Updated routinely by Dr. Michael J. Miller, our RMM blog will keep you informed of new and noteworthy technologies, reviews of recent publications and presentations, upcoming conferences and training events, and what's changing in the RMM world. You can also follow our blog posts in the Rapid Microbiology Methods LinkedIn group.
A team of researchers from Harvard Medical School, Brigham and Women's Hospital, and Massachusetts General Hospital, with others from the United States and Haiti, has determined that the strain of cholera erupting in Haiti matches bacterial samples from South Asia and not those from Latin America. These findings, which appeared in the New England Journal of Medicine, conclude that the cholera bacterial strain introduced into Haiti probably came from an infected human, contaminated food or other item from outside of Latin America.
To identify the probable origin of the cholera strain in Haiti, scientists used a third-generation, single-molecule DNA sequencing method developed by Pacific Biosciences. They determined the genome sequences of two Haitian cholera samples and three cholera samples from elsewhere around the world. Based on advanced imaging technology, the method enables researchers to observe a natural enzyme synthesizing a strand of DNA in real time. As such, the technology actually tracks and documents nature at work, a rapid approach compared to other sequencing technologies. The method allowed a comprehensive analysis and comparison of critical DNA features among the various cholera samples, which included single nucleotide variations, insertions and deletions of particular portions of the genome, and structural variations. The analysis showed a close relationship between the Haitian samples and the seventh pandemic variant strains isolated in Bangladesh in 2002 and 2008.
Genetic changes occur quickly, within hours in the lab and probably weeks within the environment, through natural modes of DNA swapping and mutation among bacteria. Their evolution is based, in part, on the acquisition, loss, and alteration of mobile genetic elements, including DNA from the CTX bacterial virus, which bears the genes encoding the cholera toxin, and other genetic sequences that may make a particular strain more adapted to a given ecosystem. The resulting heterogeneity has been used to categorize strains of the seventh pandemic and to understand their transmission around the globe.
Known nationally for her research into single-cell organisms that affect oral health, Millicent "Mimi" Goldschmidt, Ph.D., a professor of microbiology and molecular genetics at The University of Texas Health Science Center at Houston (UTHealth), has been selected to receive the 2011 American Society for Microbiology (ASM) Founders Distinguished Service Award. The award will be presented at the ASM General Meeting Awards Banquet and Dinner in New Orleans on May 22.
"Dr. Goldschmidt has furthered the understanding of the basic microbiology of the mouth," said Larry R. Kaiser, M.D., president of UTHealth. "She has done an exemplary job of serving her professional community, her scientific community and her teaching community." Microbiology is the study of cells that are invisible to the naked eye. These tiny cells include bacteria, viruses and fungi. Goldschmidt studies microbes involved with dental decay, gum disease and oral cancer, as well as rapid methods of detection (biosensors, microarrays and nanoparticles).
Before joining the UTHealth faculty in the early 1970s, Goldschmidt was the coordinator of the protocol to plan the biological tests that would be employed in the lunar receiving laboratory on the first returned moon rocks. She was also instrumental in developing isolation protocols for Apollo astronauts returning from the moon, which ensured that infectious organisms would be detected and contained.
When she started her professional career in Texas five decades ago, Goldschmidt said there were really no rapid methods to detect microorganisms. Her research contributed to the development of rapid immunological and biosensor types of detection methods to pinpoint salmonellae, E. coli and oral microbes. She consults and lectures nationally and internationally on biosensors, microarrays and nanoparticles.
The Fung Double Tube method is relatively easy to implement. The system uses one large tube and one small tube. Insert the small tube into the larger tube that holds a water sample. Then add a specially melted agar – a gelatin-like product used for solidifying culture media as a thickening agent – to create a thin layer between the two tubes that will grow C. perfringens. The unit is then placed in an incubator at 42 degrees C.
“The system makes anaerobic microbiology very simple,” Fung said. “One can see a C. perfringens colony in the Double tube in about four to five hours. We can know in five to six hours how many living colonies of C. perfringens per milliliter of water there are. That is a major improvement and it’s so cheap.”
The system works well for water testing, but more research is necessary to prepare it for use in the food industry. Fung’s research team is examining how to apply it for use with ground beef so the meat won’t have to be incubated overnight before pathogen counts can be obtained.
We have a number of compendial (USP and Ph. Eur.) and technical (PDA TR#33) documents at our disposal for the validation of rapid microbiological methods. However, specific guidance is lacking at the regulatory level, as there are no comprehensive written policies from the FDA, EMA, TGA or the Japanese PMDA. During the PDA 5th Annual Global Conference on Pharmaceutical Microbiology, a question was asked to FDA panelists when the Agency would develop a clear guidance document on RMMs. Dr. David Hussong, Director, Microbiology, CDER, responded that In the next few years there will be an internal document that the agency will follow.
The final session of this year’s PDA Global Conference on Pharmaceutical Microbiology is focused on the direction that the USP will take with the existing informational chapter 1223, Validation of Alternative Microbiological Methods. Questions were provided to the meeting attendees and here is an excerpt of some of the comments and responses:
Question: Has USP 1223 Validation of Alternative Microbiological Methods been useful or an impediment for the selection, validation and implementation of RMMs?
Response 1: Yes, the chapter has been useful as providing guidance for RMM validation.
