EMA Promotes the Use of Rapid Microbiological Methods for Water Testing

In 2005, the European Medicines Agency (EMA) explored the regulatory consequences of implementing an alternative method for rapid control of microbiological quality of water for injection (WFI) and Purified water. Discussions between the Quality Working Party, the Good Manufacturing Practices and Good Distribution Practices Inspectors Working Groups and the Committees for Human and Veterinary Medicinal Products concluded Since it is expected that the water will continue to meet Ph. Eur. specifications, if tested, no change to dossier requirements* (variations) would be involved and therefore no regulatory impact on individual products would normally be anticipated.

• *However, this will depend on the level of detail in the original dossiers concerned.

Following discussions over the last 2-3 years around the revision of the European Pharmacopoeia (Ph. Eur.) WFI monograph (0169), the Water Working Party concluded that there was evidence to support a revision of the monograph, which proposes to take account of current manufacturing practices using methods other than distillation for producing water of injectable quality.

The Ph.Eur. monograph (Monograph 169) was revised to include, in addition to distillation, reverse
osmosis (RO) coupled with suitable techniques, for the production of WFI.

In order to provide clarification and guidance in relation to the use of reverse osmosis in the manufacture of WFI and also to provide more detailed guidance on the control of biofilms, the Water Working Party developed a draft set of Questions and Answers that were sent out for public consultation on August 5, 2016, with an end of consultation date (i.e., the deadline for comments) of November 4, 2016. The draft Q&A is intended to provide preliminary guidance until such time the on-going revision of Annex I of the GMP guide is complete.

One of the questions posed by the working party is, “What testing should be employed during initial qualification and routine operation sampling?”

In answering this question, the working party states:

• Use of rapid microbiological methods should be employed as a prerequisite to the control strategy to aid with rapid responses to deterioration of the system.” 

• Methods to be considered should include, “rapid endotoxin testing – use of more sensitive and point of use test methods,” and “quantitative microbiological test methods – in line with Ph.Eur. 5.1.6 monograph Alternative Methods for control of Microbiological Quality. 

• Due consideration should be given to employing alternate methods for the rapid quantitative determination of the contamination levels existing within the water system. The validation of such system should be in line with the above referenced monograph.

As such, EMA is encouraging the industry to employ rapid microbiological methods for the quantitative assessment of viable organisms and the presence of endotoxin in WFI and purified water. Additionally, the use of such methods should be validating according to Ph. Eur. chapter 5.1.6.

In my opinion, this is long-overdo step in the right direction. Technologies for the rapid analysis of pharmaceutical grade water have been available for years, and a number of firms who have experienced water system-related contamination events could have benefited from using a rapid method to quickly detect, and most importantly, remediate issues that have directly impacted their manufacturing facilities and/or finished product.

The full Q&A draft may be viewed at:

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/08/WC500211657.pdf.

Comments on the draft are due by November 4, 2016.

CRISPR Aids New Zika Paper-Based Diagnostic Test

The rapid spread of Zika virus into South America and its link to fetal neurodevelopmental issues have underscored the need for fast, low-cost diagnostics for use in endemic regions. Now, a collaborative team of scientists from Wyss Institute for Biologically Inspired Engineering, Massachusetts Institute of Technology, Boston University, Arizona State University, Cornell University, University of Wisconsin-Madison, Broad Institute, and the University of Toronto have developed a cell-free, paper-based platform that can host synthetic gene networks and help reliable diagnosis of Zika-infected patients.

This new test, which can distinguish Zika from the very similar dengue virus within a few hours, can be stored at room temperature and read with a simple electronic reader, making it practical for widespread use.

"One of the key problems in the field is being able to distinguish what these patients have in areas where these viruses are co-circulating," explained coauthor Lee Gehrke, Ph.D., professor of microbiology and immunobiology at Harvard Medical School.

"We [now] have a system that could be widely distributed and used in the field with low cost and very few resources," added senior study author James Collins, Ph.D., professor of medical engineering and science in MIT's Department of Biological Engineering and Institute for Medical Engineering and Science (IMES).

