FDA's Proposed Changes Encourage the Use of Rapid Methods for Sterility Testing of Biologics

In a bold move by FDA's Center for Biologics Evaluation and Research (CBER), sweeping changes are now being proposed for the sterility testing of biological products. Some of the proposed changes include the elimination of culture media formulae and test requirements, sterility test requirements for most bulk material, modification of the repeat sterility test requirements, changes to the storage and maintenance requirements for cultures of test organisms used during growth promotion, and replacement of the sample size requirement. What is most relevant to this blog is that the draft changes include promoting the use of rapid microbiological methods as an alternative to the standard USP <71>. In addition, the proposed rule provides guidance on validation expectations and references the USP information chapter <1223>, Validation of Alternative Microbiological Methods. Public comments on this proposed rule must be submitted by September 19, 2011.

The proposed changes, which were published in the June 21, 2011 U.S. Federal Register (Volume 76, Issue 119) come on the heels of CBER's guidance for industry on this same topic, when, in 2008, the FDA published "Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products.” The guidance applies to somatic cellular therapy and gene therapy products, and provides a roadmap for demonstrating that an alternative, growth-based RMM is equivalent to the conventional sterility test method.

There are a number of changes that encompass a variety of topics; however, I will focus only on those changes specifically dealing with rapid or alternative methods. An overview of these proposed changes is provided below, and you can read the entire proposed rule at http://www.gpo.gov/fdsys/pkg/FR-2011-06-21/pdf/2011-15346.pdf. As you read this overview, please remember that these changes are specifically intended for biological products that are regulated by CBER, and may not apply to pharmaceutical products regulated by other FDA Centers, such as CDER.

Amendments to Sterility Test Requirements for Biological Products

Summary

The Food and Drug Administration (FDA) proposes to amend the sterility test requirements for biological products. This proposed rule is intended to provide manufacturers of biological products greater flexibility and to encourage use of the most appropriate and state-of-the-art test methods for assuring the safety of biological products. We are taking this action as part of our continuing effort to review and, as necessary, update the biologics regulations.

Background

Manufacturers of innovative products, such as cell and gene therapy products, as well as manufacturers of currently approved products, may benefit from sterility test methods with rapid and advanced detection capabilities.

Advances in technology in recent years have allowed the development of new sterility test methods that yield accurate and reliable test results in less time and with less operator intervention than the currently prescribed methods. Some examples of novel methods with the potential to detect viable contaminating microorganisms include the Adenosine Triphosphate (ATP) bioluminescence, chemiluminescence, and carbon dioxide head space measurement.

We are proposing to amend § 610.12 to promote improvement and innovation in the development of sterility test methods, to address the challenges of novel products that may be introduced to the market in the future, and to potentially enhance sterility testing of currently approved products. This proposed revision would provide manufacturers the flexibility to take advantage of modern methods as they become available, provided that these methods meet certain criteria.

Description of the Proposed Rule

This proposed rule is intended to promote improvement and innovation in the development of sterility test methods by allowing manufacturers flexibility needed for sterility testing of some novel products that may be introduced to the market, to enhance sterility testing of currently approved products, and to encourage manufacturers to benefit from scientific and technological advances in sterility test methods as they become available.

Test Methods

Currently, § 610.12(a), (b), and (e) prescribe the culture-based test method to be used for sterility testing, including the acceptable culture media (either Fluid Thioglycollate Medium or Soybean-Casein Digest Medium) and incubation conditions (time and temperature) to be used during testing, with exceptions provided in § 610.12(g). In addition, § 610.12(f) provides that a membrane filtration test method, set forth in (USP 23d revision, 1995), may be used to test bulk and final container materials or products containing oil products in water-insoluble ointments.

We propose to eliminate references to specific test methods and culture media for sterility testing, and instead require that the sterility test be appropriate to the material being tested such that the material does not interfere with or otherwise hinder the test. We believe that this revision recognizes current practices and provides manufacturers the flexibility to take advantage of suitable modern sterility test methods and keep pace with advances in science and technology. Because we are proposing to expand potentially acceptable sterility test methods to include non-culture-based methods in addition to culture-based methods, we also propose to remove the definition of a lot of culture medium currently defined in § 610.12(e)(2)(i).

