Sunday, January 20, 2013

Miller to Teach Rapid Methods Course; Sutton to Discuss USP 1116 at PDA Europe Microbiology Conference

Dr. Michael J. Miller will be teaching his two-day rapid microbiological methods course immediately following the upcoming PDA Europe Conference on Pharmaceutical Microbiology (conference is Feb. 26-27; course is Feb. 28- March 1). This comprehensive course is designed to provide an introductory review of currently available rapid microbiological method (RMM) technologies, validation strategies, applications, regulatory expectations, financial justification models and implementation plans. Taught by one of the industry’s global leaders in rapid methods, the attendee will be immersed in discussions that will provide a meaningful and understandable roadmap for how to evaluate RMMs and employ them in their own laboratory and manufacturing areas.

Additionally, Dr. Scott Sutton, The Microbiology Network and USP Expert Committee member, will provide an update to the newly revised USP chapter 1116, Microbiological Evaluation of Clean Rooms and Other Controlled Environments. An overview of some of the many sessions scheduled to be presented are listed below:
  • RMM – An Alternative Method Validation with a New Light on Statistical Tools. Vincent Beguin, Merck Millipore
  • The Response of Organisms to Stress and Injury: Exploring the Dynamics of Recovery using a Rapid Micro Method. Andrew Sage, Rapid Micro Biosystems
  • RMM Issues from a Regulatory Perspective. Andrew Hopkins, MHRA
  • Implementation of the MALDI Biotyper for Microbial Identifications in a QC Microbiology LaboratoryAmy McDaniel, Pfizer 
  • Application of Rapid Microbiology Techniques to Release a Virus Vaccine: A Case Study. Daniel Galbraith, BioOutsource
  • Rapid Microbiological Methods (RMM) – A “Stunning” Advance in the Microbiological Field, but a “Slow” Implementation in the Pharma Industry. Fulvio Tavellini, Baxter
  • Status of RMM Validation PDA Technical Report No. 33. Michael J. Miller, Microbiology Consultants, LLC
  • Remediating Pharmaceutical Water System Biofilm – What To Do After It Gets Ahead of You. T.C. Soli, Soli Pharma Solutions
  • Microbial Control Strategies in Bioprocessing Falling Short of Assuring Product Quality and Satisfying Regulatory Expectations. Anastasia Lolas, Visionary Pharma Consulting
For more information about the conference, please visit the PDA Europe website. For additional information about the two-day rapid methods course, please visit the RMM Course website

Wednesday, January 16, 2013

New Diagnostic Tool to Tackle Diarrhoea

Scientists from Bangladesh and Japan have developed a cheap and rapid diagnostic technique to identify strains of a common bug that causes diarrhoea.

Scientists had earlier identified over ten genes from five strains of the bacterium Escherichia coli that can cause diarrhoea in humans.

Earlier tests involved identifying each individual gene, a costly process that took four to five hours for each gene.

Two-step or single-step genetic tests were also developed earlier, but these could not detect all the concerned genes. These tests were based on the polymerase chain reaction (PCR) technique that amplifies genes to make their detection easier.

The new PCR test, to be published online in the Journal of Microbiological Methods, identifies ten specific E. coli genes in a single reaction, saving time and money. It was developed by a team of scientists from the University of Hirosaki, Japan, and Dhaka University (DU).

In their report the scientists said the new test would be a valuable contribution to routine diagnostic tests while also providing valuable information for physicians and researchers.

"A physician treating a patient with diarrhoea would be able to find out his ailment much faster and also, it would cost far less than the conventional method of culturing stools for 24 hours," Chowdhury Rafiqul Ahsan, one of the three authors of the report, told SciDev.Net.

Diagnostic laboratories in Dhaka now charge between US$ 23 and US$ 71, depending on how quickly gene matches are obtained. Against this, the new test identifies all ten possible causative genes in a single test that could, on commercialisation, cost less than US$ 7.

Ahsan, a senior academician at the department of microbiology at DU, toldSciDev.Net that the "biggest advantage of the highly reliable technique is (that) it enables us to detect the exact cause of the infection in less than 3–4 hours."