The findings from this study were published recently in Cell in an article entitled “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.”

Currently, patients are diagnosed by testing for reactive antibodies against Zika in their bloodstream or by looking for pieces of the viral genome in a patient's blood sample using PCR. However, these tests can take days or weeks to yield results. Furthermore, the antibody test cannot discriminate accurately between Zika and dengue.

The researcher team created a cluster of diagnostic measuring devices on a freeze-dried piece of paper the size of a stamp that employs toehold sensors and isothermal RNA amplification—coupled to a CRISPR based module. Activated by the amplified sample, the diagnostic sensors programmed into the paper provide an extremely sensitive, low-cost, programmable assay that provides rapid results.

The new test is based on technology that Dr. Collins and colleagues previously developed to detect the Ebola virus. In October 2014, the researchers demonstrated that they could create synthetic gene networks and embed them on small discs of paper. These gene networks can be programmed to detect a particular genetic sequence, which causes the paper to change color.

The sensors, embedded in the paper discs, can detect 24 different RNA sequences found in the Zika viral genome, which, like that of many viruses, is composed of RNA instead of DNA. When the target RNA sequence is present, it initiates a series of interactions that turns the paper from yellow to purple, which can be visualized by eye. The researchers also developed an electronic reader that makes it easier to quantify the change, especially in cases where the sensor is detecting more than one RNA sequence.

The investigators tested the device with samples taken from monkeys infected with the Zika virus because samples from human patients affected by the current Zika outbreak have been difficult to obtain. Amazingly, they found that in these samples, the device could detect viral RNA concentrations as low as 2 or 3 parts per quadrillion.

“The diagnostic platform developed by our team has provided a high-performing, low-cost tool that can work in remote locations," noted lead study author Keith Pardee, Ph.D., assistant professor at the University of Toronto. "We have developed a workflow that combines molecular tools to provide diagnostics that can be read out on a piece of paper no larger than a postage stamp. We hope that through this work, we have created the template for a tool that can make a positive impact on public health across the globe."

"Our synthetic biology pipeline for rapid sensor design and prototyping has tremendous potential for application for the Zika virus and other public health threats, enabling us to rapidly develop new diagnostics when and where they are needed most," Dr. Pardee added.

The researchers have envisioned that this approach could also be adapted to other viruses that may emerge in the future. Dr. Collins now hopes to team up with other scientists to develop the technology further for diagnosing Zika.

"Here we've done a nice proof-of-principle demonstration, but more work and additional testing would be needed to ensure safety and efficacy before actual deployment," Dr. Collins said. "We're not far off."

Source: Genetic Engineering & Biotechnology News

African Scientists a Step Closer to Develop Rapid Test for TB

Tuberculosis ranks alongside HIV/AIDS as a leading cause of death worldwide. According to the World Health Organisation, 1.5 million people died from TB in 2014. The challenges in tackling the disease include the facts that people are tested too late and that the turnaround for most tests is long. To remedy this a point-of-care rapid diagnostic test for TB has been developed by a multinational team of scientists led by researchers at Stellenbosch University in South Africa. One of its co-inventors, Professor Gerhard Walzl, spoke to The Conversation Africa’s health and medicine editor Candice Bailey.

How have TB tests been done up until now and what are the challenges?

There are three main tests that are currently in use.

A culture test – the most sensitive – requires people to produce a sputum sample that is sent to a centralised laboratory where a culture test is done. A positive result shows up after ten days. A confirmed negative result takes up to 42 days.

The problem with this test is that it is only available in centralised laboratories, which means patients must make several trips to a hospital or health facility to get their results. And it is very expensive.

Then there is the sputum microscopy test. This is widely used in Africa. It requires the sputum slides of each patient to be individually checked.

The test is inexpensive. But it is labour intensive, which means that only a limited number of smear tests can be assessed a day. In addition, it only has a 60% sensitivity rate.

On top of this, the test poses particular challenges for children and for people living with HIV.