Section § 610.12(e)(2)(ii) stipulates the test organisms, strains, characteristics, identity, and verification to be used. We propose to eliminate the requirement to test culture media with specific test organisms and to eliminate the requirement regarding the number of organisms that must be used to demonstrate the growth-promoting qualities of the culture media. This flexibility would allow manufacturers to use sterility test methods that are either culture-based or non-culture-based, which may necessitate different verification activities. Thus, instead of specifying the number and type of test organisms, proposed § 610.12(b) would require the following: (1) Use of a sterility test method that is appropriate to the material being tested such that the material does not interfere with or otherwise hinder the test; (2) validation studies to demonstrate that the sterility test method used is capable of consistently detecting the presence of viable contaminating microorganisms; and (3) verification that the sterility test method and test components used can detect the presence of viable contaminating microorganisms.

Due to the variety of currently available and potential future sterility test methods, we propose to eliminate specified incubation conditions (time and temperature) and visual examination requirements currently prescribed in § 610.12.

Validation

USP General Chapter 1223, ‘‘Validation of Alternative Microbiological Methods,’’ states: ‘‘Validation of a microbiological method is the process by which it is experimentally established that the performance characteristics of the method meet the requirements for the intended application.’’ For sterility testing, this means that the test can consistently detect the presence of viable contaminating microorganisms.

We propose to eliminate the prescribed sterility test methods found in current § 610.12 and instead allow the use of sterility test methods that are validated in accordance with established protocols, to be capable of consistently detecting the presence of viable contaminating microorganisms. If an established USP compendial sterility test method is used, a manufacturer must verify that this established method is suitable for application to the specific product; however, FDA considers established USP compendial sterility test methods to already have been validated using an established validation protocol, so their accuracy, specificity, and reproducibility need not be re-established to fulfill the proposed validation requirement. In contrast, novel methods and any methods that deviate from the USP compendial sterility test methods would require the detailed validation discussed (below).

Proposed § 610.12 allows the use of a material sample that does not interfere with or otherwise hinder the sterility test from detecting viable contaminating microorganisms. This requirement is crucial, because the material itself or substances added to the material during formulation may make some sterility tests inappropriate for use. A validated sterility test method is a critical element in assuring the safety and quality of the product. USP General Chapter 1223, as well as the ICH Guideline for Industry (Text on Analytical Procedures), provide general descriptions of typical validation parameters, how they are determined, and which subset of each parameter is required to demonstrate validity, based on the method’s intended use.

In the context of reviewing sterility test methods as part of BLAs and BLA supplements, FDA may decide, as appropriate, to encourage the use of the compendial method as a benchmark or starting point for validation of novel methods and certain other methods. FDA is specifically seeking comments on whether the proposed requirements are sufficient to ensure adequate validation of novel sterility test methods or whether additional criteria or guidance is needed.

It is important to consider validation principles, such as limit of detection, specificity, ruggedness, and robustness, while developing the validation protocol and performing validation studies. These terms are defined as follows:

- The limit of detection reflects the lowest number of microorganisms that can be detected by the method in a sample matrix. This is necessary to define what is considered contaminated.

- Specificity is the ability of the test method to detect a range of organisms necessary for the method to be suitable for its intended use. This is demonstrated by challenging the sterility test with a panel of relevant organisms in the sample matrix.

- Ruggedness is the degree of reproducibility of results obtained by analysis of the same sample under a variety of normal test conditions, such as different analysts, different instruments, and different reagent lots.

- Robustness is the capacity of the test method to remain unaffected by small, but deliberate variations in method parameters, such as changes in reagent concentration or incubation temperatures.

For non-culture-based methods, the feasibility of identifying microorganisms from a contaminated sample should be evaluated during validation. If a method does not have the capability to identify microorganisms to the species level, the validation protocol should require that an additional method for species identification be utilized for investigation of detected contaminants.

The test organisms selected should reflect organisms that could be found in the product, process, or manufacturing environment.

The validation study design should contain the appropriate controls to evaluate the product sample’s potential to generate false positive and false negative results. Validation of the sterility test should be performed on all new products, and repeated whenever there are changes in the test method that could potentially inhibit or enhance detection of viable contaminating microorganisms.