Azharul Islam Khan, head of the diarrhoeal diseases unit at the International Centre for Diarrhoeal Disease Research, Bangladesh, said the development was significant for public health. "Last year we found about 12 per cent of all diarrhoea patients suffering from E. coli infection, considered almost as deadly as cholera."

E. coli infection is characterised by rapid discharge of liquid stools which can be fatal if immediate rehydration therapy is not given, Khan added.

Here is the full reference and abstract:

A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli.

Miyuki Fujiokaa, Yoshimitsu Otomoa, Chowdhury Rafiqul Ahsanb.

a Hirosaki University, Graduate School of Health Sciences, Hon-cho 66-1, Hirosaki, Aomori, 036-8564, Japan.
b Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh.

Escherichia coli that causes diarrhea in humans is referred to as diarrheagenic E. coli (DEC), and has been categorized into the following 5 groups: shigatoxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), and enterotoxigenic E. coli (ETEC). In this study, we developed a novel one-step multiplex polymerase chain reaction (mPCR) for the rapid detection of 10 pathogenic genes (stx1, stx2, eae, bfpA, invE, aggR, esth, estp, elt, and astA) of DEC. Five categorized strains were used as positive controls for DEC harboring each pathogenic gene, and 828 DEC-like strains, isolated from diarrheal stool samples and assumed to be DEC on the basis of serotyping, were used in the mPCR-based detection of the pathogenic genes. To demonstrate the utility of mPCR, the 828 strains were subjected to our optimized protocol, and the results obtained were compared with those obtained by monoplex PCR. The results showed agreement for all strains. Using mPCR, we also detected 65 DEC and 41 astA-positive E. coli, and 7 of these DEC strains were “O antigen untypable” (OUT). This novel mPCR protocol allowed for rapid, convenient, and economical pathogenicity-based identification of the DEC.

Saturday, January 12, 2013

Biochip Vortex Spins to Sort Bacteria by Size

By varying laser and electric fields, scientists can use tiny centrifuge-like whirlpools to separate particles and microbes.

The technology could bring innovative sensors and analytical devices for lab-on-a-chip applications, or miniature instruments that perform measurements normally requiring large laboratory equipment.

Rapid electrokinetic patterning (REP) is a potential new tool for applications, including medical diagnostics; testing food, water, and contaminated soil; isolating DNA for gene sequencing; crime-scene forensics; and pharmaceutical manufacturing.

The researchers have used the method for the first time to collect microscopic bacteria and fungi, says Steven T. Wereley, a professor of mechanical engineering at Purdue University.

“The new results demonstrate that REP can be used to sort biological particles but also that the technique is a powerful tool for development of a high-performance on-chip bioassay system,” Wereley says.

A research paper about the technology was on the cover of the December 7 issue of Lab on a Chip magazine, and appears as a news item in the January 13 issue of Nature Photonics.

The technology works by using a highly focused infrared laser to heat fluid in a microchannel containing particles or bacteria. An electric field is applied, combining with the laser’s heating action to circulate the fluid in a “microfluidic vortex,” whirling mini-maelstroms one-tenth the width of a human hair that work like a centrifuge to isolate specific types of particles based on size.

Particles of different sizes can be isolated by changing the electrical frequency, and the vortex moves wherever the laser is pointed, representing a method for positioning specific types of particles for detection and analysis.

The researchers used REP to collect three types of microorganisms: a bacterium called Shewanella oneidensis MR-1; Saccharomyces cerevisiae, a single-cell spherical fungus; and Staphylococcus aureus, a spherical bacterium. The new findings demonstrate the tool’s ability to perform size-based separation of microorganisms, Wereley says.

“By properly choosing the electrical frequency we can separate blood components, such as platelets,” he says. “Say you want to collect Shewanella bacteria, so you use a certain electrical frequency and collect them. Then the next day you want to collect platelets from blood. That’s going to be a different frequency. We foresee the ability to dynamically select what you will collect, which you could not do with conventional tools.”

The overall research field is called “optoelectrical microfluidics.” More research is needed before the technology is ready for commercialization.

“It won’t be on the market in a year,” Wereley says. “We are still in the research end of this. We are sort of at the stage of looking for the killer app for this technology.”

REP may be used as a tool for nanomanufacturing because it shows promise for the assembly of suspended particles, called colloids. The ability to construct objects with colloids makes it possible to create structures with particular mechanical and thermal characteristics to manufacture electronic devices and tiny mechanical parts.

