Thursday, December 22, 2011

Happy Holidays from rapidmicromethods.com

From all of us at rapidmicromethods.com, we wish you and your family, friends and colleagues a very happy holiday season, and a very healthy and prosperous New Year.

Nanotechnology and Microbiology

Dr. Arti Goel, Lecturer, Amity Institute of Microbial Biotechnology, Amity University, Noida, India, recently described the opportunities that nanotechnology has on a wide range of microbiology applications, namely, within the food, clinical diagnostics and water sectors. Here is an excerpt from his white paper, which was recently published on Nanowerk's website (http://www.nanowerk.com/).

Microbiology relates to nanoscience at a number of levels. Many bacterial entities are nano-machines in nature, including molecular motors like flagella and pili. Bacteria also form biofilms by the process of self-assembly (for example the formation of Curli-film by E. coli). The formation of aerial hyphae by bacteria and fungi is also directed by the controlled and ordered assembly of building blocks. Also, the formation of virus capsids is a classical process of molecular recognition and self-assembly at the nano-scale.

Nanotechnology involves creating and manipulating organic and inorganic matter at the nanoscale. It promises to provide the means for designing nanomaterials; materials with tailor-made physical, chemical and biological properties controlled by defined molecular structures and dynamics. The present molecular biology techniques of genetic modification of crops are already forms of what has been termed nanotechnology.

Nanotechnology can provide for the future development of far more precise and effective methods of, and other forms of, manipulation of food polymers and polymeric assemblages to provide tailor-made improvements to food quality and food safety. Nanotechnology promises not only the creation of novel and precisely defined material properties, it also promises that these materials will have self-assembling, self-healing and maintaining properties.

Nanoscience does have an impact on several areas of microbiology. It allows for the study and visualization at the molecular-assembly levels of a process. It facilitates identification of molecular recognition and self-assembly motifs as well as the assessment of these processes. Specifically, there are three areas where microbiologists use nanotechnologists' techniques:

– Imaging single molecules
– Poking and pulling nanoscale objects (laser traps, optical tweezer)
– Determining spatial organization in living microbes (AFM, near/far field microscope).

Nanotechnology in food microbiology

Detection of very small amounts of a chemical contaminant, virus or bacteria in food systems is another potential application of nanotechnology. The exciting possibility of combining biology and nanoscale technology into sensors holds the potential of increased sensitivity and therefore a significantly reduced response-time to sense potential problems.

Nanosensors that are being developed by researchers at both Purdue and Clemson universities use nanoparticles, which can either be tailor-made to fluoresce different colors or, alternatively, be manufactured out of magnetic materials. These nanoparticles can then selectively attach themselves to any number of food pathogens. Employees, using handheld sensors employing either infrared light or magnetic materials, could then note the presence of even minuscule traces of harmful pathogens. The advantage of such a system is that literally hundreds and potentially thousands of nanoparticles can be placed on a single nanosensor to rapidly, accurately and affordably detect the presence of any number of different bacteria and pathogens. A second advantage of nanosensors is that, given their small size, they can gain access into the tiny crevices where the pathogens often hide.

The application of nanotechnologies on the detection of pathogenic organisms in food and the development of nanosensors for food safety is also studied at the Bioanalytical Microsystems and Biosensors Laboratory at Cornell University. The focus of the research performed at Cornell University is on the development of rapid and portable biosensors for the detection of pathogens in the environment, food and for clinical diagnostics. The bioanalytical microsystems use the same biological principles as were used in the simple biosensors, i.e. RNA recognition via DNA/RNA hybridization and liposome amplification. The bioanalytical microsystems that are studied focus on the very rapid detection of pathogens in routine drinking water testing, food analysis, environmental water testing and in clinical diagnostics.

Nanotechnology in medical biology – application of nanodiagnostics in infectious diseases

The rapid and sensitive detection of pathogenic bacteria at the point of care is extremely important. Limitations of most of the conventional diagnostic methods are the lack of ultrasensitivity and delay in getting results. A bioconjugated nanoparticle-based bioassay for in situ pathogen quantification can detect a single bacterium within 20 minutes.