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Blogging at 35,000 Feet and Thinking About E. coli

As I sit on a flight from Tucson to Dallas, I am making use of the onboard WI-FI service and, believe it or not, thinking about rapid micro methods. If you have ever used the lavatory during flight, or asked for a coffee from the flight attendant, I am certain that many of you (especially the microbiologists reading this post) have wondered how contaminated the airplane water really is.

In the United States, drinking water safety on airlines is jointly regulated by the EPA, the Food and Drug Administration (FDA), and the Federal Aviation Administration (FAA). EPA regulates water quality in public water systems in accordance with the Safe Drinking Water Act (SDWA). EPA regulates the public water systems that supply water to the airports and the drinking water once it is onboard the aircraft. The regulatory structure for all public water systems, including aircraft, relies upon self-monitoring and reporting of results to the primacy (primary enforcement) agency. The primacy agency for aircraft public water systems is EPA.

In 2004, EPA found all aircraft PWSs to be out of compliance with the national primary drinking water regulations (NPDWRs). According to the air carriers, it is not feasible for them to comply with all of the monitoring that is required in the existing regulations. The existing NPDWRs were designed for traditional, stationary public water systems, not mobile aircraft water systems that are operationally very different.

For example, aircraft must maintain rigorous operating schedules. They fly to multiple destinations throughout the course of any given day and may board drinking water from sources at any of these destinations. Aircraft board water from airport watering points via temporary connections.

Aircraft drinking water safety depends on a number of factors including the quality of the water that is boarded from these multiple sources, the care used to board the water, and the operation and maintenance of the onboard water system and the water transfer equipment (such as water cabinets, trucks, carts, and hoses).

These unique operational characteristics present different challenges, which EPA addressed in the Aircraft Drinking Water Rule (ADWR), which went into effect in 2009.

The primary purpose of the ADWR is to ensure that safe and reliable drinking water is provided to aircraft passengers and crew. This entails providing air carriers with a feasible way to comply with the SDWA and the NPDWRs. The existing regulations were designed primarily with traditional, stationary public water systems in mind. Some of these requirements have proven difficult to implement when applied to aircraft water systems, which are operationally very different. Therefore, using a collaborative rule making process, EPA developed the ADWR that is tailored to aircraft public water systems. The final rule combines coliform sampling, best management practices, corrective action, public notification, operator training, and reporting and record keeping to improve public health protection.

In reading the rule, the frequency of coliform monitoring is tied to the frequency of disinfection and flushing of the aircraft water system. For example, if an aircraft is disinfected and flushed on a quarterly basis, the minimum frequency of routine sampling is only one time within a 12-month period. If the disinfection and flushing schedule is only one time per year or less, then the frequency of monitoring is increased to 12 times per year (monthly).

Two coliform samples are taken per monitoring period: One sample must be taken from a lavatory and one sample from a galley. Any total coliform-positive sample must be further analyzed for the presence of E. coli. If any routine sample is E. coli-positive, public access to the water system is restricted no later than 24 hours after being notified by the testing lab of the E. coli-positive result, and all public access restrictions must remain in-place until the aircraft water system has been disinfected and flushed and a complete set of follow-up samples is total coliform-negative.

Here's the big question: if the samples are sent to a lab, how long does it take for positive results to get back to the airline in order to implement these restrictions and perform follow-up testing after the system has been disinfected and flushed? Wouldn't this be a great place to implement rapid testing methods for the airline industry? There are a number of presence/absence tests for E. coli and other potential water-borne pathogens that could be used today.

Just something to think about the next time you get on an airplane. I am thinking about it right now!

To read the entire EPA Final Rule, please Click on This Link to download the PDF file.

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New MRSA strain detected among humans, cows in UK, Denmark

Researchers have discovered a new strain of methicillin-resistant Staphylococcus aureus, a mecA homologue that is genetically different compared with existing strains, in both human and bovine populations in the UK and Denmark. Standard molecular tests failed to identify the strain, therefore new diagnostic guidelines for the detection of MRSA should consider tests for the mecALGA251 gene, according to data presented at a press conference in advance of publication in The Lancet Infectious Diseases.