Purdue researchers are pursuing the technology for pharmaceutical manufacturing, Wereley says, because a number of drugs are manufactured from solid particles suspended in liquid. The particles have to be collected and separated from the liquid. This process is now done using filters and centrifuges.

REP also might be used to diagnose the presence of viruses, as well, although it has not yet been used to separate viruses from a sample, Wereley says.

Unlike conventional tools, REP requires only tiny samples, making it potentially practical for medical diagnostics and laboratory analysis.

Mechanical engineering doctoral student Jae-Sung Kwon, working extensively with Sandeep Ravindranath, a doctoral student in agricultural and biological engineering, is lead author of the Lab on a Chip paper. Researchers from Purdue, Oak Ridge National Laboratory, and Bindley Bioscience Center also contributed.

Source: Purdue University

Breath Test Could Sniff Out Infections in Minutes

Bacteria hiding in the lungs might not be able to hide much longer. Although traditional tests can take days or weeks to culture to determine the presence of certain harmful bacteria—such as those that cause tuberculosis—a much more rapid technique for detecting lung infections might be on the horizon.

Researchers have developed a test that can detect the presence of common infectious bacteria based just on the breath. The test picks up signature volatile organic compound (VOC)—particles emitted in gasses—profiles that the bacteria produce that are distinct those that the body—or other bacteria—give off. The findings were published online January 10 in the Journal of Breath Research.

The researchers, led by Jiangjiang Zhu of the University of Vermont, conducted the studies in lab mice that were infected with different types of common bacteria: two different strains of Pseudomonas aeruginosa, which can cause pneumonia, and one strain of Staphylococcus aureus, which can cause respiratory infections. The next day, the researchers tested the animals’ breath by ionizing breath samples then shooting them through a mass spectrometer to analyze concentrations of various VOCs in a process called secondary electrospray ionization mass spectrometry (SESI-MS).

The test detected the different bacterial infections as well as differentiated between healthy and infected mice. It also located the difference between the two strains of P. aeruginosa. The researchers call these different VOC signatures a bacteria’s “breathprint.”

“We have strong evidence that we can distinguish between bacteria infections of the lung in mice very effectively using the breathprint SESI-MS approach,” Jane Hill, also of the University of Vermont and study co-author, said in a prepared statement. This technique will have to be tested in large human trials before it makes an appearance in the clinic. But the rapidity of the test is appealing. And it could at least make it a good first step in detecting bacterial infections, with a follow-up culture coming later if deemed necessary—to detect drug-resistant TB, for example.

Hunting for compounds in the breath can be tricky because individuals have different collections of compounds, creating some 4,000 to 6,000 potential compounds to contend with. “This high degree of variability in the composition of human breath underscores the difficulty inherent in choosing a small number of biomarkers for diagnostic purposes,” the researchers noted in their paper. But the SESI-MS technique can find even very low concentrations of particular VOCs—all the way down to parts per trillion.

This testing technique is also promising because it samples the VOC fingerprint of a bacterial infection in a host—rather than comparing a host sample to a profile from bacteria cultured in the lab. This real-life proof-of-principle is important because, “there will be VOCs that are unique to the host-pathogen interaction,” the researchers hypothesized in their paper.

Similar breath tests have also been studied for detecting other ailments, such as diabetes and cancer. And Hill and her colleagues think that they might be able to extend the “breathprint” approach to identify other infections. “I suspect that we will also be able to distinguish between bacterial, viral and fungal infections of the lung,” she noted.

Source: Scientific American

Thursday, January 10, 2013

UMDNJ Develops Rapid Detection Test for Exserhilum rostratum in Response to Compounding Pharmacy Deaths

A rapid detection test for Exserhilum rostratum, the fungus primarily responsible for 39 deaths among patients injected last year with a contaminated steroid medication, has been developed by a research team led by David S. Perlin, PhD, Executive Director of the Public Health Research Institute (PHRI) at the University of Medicine and Dentistry of New Jersey-New Jersey Medical School. Details of the test have been published online by the Journal of Clinical Microbiology.

