minutes instead of multiple hours,” Kaushik says.

That’s time well-spent.

Source: Florida International University (Ileana Varela and Robyn Nissim)

Sunday, November 1, 2015

Recent Report Calls for Rapid Diagnostics to Slash Antibiotics Use

Faster diagnostic tools that can distinguish between bacterial and viral infections are urgently needed in the UK to help reign in unnecessary prescriptions for antibiotics, a report by the Review on Antimicrobial Resistance has concluded.

Current diagnostic approaches can take up to 36 hours to get a result, and in many cases antibiotics are prescribed ‘just in case’ they are needed, fuelling the resistance issue.

The review group - established by Prime Minister David Cameron last year to address the ticking timebomb of antimicrobial resistance - is calling for use of new technologies to develop tests that will enable more precise prescriptions and help preserve the effectiveness of the current antibiotics arsenal.

“To avoid the tragedy of 10 million people dying every year by 2050, the world needs rapid diagnostics to improve our use of antibiotics. They are essential to get patients the right treatment, cut down on the huge amount of unnecessary use and make our drugs last longer,” said AMR lead and economist Jim O’Neill.

While some technology that could improve antibiotic use already exists, “it is used too little; and where it is under development, the lack of viable commercial markets and reimbursement mechanisms for the end product means the innovation risks dying on the vine”, O’Neill warned.

'No interest' from drugmakers

Explaining the rather stagnant market, the report notes that drugmakers have “no commercial interest in the advent of rapid diagnostics, which would act to limit the number of antibiotics prescribed,” and compounding the issue further, it is more expensive and more time‑consuming to use a diagnostic rather than simply prescribe a drug ‘just in case’ it is needed, “even if a test could help save costs and reduce waste at a health system-wide level.

To help fuel innovation and activity in the area, the AMR is proposing “a bold, globally-coordinated Diagnostic Market Stimulus pot (DMS)” that would secure a market-based revenue stream for developers of products that match a recognised area of need, and it also calls for greater funding for product developers to support early-stage R&D activities, possibly from the $2-billion, five-year Global Innovation Fund it proposed earlier this year to promote antibiotics research.

The Review will now spend the coming months engaging with governments, NGOs and industry globally to discuss and develop its proposals further, with a more detailed final package of actions to be published in Spring next year covering the whole antimicrobial resistance landscape.

OJ’s Bio’s test

Meanwhile, UK-based OJ Bio says it is developing exactly the sort of rapid diagnostic test technology highlighted the new report.

The group is working on a new point of care diagnostic testing device, Xtalline, that uses special biosensors to identify the presence of various diseases in patient samples, with the results of the tests being displayed on a mobile phone app or healthcare systems.

The new product development programme is already underway that will see the device being used for the detection of C-reactive protein, a protein biomarker of inflammatory disease that can be used to rule out serious bacterial infections and so has potential an effective control tool reducing inappropriate prescribing of antibiotics.

Tuesday, June 16, 2015

Stratos Genomics Develops Disruptive Gene Sequencing Technology

Seattle–based, Stratos Genomics Inc, an innovator in molecular engineering and gene sequencing techniques, announced today the successful demonstration of nanopore sequencing with their proprietary expandable nucleotides (X-NTP™) utilizing a novel single molecule detection method.

This advance–single molecule detection of long DNA sequences–builds upon previous success in which longer 210 and 36 base template sequences were the foundation of earlier work by the company.

Stratos’ Sequencing by Expansion technology (SBX™) represents a new platform for a low cost and more rapid method for whole genome sequencing. In a nutshell, SBXTM “is an efficient, low-cost DNA preparation method that rescales a DNA target into a longer surrogate polymer,’’ according to the company’s recent press release.


This surrogate–referred to as an Xpandomer™–is a reproduction of sequence data using what is known as “high signal-to-noise reporters,” thus allowing rapid identification of specific base sequences. This approach allows for single molecule detection of long sequences using small, low cost, nanopore instruments, also allowing for alternative measurement approaches. Representing a major advance, Stratos’ X-NTP sequencing was developed using a proprietary DNA polymerase to incorporate the expandable nucleotides.



“One year ago, we set a challenging goal to sequence X-NTP based Xpandomers in a nanopore prior to June and we accomplished it,” said Mark Kokorois, Stratos Genomics President and Chief Scientific Officer. “With the fundamental processes in place, our focus is now on optimizing for commercial level performance,” added Kokoris.

Roche , in June 2014, made a key investment in Stratos Genomics embarking on a research collaboration to pursue additional development of the specific technology enabling a transition to single molecule sequencing of DNA fragments with the aid of protein nanopores.

Over the past year, Roche has been partnering with Stratos to develop low-cost and rapid preparation techniques for DNA Xpandomers™. Roche’s expertise in polymerase mutagenesis, design of protein nanopores, modified nucleotide chemistries and rare reagent manufacturing was invaluable to the joint effort.

“The milestone sequencing results are promising,” explained Vinod Makhijani, Vice President and Project Leader, Roche Sequencing Business Development. “While several technical challenges remain on the path towards commercial readiness, we’re optimistic about our ability to tackle them with the joint expertise and resources of the Roche-Stratos team.”

“Roche Ventures and Fisk Ventures both have exercised their right under last year’s Series B financing to purchase additional shares based on our milestone success. Roche will invest an additional $10 million and Fisk Ventures $5 million. This will complete our $30 million Series B funding,” said Allan Stephan, CEO, Stratos Genomics.

The aim of Stratos Genomics, according to Stephan, is to make SBX the preferred method of DNA sequencing by ultimately making the ‘Sequencing by Expansion’ (SBX™) method low cost, rapid and widely available. SBX represents ground-breaking technology, utilizing a single-molecule detection method that removes the limitations of current, potentially error-prone sequencing technologies, essentially by promoting allowing a low-cost, rapid alternative to whole genome sequencing.

There are some companies that have attempted single molecule sequencing in different variations, including Pacific Bio and Oxford Nanopore, explains Stephan. However, Stratos’ patented single molecule detection technology, remains unique in their field of competitors at this stage.

One of the most widespread techniques currently available for genome sequencing–known as sequencing by synthesis (SBS)- was developed by Ilumina, based in Menlo Park, California.

SBS relies on creation of a large “forest” of uniform DNA segments, “almost like a field of the exact same pieces of DNA you are going to be measuring”, explains Stephan. The process involves floating in individual nucleotides with fluorescent capability. When a specific nucleotide is incorporated, your get a burst of light that optics can detect. “It’s a serial process of flooding in these nucleotides and reading the optical signatures that comes out of it”–which can be cumbersome “and involve big machines” described Stephan.

In comparison– in single molecule detection—as Stephan explains, “you don’t have to rely on an enormous ‘forest’ to give you a signal, but instead are getting a signal from the individual expandable base–using an Xpandomer.”

The single molecule detection approach to sequencing clearly represents a potentially disruptive technology in gene sequencing, as Stephan outlines.