“The test that we use at the moment to confirm MRSA will be falsely negative if we don’t take into account the new mecA gene,” Mark A. Holmes, PhD, of the department of veterinary medicine at the University of Cambridge, said during the press conference. “We are concerned that there is circumstantial evidence that MRSA is moving between cattle and people. The new MRSA is found in strains or family types of S. aureus that previously were only thought to exist within cows.”

In 2007, Holmes and colleagues identified a pair of isolates (strains LGA251 and LGA254) resistant to methicillin-like drugs in milk from dairy cows and set out to determine the cause of resistance in the bacteria. The researchers tested for mecA, the methicillin-resistant gene, using the standard polymerase chain reaction (PCR).

Compared with PCR test results, which yielded negative results for methicillin resistance, whole genome sequencing revealed that there was, in fact, a mecA gene, a homologue with only 60% similarity to the conventional mecA gene.

Subsequently, the researchers searched for human MRSA isolates, and similar families in both cows and people in different parts of the countries were found with this strain, suggesting geographical clustering.

Approximately 51 isolates tested positive for mecA; 15 of 26 from England; 12 of 16 from Scotland; and 24 of 32 from Denmark. Further, after assessing trends in annual incidence of mecA detection, the researchers found that rates of these MRSA increased substantially between 2007 and 2010.

“The discovery of this new mecA homologue raises issues about the detection and confirmation of MRSA,” according to the researchers. “Irrespective of whether an infection is caused by mecALGA251 MRSA or conventional MRSA, after culture and antimicrobial susceptibility testing, appropriate decisions about care of patients can be made. However, when existing PCR or monoclonal antibody methods are used as the only method to detect MRSA, or when these methods are used to confirm provisional detection of MRSA, then mecALGA251 S. aureus will be wrongly diagnosed as methicillin-susceptible.”

Although the discovery of this previously undetected mecA homologue is of public health importance, Holmes said drinking milk and consuming dairy products is not a public health concern.

“There is no survival of MRSA in milk or dairy products, as long as the milk is pasteurized,” he said. “The main worry would be that these cows represent a pool of the bacteria, that these bacteria end up colonizing people who work or live on farms and they take it out to the wider community.”

The researchers said the ramifications of not detecting S. aureus strains that carry this new strain should be considered in diagnostic protocols.

Disclosure: This research was funded by the Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

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Scientists Shed Light on Germany's Deadly Pathogen

Foodsafetynews.com has recently provided an interesting overview of the E. coli outbreak in Germany:

As the devastating outbreak of E. coli began unfolding in Germany, there was an urgent need to learn more about the unusual pathogen that was making hundreds of people sick.

So data from the genome sequencing of the rare E. coli serotype O104 were instantly released via the Internet to researchers around the world, and a flurry of information-swapping followed. Discussions about this strange bacterium went viral, rapidly communicated via blogs, Twitter and private web pages.

This combination of advanced molecular typing technology and electronic communication has led to what the authors describe as intriguing, preliminary analyses "outside the standard peer-reviewed scientific publication route."

The accompanying report in Eurosurveillance, written by an international team of investigators, emphasizes the importance of working collaboratively to quickly unlock the secrets of an infectious organism.

"The rapid exchange of information, strains and DNA fingerprints within national and international public health and food safety networks has been vital in the quick and alternative assessment of the public health significance causing the outbreak of HUS in Germany in May and June 2011," the authors state.

The contributions have resulted not only in "important new findings on the nature and possible origin of the epidemic strain," but the microbiology has helped to devise new lab screening tools for hospitals diagnosing patients and for public health authorities investigating the outbreak.

Among the findings:

The outbreak strain is not a typical virulent Shiga toxin-producing (STEC) strain, the authors write, but a much rarer and "hypervirulent" hybrid -- an enteroaggregative E. coli (EAggEC) now armed with the ability to produce Shiga toxin.

Adding to its virulence is a receptor for iron-chelating aerobactin, which is associated with extraintestinal E. coli strains, like those that cause diseases such as meningitis, sepsis and urinary tract infections outside the intestinal tract.