To date, clinicians and public health officials have been limited in their ability to detect the fungus in clinical samples such as cerebrospinal fluid, because the fungus tends not to be free-floating in the samples. The newly developed test improves on existing detection methods by using molecular beacon technology in a real-time polymerase chain reaction (PCR) assay. Through this approach, investigators have been able to detect a wide range of genomic DNA from E. rostratum, even in samples where abundant human DNA was also present. Fungal DNA has been reliably detected in amounts as small as 100 fg (femtograms, which are quadrillionths of a gram). The assay also detected DNA sequences from several related species of fungi, while tests of lab samples containing other species of fungi produced negative results, indicating a high level of specificity for the assay.

Perlin says, "The assay is a highly sensitive and robust method for early detection of the fungus in patients who might be infected but do not yet show symptoms, and also to monitor the progress of ongoing medical treatment." For critically ill patients, rapid diagnosis is essential and this assay has the potential to detect the presence of infecting fungus in less than two hours. "It is estimated that some 13,534 people received injections that might have exposed them to this fungus during the recent outbreak," notes Perlin. "We don't know the exact timeline for development of disease, and so we are not sure whether these patients are still are at risk. Using an assay such as this to detect possible infection in those patients could ease their minds if results are negative, or lead them to receive therapy in time to prevent future illness if results are positive."

The assay also can detect the fungus in vials of medication such as the steroid preparation that was the source of infection in the more than 600 patients who were sickened by the tainted injections. With that ability, drug manufacturers, health officials and even clinical practitioners could potentially check medication samples for presence of the fungus, says Dr. Perlin. In addition, the assay can be a valuable tool for future research on fungal infections.

Dr. Perlin and Yanan Zhao, PhD, developed the assay at PHRI in collaboration with Thomas J. Walsh, MD, Professor of Medicine, Pediatrics, and Microbiology & Immunology at Weill Cornell Medical Center, and his colleague Ruta Petraitiene, MD, Senior Research Associate. Dr. Walsh is coordinating a multicenter collaborative consortium to advance understanding of the diagnosis, treatment, host pathogenesis, and treatment of Exserohilum meningitis. Led by Dr. Perlin, this study is the first publication from this consortium.

Earlier molecular assays produced by Dr. Perlin and colleagues detect a wide range of fungal infections including Candida and Aspergillus species and drug resistant variants. The investigators have published research on these assays, and several have been commercialized.

Here is the full reference:

Real-Time PCR Assay for Rapid Detection and Quantification of Exserohilum rostratum, A Causative Pathogen of Fungal Meningitis from Injection of Contaminated Methylprednisolone. Yanan Zhao, Ruta Petraitiene, Thomas J. Walsh, and David S. Perlin. J. Clin. Microbiol. published ahead of print 9 January 2013 doi:10.1128/JCM.03369-12

Source: Journal of Clinical Microbiology and

Monday, January 7, 2013

FDA Proposes New Food Safety Standards for Foodborne Illness Prevention and Produce Safety

On January 4, 2013, the U.S. Food and Drug Administration (FDA) proposed two new food safety rules that will help prevent foodborne illness. The proposed rules implement the landmark, bipartisan FDA Food Safety Modernization Act (FSMA) and are available for public comment for the next 120 days. The FDA encourages Americans to review and comment on these important proposed rules.

Part of the rules recommend the use of rapid and nucleic acid-based methods for the detection of pathogens such as E. coli, Salmonella and Listeria

The proposed rules build on significant strides made during the Obama Administration, including the first egg safety rule protecting consumers from Salmonella and stepped up testing for E. coli in beef as well as existing voluntary industry guidelines for food safety, which many producers, growers and others currently follow.

The rules follow extensive outreach by the FDA to the produce industry, the consumer community, other government agencies and the international community. Since January 2011, FDA staff have toured farms and facilities nationwide and participated in hundreds of meetings and presentations with global regulatory partners, industry stakeholders, consumer groups, farmers, state and local officials, and the research community.

“The FDA Food Safety Modernization Act is a common sense law that shifts the food safety focus from reactive to preventive,” said Health and Human Services Secretary Kathleen Sebelius. “With the support of industry, consumer groups, and the bipartisan leadership in Congress, we are establishing a science-based, flexible system to better prevent foodborne illness and protect American families.”