“It can enable low cost, rapid, more accurate and targeted and whole genome sequencing, dropping the price into a range that is accessible by essentially everyone,” he concludes.

Source: Forbes

Monday, June 8, 2015

Intelligent Bacteria for Detecting Disease

Another step forward has just been taken in the area of synthetic biology. Research teams from Inserm and CNRS (French National Centre for Scientific Research) Montpellier, in association with Montpellier Regional University Hospital and Stanford University, have transformed bacteria into “secret agents” that can give warning of a disease based solely on the presence of characteristic molecules in the urine or blood. To perform this feat, the researchers inserted the equivalent of a computer programme into the DNA of the bacterial cells. The bacteria thus programmed detect the abnormal presence of glucose in the urine of diabetic patients. This work, published in the journal Science Translational Medicine, is the first step in the use of programmable cells for medical diagnosis.



Bacteria have a bad reputation, and are often considered to be our enemies, causing many diseases such as tuberculosis or cholera. However, they can also be allies, as witnessed by the growing numbers of research studies on our bacterial flora, or microbiota, which plays a key role in the working of the body. Since the advent of biotechnology, researchers have modified bacteria to produce therapeutic drugs or antibiotics. In this novel study, they have actually become a diagnostic tool.

Medical diagnosis is a major challenge for the early detection and subsequent monitoring of diseases. “In vitro” diagnosis is based on the presence in physiological fluids (blood and urine, for example) of molecules characteristic for a particular disease. Because of its noninvasiveness and ease of use, in vitro diagnosis is of great interest. However, in vitro tests are sometimes complex, and require sophisticated technologies that are often available only in hospitals.

This is where biological systems come into play. Living cells are real nano-machines that can detect and process many signals and respond to them. They are therefore obvious candidates for the development of powerful new diagnostic tests. However, they have to be provided with the appropriate “programme” for them to successfully accomplish the required tasks.

The transcriptor: the cornerstone of genetic programming

The transistor is the central component of modern electronic systems. It acts both as a switch and as a signal amplifier. In informatics, by combining several transistors, it is possible to construct “logic gates,” i.e. systems that respond to different signal combinations according to a predetermined logic. For example, a dual input “AND” logic gate will produce a signal only if two input signals are present. All calculations completed by the electronic instruments we use every day, such as smartphones, rely on the use of transistors and logic gates.

During his postdoctoral fellowship at Stanford University in the United States, Jérôme Bonnet invented a genetic transistor, the transcriptor.

The insertion of one or more transcriptors into bacteria transforms them into microscopic calculators. The electrical signals used in electronics are replaced by molecular signals that control gene expression. It is thus now possible to implant simple genetic “programmes” into living cells in response to different combinations of molecules[2].

In this new work, the teams led by Jérôme Bonnet (CBS, Inserm U1054, CNRS UMR5048, Montpellier University), Franck Molina (SysDiag, CNRS FRE 3690), in association with Professor Eric Renard (Montpellier Regional University Hospital) and Drew Endy (Stanford University), applied this new technology to the detection of disease signals in clinical samples.

Clinical samples are complex environments, in which it is difficult to detect signals. The authors used the transcriptor’s amplification abilities to detect disease markers, even if present in very small amounts. They also succeeded in storing the results of the test in the bacterial DNA for several months.

The cells thus acquire the ability to perform different functions based on the presence of several markers, opening the way to more accurate diagnostic tests that rely on detection of molecular “signatures” using different markers.
Principle of the use of modified bacteria for medical diagnosis. ©J. Bonnet/ Inserm.

“We have standardised our method, and confirmed the robustness of our synthetic bacterial systems in clinical samples. We have also developed a rapid technique for connecting the transcriptor to new detection systems. All this should make it easier to reuse our system,” says Alexis Courbet, a postgraduate student and first author of the article.

As a proof of concept, the authors connected the genetic transistor to a bacterial system that responds to glucose, and detected the abnormal presence of glucose in the urine of diabetic patients.

“We have deposited the genetic components used in this work in the public domain to allow their unrestricted reuse by other public or private researchers,[3]” says Jérôme Bonnet.

“Our work is presently focused on the engineering of artificial genetic systems that can be modified on demand to detect different molecular disease markers,” he adds. In future, this work might also be applied to engineering the microbial flora in order to treat various diseases, especially intestinal diseases.

This work received financial support from Inserm, CNRS, the Stanford-France Center for Interdisciplinary Studies, and Stanford University. Jérôme Bonnet is a recipient of the Atip-Avenir programme award, and is supported by the Bettencourt-Schueller Foundation.

[1] aimed at the rational engineering of artificial biological systems and functions
[2] (Bonnet et al. Science, 2013).
[3] available on: https://biobricks.org/bpa/

Source: Inserm

Thursday, May 28, 2015

New Chip Makes Testing for Antibiotic-Resistant Bacteria Faster, Easier

It’s a device that could transform a doctor’s ability to treat infections: a test for antibiotic resistance that works in just one hour – instead of several days.

We live in fear of ‘superbugs’: infectious bacteria that don’t respond to treatment by antibiotics, and can turn a routine hospital stay into a nightmare. A 2015 Health Canada report estimates that superbugs have already cost Canadians $1 billion, and are a “serious and growing issue.” Each year two million people in the U.S. contract antibiotic-resistant infections, and at least 23,000 people die as a direct result.

But tests for antibiotic resistance can take up to three days to come back from the lab, hindering doctors’ ability to treat bacterial infections quickly. Now PhD researcher Justin Besant and his team at the University of Toronto have designed a small and simple chip to test for antibiotic resistance in just one hour, giving doctors a shot at picking the most effective antibiotic to treat potentially deadly infections. Their work was published this week in the international journal Lab on a Chip.

Resistant bacteria arise in part because of imprecise use of antibiotics – when a patient comes down with an infection, the doctor wants to treat it as quickly as possible. Samples of the infectious bacteria are sent to the lab for testing, but results can take two to three days. In the meantime, the doctor prescribes her patient a broad-spectrum antibiotic. Sometimes the one-size-fits-all antibiotic works and sometimes it doesn’t, and when the tests come back days later, the doctor can prescribe a specific antibiotic more likely to kill the bacteria.

“Guessing can lead to resistance to these broad-spectrum antibiotics, and in the case of serious infections, to much worse outcomes for the patient,” says Besant, pictured at right (photo courtesy U of T Engineering). “We wanted to determine whether bacteria are susceptible to a particular antibiotic, on a timescale of hours, not days.”

The problem with most current tests is the time it takes for bacteria to reproduce to detectable levels. Besant and his team, including his supervisor Professor Shana Kelley of the Institute for Biomaterials & Biomedical Engineering and the Faculties of Pharmacy and Medicine, and Professor Ted Sargent of The Edward S. Rogers Sr. Department of Electrical & Computer Engineering, drew on their collective expertise in electrical and biomedical engineering to design a chip that concentrates bacteria in a miniscule space – just two nanolitres in volume – in order to increase the effective concentration of the starting sample.