The genome of the outbreak strain "clustered closest" to an EAggEC strain isolated in 2002, with the addition of the Shiga toxin factor and antibiotic resistance genes.

EAggEC causes diarrhea in travelers and persistent diarrhea in infants and young children in countries with poor sanitation, but is common in all parts of the world, industrialized and developing nations alike, the report explains. Unlike STEC, which have an animal reservoir (primarily ruminants), EAggEC have a human reservoir, so E. coli O104 likely does too.

"This observation suggests the startling possibility," the authors note, "that this new O104 strain may have the capacity to persist among human populations, perhaps indefinitely."

EAggEC is capable of causing diarrhea in adults and children, even in the absence of Shiga toxin, notes the report, which cites a 1993 outbreak of the bacteria among Japanese children that sickened 2,700.

The authors also state that "the role of EAggEC as an important pathogen in AIDS patients continues to develop, and EAggEC now ranks among the most important enteric pathogens in this population group."

While most STEC are eae positive, the outbreak O104 strain in Germany is eae negative -- eae being genetic coding for the protein intimin, which the bacteria use to attach to the intestinal wall. The Eurosurveillance report says, " ... it is indeed conceivable that the enteroaggregative adherence phenotype could have allowed these E. coli O104 strains to colonise the intestinal mucosa of the affected patients as efficiently as typical eae-positive STEC/VTEC strains.

"The different mechanism of adhesion might also explain why this strain is more likely to cause severe disease in adults rather than children ..." But the authors say this factor needs more study, and that the different rates of hemolytic uremic syndrome (HUS) between adults and children in Germany might just reflect a difference in exposures to the outbreak source.

Hybrid strains of E. coli such as O104 aren't new, the researchers suggest. For example, one HUS outbreak caused by a "mixed pathotype," but a different serotype, was reported from France in 1998. But the authors found five previously reported cases of diarrhea or HUS caused by O104:H4: Germany in 2001, France in 2004, South Korea in 2005, Georgia in 2009 and Finland in 2010.

The 2011 German outbreak strain most closely resembles the Georgian strain genetically, they said, but there is no known epidemiological link between the two so "the meaning of this finding remains elusive."

The Eurosurveillance editorial says that since June 10, the number of newly reported cases of infection and HUS in Germany has been gradually decreasing, "which suggests we may finally be reaching the end of the outbreak."

The search for the source and vehicle of the outbreak has been "long and arduous," the editorial went on, adding that it was "extensive investigations" that finally implicated the organic sprout farm in Lower Saxony near Hamburg.

"Sprouts produced at this farm had been distributed to many of the incriminated restaurants and catering facilities, and thus identified as the likely vehicle of infection," the authors wrote.

As of last week, the outbreak toll was 3,335 ill, 817 with HUS and 37 dead, including a 2-year-old boy, the first child to die.

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Rapid Micro Biosystems Announces Opening of New Demo Center in Europe

More RMM companies are expanding their training centers in Europe. This time, Rapid Micro Biosystems announced the opening of a state-of-the-art demo center located in Darmstadt, Germany.

The new space provides a dedicated demonstration environment for showcasing the Growth Direct™ system -- the company's automated, rapid, non-destructive microbial enumeration device -- in a simulated lab environment.

The center will exhibit automated rapid detection and its value to the microbial QC user. In addition to the demonstration space, the facility also includes a spacious conference room and reception area for visitors.

"We are excited about the new facility and look forward to the opportunity to demonstrate to those companies that want to accelerate and automate their microbial testing the unique value that Growth Direct™ can bring to businesses," said Christof Hasse, Rapid Micro Biosystems' European sales manager.

"The European Market is integral to our success, and this demo center enhances our commitment to Europe," said Steve Delity, CEO of Rapid Micro Biosystems.

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UK Court Trial Starts Over 3M's Killing Of MRSA Superbug Test

The development of a rapid method for the detection of MRSA in hospitals has been scrapped, and now the UK government is looking for compensation from 3M. The following is an overview of the unfolding story from PRNewswire:

A company owned by the Ministry of Defence today (Wednesday June 15) will launch a High Court lawsuit against the American giant 3M, claiming that it failed properly to market an innovative MRSA diagnostic invented by the MoD at its Porton Down research laboratories.