In the case of young children, samples need to be taken from their stomachs as they cannot follow instructions to produce a good quality sputum sample. This requires the use of a nasal tube, which is not pleasant for the child or the health-care worker.

The test also isn’t effective for people living with HIV. This is because their sputum often has low levels of the bacteria, which can lead to a false negative test result.

There is also a molecular test that detects bacterial DNA in the sputum sample. This test only takes two hours to produce a result and although it speeds up the detection of TB, it is not widely available to people in rural areas as instruments are placed in a centralised manner.

How will your test change this?

If our test is accepted after clinical trials are completed it will be able to provide almost immediate results. People will be able to be diagnosed and start treatment in a single visit to a health-care facility.

The test is done with blood obtained from a finger prick and can make a TB diagnosis in less than an hour. The diagnostic test is a hand-held, battery-operated instrument that will measure chemicals in the blood of people with possible TB. This test will not have to be done in a laboratory and health-care workers will be able to perform it with minimal training.

It is a low-cost screening test and has the potential to significantly speed up TB diagnosis in resource-limited settings.

At what stage is the test?

The test is still in development. We have patented the biosignature, which identifies the levels of chemicals in the blood of a patient. A biosignature consists of a combination of chemicals and indicates a disease state. This signature was discovered by African scientists. The inventors included South African, Cameroonian and Ethiopian scientists.

The test’s accuracy and efficacy will be tested in five African countries over the next three years. We will recruit 800 people who have TB symptoms from Namibia, the Gambia, Uganda, Ethiopia and South Africa.

Clinical research sites will be set up or strengthened in all five countries. And participating countries will be able to use the data generated from this project.

We are still trying to improve the signature by adding additional markers. In addition, we would like to optimise and fine tune the device to enable it to measure the signature on a strip similar to a pregnancy test or a glucose test strip.

Why is the test important for South Africa?

South Africa has the highest TB rates in the world. Each year between 450,000 and 500,000 people develop TB. This gives the country an incidence rate of 834 infections for every 100,000 people. On the rest of the continent, the incidence rate is between 300 and 600 infections for every 100,000 people. In China the incidence is 68 for every 100,000 people and in most European countries it is less than ten for every 100,000 people.

One of the challenges in South Africa is that people in remote areas with high TB incidence still do not benefit from newer developments in TB testing. As a result they face long diagnostic delays and often need to come back to clinics on several occasions before they are diagnosed.

This test will mean that health-care workers with minimal training can use the test at grassroots level and get immediate access to screening test results.

It would also reduce the cost of testing for TB. Our test would initially cost US$2.50 per test. With commercialisation that price could drop significantly. Currently the culture test costs $45 per test while the DNA sample test costs $12 per test.

How does this test fit into the bigger picture of dealing with TB?

The test would be used best as a screening test. This is because it can identify people who need further investigation and can screen out those who don’t. So far we have been able to identify 70% of patients who do not need further testing.

The World Health Organisation has identified a screening test as important for high-prevalence areas, for those who are in contact with people who have TB, those living with HIV, homeless people, immune-compromised people and those living in areas with poor access to diagnostic services.

Source: The Conversation

White House and ASM Microbiome Research Project Includes Rapid DNA Sequencing

The White House, along with the American Society of Microbiology (ASM), announced the formation of several initiatives designed to further knowledge of the microbiome's benefit to human life and planetary function.

And in an additional concerted effort to advance microbial research, a diverse group of scientists writing today in mBio advocated for new technologies, systems, and philosophical approaches to understanding and harnessing the microbiome.

National research initiative

The White House's Office of Science and Technology Policy (OSTP) today announced the genesis of an interdisciplinary National Microbiome Initiative (NMI). The program will boost federal support of research that attempts to understand the function of microorganisms that live on or in all living species and are necessary to the well-being of living creatures and ecosystems.

Imbalanced microbiomes caused by human disruption and environmental degradation contribute to a host of issues, including chronic and infectious diseases, the spread of harmful microbes, reduced agricultural yields, and excesses of atmospheric carbon, the OSTP said.