Detection of single-molecule hybridization has been achieved by a hybridization-detection method using multicolor oligonucleotide-functionalized QDs as nanoprobes. In the presence of various target sequences, combinatorial self-assembly of the nanoprobes via independent hybridization reactions leads to the generation of discernible sequence specific detection of multiple relevant sequences.

A spectroscopic assay based on SERS using silver nanorods, which significantly amplify the signal, has been developed for rapid detection of trace levels of viruses with a high degree of sensitivity and specificity. The technique measures the change in frequency of a near- infrared laser as it scatters viral DNA or RNA. That change in frequency is as distinct as a fingerprint. This novel SERS assay can detect spectral differences between viruses, viral strains, and viruses with gene deletions in biological media. The method provides rapid diagnostics (60 s) for detection and characterization of viruses generating reproducible spectra without viral manipulation. This method is also inexpensive and easily reproducible.

The use of nanoparticles as tags or labels allows for the detection of infectious agents in small sample volumes directly in a very sensitive, specific and rapid format at lower costs than current in-use technologies. This advance in early detection enables accurate and prompt treatment.

Quantum dot technology is currently the most widely employed nanotechnology in this area. The recently emerging cantilever technology is the most promising. The technology strengthens and expands the DNA and protein microarray methods and has applications in genomic analysis, proteomics, and molecular diagnostics.

Waveguide technology is an emergent area with many diagnostic applications. Nanosensors are the new contrivance for detection of bioterrorism agents. All these new technologies would have to be evaluated in clinical settings before their full import is appreciated and accepted.

Nanotechnology in water microbiology – water treatment by detection of microbial pathogens

An adequate supply of safe drinking water is one of the major prerequisites for a healthy life, but waterborne diseases is still a major cause of death in many parts of the world, particularly in young children, the elderly, or those with compromised immune systems. As the epidemiology of waterborne diseases is changing, there is a growing global public health concern about new and reemerging infectious diseases that are occurring through a complex interaction of social, economic, evolutionary, and ecological factors.

An important challenge is therefore the rapid, specific and sensitive detection of waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. However, newer enzymatic, immunological and genetic methods are being developed to replace and/or support classical approaches to microbial detection. Moreover, innovations in nanotechnologies and nanosciences are having a significant impact in biodiagnostics, where a number of nanoparticle-based assays and nanodevices have been introduced for biomolecular detection.

Monday, December 19, 2011

Scientists Develop Nanomechanical Biosensor Based on Photonic Crystal Nanowire Array

Yuerui Lu, a student of Amit Lal, Cornell University’s Professor of electrical and computer engineering, has developed a photonic crystal nanowire array-based biosensor, which is capable of performing low-cost, highly sensitive and rapid test for detecting disease markers and similar molecules at ultra-low concentrations, paving the way to identify diseases at an early stage.

The nanomechanical biosensor is a 50-┬Ám diameter mechanical resonator made of a thin membrane of silicon-silicon dioxide comprising closely stacked, neatly arranged vertical nanowires over it. This pattern provides a high surface-to-volume ratio for delivering high sensitivity to detect biomolecules at ultra-low concentrations down to fM.

A schematic drawing of the biosensor, which consists of ordered nanowires on top of a silicon-silicon dioxide membrane, may be viewed by clicking on the image at the left (Credit: Yuerui Lu).
The biosensor operates by bonding the nanowires with the single-stranded probe DNA molecules. When the molecules are brought in touch with a target single-stranded DNA, the respective molecules join together, causing a change in the mass recorded by the device, which in turn changes the device’s resonance frequency.

When the device is irradiated by a laser beam, the novel design of the device’s nanowires enables the absorption of over 90% of the light, causing an effective opto-thermo-mechanical agitation of the resonator. The change in the resonance frequency can be optically recorded rapidly and remotely without the use of electrical wires, facilitating the fabrication of the device at a lower cost.