The MoD company, Ploughshare Innovations, and its financial partner, private equity firm Porton Capital, are claiming up to £41m from 3M for failing to 'actively and diligently' market their MRSA test, named BacLite. 3M denies liability and is defending the claim.

BacLite was a high-speed test which enabled hospitals to identify MRSA infections within five hours, compared to the more usual 48-hour test procedures. Originally discovered in the nineties at the MoD's Porton Down research establishment in Wiltshire, it was subsequently approved by the UK health authorities and was first marketed to hospitals here in 2005.

Following early sales success in the UK, in 2007 3M bought BacLite and the company which the two partners had formed to market it, Acolyte, for an initial £10.4m. An earn-out clause in the contract meant that the partners could have been entitled to up to an additional £41m depending on BacLite's sales between 2007 and 2009.

Ploughshare and Porton's sales agreement with 3M committed the American group to develop and market BacLite to health authorities and hospitals inEurope, North America, and Australia. However, just over a year later 3M closed the business, following intervention by its chairman, CEO and President, British-born George Buckley.

The High Court lawsuit launched yesterday will accuse 3M of deliberately allowing BacLite to fail commercially by starving it of competent staff and funds. Additionally, the lawsuit alleges that 3M mismanaged its application for regulatory approval for BacLite with the US health authorities, and failed to seek similar approval at all in other territories.

Harvey Boulter, CEO of Porton Capital, commented - "The failure of 3M to carry out its contractual obligations and commercialise an already-proven British invention is a disgrace. MRSA is a potentially fatal germ which affects hospital patients across the world. Not only could BacLite have helped reduce patients' exposure to MRSA, but the British taxpayer could and should have benefited financially from the MoD's invention. Instead, they have been left out of pocket by 3M's negligence."

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The Nanomechanics Revolution in Medicine and Microbial Detection

When I teach my comprehensive training course on Rapid Microbiological Methods, I always include novel and next generation technology platforms in my discussions. One of my favorite topics is the future of microbial detection using nanotechnology, in particular, micro and nano cantilevers. I have recently come across a nice review of microcantilevers from the Medical technology Business Europe website. In this article, the authors discuss the use of microcantilevers for the assessment of drug-target interactions for a broad range of antibiotics and drugs under development. However, the technology can also be used for microbial detection. For example, scientists at Purdue University are developing advanced nanocantilever sensors capable of detecting minute quantities of viruses, bacteria and other contaminants in air and fluids by coating the cantilevers with proteins and antibodies that attract these contaminants.

I am providing a condensed version of the online article below, but if you would like to read the full paper, please visit www.mtbeurope.info/content/ft1106001.htm.

Within all areas of medical science, there is a constant need for new techniques, drugs, and ideas. One crucially urgent area is that of anti-bacterial drugs. The current international portfolio of antibiotics is becoming increasingly redundant as healthcare acquired infections (HAI) are on the up, and an increasing proportion of these are with Multi-Drug Resistant (MDR) bacterial strains, which are resistant to normal antibiotics.

However, a novel technique exploiting nanotechnology used in the micro-electronics industry presents the opportunity to speed up the discovery process for antibiotics and other pharmaceutical products, as well as many other diagnostic and therapeutic processes.

The nanotechnology in question uses microcantilevers, and their bending properties to evaluate the binding interactions between small molecules such as drugs or DNA. Microcantilevers are thin (<10µm) strips of silicon (0.5mm long and 0.1mm wide) which can detect the binding of molecules, target analytes, to ligands attached to the cantilever.

Each cantilever is ‘functionalised’ with particular surface coatings: these comprise of a primary layer (a self-assembled monolayer, SAM) and ligands that bind to specific target analytes. The ligand is immobilised on one side of the cantilever and the relevant target analyte is added to the system in solution. Binding interactions of the ligand and target analytes results in bending of the cantilever.

These highly sensitive biosensors detect minute changes in surface stress as the drug binds specifically to the ligands tethered on the cantilever (click on the illustration at the top of this blog post for a closer look). They can also be operated in a dynamic mode, when the added mass of bacteria or other biological substances can be detected.