They achieve this high concentration by ‘flowing’ the sample, containing the bacteria to be tested, through microfluidic wells patterned onto a glass chip. At the bottom of each well a filter, composed of a lattice of tiny microbeads, catches bacteria as the sample flows through. The bacteria accumulate in the nano-sized well, where they’re trapped with the antibiotic and a signal molecule called resazurin.
Living bacteria metabolize resazurin into a form called resorufin, changing its electrochemical signature. If the bacteria are effectively killed by the antibiotic, they stop metabolizing resazurin and the electrochemical signature in the sample stays the same. If they are antibiotic-resistant, they continue to metabolize resazurin into resorufin, altering its electrochemical signature. Electrodes built directly into the chip detect the change in current as resazurin changes to resorufin.

“This gives us two advantages,” says Besant. “One, we have a lot of bacteria in a very small space, so our effective starting concentration is much higher. And two, as the bacteria multiply and convert the resazurin molecule, it’s effectively stuck in this nanolitre droplet – it can’t diffuse away into the solution, so it can accumulate more rapidly to detectable levels.”

“Our approach is the first to combine this method of increasing sample concentration with a straightforward electrochemical readout,” says Sargent. “We see this as an effective tool for faster diagnosis and treatment of commonplace bacterial infections.”

Rapid alternatives to existing antibiotic resistance tests rely on fluorescence detection, requiring expensive and bulky fluorescence microscopes to see the result.

“The electronics for our electrochemical readout can easily fit in a very small benchtop instrument, and this is something you could see in a doctor’s office, for example,” says Besant. “The next step would be to create a device that would allow you to test many different antibiotics at many different concentrations, but we’re not there yet.”

Source: University of Toronto

AgriLife Research Engineer Develops Real-Time Listeria Biosensor Prototype

A Texas A&M AgriLife Research engineer and a Florida colleague have developed a biosensor that can detect listeria bacterial contamination within two or three minutes.

“We hope to soon be able to detect levels as low as one bacteria in a 25-gram sample of material – about one ounce,” said Dr. Carmen Gomes, AgriLife Research engineer with the Texas A&M University department of biological and agricultural engineering, College Station.

The same technology can be developed to detect other pathogens such as E. coli O157:H7, she said. But listeria was chosen as the first target pathogen because it can survive even at freezing temperatures. It is also one of the most common foodborne pathogens in the world and the third-leading cause of death from food poisoning in the U.S.

“It can grow under refrigeration, but it will grow rapidly when it is warmed up as its optimum growth temperature ranges from 30 to 37 degrees Celsius — 86 to 98 degrees Fahrenheit,” Gomes said. “This makes it a particular problem for foods that are often not cooked, like leafy vegetables, fruits and soft cheeses that are stored under refrigeration.”


Currently, the only means of detecting listeria bacteria contamination of food requires highly trained technicians and processes that take several days to complete, she said. For food processing companies that produce and ship large quantities of foodstuff daily, listeria contamination sources can be a moving target that is often missed by current technology.

The biosensor she is working on is still in the prototype stage of development, but in a few years she envisions a hand-held device that will require hardly any training to use.

Gomes is collaborating with Dr. Eric McLamore at the University of Florida at Gainesville.

“I do the biological and polymer engineering; he does the electrochemistry and nanostructures,” she said.

As for the biological component, Gomes said she is using “nanobrushes” specially designed to grab particular bacteria.

The nanobrushes utilize “aptamers,” which are single-stranded DNA or RNA molecules that bind to the receptors on the target organism’s cell outer membrane, Gomes said. This “binding” is often compared to the way a key fits into only one lock.

In this manner, the nanobrushes select for only a specific type of cell, which in the case of her work is the listeria bacterium.

Gomes noted that the inspiration for the nanobrushes comes from the Hawaiian bobtail squid, a football-sized creature that forms a symbiotic relationship with bioluminescent bacteria. Microscopic, hair-like structures, called cilia, on the squid’s light organ select and capture the bacteria from a very complex microbial soup of the ocean.

“The squid feeds the bacteria sugar and amino acids and in return, the bioluminescent bacteria allow the squid to produce light, which then allows the squid to escape from things that might want to eat it,” she said. “To predators, the bioluminescence is very similar to the light coming from the moon and stars at night, which acts as a ‘camouflage’ when observed from below.

“The selection process the polymers use to select for specific bacteria in the listeria biosensor is very similar to the squid’s cilia. We are trying to mimic the same mechanism of bacteria’s capture used by the squid’s cilia.”

Currently, the listeria biosensor is about the size of a postage stamp, with two wires leading to two etched conductive areas. After a few minutes, when the polymer nanobrushes have had time to grab the selected bacteria, the rest of the sample is washed away and the impedance, or resistance, between the two surfaces is measured electronically.

Gomes and McLamore are moving on to refining the electronics to something that can be handheld and easily used. Also in the works is a disposable paper-based biosensor that can be disposed of after one use.

In early April, they were awarded a three-year $340,000 National Science Foundation grant to continue their work on nanobrushes for pathogen detection.

Source: AgriLife TODAY

Sunday, May 24, 2015

Colorado Researchers Fighting to Get Ahead of the Next Ebola Outbreak

ReEBOV may be one of the most important technologies developed by Corgenix Medical Corp., but it's not a financial boon for the 25-year-old diagnostic test firm.

Company officials would just as soon keep it that way.

ReEBOV this year became the first rapid diagnostic test to gain U.S. and international approval for use to detect the deadly Ebola virus.

In the past couple of weeks, more than 10,000 of the kits — which bear similarity to pregnancy tests and can detect the presence of Ebola in blood in less than 15 minutes, instead of hours or days — were shipped to clinics and hospitals across the globe.


"It would be highly unlikely that this product would ever be an enormous commercial success," said Douglass Simpson, the former Corgenix chief executive who now oversees the Broomfield firm's infectious-diseases group. "Certainly, we would hope not."

Since last summer, Ebola, which has no known cure or vaccine, has killed more than 11,000 people, mostly in West Africa, according to the Centers for Disease Control and Prevention.

But the worst-ever outbreak is waning, and that has Corgenix and other Colorado researchers racing to test their products before the disease goes dormant again.

Their aim: to develop an artillery of therapies and technologies along with a robust, data-driven network to track and combat Ebola the next time it rears its head. (Some countries, including Liberia, have been declared Ebola-free.)

"You can imagine that even before (the Ebola outbreak) that the health care infrastructure there needed some upgrading," said Asher Greenberg, Ebola project coordinator and communications officer for Fio Corp., a mobile diagnostics firm based in Canada. "The crisis has brought out a number of companies with solutions, but all of this needs to be integrated."

Corgenix's work, funded mostly by grants, includes adapting its ReEBOV test to be compatible with Fio's Deki Reader. The reader is an 8-inch-by-4-inch mobile device that analyzes and transmits data from immunoassay tests, like ReEBOV, that have been fit into a plastic housing about the size of a microcassette tape.

