PDA Microbiology Conference Update: Real Time Microbial Detection and Quality Control

Rapid microbiological methods (RMM) are utilized for a variety of applications, including in-process bioburden testing, environmental monitoring and finished product release for both sterile and non-sterile product. Recent advances in optical spectroscopic techniques now allow for the real-time detection, sizing and enumeration of microorganisms during volumetric air sampling. For the very first time, one of today’s sessions brought together three technology innovators that provide this type of instantaneous microbial detection capability. Following an introduction into the need for real-time monitoring that will support a QbD and PAT model for the parametric or real-time release of aseptically-filled, each innovator discussed the scientific and quality benefits, as well as contamination control opportunities that their technologies can provide.

I opened the session with a future vision for the real-time release of aseptically filled product. To realize this vision, I discussed the need for RMMs to be incorporated into the manufacturing process stream, providing real-time and continuous monitoring of in-process bioburden (e.g., pre- and post-filtration, and especially at the point of filling), in addition to real-time environmental monitoring. Additional considerations included the utilization of a robust manufacturing barrier system (e.g., an isolator or closed RABS), which would eliminate human-borne contaminants, as well as the incorporation of advanced aseptic filling technologies, such as blow-fill-seal and closed-vial filling technologies.

The next three presentations focused on real-time viable air monitoring technologies that are currently available to the pharmaceutical industry. Brief summaries and conclusions from each speaker are provided below.

IMD-A Systems for Instantaneous, Data-Rich Detection. Scott Morris, Applications Engineering Manager, BioVigilant

• A technical overview of two IMD-A instruments, validation data and potential applications were provided
• Continuous monitoring and technological sensitivity are a paradigm shift from traditional methods; these come with great benefits but will also require validation 
• Different applications and environments are unique; therefore, in-situ testing and qualification of optical spectroscopic/intrinsic fluorescence RMMs is key to success
• Data-rich feedback and software tools empower the end-user to extract relevant and actionable process knowledge, facilitating PAT and QbD, and allows the industry to move closer to real-time release

Real Time Viable Particle Detection: Key Capability and Application Considerations. Darrick Niccum, Global Product Manager-Biotechnology, TSI

• A technical review of TSI’s BioTrak Real Time Viable Particle Detector was provided
• This was followed by a review of laser induced fluorescence 
• Real time viable particle detection has great potential to improve pharmaceutical manufacturing process
• Some key performance parameters to consider include sample flow rate, aerosol efficiency, aerosol concentrator performance, an effective sampling rate, total particulate counting performance and discrimination capability

Application of Real-Time Microbial Monitoring in an Environmental Monitoring Program. Elizabeth Bennett, Microbiologist Application Scientist, Particle Measuring Systems

• A technical review of Particle Measuring System’s BioLaz Real-Time Microbial Monitor was provided
• Applications within filling lines, sterility test isolators, biosafety cabinets and during aseptic transfers was then discussed
• The benefits for using real-time environmental monitoring (EM) technologies include immediate notification for alarm response, the ability to partition finished product in the even of a microbial excursion (based on the timing of alarms), verification of acceptable biological levels prior to filling, faster batch release, reduced operator error and paperless data management
• Incorporation of this type of RMM into the existing EM program was further examined

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PDA Microbiology Conference Update: Revision of USP 1223

USP Chapter 1223, Validation of Alternative Microbiological Methods, has been under revision for at least the past year. During today’s conference, Tony Cundell, Director, Analytical Sciences Microbiology, Merck Research Laboratories and Vice-Chair, USP General Chapters – Microbiology Expert Committee, provided an update to the chapter’s revision process and what we should see when the draft is published in a future issue of the Pharmacopeial Forum.

The revised chapter will most probably reduce or eliminate the prescriptive guidance that is currently found within the existing chapter. This will allow users of alternative or rapid technologies to have more flexibility in the validation and use of these novel systems. Here is an overview of the proposed changes:

• Will include wider discussions of instrument and method validation and address the relationship of alternate methods to the USP General Notices and other relevant chapters
• Address regulatory requirements
• Will introduce concepts of performance, results and equivalence to existing methods
• Considerations for QC product release assays versus referee tests
• Provide guidance on alternate methods for compendial microbial tests
• Will consider equipment selection and qualification with actual product
• User specifications
• Installation, operational and performance qualification considerations
• Better define specificity, limit of detection, ruggedness and robustness, and other validation criteria for qualitative and quantitative technologies
• The responsibility of end-users versus instrument suppliers
• Method suitability
• Statistical tools
• References

And don't look for recommendations on the best rapid method for a specific application, as there is no plan to include any additional discussions regarding scientific principles or proprietary technologies.