Lal stated that the device can be used for clinical analysis, for instance in DNA testing wherein current methods compare DNA against a typical sequence, which are expensive and time consuming. The novel device can be coded with specific DNA series based on relevance, and those particular molecules can be identified in early stages when at lower concentrations, he said. Doctors can have a cartridge comprising a sequence of membrane sensors that allow the detection of DNA defects rapidly, he added.

The biosensor can be utilized for environmental monitoring purposes such as monitoring of water quality. The research team expects to upgrade the sensitivity of its device to some protein molecules, which is a challenge to the team, as those molecules do not attach as efficiently as DNA molecules do.

Wednesday, December 14, 2011

HPLC of PCR Products Now Being Used to Detect Pathogens in Food


Recent developments in rapid methods have led researchers to utilize multiplex polymerase chain reaction (PCR) and high-performance liquid chromatography (HPLC) for the identification of food borne pathogens, such as diarrheagenic E. coli. A recent overview from separationsnow.com discusses this new application, and I have provided an excerpt below.

Dangers of Diarrhoea

Children are at higher risk of contracting diarrhoea than adults due to their underdeveloped immune systems and they are likely to be affected for longer periods. In the Western world, most cases are easily treated but it is a different story in developing countries, where infection rates are higher and death is a common outcome.

Acute diarrhoea is generally caused by bacterial, viral, or parasitic infection and one of the key bacteria is diarrhoeagenic Escherichia coli, which can enter the body via contaminated food. However, it is not simply one strain that is responsible. In foods, four main categories can cause diarrhoea: enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorraghic (EHEC) and enteroinvasive (EIEC) E. coli.

Each of these categories contains similar but individual DNA, which proved a blessing for a team of Chinese scientists who have exploited the differences in a novel detection method. Lichun Cui from the Northeast Forestry University, Harbin, with colleagues from the Northeast Agricultural University, Harbin, and the Heilongjiang and Hainan Entry-Exit Inspection and Quarantine Bureaus, devised a procedure based on denaturing HPLC that could detect single or mixed E. coli infections in food.

Denaturing HPLC

In denaturing HPLC, mismatches in the DNA bases of double-stranded DNA allow their separation provided certain criteria are met. At elevated temperatures, the hetero and homo duplex chains unwind, or denature, to their individual strands which are resolved on the HPLC column.

The HPLC stationary phase must be inert and electrically neutral. Under these conditions, DNA cannot bind due to its inherent negative charge but the addition of an ion pairing agent to the mobile phase changes the properties and binding is achieved via electrostatic interactions.

The hetero duplexes are denatured to a greater extent than the homo duplexes, so that they are retained less strongly on the column and elute first. So, pairs of hetero and homo chains are observed in the HPLC chromatograms.

In the case of E. coli, the researchers employed a poly(styrene-divinylbenzene) column and added triethylammonium acetate to the mobile phase for ion pairing. Separation was effected with a gradient of acetonitrile.

DNA was extracted from E. coli bacteria and subjected to polymerase chain reaction (PCR) amplification using unique primers based on the conserved regions of each of the four bacteria. Sufficient sample was generated for HPLC analysis and only the expected products were produced, as proven by agarose gel electrophoresis. The average size of the products was 220, 300, 330 and 500 base pairs for ETEC, EPEC, EIEC and EHEC, respectively.

Bacterial Strains Detected Together in Food

The HPLC separations were carried out at 50°C under non-denaturing conditions, which produced a single peak for each amplified fragment. They eluted at different retention times over 4-9 minutes, allowing them to be distinguished from each other.

The PCR products from all four E. coli categories were then mixed together for HPLC, confirming that they can be separated and distinguished using their retention times.

The critical step in the process is the specificity towards each category. This was assured by subjecting the genomic DNA from 34 bacterial strains to the same amplification and analysis process using the unique primers. Only the ETEC, EPEC, EIEC and EHEC strains and their isolates gave positive results.

The novel procedure was used to test 189 samples of faecal matter from patients as well as 690 import and export food samples, including beef, pork, chicken, sausages and milk. Blind testing was carried out and the results were compared with those from the conventional method which involves bacterial culture over 2-3 days and biochemical reactions.