The burden of foodborne illness in the United States is substantial. One in six Americans suffer from a foodborne illness every year. Of those, nearly 130,000 are hospitalized and 3,000 die from their illness. Preventing foodborne illnesses will improve public health, reduce medical costs, and avoid the costly disruptions of the food system caused by illness outbreaks and large-scale recalls.

These two FSMA rules are part of an integrated reform effort that focuses on prevention and addresses the safety of foods produced domestically and imported, with additional rules to be published shortly.

The first rule proposed today would require makers of food to be sold in the United States, whether produced at a foreign- or domestic-based facility, to develop a formal plan for preventing their food products from causing foodborne illness. The rule would also require them to have plans for correcting any problems that arise. The FDA seeks public comment on this proposal. The FDA is proposing that many food manufacturers be in compliance with the new preventive controls rules one year after the final rules are published in the Federal Register but small and very small businesses would be given additional time.

The FDA also seeks public comment on the second proposed rule released today, which proposes enforceable safety standards for the production and harvesting of produce on farms. This rule proposes science- and risk-based standards for the safe production and harvesting of fruits and vegetables.

The FDA is proposing that larger farms be in compliance with most of the produce safety requirements 26 months after the final rule is published in the Federal Register. Small and very small farms would have additional time to comply, and all farms would have additional time to comply with certain requirements related to water quality.

“The FDA knows that food safety, from farm to fork, requires partnership with industry, consumers, local, state and tribal governments, and our international trading partners,” said FDA Commissioner Margaret A. Hamburg, M.D. “Our proposed rules reflect the input we have received from these stakeholders and we look forward to working with the public as they review the proposed rules.”

Before issuing the two rules, the FDA conducted extensive outreach that included five federal public meetings and regional, state, and local meetings in 14 states across the country as well as making hundreds of presentations to ensure that the rules would be flexible enough to cover the diverse industries to be affected. The FDA also visited farms and facilities of varying sizes.

“We know one-size-fits-all rules won’t work,” said Michael R. Taylor, the FDA’s deputy commissioner for foods and veterinary medicine. “We’ve worked to develop proposed regulations that can be both effective and practical across today’s diverse food system.”

Additional rules to follow soon include new responsibilities for importers to verify that food products grown or processed overseas are as safe as domestically produced food and accreditation standards to strengthen the quality of third party food safety audits overseas. Improving oversight of imported food is an important goal of FSMA. Approximately 15 percent of the food consumed in the United States is imported, with much higher proportions in certain higher risk categories, such as produce. The FDA will also propose a preventive controls rule for animal food facilities, similar to the preventive controls rule proposed today for human food.

The FDA plans to coordinate the comment periods on the major FSMA proposals as fully as possible to better enable public comment on how the rules can best work together to create an integrated, effective and efficient food safety system.

For more information, please see the following FDA links:
An additional resource for staying up-to-date on the FSMA, including statistics and links, is provided in the Katom Restaurant Supply, Inc. Learning Center

Tuesday, January 1, 2013

Real-Time Outbreak Sequencing

When an infectious outbreak occurs, hospital investigators combine epidemiological data with bacterial typing to trace the source and path of the pathogen in the hopes of preventing further infections. Current methods are slow and offer limited resolution, meaning they can’t always differentiate between strains originating from the same bacterial clone. But by dramatically increasing the speed and accuracy of strain discrimination, a new generation of rapid, low-cost whole-genome sequencing (WGS) technologies promises to revolutionize outbreak surveillance and investigation.

With full genome sequences, researchers can spot the mutations that accumulate every time a bacterium divides down to the single nucleotide level. This allows them to track the evolution and movement of microbes with unprecedented precision, potentially leading to life-saving interventions and improved infection control strategies.

“I expect whole-genome sequencing will be transformative, in particular in outbreak investigations, within the next few years,” said Kathryn Holt, a microbiologist at the University of Melbourne in Australia. “The key advance is the dramatic increase in resolution, which enables us to be much surer about transmission pathways.”