PDA Technical Report #33, Evaluation, Validation and Implementation of New Microbiological Testing Methods, which is also under revision, will fill in the gaps that the future USP 1223 will leave. For example, TR#33 will provide detailed guidance on every step of the validation and implementation process, regulatory guidance from the FDA. EMA and other agencies, business and return on investment considerations, as well as a review of the technical and scientific aspects of technologies that are currently available. The revised TR#33 is planned for completion in 2013.

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Blogging from the PDA Microbiology Conference: Compounding Pharmacies and Fungal Meningitis

Another year has passed and once again, I am blogging from the PDA Global Conference on Pharmaceutical Microbiology. This is the 7th year for this signature scientific meeting, where microbiologists from around the world gather to learn about the latest technologies, industry best practices and regulatory expectations with regard to contamination control and microbiological considerations during the manufacture of drug product.

The conference began with an outstanding first keynote address entitled Outbreaks Associated with Pharmaceutical Product: Steps for Prevention presented by Matthew J. Arduino, Dr. Ph., Lead Microbiologist, Chief Clinical and Environmental Microbiology Branch, Centers for Disease Control and Prevention.

During his presentation, Dr. Arduino reviewed the role of the CDC, key outbreaks that have occurred over the past few decades, where contamination has occurred, and how to prevent outbreaks and adverse events. It is interesting to note, that in light of very recent events associated with the fungal contamination of pharmacy compounded injectable product (see below), the number of products that have been contaminated by compounding pharmacies have increased since the 1990's. Microbial contaminants isolated from patients and products (contaminated at the compounding pharmacy level) have included Burkholderia cepacia, Pseudomonas aerugnosa, P. fluorescens, P. putida, Serratia marcescens, Exophiala dermatitidis, Elizabethkingae meningosepticum, Enterobacter cloacae, Fusarium spp, Bipolaris, Bullera spp and Rhodotorula.

Dr. Arduino concluded that outbreaks highlight the potential of certain products, manufacturing processes and infection control practices to cause high morbidity and mortality. There continues to be unsafe injection practices, and medication handling. Finally, we continue to see problems associated with compounding pharmacies.

Of course, the session wold not have been complete without a discussion of the fungal (Exserohilum rostratum) meningitis outbreak that has now affected patients in at least 17 states within the U.S. Nearly 14,000 patients may have received contaminated steroid injections since May 21, 2012, which have been produced by the New England Compounding Center (NECC) of Framingham, Mass. The potentially tainted drugs were sent to pain clinics and health care facilities in 23 states. As of today, infections related to the outbreak reached 308, with 23 deaths. That includes 304 cases of meningitis, stroke or other nervous system problems tied to epidural injections of contaminated steroids (methylprednisolone acetate), plus four infections in patients who received pain shots in joints such as the hip, knee shoulder or elbow. The death toll has held steady for a few days, but officials with the CDC said that doesn't necessarily mean that the outbreak is waning.

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Dutch Bio-Hackers Mobilize Malaria Testing

Amplino may be the ultimate garage project. Three DIY bio-hackers have created a mobile malaria testing kit they claim can identify different strains of malaria with higher accuracy, and at lower levels of parasite concentrations than existing rapid diagnostic tests.

The testing device is connected via Bluetooth to a mobile phone, making it possible to track malaria outbreaks and the spread of particular strains. The team just won 40,000 EUR ($52,000) in the Vodafone Mobile for Good competition to further develop the kit.

Amplion’s young founders Jelmer Cnossen, Wouter Bruins, and Pieter van Boheemen have backgrounds in bio-informatics/mechanical engineering, cell biology, and functional genomics respectively. The group became fascinated by the DIYBio movement, which blends biology expertise with electronics, software development, and open source principles. Amplino’s team started to look at a technique called PCR, which copies a segment of DNA billions of times so that it can be analyzed. PCR is a well-established technique used in criminal DNA testing, disease diagnosis, and even testing whether food is halal.

A $600 “Build it yourself” OpenPCR machine already existed. The team decided to go one step further and develop a mobile device to do real-time PCR plus diagnosis of malaria. When you add a DNA binding dye to the multipled DNA mixture and shine light of a specific wavelength on it, the mixture will emit light when the malaria parasite is present.

In fact, the technology can detect any kind of pathogen, not just malaria, depending on the selection of a chemical component called a primer used in the device. A commercial real-time PCR setup can cost up to $30,000. Amplino built one for $60.