Researchers from Fio and Corgenix are in West Africa now, testing the new products in the lab and the field.

"This is a great time, with all of the attention still on West Africa, to update the infrastructure so that the next outbreak can be handled with less loss of life," Greenberg said.

Although the Ebola epidemic was thousands of miles away from the U.S., its severity spurred agencies such as the National Institutes of Health and organizations including the Bill & Melinda Gates Foundation and the Paul G. Allen Family Foundation to pour billions of dollars into the fight.

Several million dollars went to Corgenix, a 55-employee company that has developed a cache of more than 50 diagnostic products spanning a variety of diseases.

Financial backing

Corgenix, in partnership with the Viral Hemorrhagic Fever Consortium, had success in bringing to market a rapid diagnostic test for Lassa, another deadly hemorrhagic fever, and dipped its toes in developing a similar test for Ebola.

When the latest Ebola outbreak worsened, Corgenix's Ebola research was brought to the frontline.

The Gates and Allen foundations both provided financial backing to integrate the Corgenix and Fio technologies. The aim not only was to identify and then isolate Ebola-infected individuals but also to gather data that would allow health officials to monitor potential hot spots and get a leg up on containment.

Matt Boisen, Corgenix's program director for infectious disease and emerging technologies, last week began his ninth research trip to West Africa. Before the Ebola outbreak, Boisen conducted research in Kenema, Sierra Leone, related to Corgenix's Lassa rapid test.

Boisen and other Corgenix employees and consortium research partners volunteered for the work, said Simpson, who moved from the role of CEO to consultant after Corgenix's acquisition last year by Orgentec.

"Thank goodness for people who are willing to go," said Simpson, who also has traveled to the region on behalf of Corgenix. "I don't think anyone is not fully aware of the dangers. If something were to happen, I'd carry that. I'd blame myself forever."

There are elements of risk in traveling to countries roiled by political unrest and to regions battling deadly diseases with limited health infrastructure. Despite a strict regimen of taking medications and applying skin protection from mosquitoes, Boisen returned from one of his trips and landed in a hospital with a case of malaria, said his wife, Shelly.

"He did go to Nigeria one time, and Matt and (other researchers) were escorted by several men with AK-47s," she said. "The whole time, all I'm thinking of is 'Are you safe? Are you safe? Are you safe?' "

On his trip earlier this year, Boisen saw how Ebola's spread overwhelmed the Kenema Government Hospital, effectively shutting it down temporarily.

Ebola killed several of his friends and associates at the Kenema hospital and lab.

"But that's why we take the precautions that we do," he said.

Boisen's work in Kenema will be centered entirely in the Lassa Fever Laboratory at the Kenema hospital, and any samples tested will take place under a protective hood.

Boisen will be wearing protective gear as he works, which helps lift some of the emotional weight that builds when he travels to hot zones, his wife of 14 years said.

"I know he's safe," said Shelly Boisen, who works as a certified nursing assistant at Longmont United Hospital. "He actually did a video for us the last time he went down. I'm going to give it to our hospital (as an example of) precaution training. It took him about 20 minutes to put on the first layer; he duct-tapes the shirt, puts on gloves, duct-tapes the gloves onto the sleeves, and puts on the helmet."

She's also buoyed by the understanding that the dangerous work being done by her husband and others is for the greater good.

"There are good people over there that are also helping and putting their lives on the line, and he's just one of them," she said. "He's done a lot of this in the company, too, in the lab — test after test after test after test to perfect it."

Complementing the on-the-ground work in Africa by Corgenix, Fio and others is Ebola-related domestic research such as that occurring at Colorado State University.

Grants for research

Mathematics professor Michael Kirby and the university's Biopharmaceutical Manufacturing and Academic Resource Center, or BioMARC, each received grants to conduct Ebola-related research.

BioMARC was awarded a $2 million subcontract from the U.S. Department of Defense in October to develop and manufacture a vaccine to protect against filoviruses, including Ebola and Marburg.

"The project is moving forward with successes that are in-line with product expectations," Dennis Pierro, BioMARC director, said in a statement. "We are advancing the product toward the objective of the U.S. Department of Defense."

BioMARC officials said they expect to provide more information in the coming months.

The separate mathematics-focused study has generated some positive initial results, said Kirby, a professor in CSU's departments of Computer Science and Mathematics.

Kirby previously applied mathematical algorithms to data collected from patients infected with the H1N1 influenza virus to try to understand how the flu virus moved through and took root in the immune system.

Analyzing the behavior of the more than 1,400 genes involved, Kirby helped to show the genetic pathway of the virus and identify the point at which an affected person became symptomatic.

"What we discover with influenza we hope will carry over to the Ebola virus," Kirby said.

Kirby's team does not collect the data but rather uses data sets that already are in the public domain, including that of infected mice and nonhuman primates.

The early results from the mice data showed some pathways that were comparable to human influenza infection.

"For Ebola, it's very interesting," he said. "There are no tests for Ebola until you've become symptomatic."

And it can be weeks after infection before symptoms such as a fever start to show.

Finding those pathways could bolster diagnostic and early warning tests and, ideally, limit the spread of the disease, he said.

"It's kind of like a canary in a coal mine," he said.

SOURCE: The Denver Post

Thursday, April 16, 2015

New Biosensing Platform Could Quickly and Accurately Diagnose Disease and Monitor Treatment Remotely

In much the same way that glucometers and pregnancy tests have revolutionized in-home diagnostic testing, researchers from Florida Atlantic University and collaborators have identified a new biosensing platform that could be used to remotely detect and determine treatment options for HIV, E-coli, Staphylococcus aureas and other bacteria. Using a drop of blood from a fingerprick, this novel biosensing platform provides clinically relevant specificity, sensitivity and detection of pathogens from whole blood and plasma.

The thin, lightweight and flexible materials developed by these researchers can be fabricated and operated without the need for expensive infrastructure and skilled personnel, potentially solving real-world healthcare problems for both developed and developing countries. Using this technology, they also have developed a phone app that could detect bacteria and disease in the blood using images from a cellphone that could easily be analyzed from anywhere in the world. Please click on the top image to enlarge. 

Waseem Asghar, Ph.D., assistant professor of electrical engineering in the College of Engineering and Computer Science at FAU, co-first author on the study, along with Hadi Shafiee, Ph.D., instructor in medicine at the Division of Biomedical Engineering at Brigham and Women's Hospital, Harvard Medical School; Fatih Inci, Ph.D.; and Utkan Demirci, Ph.D., Stanford School of Medicine, senior authors on the study, have published their findings in Nature Scientific Reports in an article titled “Paper and Flexible Substrates as Materials for Biosensing Platforms to Detect Multiple Biotargets.” Other team members on the study include Mehmet Yuksekkaya, Ph.D.; Muntasir Jahangir; Michael H. Zhang; Naside Gozde Durmus, Ph.D.; Umut Atakan Gurkan, Ph.D., and Daniel R. Kuritzkes, M.D.