Genomic detectives

Two studies from this year demonstrate the power of this approach. In 2011, the National Institutes of Health (NIH) Clinical Center in Bethesda, Maryland, experienced an outbreak of a highly resistant form of Klebsiella pneumoniae, which causes urinary, respiratory, and blood infections in people with weakened immune systems, and kills more than half of it infects. The doctors knew one patient was carrying the pathogen but she was carefully isolated, so they thought it had been contained. Then, 3 weeks after she was discharged, another patient was diagnosed and more cases followed. In total, 18 patients were infected and 6 died as a direct result.

The conventional method for bacterial typing is pulse-field gel electrophoresis, in which large DNA fragments are separated by an electric field to create genetic fingerprints for each bacterial sample. But the technique “was too coarse to tell us whether our first two cases were two separate introductions or [if] one was transmitted from the other,” said Tara Palmore, an infectious disease physician at the Clinical Center. Doctors and researchers were at a loss to explain the spread—and powerless to stop it.

Once the outbreak was under control, colleagues from the National Human Genome Center sequenced the genomes of the bacteria isolated from affected patients. Genetic similarities between samples revealed that all the strains had come from the first patient (patient 1), which had seemed unlikely from the epidemiological data—because there was no direct contact between that patient and those that were subsequently infected. They also found that patient 1 harbored three genetically distinct versions of Klebsiella, and that each had been transmitted on separate occasions to start three different lineages of infection.

Using an evolutionary tree to reconstruct the likely route, the researchers showed that “the dynamics of transmission were far more complex than we first realized,” said Palmore. “It was mostly being spread by asymptomatically colonized patients,” or “carriers.” For example, the bacteria first jumped from patient 1 to two patients that remained asymptomatic for some time—and it was those asymptomatic patients that passed the infection on to “patient 2,” the second patient to show signs of infection. Such a counterintuitive path could not have been predicted, and would not have been detected without genetic data.

Over in the United Kingdom, as a strain of methicillin-resistant Staphylococcus aureus (MRSA) spread through a neonatal unit at Rosie’s Hospital in Cambridge, infecting 12 infants over a 6-month period in 2011, researchers also turned to WGS for answers. A team at Cambridge University and the nearby Wellcome Trust Sanger Institute compared full-genome sequences of bacteria from infected infants, and revealed that all the strains were descended from a common source. Then, when another baby was infected 2 months after the previous case, sequencing showed it was part of the same outbreak. That led the team to screen all the staff, which identified one person who was carrying MRSA—and was likely involved spreading it to the babies.

“It’s impossible to prove that this epidemic was stopped by this intervention, but we believe it prevented further transmission,” said Julian Parkhill, a microbiologist at the Sanger Institute and co-author of the study. But either way, he added, it demonstrates the power of whole-genome sequencing for elucidating the movements of pathogens.

Routine practice?

With the latest bench-top machines capable of sequencing a bacterial genome in just a few hours, outbreak analyses can now be performed in real time—with obvious benefits. “It will enable rapid identification of a [real] transmission, and save a lot of time and effort in not having to chase down spurious transmissions,” said Parkhill. And more efficient tracking of transmission allows for much more targeted and effective infection-control measures, Palmore added.

In addition, WGS will help to track antibiotic resistance mutations as they evolve. “It can be quicker than phenotypic testing, which requires growing the bacteria in the presence of a range of antibiotics,” said Holt. “With sequencing, we can quickly see which resistance mechanisms the bug has encoded in its DNA,” which can guide treatments.

Real-time sequencing of hospital pathogens is unlikely to become a routine practice quite yet, however, largely because the interpretation of genetic data requires a level of expertise that lies beyond most clinicians. To overcome that obstacle, the development of analysis tools that can provide clinically relevant information in a manner that infectious-disease physicians can understand is absolutely critical, said Parkhill. “The average clinician in a hospital is not going to be able to do this [analysis], so it has to be automated.” Parkhill’s group is working to develop such a system.

Meanwhile, the UK Health Protection Agency is already exploring whether it is cost-effective to use the approach to supplement existing methods in a select few English hospitals. Indeed, although only a handful of proof-of-principle case studies exist, it’s clear that WGS is going to have a big impact on infection control and public health in the future, said Derrick Crook, a microbiologist at the University of Oxford who worked on a pilot study for tracking MRSA and C. difficile in three UK hospitals. “We’re looking at this being used [in a clinical setting] over the next few years.”