Response 2: Yes, but we would like to have more guidance on sensitivity and limit of detection strategies. For example, methods for developing very low inoculum levels, such as 1 cell.
Response 3: The use of statistics for each validation criteria should be more clearly defined and relevant.
Response 4: Much work has been done on limit of detection for sterility testing and these methods are appropriate for use and should be incorporated into the USP guidance.
Response 5: There needs to be a good balance between specific guidance, such as acceptance criteria, and background information into the reasons why the guidance is provided. The chapter should not be a white paper.
Response 6: We need a simple benchmark on how to validate RMMs. The food industry and AOAC have addressed this 20 years ago!
Question: Do you like to see examples?
Response 1: In the EU, regulators took the example in Ph. Eur. 5.1.6 as gospel and that caused many problems for companies validating RMMs. As a result, the next revision of 5.1.6 will not contain an example but will be published in PharmEuropa.
Response 2: I want to see real, practical and successful case studies of how RMMs have been validated, not theoretical examples.
Response 3: Recommendation is not to have an example but to put this information in Pharmacopeial Forum and/or reference other guidance documents that will provide examples, such as PDA Technical Report #33.
Response 4: I want to see guidance on controls based on technology platforms and applications (e.g., negative and positive controls). But don’t provide specific examples, but more suggestions on good controls that should be run.
Question: Should compendial guidance documents (JP, USP, and EP) on the validation of RMMs be harmonized?
Response: By a show of hands, many in the audience said yes.
Final summary statements from the USP based on these discussions:
We may be making this much harder than it should be. We need to go back and take a closer look at our recommendations in this chapter and provide a more “user-friendly” set of recommendations. Because there are many different processes and products that would utilize RMMs, it is really up to you, the users of RMMs, to define how to best validate these new systems. USP intends to provide better guidance on the use of alternative methods with input from stakeholders.
USP 1223 needs to be revised. There needs to be a clarification of sensitivity, limit of detection and limit of quantification, with specific validation criteria and not sole reliance on parallel testing. The use of CFUs is difficult because there is no good quantitative definition of what a CFU is. Statistical models used with validation criteria needs to be revisited. Microorganisms chosen during validation should be appropriate for the intended applications, process and product. We need to consider how to handle slow growing microorganisms, and address the use of appropriate controls.
Next, it appears that specific examples should not be included to avoid regulatory expectations that may not be appropriate. Instead, maybe we should reference PDA TR #33 and/or submit a Stimuli Article. Any reference to specific RMM technologies should not be included.
We should address the relevance of referee tests for short shelf-life products such as biologics.
We should work toward harmonizing all RMM guidance documents; however, this may be easier said than done. This is not a simple matter and could be time consuming. However, we will discuss how we can interact with other organizations to determine if our revisions can take into account other guidance documents.
Finally, we need to have more discussions with the stakeholders of RMMs more frequently than we have in the past, and we will discuss how we can seek out mechanisms to make this happen.
Jennifer Gray of Novartis Pharma AG, Switzerland, presented their strategy for validating the Millipore Milliflex Rapid as an alternative ATP bioluminescence RMM to the traditional compendial sterility test. The drivers for a rapid sterility test included the early identification of product contamination events, a reduction of through put time for sterile drug product release, and to increase the company’s level of expertise in the field of rapid microbiological methods. Novartis validated a 5-day sterility test using 22 heat-stressed cultures (7 ATCC strains and 15 environmental isolates). The cultures were also used to determine the most optimal medium to be used in the system. The FDA approved comparability protocols outlining the validation strategy for multiple products, EMA approval was obtained in February 2010 and MHRA approval was obtained in May 2010.
Amelia Tait-Kamradt, Pfizer, discussed their assessment of Pall’s new GeneDisc system. They conducted a number of studies addressing specificity, limit of detection, ruggedness, robustness, and the impact that excipients may have on the ability of the system to detect microorganisms. More information on the GeneDisc system may be found on our website on the Technology page.
Dr. Geert Verdonk of Merck presented his validation studies using the Charles River Laboratories Endosafe PTS. Dr. Verdonk explored the use of this rapid and portable endotoxin detection system as part of Merck’s PAT-RMM program. A variety of validation studies were also presented.
Injectable product manufacturing is booming because of the growth of new biopharmaceuticals and small molecule anticancer drugs. The requirements for contamination control will become even more stringent than today. Isolators or blow-fill-seal equipment have already replaced the conventional clean rooms and LAF-hoods in many production facilitities. Conventional microbiological monitoring methods, requiring 3 to 5 days of incubation will become inappropriate. Equipment is already available allowing real time, simultaneous viable and non-viable counting.
This book is a useful reference guide for the SMB (Small and Medium Business) pharmaceutical sector which does not have the resources to have access to such top-quality information in this field. This book therefore represents an unparalleled and unprecedented text in the field of pharmaceutical and medical device microbiology. Perhaps even more outstanding is the fact that this book not only covers subject matter and technical content which is established as best and expected practice, but also includes content regarded as possible, future and emerging technology or processes.
The results of 45 years of scientific and technological development are laid down in these 33 chapters. These chapters, all written by international experts, give a vivid picture of today’s pharmaceutical microbiology. The high standard of the chapters makes it an essential reference guide that should be on the shelf of everyone who is involved or interested in this field.