In the article, the researchers address the limitations of current paper and flexible material-based platforms and explain how they have integrated cellulose paper and flexible polyester films as new diagnostic tools to detect bioagents in whole blood, serum and peritoneal fluid. They employed three different paper and flexible material-based platforms incorporated with electrical and optical sensing modalities. They were able to demonstrate how these new materials can be widely applied to a variety of settings including medical diagnostic and biology laboratories.

Using paper and flexible substrates as materials for biosensors, Asghar and his colleagues have identified a new rapid and cost-effective way to diagnose diseases and monitor treatment in point-of-care settings. They have been able to show how their new platforms are uniquely able to isolate and detect multiple biotargets selectively, sensitively, and repeatedly from diverse biological mediums using antibodies.


Please click on the image to enlarge.

“There is a dire need for robust, portable, disposable and inexpensive biosensing platforms for clinical care, especially in developing countries with limited resources,” said Asghar.

Existing paper and flexible material-based platforms use colorimetric, fluorometric and electrochemical approaches that require complex labeling steps to amplify their signal, are very costly to fabricate and also require expensive equipment and infrastructure.

“The future of diagnostics and health monitoring will have potentially cell-phone based or portable readers sipping saliva or blood and continuously monitoring human health taking it way beyond where we are with counting steps today,” said Demirci, who is the corresponding author.

Asghar notes that because their materials are easy to make, easy to use, and can easily and safely be disposed by burning, they provide appealing strategies for developing affordable tools that have broad applications such as drug development, food safety, environmental monitoring, veterinary medicine and diagnosing infectious diseases in developing countries.

“Our paper microchip technologies can potentially have a significant impact on infectious diseases management in low- and middle-income countries where there is limited laboratory infrastructure,” said Shafiee.

Demirci notes that these platforms could potentially be adapted and tailored to detect other pathogens and biotargets with well-known biomarkers.

Source: Florida Atlantic University

Sunday, March 29, 2015

A Review of the USP Workshop on Alternative Methods

Last week, the USP hosted a workshop on alternative microbiological methods. Dr. Tony Cundell, a member of the microbiology expert committee, briefly summarized the workshop and the status of the revision to chapter 1223 in the Rapid Microbiology Methods LinkedIn Group:

The USP Alternative Microbiological Methods Workshop, March 16-17 at USP Headquarters attracted 66 registrants. The USP Microbiology Expert Committee presented revisions to USP<1223> 
The revisions included a discussion on the limitations of the CFU, acceptable procedures, and performance, results and decision equivalence option. Also the concept of the non-inferiority test was promoted as a statistical tool. The revision will be published June, 2015 with an official date of December, 2015. 
Invited speakers from the FDA, JP, the pharmaceutical industry and academia generally supported the revisions.

I appreciate Tony’s post.  I also attended this workshop and although the invited speakers, as he stated, generally agreed with the revision, a number of attendees (including myself) were left with unanswered questions and an uncertainty of the appropriateness of the revised chapter 1223 as well as a proposed chapter on a rapid sterility test.  But there were also productive presentations that provided an overview of current regulatory expectations, the use of statistics and industry’s perspectives on validation.  A brief summary of my take away messages from the meeting is presented below.

Erika Pfeiler (CDER, FDA) described the Agency’s focus on alternative microbiology methods (AMMs). She reviewed FDA’s policies and stated that CDER has approved AMMs for water testing, environmental monitoring, bioburden testing, microbial limits (for release and stability) and sterility testing (for release and stability). She also stated that FDA welcomes submissions for the use of AMMs, they are routinely approved, different approaches to validation are acceptable and validation studies should depend on your product and process.  I agree with Erika’s position and continue to appreciate the Agency’s support for the implementation of new microbiology technologies.

Stephen Wicks (EDQM) reviewed the current revision to Ph. Eur. chapter 5.1.6. This draft is currently available for public review (through Pharmeuropa) and you can provide comments until March 31. I am encouraged by Stephen’s statement that the revised chapter 5.1.6 will be fairly aligned with PDA’s Technical Report No. 33 (TR33).

Nobuyasu Yamaguchi (Osaka University) provided an overview of the Japanese Pharmacopeia’s (JP) new informational chapter on rapid microbiological methods (RMM). The chapter will be published in the 17th edition of the JP with an English version being provided in the summer of 2015. When speaking on validation, he stated that the RMM must be equivalent or better than the current method. However, he also stated that a correlation between the RMM and the current method would not be necessary. When I asked him how you could demonstrate the RMM is equivalent or better than the current method without correlating the two, his response was that the validity of the new method should be confirmed. When asked for an example, he could not think of a RMM that would fit this scenario.  Therefore, in my opinion, the JP should consider providing additional clarification regarding the demonstration of equivalence.

Edwin van den Heuve (Eindhoven University of Technology) presented a detailed review of how to use non-inferiority models when statistically comparing data sets arising from AMM validation studies.  I thought his presentation was well received and his recommendations offer the industry additional statistical tools when validating AMMs.

Separately, representatives from the USP expert committee provided their overviews of changes to USP 1223 and a new proposed chapter, 71.1.

James Akers summarized the purpose for the changes to USP 1223. He stated that microbiology analysis is transitioning from largely growth-based methods to methods that are based more on molecular biology. The term “molecular biology” was also used in the revision process.  When questioned to clarify if the term “molecular” meant nucleic acid amplification techniques (NAAT), he responded that the term was not limited to NAAT but to methods that worked “at the molecular level.” It is my opinion that this term should be better defined in chapter 1223 as many technologies may work at the molecular level.

He then described the CFU as a cell count estimate, which we all agreed on, since the CFU may arise from a single cells or a clump of cells. Following an extended discussion on the CFU, he stated that the CFU can no longer be used as the “Gold Standard” for microbial enumeration, and that the true nature of the CFU be considered when working with AMMs that provide alternate signals. I generally agree with his position.

He mentioned that a technology that provided enhanced colony detection (based on growth) would not require validation as a completely new method. I also agree, and PDA’s TR33 described these types of methods as automated technologies.

Next, he stated as we move away from growth-based methods the traditional route to microbial identification is not possible and that a laboratory can use specific PCR probes for this process.  I do not necessarily agree with this position because today’s PCR probes are generally used for presence/absence testing for a specific microbial target of interest (e.g., E. coli) and not for microbial ID unless PCR is used in conjunction with gene sequencing. Furthermore, if the starting material is DNA, the potential for detecting residual DNA or DNA from dead cells can lead to a false positive response. This is why most PCR-based systems require a sufficient amount of viable cells (e.g., from an isolated colony) to avoid this phenomenon. We may have to wait for the publication of the final revision of chapter 1223 to understand the direction the USP is taking.

