Source: http://blog.rapidmicromethods.com/2011/06/
Timestamp: 2019-04-19 04:32:07+00:00

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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.
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.
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.
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.
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.
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.
- 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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].
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.
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.
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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