The NMI will focus on encouraging comparative study of microbiomes across ecosystems to arrive at a better understanding of the conditions under which microscopic life thrives. The initiative's objectives were distilled during a year-long fact-finding process and will support interdisciplinary research, develop technologies that organize and increase access to microbial data, and expand the microbiome workforce through education and citizen science, the OSTP said.

The federal government will invest more than $121 million in the NMI, adding to the 2012 to 2014 investment of $922 million in microbiome research. The OSTP is partnering with groups including the Bill and Melinda Gates Foundation, the University of Michigan, and The BioCollective, LLC, to advance understanding of the microbial interface between humans and agricultural practices, the effect of the microbiome on Type 1 diabetes, and the establishment of a microbial data and sample bank, the White House said. Stakeholders will provide an additional $400 million in funding.

"We expect that by accelerating progress in this important field, the NMI will deliver considerable benefits to our planet and those who inhabit it," said Jo Handelsman, PhD, OSTP associate director for science.

Multisector collaboration and funding

Despite growing scientific knowledge about the ubiquity of microscopic life and the benefits that microorganisms confer to human and animal health, little is known about how microbes function and communicate.

In response to this gap in knowledge, the ASM is partnering with the American Chemical Society, the American Physical Society, and the Kavli Foundation to offer $1 million in research support as part of the Kavli Microbiome Ideas Challenge, the ASM said.

Researchers in all scientific disciplines are invited to submit ideas for experimental research into how microbial life forms communicate, interact within communities, and regulate health and bodily processes with multicellular hosts, the ASM said.

"This initiative is an important step to expand our understanding of the pivotal role of microbes in the ecosystem," said Lynn Enquist, PhD, ASM president.

The future of microbiome research

An interdisciplinary group of scientists writing today in mBio propose additional collaborative research that reflects the microbiome's effects, not only on human health, but on all aspects of the environment.

Microbes have shaped the planet and its atmosphere for more than 3.5 billion years, yet scientific research has only recently prompted "the realization that our species, Homo sapiens, is at least as microbial as human in terms of cell numbers and much more so in terms of genetic potential," the authors said.

The microbiome affects numerous systems necessary for life, including the atmospheric oxygen-carbon cycle, soil quality, and human digestive and immune system function. Disruptions or imbalances to microbial life can result in environmental degradation and temperature change, increases in human and animal disease, and the evolution of microbes that can cause harmful infections.

"Our planet's natural biomes and those that we manage for food and fuel will likely experience conditions beyond their contemporary climate boundaries, and our current understanding of the sensitivities of their microbial components limits our ability to predict how they will respond," the authors said.

The authors advocate for cross-sector, collaborative efforts to advance understanding of microbial function and its benefits to human and ecosystem health that go beyond microbial DNA sequencing and move toward an understanding of function in microbial communities and in different physical environments.

The authors' recommendations for research priorities include development of technology for rapid microbial DNA sequencing, understanding of genetic diversity, and analysis of gene expression and function; establishment of a microbial data and sample reference catalog; advances in imaging technology that can penetrate soil and water without damaging microbes (eg, silicon-based sensor arrays); and better studies that evaluate microbial function and ecosystem dependencies.

A growing global population, increased exposure to new microbes in urban environments, and the threat of resource shortages and climate change makes these areas of study more crucial than ever, the authors said. Though they encourage cross-sector work to discover solutions to global challenges, the authors acknowledge that the most important partnership is the one between human and animal life and the microbiome.

"This intermingling of genes and functions across the tree of life continues, allowing multicellular organisms to adapt more rapidly to new environments, using the versatility of their microbial partners," the authors said.

MIT Develops a Rapid, Paper-Based Zika Diagnostic Test

A new paper-based test developed at MIT and other institutions can diagnose Zika virus infection within a few hours. The test, which distinguishes Zika from the very similar dengue virus, can be stored at room temperature and read with a simple electronic reader, making it potentially practical for widespread use.

