Wednesday, December 28, 2016

New Paper on Using Statistics When Validating Rapid Microbiological Methods

From 2010 to 2013, European Pharmaceutical Review published a very successful series on rapid microbiological methods (RMM) that included such topics as how to demonstrate a rapid method is equivalent to a conventional or pharmacopoeial method. PDA Technical Report #33, the recently revised USP chapter <1223> and the proposed changes to Ph. Eur. Chapter 5.1.6 all recommend the use of statistical analysis when validating rapid methods and establishing equivalence. However, many microbiologists find the concepts of statistical analysis and hypothesis testing intimidating and, in some cases, unapproachable. Once and for all, it was time to demystify statistics and provide a framework for embracing the mathematical models required when validating novel RMM technologies. 

To accomplish this, I have teamed up with two of the industry’s leading experts on statistical analysis and the validation of rapid microbiological methods: Edwin R. van den Heuvel, PhD (Eindhoven University of Technology) and David Roesti, PhD (Novartis Pharmaceuticals).

Our paper, The Role of Statistical Analysis in Validating Rapid Microbiological Methods, has recently been published in this month’s European Pharmaceutical Review. Clicking on the publication title (above) will give you online access to the paper (this is free to view but you may need to sign up for access). Alternatively, please copy and past the following address into your web browser:

http://www.europeanpharmaceuticalreview.com/46555/european-pharmaceutical-review-magazine/past-issues/issue-6-2016/rmms-supplement/

This comprehensive article discusses the concepts of statistics and hypothesis testing, including:
  • How to formulate a null and alternate hypotheses as it relates to rapid method validation 
  • Identifing the appropriate statistical test
  • How to calculate a p-value
  • Comparing the p-value with a chosen alpha level
Next we, dive into equivalence and non-inferiority tests, which we believe is the preferred strategy for demonstrating a rapid method is as good as and/or no worse than the existing microbiological method you wish to replace. The article specifies how to select lower and upper equivalence levels and also provides case studies on how to utilize these statistical tools.

Wednesday, August 24, 2016

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.

Wednesday, May 18, 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.

Friday, May 6, 2016

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