Edward Tidswell subsequently went into greater detail on the changes to chapter 1223. Essentially, his presentation was a comparison of the original USP 1223 with the proposed revision that was published in 2014 for public comment.  He stated the core intent of the revision was about patient safety and a paradigm shift to unshackle ourselves from the CFU and to embrace a concept of “decision quality.” That’s good. He also stated the revision simplifies the validation process. I disagree. The draft was very confusing to follow and was the reason for my submission of a comprehensive list of comments and questions to the committee last year. I also understand that others in the industry, including organizations such as the PDA, responded with similar comments.  Unfortunately, Ed did not address any of the industry’s responses sent to USP during the public comment period, and now we must wait until the final revision is published mid-year to determine if our comments were adequately addressed.

Following Ed’s presentation, one attendee asked what would happen if the published chapter still requires clarification in order to understand the document’s guidance and recommendations. The response from USP was as follows:  the chapter can be revised in the next revision cycle (i.e., 2015-2020). In my opinion, this is not an efficient process considering the industry voiced its opinions last year and attendees repeated a number of their submitted comments during the workshop. For example, David Roesti (Novartis) commented (during this year’s workshop and during last year’s public comment period) that the recommended use of a 0.2 delta value when calculating non inferiority was too strict. He also stated that they successfully used a 0.3 value when validating a rapid sterility test and FDA and EMA approved their approach.  However, if they would apply the 0.2 value to their test data, the test for non inferiority would fail. USP’s response to his comments was as follows: we should address this ASAP…but it probably won’t happen until after the final revision is published. I should note that David Roesti also presented his company’s work on validating a rapid sterility test that was approved by more than 50 countries including the US and EU. It is my opinion that the USP should align their recommendations (sooner rather than later) with the validation strategies that have been accepted by the global regulators.

Tony Cundell followed with an overview of the equivalence section of the draft 1223 chapter. He opened his presentation by stating that existing method validation approaches are limiting RMM implementation. I disagree. A number of companies around the world have successfully validated alternative and rapid methods, including those used for sterility testing, which have gained FDA and EMA approvals by following the validation guidance in documents such as PDA’s TR33.

He then walked the attendees through the same table for equivalence that was presented in the 2014 draft chapter.  The four equivalency options are still extremely confusing and it is not clear from his presentation whether the final draft will provide additional clarification on how each of these options are to be applied.

The next topic focused on a proposed rapid sterility test chapter numbered 71.1. According to James Akers, the purpose of such a chapter is to encourage the development of a rapid sterility test for stakeholders that require a time-to-result that is much faster than the conventional 14-day assay.  Stakeholders were identified as compounding pharmacies, cell therapy companies and firms that produce radiopharmaceuticals. During presentations by James Akers and Tony Cundell, it was disclosed that the USP will advocate a limit of detection (LOD) of 10-100 cells for a rapid sterility test, which, as the presenters explained, will provide an acceptable level of patient safety for those products that have a very limited shelf life. In fact, the USP expert committee is working towards writing a new chapter describing a compendial rapid sterility test based on reverse transcriptase (RT)-PCR (using universal primers and probes) and flow cytometry that would take between 6 to 48 hours to complete. Tony also stated that we cannot have a rapid sterility test that only specialized labs can perform.  I agree; however, I do have additional concerns regarding their position.

First, the selection of RT-PCR and flow cytometry may be premature at this time. The USP based the selection of these two platforms on a stakeholder survey in which six respondents recommended RT-PCR and two respondents recommended flow cytometry. That’s a total of eight (8) stakeholders. In my opinion, the numbers of respondents does not necessarily reflect the views of the industry as a whole.

Furthermore, I do not believe that RT-PCR may be as sensitive or robust as what may be required for a rapid sterility test.  The supplier of a commercial, off-the-shelf (COTS) RT-PCR system has previously reported their LOD at 100-1000 cells, which may be too high for an acceptable rapid sterility test. Interesting, this same system was removed from the market two years ago due to issues associated with robustness and sensitivity.  Additionally, flow cytometry may not possess the required LOD for a rapid sterility test (e.g., some flow cytometry systems report a sensitivity level of at least 100 cells with good repeatability). Therefore, additional work in this area is warranted. It is also my opinion that a rapid sterility test that is longer than 48 hours (e.g., 72 hours) may still be acceptable for the stakeholders while meeting the needs of the patient. For this reason, the end-user should dictate the required time-to-result, not the USP.

Second, it is my opinion that an acceptable LOD of 10-100 cells may not necessarily be aligned with regulatory expectations. During the same workshop, Simleen Kaur (CBER, Office of Compliance and Biologics Quality, FDA) presented an overview of technologies that the Agency examined while developing a rapid sterility test. She stated that studies were conducted using 1 and 10 cells, but that she did not know at what level FDA would ultimately accept in terms of LOD for a rapid sterility test (i.e., 1 or 10 cells).  Interestingly, James Akers was quick to interject that we need to be very careful about using a single cell level as an appropriate LOD for a rapid sterility test. Only time will tell what organization will really take the lead in this debate.  I will say; however, that multiple companies around the world have successfully validated (and have gained regulatory approval for) rapid sterility tests by challenging their products with single cell concentrations of stressed organisms (and even below this level, such as 0.5 and 0.05 cells).  The approved technologies have included solid phase cytometry and ATP bioluminescence.

Lastly, if the USP wants to avoid “a rapid sterility test that only specialized labs can perform,” how can they recommend RT-PCR at this time? In the absence of a COTS RT-PCR system, I would expect that a lab wanting to validate a rapid sterility test would have to acquire all the instrumentation, probes and primers and conduct the necessary studies with expertise that have significant molecular biology skills to qualify the method for its intended use. Wouldn’t this be a considered a “specialized lab?”

Given all of these points, it is my opinion that the USP expert committee may need to rethink its position regarding a rapid sterility test chapter and to utilize the appropriate experts (e.g., in the form of an advisory panel) to help guide the process forward.

In conclusion, the workshop provided for good discussions and consideration for the implementation of rapid and alternative methods across our industry. Unfortunately, we will have to wait for the publication of the final chapter 1223, which is due out this summer, to see what changes the expert committee has made, if any, to the comments that were submitted last year. And if the chapter does not provide additional clarity and direction, the industry can always rely on PDA’s TR33 and Ph. Eur. 5.1.6.

Saturday, February 21, 2015

EU Suports Development of New Detection Technologies for Bacterial Pathogens

RAPTADIAG is a small or medium-scale focused research project supported by the European Commission under the 7th Framework Programme with Grant agreement no. 304814. It contributes to the programme FP7-HEALTH-2012-INNOVATION-2, topic HEALTH.2012.2.3.0-1: Diagnostics for infectious diseases in humans.

The past two years have seen the consortium turn a novel diagnostic test for bacterial meningitis into what is likely to become a full-blown set of sensor technologies for detecting bacterial pathogens of all kinds.

Whilst the sector has made some giant leaps over the past few years, much contemporary medicine still revolves around symptom-based treatments and costly diagnosis methods. In the case of ‘bacterial meningitis’ (BM), symptoms would usually develop within three to seven days after initial exposure if at all, as some people can carry the bacteria without getting sick. No treatment means a 50 % chance of dying, and the treatment’s effectiveness depends on how soon it is administered.