A total of 60 positive samples were identified with all four strains being detected. The results were in perfect agreement with the conventional method, confirming the validity of the approach. However, the PCR denaturing HPLC method is much faster and provides a valid alternative. It could also replace the basic PCR assay which requires a gel electrophoresis step.

The multiplex method is capable of detecting all four strains in one procedure, so can identify one or more of the infections in contaminated food and in patient faeces, permitting rapid detection and diagnosis and reducing the time before appropriate action can be taken.

The original article appears here. Image by Renjith Krishnan.

Monday, December 5, 2011

A Fast Nanotechnology Platform to Detect/Capture Bacteria in Clinical Samples


The folks at Nanowerk (http://www.nanowerk.com/) recently highlighted a novel use of Surface-Enhanced Raman Spectroscopy (SERS) that can be used for label-free sensing of bacteria. A dual function biochip is now being utilized to capture bacteria in blood samples, followed by analysis of the bacteria using Raman spectroscopy. The image on the left (click on the image for a larger picture) illustrates the core of the biochip, where an array of silver nanoparticles (silver) coated by Vancomycin (green) selectively capture bacteria (white) while blood cells (red) are excluded. The following discussion is excerpted from the Nanowerk article, in which Dr. Yuh-Lin Wang, Distinguished Research Fellow, Institute of Atomic and Molecular Sciences, Academia Sinica, and Professor in the Department of Physics at National Taiwan University, describes the technology (Image: Dr. Wang, Academia Sinica. Correspondence and requests for materials should be addressed to Prof. Wang, email: ylwang@pub.iams.sinica.edu.tw).

Surface-enhanced Raman spectroscopy (SERS) is a powerful research tool that is being used to detect and analyze chemicals as well as a non-invasive tool for imaging cells and detecting cancer. It also has been employed for label-free sensing of bacteria, exploiting its tremendous enhancement in the Raman signal.

SERS can provide the vibrational spectrum of the molecules on the cell wall of a single bacterium in a few seconds. Such a spectrum is like the fingerprints of the molecules and therefore could be exploited as a means to quickly identify bacteria without the need of a time-consuming bacteria culture process, which typically takes a few days to several weeks depending on the species of bacteria.

To practically apply SERS to the early diagnosis of bacteremia – the presence of bacteria in the blood – it is most desirable to be able to capture bacteria in a patient's blood onto the SERS substrate.

"A typical SERS-active substrate consists of arrays of nanoscale metallic objects, for example, silver nanoparticles and etch-pits on silver surfaces, which can sustain surface plasmon polariton resonance and enhance the Raman signal of molecules on or near the substrate," Dr. Yuh-Lin Wang, explains. "In our recent work, we found that coating a thin layer of vancomycin on a SERS substrate drastically increases its capability to capture bacteria in the blood samples without introducing significant spectral interference to SERS spectrum of the captured bacteria."

Previously, researchers already used vancomycin-coated magnetic nanoparticles to capture bacteria in water. Wang and his team therefore asked the question whether it is possible to endow the vancomycin-coated SERS substrates with the concurrent functionalities of bacterial capturing and sensing.

Reporting their work in the November 15, 2011 issue of Nature Communications ("Functionalized arrays of Raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood"), first-authored by Ting-Yu Liu, they demonstrate that functionalization by vancomycin of substrates of silver nanoparticles on arrays of anodic aluminum oxide nanochannels not only dramatically enhances their ability to capture bacteria in liquid but also significantly increases their SERS signal.

Wang notes that the team's findings took them by surprise since the signals from the vancomycin coating were expected to be larger than that from the bacteria cell wall since the later is closer to the SERS substrate.
"This unexpected discovery opens up many possibilities for the creation of SERS-based multifunctional biochips for rapid culture-free and label-free detection and drug-resistant testing of microorganisms in clinical samples," he says.

Coating SERS substrates by vancomycin, which is an antibiotic by itself, in order to add the function of capturing bacteria to the substrates is a brand-new approach to overcome a major obstacle facing the practical application of SERS in clinical diagnosis.