Malaria is a massive problem in the developing world. WHO estimates that up to 1 million people die each year from the disease, the majority of which are children in sub-Saharan Africa. The main methods of testing in the developing world are rapid diagnostic blood tests, which look like a pregnancy test and can be easily used in the field, and microscopy of a blood smear. The basic measure of accuracy of a malaria test is its sensitivity, in other words what percentage of infected people are correctly identified. Other factors in assessing a testing system include the concentration of parasites which need to be present in the blood for the test to correctly detect malaria, the number of false positives, storage life, or whether refrigeration,  clean water or trained medical staff are required.

Rapid diagnostic tests (RDTs) are less sensitive than lab tests like microscopy and cannot identify different types of malaria. Microscopy requires samples to be sent to a laboratory where trained health professionals review the blood samples. Incorrect diagnosis means that the limited supply of malaria drugs is not allocated optimally. However, testing at the point of care can still result in higher levels of correct patient treatment than more accurate, but slower, lab testing.  The World Health Organisation tests available RDTs each year to determine their sensitivity and PCR technology is used as the benchmark. So PCR can be regarded as the gold standard of malaria diagnostics.

Amplino’s test combines the accuracy of PCR with the mobility and ease of use of rapid diagnostic tests. It can be used by non-medical staff for immediate diagnosis of different types of malaria in the field. It can also detect malaria in pregnant women. During pregnancy, a woman’s immune system is suppressed, making her twice as likely to die from malaria. The parasites that cause the disease can hide in the placenta, making them much harder to detect. PCR can detect malaria at up to 10x lower concentrations of parasites in the blood than RDTs, making it suitable to identify re-infections as well as malaria in pregnancy.

Amplino estimates that the final mobile testing device will cost about $250, while the cartridge required per test costs between 50 cents and $1. Getting the device to market is still a long road. While the core PCR technology is not new, all medical devices need to be certified. This process, plus getting the prototype ready for manufacturing, could cost up to 1.5 million EUR over the next few years.

There are not many competing devices. InstantLabs makes a portable, real-time PCR system, but the company seems to focus mainly on the food safety industry. Lava Amp makes a $300 machine to run PCR but not to diagnose a particular disease.

“Our ultimate goal would be to get money from the Gates foundation,” said Bruins. ”We need strategic, not purely financial investors.” The Vodafone prize will allow the team to produce a version of the prototype that can be manufactured on a large scale (they estimate this will take six months) and test it in the field in Burkina Faso.

Amplino is not seeking to patent the technology. “I actually have two patents pending (for other technologies), ” Bruins explained. “But is a patent really fit for our mission? Maybe we need to ditch the whole patent approach.”

Amplino was founded in 2012, is based in Leiden in the Netherlands, has three employees, and is privately funded.

Source: VentureBeat (http://venturebeat.com)

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Rapid Methods in the Meat and Poultry Industries

Meat and poultry processors want faster, more accurate rapid-test results to protect their products, customers, consumers and their own companies from potential food-safety dangers. And rapid-test suppliers are endeavoring to satisfy this demand.

Stronger regulations from the US Dept. of Agriculture’s Food Safety and Inspection Service, Food and Drug Administration as well as the Food Safety Modernization Act are leading to increased rapid-test usage by industry. Growing food-safety awareness from the public and government also are playing a role in the demand for faster, more accurate tests, says Chris Lopez, technical sales and pricing analyst with San Antonio-based Food Safety Net Services. 

“Microbiological testing and high quality programs are a good starting point for a foundation to ensure the protocols of meat and poultry companies are protecting their products and their business,” he says. 
Various platforms of rapid testing use different scientific approaches to detect the microorganism of interest, Lopez says. These platforms usually fall under three main approaches: antibody-based, nucleic acid-based or enzymatic. 

Processors’ priorities

Many rapid-test providers tout their tests are user-friendly and that anyone from a trained molecular microbiologist to entry-level lab technician can conduct them. 

“When we chose our ATP [adenosine triphosphate] testing system, we looked for the system with accuracy, ease of use and understandable results,” says Darren Toczko, senior manager of food safety with Bar-S Foods Co., Phoenix, Ariz. 

“I want accuracy, sensitivity, reproducibility, costs, time-saving and ease of use,” adds Carl Zerr, director of international food safety and quality assurance with Rastelli Foods Group, Swedesboro, NJ. “The most important thing to me is accuracy.” 

Bar-S plants use ATP testing during pre-op inspections. At its corporate off-site laboratory, it uses a Listeria spp. test for processing environmental swabs. 