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The Search for E. coli Gains Speed

The fever is heating up in Europe over the recent E. coli scare, and strain-specific tests quickly ruled out bean sprouts as the cause of the deadly outbreak. The following update comes from http://technologyreview.com, which is published by MIT.

German bean sprouts were declared dangerous on Monday morning and safe by Monday afternoon.

Bean sprouts were briefly added to the list of foods seen as potential sources of E. coli 0104—a new strain that has infected more than 2,300 people and killed 23 in Germany over the past three weeks. The list of potentially dangerous foods includes cucumbers, lettuce, tomatoes, and other raw salad vegetables.

The rapid reprieve for sprouts can be attributed to the availability of high-speed pathogen-specific tests. These assays, laboratory procedures that measure the biochemical activities of food samples, can now return reliable results in as little as 10 to 24 hours.

"As soon as we finished sequencing the DNA of this strain, we made the results public, like any ethical company would," says Nir Nimrodi, head of food safety at Life Technologies, which has developed a custom test for E. coli 0104.

Life Technologies, a California-based biotechnology company with laboratories in Darmstadt, Germany, started shipping custom testing kits to European laboratories this week so that food suspected of carrying the pathogen could be tested as quickly as possible.

"We all have E. coli right now in our bellies," Nimrodi says, "but not all the strains are dangerous, and even fewer of them are deadly to those infected. And if you use a general E. coli test, even if it tests positive, it does not mean you have discovered a source. With a strain-specific test, you can find out if you have been watering plants with infected water and need to stop, or if you have a specific crop of vegetables that are affected and need to be recalled."

It can take as long as 10 days to gather results using traditional laboratory testing methods, but the new test can determine the presence or absence of the hybrid E. coli pathogen in hours.

"It has implications for public health and epidemic monitoring," says Michael Somers of Children's Hospital Boston. "The less time you have to spend growing patient specimens and subtyping something like E. coli, the easier it is to find the source of an epidemic."

He added that "about 10 percent of the people infected with E. coli develop hemolytic uremic syndrome (HUS)." The kidney infection develops four to 10 days after bloody diarrhea appears. Some 90 percent of HUS cases in children are attributed to E. coli infections.

According to researchers, the new strain is a hybrid of enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC).

EAEC was first identified in 1987 and is well-documented as a cause of persistent diarrhea, especially in children in the developing world, and as a food pathogen that is increasingly resistant to antibiotics in industrialized countries. EHEC is associated with hemorrhagic colitis and HUS. It is the leading cause of acute renal failure in children.

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FDA Clears Idaho Technology-Developed Q Fever Test

This week the U.S. Food and Drug Administration cleared the first nucleic acid amplification In vitro diagnostic (IVD) test that detects Coxiella burnetii, the bacteria that causes Q fever. The test developed by Idaho Technology, Inc. (ITI) will be used to test military personnel suspected of contracting the disease and run on the Joint Biological Agent Identification and Diagnostics System (JBAIDS), utilized across all branches of the military for diagnostic testing. Use of the test is limited to designated Department of Defense laboratories equipped with the JBAIDS.

Q fever is an emerging infectious disease among U.S. soldiers serving in Iraq and other countries around the globe. The disease is usually transmitted to humans after contact with infected animals or exposure to contaminated environments. The bacteria is extremely hardy and resistant to heat, drying and many common disinfectants which can enable it to survive for very long periods in the environment. Early detection and identification of the bacteria will enable faster treatment and recovery.

“We are very pleased that the JBAIDS capability continues to evolve. Q fever marks the fifth FDA-approved JBAIDS assay kit with many more in the pipeline and expected out soon,” said Kirk Ririe, chief executive officer of ITI.

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DNA Sequencing Data Reveals New Hybrid E. coli Strain is Cause of German Outbreak

As a follow up to my posting regarding the recent E. coli outbreak in Europe, Life Technologies Corporation announced that preliminary data from DNA sequencing performed in cooperation with the University Hospital Muenster, Germany, on the Ion Personal Genome Machine (PGM™) strongly suggests that the bacterium at the root of the deadly outbreak in Germany is a new hybrid type of pathogenic E. coli strains.