According to Morten A. Geday, coordinator of the RAPTADIAG (Rapid Aptamer based diagnostics for bacterial meningitis) project and professor, treatment effectiveness is dragged down by the fact that early diagnosis is currently possible only through use of very expensive technologies. Not only are these methods taking too long to give an accurate result, but they are also too complex to be used outside major hospital facilities.

Together with partners from Switzerland and Denmark and thanks to EUR 2.2 million of EU funding, Prof. Geday set out to overcome these obstacles with a fast, easy-to-use and inexpensive diagnostic test for Neisseria meningitides (aka meningococcus) and Streptococcus pneumonia, which are responsible for 80 % of BM cases. He and his team have already developed three groundbreaking technologies, including a microacoustic-resonating sensor and a liquid crystal-based sensor, and are now planning to take their project to the next level.

In this interview, Prof. Geday explains his consortium’s journey since the project started in 2011. He also elaborates on the findings that made them reconsider the project’s raison d’être, from better diagnosis for BM to detection of a much larger spectrum of bacteria, in contexts as varied as food or water borne pathogens entering the food chain, water resources, or even air conditioning units.

What’s so new or innovative about this test? How does it work?

The new diagnostic tests will be faster (minutes rather than hours or days) and cheaper (euros rather than several 10s of euros) than the currently-available technologies. They were intended to address the clinical need for a diagnosis of these diseases with a high degree of morbidity, reducing the possibility of misdiagnosis and abuse of antibiotics.

To enable microorganism recognition, we use novel aptamer receptors rather than conventional antibodies. In a nutshell, aptamers are short single-stranded DNA/RNA molecules which can undertake a three-dimensional structure by intra-strand pairing of the nucleic bases. This structure is then selected based on its high affinity and specificity towards the desired antigen or target.

Three different sensor technologies are being developed in parallel. The first technology is the adaptation of the commercial evanescent biosensor technology (Eva-sensor) using aptamer receptors instead of antibodies. Two more experimental (university-developed) technologies are being employed to develop a rapid test at a significantly lower cost, i.e. a microacoustic-resonating sensor and a liquid crystal-based sensor. The challenge in developing these two sensors was first of all to show that it is possible to develop microacoustic-resonating sensors with the necessary sensitivity, and then that we could develop liquid crystal-based sensors with the potential for single cell detection.

What were the main difficulties you faced and how did you resolve them?

The project has been marred by two problems, one technical and one scientific. Shortly after the kick-off, one of the principal partners went bankrupt. This meant that the project found itself without the possibility of developing the key receptor molecules, i.e. the aptamers. The solution eventually came from one of the partners who took on this responsibility by employing key staff members from the bankrupted partner. The handling of the bankruptcy, the redefinition of responsibilities, and getting the project back up to speed has led to a six-month delay in execution. However, the highly successful development of both the liquid crystal-based sensor and the microacoustic resonators is closely related to the choices we made then.

The second scientific problem is the development of the BM-specific aptamers. As the project is progressing, it is becoming increasingly clear that the necessary affinity and specificity towards the targets will reach the limits of the consortium’s abilities as it stands. To what extent this reflects the limitations of the consortium or the limitations of the aptamer technology is not entirely clear. The workaround is the employment of BM-specific antibodies and existing aptamers targeting alternative pathogens in the testing and validation of the developed technologies.

So you progressively moved away from BM to focus on other types of pathogens. How did that happen?

During the execution of the project, it has become increasingly clear that while the development of cheaper and faster BM detection could impact the detection and subsequent limitation of a BM epidemic in the Third World, the clinical impact in the West would probably be limited.

At the same time, we have realised that the technologies being developed for BM detection have a significant impact on the detection of bacterial pathogens in a large number of contexts, most notably food or water borne pathogens either in the food chain, in water resources or in air conditioning units. Similarly, these technologies may pave the way for novel means of detection of human pathogens in saliva or other bodily fluids.

As a consequence, various proposals aiming to further mature these technologies were presented in the last round of FP7, and a much more ambitious project — which to some extent is building on the experiences gained during RAPTADIAG — is currently being evaluated in a Horizon 2020 Call.

Where do you stand with your objective of delivering at least one commercial product by the end of the project?

The project is well on track. The Eva-sensor can already be purchased, and Davos Diagnostics have proven that their technology is suitable for bacterial detection using aptamer recognition or otherwise. On the other hand, both the microresonators and the liquid crystal sensors are still too immature. These technologies require a strong industrial partner. In the light of the financial situation in Spain, it is unlikely that funding for a spin-off involving the participating scientists can be found, and thus the technology must be transferred to an existing entity. We will, together with the technology transfer office at the University, start looking for potential partners in the near future.

Would you say that the project results meet your expectations?

The project, originally scheduled to finish in June 2015, has already achieved a great number of its objectives. We have proven the use of the aptamers as receptor molecules for bacterial pathogens in the Eva-sensor, resulting in fast and easy pathogen detection (patents pending). At the same time, the microacoustic-resonating biosensor technologies are already approaching the sensibility needed to potentially detect the binding of one microorganism alone, which is the ultimate detection limit, while the liquid crystal sensor is opening the way for an exceedingly simple and inexpensive detection method, with either visual (without the need for any instrumentation!) or simple optoelectronic inspection with miniature readers or even mobile phone cameras. The microacoustic resonators have already been published in various peer-reviewed journals, while a patent has been submitted in order to protect the liquid crystal sensor technology.

Thus from a technological bio-sensor development point of view, the project has vastly exceeded even the participants’ expectations.

When do you think patients and health workers could realistically start benefiting from your findings?

The payback to society will depend to a large extent on the conservatism of the medical sector. It will be immensely difficult even for our finished product, Eva-sensor, to have a significant impact over the next two years, even though Davos Diagnostics, during — and to some extent, thanks to — this project, has become ISO certified. Over the longer term (three to five years), we expect the Eva-sensor to become widespread in hospital wards, providing faster and easier detection of a large number of pathogens and other biological targets. The future of both the liquid crystal and the micro-resonating sensors will entirely depend on the industrial partners that the consortium gets interested in its technologies.

Source: EU Community Research and Development Information Service

WHO Approves Rapid Ebola Test

The United Nations World Health Organization (WHO) today announced that it has approved for use a rapid diagnostic test kit for Ebola that can provide results in 15 minutes and correctly identify 92 percent of patients infected by the disease that has killed more than 9,400 people, mainly in West Africa.

Meanwhile at UN headquarters, Dr. Bruce Aylward, who leads WHO's response on Ebola, and Dr. David Nabarro, the UN Secretary-General's Special Envoy on Ebola, briefed Member States on the need to maintain the robust response to get the number of cases to zero.

“As long as there is even one case of Ebola active in the human population, it's a danger for everybody – it's a problem for West Africa, it's a problem for [wider] Africa and it's a problem for the world, Dr. Nabarro told reporters after their briefing. “We must be fully engaged, all of us, until the last person with Ebola is treated and is cured.”