“If the surface is not clean, we know it within seconds,” Toczko says. “ATP testing does not replace Aerobic Plate Count/total plate count or Listeria testing. It is an indication of cleanliness, not of microbe level or type.” 

The Listeria spp. tests used in the Bar-S lab are fast, accurate and flexible to company sampling needs. This enables the processor to find and respond to any issues in a timely manner with confidence, he says. Bar-S’ Listeria spp. testing must be an AOAC method performed in an ISO 17025 laboratory. 

“Our company lab, which processes samples for four plants, has been ISO17025-accredited since 2006, with Listeria spp. on its scope of accreditation,” he adds. “Any Listeria method without an enrichment step to allow very low numbers of cells to grow would not be considered. Since Listeria is a slow grower, faster is not always better.” 

The test Bar-S uses requires a single-step enrichment and incubation for 40 to 48 hours, Toczko continues. “We stay away from methods which would lead to FSIS scrutiny of our results,” he adds. “It must also be flexible for increases in numbers of samples to be tested without excess increase in time, people or equipment,” he says. 

Rastelli Foods Group, a major national US foodservice distributor of beef, lamb, veal, pork, poultry and seafood products, incorporates rapid testing for E. coli O157 and Salmonella. It currently uses the FoodChek MICT system for the E. coli O157 test and upon AOAC certification, will also use the Listeria spp test. 

“I like the accuracy of the results, the ease of preparation and sample protocols to put everything together,” Zerr says of his E. coli O157 rapid test. “I didn’t need to hire a microbiologist to do the testing,” he adds. “The cost of the FoodChek Reader unit, test cassettes and pickup transportation savings I get by not sending every test to an independent lab is another factor.” 

The company conducts many in-house screening tests. Zerr still sends bi-weekly samples to an independent lab to confirm that his in-house test results are consistent, but he has reduced this practice by about 90 percent. 

Rastelli Foods now samples and tests products for the presence of E. coli O157 pathogens in its ground beef at 15-minute intervals. Before, tests may have been done two or three times a day. But over his first year despite increasing their testing, the company has saved around $14,000 with FoodChek’s system, Zerr says. 

Examine each technology 

A gap exists between current microbiology diagnostic products and technologies and what is ideally desired in rapid testing by plant QC managers, says William Hogan, president and CEO of FoodChek Systems Inc., Calgary, Alberta, Canada. Faster time-to-results (TTR) is the key requirement that enables better decision-making, improved economics and reduced risks, he adds. 

FoodChek’s patented MICT magnetic nanotechnology system eliminates human error by using a testing process that includes a bench-top electronic magnetic diagnostic reader, Hogan says. The FoodChek system is the only food-safety pathogen screening test that uses nanotechnology, which consistently produces accurate results in as little as eight hours, including the sample enrichment growth phase, he adds. 

FoodChek’s MICT Assay cassettes for E. coli O157 and Listeria spp provide very fast and accurate results in hours not days; are easy, five-step processes; and offer savings of up to 50 percent on the cost of pathogen testing, he continues. The company’s patent-pending Actero Enrichment Media also claim to have the fastest time-to-results in the bacteria growth phase; making the timeline to results up to 30 percent faster for E. coli and Listeria, and up to 70 percent faster for Salmonella. “Our MICT test cassettes give a quantitative result with a hard copy printout,” Hogan says. 

Processors should use rapid tests as insurance to safeguard their business, Hogan insists. “I see [rapid tests] as a huge marketing tool,” he adds. “If you test more than your competitors, why would a consumer or retailer not buy from you? We’re creating the FoodChek symbol/brand for food safety to be like the Nike Swoosh. Rastelli Foods intends to be the first to put the FoodChek brand on its packaging.”

“[After the enrichment growth phase], our tests take 30 minutes to set up and 48 seconds to get results. We can find the results in the same production shift; nobody else can do this,” Hogan says. 

FoodChek’s E. coli O157 rapid test is AOAC-approved and USDA/FSIS comparable. A new AOAC-approved, USDA/FSIS comparable Listeria spp rapid test is due for release this October; its Campylobacter test is expected out in January 2013; and its Salmonella spp; and its new STECs (shiga-toxin producing E. coli) tests are expected to be released in the second quarter of 2012. 

More meat and poultry processors and testing labs are looking for technologies with faster turnaround times for their pathogen screening so decisions can be made earlier regarding releasing inventory or in-process product, concurs Christine Aleski, US pathogen specialist, 3M Food Safety Department, St. Paul Minn. 