“We have a system that could be widely distributed and used in the field with low cost and very few resources,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute for Medical Engineering and Science (IMES) and the leader of the research team.

An outbreak of the Zika virus that began in Brazil in April 2015 has been linked to a birth defect known as microcephaly. Many infected people experience no symptoms, and when symptoms do appear they are very similar to those of related viruses such as dengue and chikungunya.

Currently, patients are diagnosed by testing whether they have antibodies against Zika in their bloodstream, or by looking for pieces of the viral genome in a patient’s blood sample, using a test known as polymerase chain reaction (PCR). However, these tests can take days or weeks to yield results, and the antibody test cannot discriminate accurately between Zika and dengue.

“One of the key problems in the field is being able to distinguish what these patients have in areas where these viruses are co-circulating,” says Lee Gehrke, the Hermann L.F. von Helmholtz Professor in IMES and an author of the paper.

Collins, Gehrke, and colleagues from Harvard University’s Wyss Institute for Biologically Inspired Engineering and other institutions described the new device in the May 6 online edition of Cell. The paper’s lead authors are Melissa Takahashi, an IMES postdoc; Dana Braff, an MIT graduate student; Keith Pardee, an assistant professor at the University of Toronto and former Wyss Institute research scientist; Alexander Green, an assistant professor at Arizona State University and former Wyss Institute postdoc; and Guillaume Lambert, a visiting scholar at the Wyss Institute.

Paper-based detection

The new device is based on technology that Collins and colleagues previously developed to detect the Ebola virus. In October 2014, the researchers demonstrated that they could create synthetic gene networks and embed them on small discs of paper. These gene networks can be programmed to detect a particular genetic sequence, which causes the paper to change color.

Upon learning about the Zika outbreak, the researchers decided to try adapting their device to diagnose Zika, which has spread to other parts of South and North America since the outbreak began in Brazil.

“In a small number of weeks, we developed and validated a relatively rapid, inexpensive Zika diagnostic platform,” says Collins, who is also a member of the Wyss Institute.

Collins and his colleagues developed sensors, embedded in the paper discs, that can detect 24 different RNA sequences found in the Zika viral genome, which, like that of many viruses, is composed of RNA instead of DNA. When the target RNA sequence is present, it initiates a series of interactions that turns the paper from yellow to purple.

This color change can be seen with the naked eye, but the researchers also developed an electronic reader that makes it easier to quantify the change, especially in cases where the sensor is detecting more than one RNA sequence.

All of the cellular components necessary for this process — including proteins, nucleic acids, and ribosomes — can be extracted from living cells and freeze-dried onto paper. These paper discs can be stored at room temperature, making it easy to ship them to any location. Once rehydrated, all of the components function just as they would inside a living cell.

The researchers also incorporated a step that boosts the amount of viral RNA in the blood sample before exposing it to the sensor, using a system called NASBA (nucleic acid sequence based amplification). This amplification step, which takes one to two hours, increases the test’s sensitivity 1 million-fold.

Julius Lucks, an assistant professor of chemical and biomolecular engineering at Cornell University, says that this demonstration of rapidly customizable molecular sensors represents a huge leap for the field of synthetic biology.

“What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” says Lucks, who was not involved in the research.

Distinguishing viruses

The team tested the new device using synthesized RNA sequences corresponding to the Zika genome, which were were then added to human blood serum. The researchers showed that the device could detect very low viral RNA concentrations in those samples and could also distinguish Zika from dengue.

The researchers then tested the device with samples taken from monkeys infected with the Zika virus. (Samples from human patients affected by the current Zika outbreak are very difficult to obtain.) They found that in these samples, the device could detect viral RNA concentrations as low as 2 or 3 parts per quadrillion.

The researchers envision that this approach could also be adapted to other viruses that may emerge in the future. Collins now hopes to team up with other scientists to further develop the technology for diagnosing Zika.

“Here we’ve done a nice proof-of-principle demonstration, but more work and additional testing would be needed to ensure safety and efficacy before actual deployment,” he says. “We’re not far off.”