The two doctors expressed their concerns about the recent slowdown in the pattern of decline in cases over the last four weeks in the three most affected countries of Guinea, Liberia and Sierra Leone.

Referring to a graph showing that the last four weeks has seen more than 120 Ebola cases a week, Dr. Aylward said “this is not what you want to see,” and described the trend as “a very bumpy road” on the wary to zero cases.

They also told reporters that the upcoming rainy season starting in about two months posed as a complicating factor as it could give the virus a chance to get ahead of the response.

Earlier in Geneva, the UN health agency announced that it had “assessed and today listed the ReEBOV Antigen Rapid Test Kit [manufactured by Corgenix Medical Corp of the United States] as eligible for procurement to Ebola affected countries.”

“The test was evaluated under WHO's Emergency Assessment and Use, a procedure established to provide minimum quality, safety and performance assurance for diagnostic products in the context of the Ebola emergency, the announcement said.

The new test, which can provide results within 15 minutes, “is able to correctly identify about 92 per cent of Ebola infected patients and 85 per cent of those not infected with the virus,” according to WHO.

In comparison, Ebola is currently being tested in laboratories largely through the detection of the virus’s nucleic acid (genetic material), using commercial or in-house tests which employ conventional PCR techniques. Nucleic acid tests (NATs) are more accurate but are complex to use and require well-established laboratories and fully trained personnel. In addition, turn-around time can vary between 12 and 24 hours.

WHO Spokesman Tarik Jašareviæ told reporters in Geneva that the new test was a little bit less accurate than the test that WHO was currently using, but it was easy to perform, it did not require electricity and it could be used in lower level health care facilities or in mobile units for patients in remote settings.

The WHO spokesman also said that a number of agencies, such as the Médecins Sans Frontières (MSF), have expressed interest in purchasing it.

The current Ebola outbreak in West Africa has affected more than 23,000 people with over 9,400 deaths, mostly in Guinea, Liberia and Sierra Leone.

Friday, February 13, 2015

Harvard, MIT Researchers Develop Rapid Diagnostic for Virus Detection

Thanks to research by Lee Gehrke, a professor of health sciences and technology, and other researchers at Harvard and MIT, viruses like Ebola may be more rapidly detected and tracked than ever before. The rapid diagnostic, which can detect deadly pathogens in under 30 minutes, is part of an interdisciplinary project supported by the National Institutes of Health.

This invention has grown out years of research at Gehrke’s lab on RNA viruses, viruses that have one-stranded RNA rather than DNA as their genome. Ebola is an example of an RNA virus. The researchers are pairing the rapid diagnostic with a phone application to allow the spread of the viruses to be tracked via real-time maps.

30 MINUTES OR LESS

Gehrke said that the goal of the researchers was to develop a rapid diagnostic test that can perform in under 30 minutes and can be used as a point-of-care device to evaluate patients with fevers, which can indicate many different underlying diseases.

“If a patient comes into a clinic and has a fever, you want to be able to rule out some pathogens,” he said. “In West Africa, there are a number of viruses that would present as fever, including Ebola, Lassa, and Marburg, so it is very useful to get a very quick idea of what the patient is suffering from.”

While the Ebola outbreak in West Africa has ascended to international media headlines in recent months, Gehrke warned against ignoring other dangerous viruses.

“Ebola is a terrible disease, but it is not the only one that I think we should be prepared for,” Gehrke added. “There are other emerging viruses that we also need to have a great sense of preparedness for.”

The device is specific for several viruses, including Ebola and dengue fever, and it can detect more than one pathogen at once, according to Gehrke.

“By detecting whether a person’s fever is in fact a symptom of Ebola, the rapid diagnostic can help better quarantine people,” said Kimberly Hamad-Schifferli, MIT professor and senior collaborator on this project.

Gehrke also attested to the value of rapid detection of the virus.

“Being able to detect the virus and identify the people or environments that are contaminated is very important for reducing or stopping the spread. Early detection is important for treatment,” he said.

NUTS AND BOLTS

Unlike other diagnostic tools, the Harvard and MIT-developed device can be operated cheaply by personnel with little training in a variety of settings. Competing approaches, like polymerase chain reaction, require more time and expensive equipment.

“This approach gives much greater flexibility, cost control, and adaptability in responding to outbreaks of different emerging pathogens in different parts of the world,” Gehrke wrote in an email.

Gehrke attributed this flexibility in part to the ability of the device to operate without power or refrigeration. The device does not require specialized chemicals, equipment, or training, researchers said.

“We are interested in making something that can detect disease rapidly and can be operated by an end user, or someone who is not medically trained or a technician,” Hamad-Schifferli said.

The device uses a sample of a body fluid, such as blood, serum, or saliva, to test a patient, Gehrke said. The test operates by diverting the sample in a maze-like structure and making it run into chemicals, said José Gómez-Márquez, director of the Little Devices Lab at MIT and senior collaborator on this research.

According to MIT postdoctoral fellow Justina Tam, who is working on visual markers, or detection agents, for the rapid diagnostic, “the detection agents are agents that have some visible color if they accumulate in a certain area of the test, which means a protein for a specific virus is present in the sample.”

Not only does this visible color indicate whether a patient is infected with a virus, but it also reveals the virus’s specific strain.

REAL-TIME RESULTS

According to Gómez-Márquez, aggregating the results of the rapid diagnostic through an accompanying phone app allows researchers to track the spread of viruses in real-time, which he hopes will prevent their spread in the future.

He called the device a crowdsource diagnostic because it relies on many users to send images of their results via the phone app. The diagnostic itself gives a result, but the phone app quantifies it and measures the location of the infectious diseases so that the spread of viruses can be tracked rapidly, Gómez-Márquez said.

“If we can do that for hundreds of patients, then we can get a real-time map of the spread of the disease,” he added.

He said that up to now, maps of the spread of viruses include information of what occurred in the past, based on death records and hospital records that are weeks old. The new technology gives health providers a better understanding of the path viruses could take in the future.

“In public health, we don’t have real-time maps for epidemiology, but that’s important for saving the next lives.The prevailing attitude is looking at the past, which doesn’t allow us to help the future,” he said.

According to Gómez-Márquez, several people are working on the phone app for different platforms, including the Android phone.

According to JN Fang ’16, who is working on an Android version of the app, health workers administering the diagnostic will be able to use the app to read and aggregate the results of the test. The app can determine “if...the change in test strips is enough of a change to indicate that the patient has tested positive for either of the four strains of dengue fever or Ebola,” she said.

After testing the diagnostic in the laboratory, Gehrke said his lab is currently field-testing the device. According to Gehrke, the commercial launch of the device will depend on the regulatory process for approval by the FDA.

This rapid diagnostic will be another tool among “a number of different methods [that] are going to be necessary for detecting and hopefully stopping this virus and other infectious viruses,” Gehrke said.

Source: The Havard Crimson