The data obtained from the DNA sequencer shows the presence of genes typically found in two different types of E. coli: enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results, which are being confirmed by further data analysis on the Ion PGM™, may provide insight into this bacterium's aggressiveness and help prevent further outbreaks.

"The rapid whole genome sequencing results enabled us to discover within days a unique combination of virulence traits ... and makes this German outbreak clone a unique hybrid of different E. coli pathovars," said Dr. med. Alexander Mellmann, scientist at the German National Consulting Laboratory for Hemolytic Uremic Syndrome (HUS) at the Institute of Hygiene, University Hospital Muenster.

The data gathered at Life Technologies' laboratories in Darmstadt, Germany, will be used by scientists at the University Hospital Muenster, to develop better tests to positively identify the illness in people showing symptoms of the infection, which includes kidney failure and bloody diarrhea. For the first time since the outbreak, it is now possible to study in detail the basis for what makes this strain so aggressive.

Life Technologies' Simone Guenther, Ph.D., who carried out the sequencing work, said: "The severity of this outbreak meant that speed was of the essence. We were able to provide the data in record time to University Hospital Muenster. In previous outbreaks it would have taken much longer to reach this stage."

To prevent further spreading of the bacterium, Life Technologies began shipping its custom E. coli testing kits to European laboratories this week to screen contaminated food thought to be at the center of the outbreak that has killed 17 people and affected more than 1,000 in Europe.

The kits serve as a first line of defense to detect the presence of pathogenic E. coli. Secondary testing is then performed using more specific kits. Life Technologies will develop new customized kits specifically designed to detect the hybrid strain inGermany once the sequencing data has been fully analyzed in the next few days. The company can design custom assays in less than one week.

The kits shipped to Europe are part of a large offering of molecular tests developed by Life Technologies capable of accurately detecting most pathogens that are dangerous to humans. The Ion PGM™ is a DNA sequencing instrument featuring semiconductor technology that can complete a highly accurate sequence of DNA in just two hours. The DNA sample used for the sequencing work was collected from a patient with the illness.

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Clean water tech firm gets funding from Ontario

An Ottawa tech company will get a $2.7 million injection from the province to help it develop a new water and food pathogen detection product, Infrastructure Minister and Ottawa West-Nepean MPP Bob Chiarelli announced yesterday.

Chiarelli visited Spartan Bioscience yesterday to learn about the company’s water testing technology and to announce that 50 new clean water jobs will soon be created in Ottawa thanks to the province's funiding.

“We will develop a system for the rapid detection of pathogens in food and water,” said Spartan Bioscience CEO Larry D’Andrea. “The system that we developed right here in Ottawa will help to strengthen the safety of the food and water supply.”

The on-site water testing system will reduce analysis times from 72 hours to about two to four hours and help reduce the risk of bugs such as Listeria, E. coli, and salmonella. The government funding will go toward the $7.8 million dollar project, as the company immediately expands the number of employees from 20 to 43.

“We have left in our wake the terrible legacy of Walkterton. Today, Ontario has the highest standards of drinking water in North America,” said Chiarelli.

He also said the high-tech is in revival mode in Ottawa and looks forward to see the Spartan system expand globally.

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PCR methods for the rapid detection and identification of pathogenic Legionella spp. in water

Scientists from the College of Life Sciences, Nankai University, Tianjin, China have demonstrated a multiplex PCR method to be highly specific and reproducible when tested against 41 target strains of Legionella and 17 strains of other bacterial species in mock water samples. The results of their studies were published in Applied Microbiology and Biotechnology.

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB. Zhou G, Cao B, Dou Y, Liu Y, Feng L, Wang L. Appl Microbiol Biotechnol. 2011 May 29. [Epub ahead of print].

Abstract

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.

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Comparison of gene amplification and culture-based methods for Salmonella detection

A recent publication in the Journal of Food Protection compares the detection sensitivity of Salmonella typhimurium by reverse transcriptase PCR, loop-mediated isothermal amplification and culture-based assays. The authors conclude that the 1-day RT-LAMP assay can be used for routine Salmonella screening by the pork industry. The full reference and abstract follows.

Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments. Techathuvanan C, Draughon FA, D'Souza DH. J Food Prot. 2011 Feb; 74(2): 294-301.

Abstract

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry.

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