“Time is money and this axiom rules in the quality control lab as well,” she adds. “The sooner the results are available and the more accurate those results, with fewer retests required, the better financial circumstances for the company.” 

Pathogen screening for Salmonella, E. coli O157(H7) and STECs are used most by 3M’s meat and poultry clients. 

Rapid molecular testing offers earlier release from test-and-hold on inventories; increased specificity and sensitivity to help reduce the number of repeat tests; streamlined workflow to increase lab efficiency and technician productivity; real-time results to help make critical decisions faster; robust hardware with minimal maintenance requirements and less downtime for the lab; and powerful software that is easy to use and operate, she says. 

3M’s Molecular Detection System is a simple, rapid, specific and cost-effective nucleic acid amplification and detection method, she adds. 

“3M combined two unique technologies – Isothermal DNA amplification and bioluminescence detection – to offer the specificity and sensitivity required in a pathogen test solution that is also fast, simple and cost-effective,” she adds. “Compared to other rapid-detection methods, the system improves efficiencies in the lab process by offering users only one preparation protocol across all assays and all matrices allowing for batch processing, easier training and less chance for human error.” 

Because the DNA amplification is detected via bioluminescence, the 3M Molecular Detection System offers the unique use of color-coded assay tubes to differentiate pathogen assays making it easier for technicians work with its system, she says. Bioluminescence provides real-time detection of the DNA amplification and simultaneous amplification and detection allows for detection of positive results before the end of the run (as early as 15 minutes). 

With a smaller footprint that a standard notebook computer, the 3M Molecular Detection Instrument requires significantly less space than the market leading rapid methods, she says. 

3M’s technology instrument is robust and portable with no need for recalibration, requires minimal maintenance and provides automatic diagnostics at start up. Isothermal DNA amplification proceeds at a constant temperature, removing the need for complicated instrumentation. Bioluminescence detection eliminates the need for high-cost excitation sources, fluorophores, fluorescent filters and detectors. Assays are available for testing Salmonella, Listeria and E. coli including O157 H7. 

Meat and poultry customers of Bio-Rad Laboratories Inc., Hercules, Calif., are using the company’s real-time PCR assays, says Wendy Lauer, senior product manager, food science division. These tests typically are for Salmonella and E. coli O157:H7. The company also offers an assay for STECs. 

However, its most popular assay is its Listeria assay for environmental samples. “That’s where they test the processing environment instead of the food to use Listeria as an indicator of hygiene,” Lauer says. 

Doing the culture method could be less expensive than a rapid method, but, regarding time-to-results – cost is a relative term. “If you have your food sitting in a warehouse waiting for test results, that’s an additional cost on top of your cost of testing,” Lauer says. “If you use a rapid test and get a result after 24 hrs. as opposed to 48 hrs., that’s one day more you have shelf-life on that product.” 

Another rapid-test benefit is the ability of high-throughput screening. If a company is processing 100, 200 or more samples at one time, it needs something that can fit into that workflow. Usually, rapid methods are designed for that, Lauer says. 

Sensitivity is key. “You want to have the right answer to your rapid testing [and not false negatives or positives],” Lauer says. “PCR methods detect DNA of the target, they’re extremely sensitive, which yield better results.” 

iQ-Check is the company’s line of real-time PCR kits. “We have a full menu of different tests,” Lauer says. “We have iQ-Check Salmonella; iQ-Check Listeria (species and monocytogenes); iQ-Check Campylobacter; iQ-Check O157; and iQ-Check-STEC. All of our tests can all be run at the same time because chances are people aren’t just running one pathogen. Most people are running Listeria for their environmentals and Salmonella for their products, and they’re possibly running an O157 as well. 

Enrichment times depend on the organism. Bio-Rad’s O157 test is validated for an eight hr. enrichment; STEC is 10 hours; Salmonella is 20 hrs. with no regrowth step; and Listeria 24 hrs. After the enrichment, the sample processing time is about 30-45 minutes and the run time is about an hour- and-a-half to two hrs. For the Listeria test, complete results are available between 26-27 hrs.; for O157 complete results are within 10-11 hrs.; Salmonella is less than 24 hrs. For all tests, the enrichment times are 24 hrs. or less.” 

‘Time is money’

Expect rapid testing to increase among US meat and poultry processors in the future, all sources agree. 

“Processors will be looking for ‘flawless information instantly,’” Hogan says. “It’s about gaining the fastest time-to-results. You’re going to see an entire movement towards technologies being the fastest because time is money.”

Source: MeatPoultry.com (http://www.meatpoultry.com)

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