The research was funded by the Wyss Institute for Biologically Inspired Engineering, MIT’s Center for Microbiome Informatics and Therapeutics, the Defense Threat Reduction Agency, and the National Institutes of Health.

SOURCE: MIT News

Laser Tool Effective at Identifying Mutant Listeria Bacteria

The folks at Purdue University continue to develop next generation technologies for the detection of pathogenic bacteria. The latest involves the use of light scattering.

A Purdue University-developed laser tool already effective in quickly detecting harmful bacteria has been shown to detect mutant varieties of listeria - and in the same amount of time.

The BARDOT (pronounced bar-DOH') laser scans bacteria colonies looking for unique patterns that each bacterium makes. When the light penetrates a bacteria colony, it produces a scatter pattern that can be matched against a library of known bacteria patterns to identify a match. The system can identify bacteria such as salmonella, listeria, bacillus, vibrio and E. coli within 24 hours.

Now, Arun Bhunia, professor of food microbiology, and Atul Singh, research scientist, have shown that BARDOT (acronym for "bacterial rapid detection using optical scatter technology") can pinpoint small genetic mutations in listeria just as quickly, significantly reducing the time it would take scientists to identify those mutations in bacterial strains used for research. Their study was published in the journal Applied and Environmental Microbiology.

"This is a versatile microbiology tool, and we wanted to see if we can use it for mutant strains," Bhunia said. "This is a really powerful tool to help researchers find those mutant strains much easier on a petri plate. You can avoid the laborious techniques required to screen or detect these mutant strains."

Scientists use mutant bacteria to understand biology of pathogens and how they can combat outbreaks in food that can cause illnesses or death. Current methods of identifying mutants can take several days, whereas BARDOT can do the same work in less than a day.

Singh said he visualized the changes in bacteria using the laser system.

"It's like if you compare a wild type that is a normal bacteria, you get a scatter pattern. And then if you delete a certain gene, you get a new picture," he said.

The reverse was also true. By restoring the deleted gene, the BARDOT system recognized the bacteria as a regular wild type of strain.

Bhunia said his lab will continue to study BARDOT's ability to identify mutants of other bacteria and build libraries so the tool can be used for that work. He will also test the system's ability to identify mutant bacteria from natural settings such as from contaminated food.

The U.S. Department of Agriculture funded this study through the Center for Food Safety Engineering.

ABSTRACT

Virulence Gene-Associated Mutant Bacterial Colonies Generate  Differentiating Two-Dimensional Laser Scatter Fingerprints

Atul K. Singh, Lena Leprun a, Rishi Drolia, Xingjian Bai,
Huisung Kim , Amornrat Aroonual, Euiwon Bae, Krishna K. Mishra, Arun K. Bhunia

In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid detection using optical scattering technology) could be used to directly screen colonies of Listeria monocytogenes, a model pathogen, with mutations in several known virulence genes, including the genes encoding Listeria adhesion protein (LAP; lap mutant), internalin A (ΔinlA strain), and an accessory secretory protein (ΔsecA2 strain). Here we show that the scatter patterns of lap mutant, ΔinlA, and ΔsecA2 colonies were markedly different from that of the wild type (WT), with >95% positive predictive values (PPVs), whereas for the complemented mutant strains, scatter patterns were restored to that of the WT. The scatter image library successfully distinguished the lap mutant and ΔinlA mutant strains from the WT in mixed-culture experiments, including a coinfection study using the Caco-2 cell line. Among the biophysical parameters examined, the colony height and optical density did not reveal any discernible differences between the mutant and WT strains. We also found that differential LAP expression in L. monocytogenes serotype 4b strains also affected the scatter patterns of the colonies. The results from this study suggest that BARDOT can be used to screen and enumerate mutant strains separately from the WT based on differential colony scatter patterns.

SOURCE: Purdue University

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.

Source: Institute of Physical Chemistry of the Polish Academy of Sciences  

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)

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.

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

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