"We have been making various attempts to overcome this obstacle in the last two years" says Wang. "This exciting result is the outcome of an experiment that makes some sense superficially but is considered unlikely to work on second thought because of the likely interference from the vancomycin coating. After we saw the surprisingly good result that the SERS signals from the vancomycin coating do not interfere with that of the captured bacteria, we pondered on the question "why" for months and finally came up with some qualitative explanation as given in our paper."
"In retrospect, this is an interesting case in which the original motivation was to tackle the conceptually simple and practically challenging problem of trying to capture bacteria onto a small area of a biosensor without compromising its original sensing function. It turns out that our solution not only allows us to capture bacteria ~1000 times more effectively but also enhances the sensitivity of the sensor by several times rather than decreasing it."

To demonstrate the bacterium-capturing capability of their substrate, the researchers immersed it in a water sample with ultra-low concentration (100 cfu/ml) of bacteria for 1 hour and then rinsed it in deionized water. Examining the substrate with scanning electron microscopy (SEM), they were then able to detect a concentration of bacteria on their substrate.
Wang and his team point out that their findings are a major step towards the development of a high-speed and -sensitivity nanotechnology platform that has high potential to capture/detect bacteria in clinical or environmental samples.

This SERS-based rapid detection method is a very promising approach to dramatically reducing the time needed to detect bacteria in the blood of bacteremia patients to within an hour. By contrast, a conventional biological assay usually requires the sample preparation time ranging from days for fast growing bacteria to weeks for slow growers.

The researchers point out that, in principle, this sensing platform could be exploited for the detection of various microorganisms such as virus and bacteria in various clinical samples, e.g., water, phlegm, sputum, blood and marrow, as well as food, and environmental samples.

"Culture-free and label-free detection of microorganisms remain among the most exciting directions in the development of rapid biosensing technology," says Wang. "One of the most difficult challenges is the development of complementary sample preparation technologies. In order for a new method to be accepted by the research community, scientists and practitioners are looking for a total rather than partial solution."

Friday, December 2, 2011

NEW RMM Tutorial Pages Launched at rapidmicromethods.com


In connection with the launch of the new RMM Product Matrix, rapidmicromethods.com has developed a series of new scientific tutorial pages. The tutorial pages provide in-depth reviews of the benefits of implementing RMMs, the science behind the technologies, why they differ from conventional microbiology methods, and how they can be applied in the modern microbiology lab and manufacturing environment. Separate pages discuss RMM technologies based on growth, viability staining and laser excitation, the detection of cellular components, optical spectroscopy, nucleic acid amplification and gene sequencing, and Micro-Electrical-Mechanical Systems such as biosensors and microarrays. Visit the new RMM Tutorial pages at http://rapidmicromethods.com/files/tutorial.html.

NEW Rapid Methods Product Matrix Launched on rapidmicromethods.com


Rapidmicromethods.com has launched an innovative resource for directly comparing more than 45 different rapid method technologies. The RMM Product Matrix provides details on scientific methods, applications, time to result, throughput, sensitivity, organisms detected, identification libraries and product workflow in three separate comparison tables (microbial identification, qualitative and quantitative methods). Never before has this much information been available in one place anywhere online or in print. Users can now use the Product Matrix to assist in matching the right RMM technology with their microbiology applications. Visit the new RMM Product Matrix at http://rapidmicromethods.com/files/matrix.html.

Friday, November 11, 2011

Our November Newsletter is now available


The rapidmicromethods.com November Newsletter is now available. In this issue, we discuss upcoming rapid micro method conferences including Pharmig and the ECA annual RMM meeting. Our newsletter also provides updates from our RMM news page, Blog and Calendar of Events. Please visit http://rapidmicromethods.com/newsletter/ to sign up for the free Newsletter.

Saturday, October 22, 2011

Yale Students Develop Rapid Bacteria Detector


A team of engineering graduate students at Yale is working to create a new technology that could help prevent widespread food-borne illness and lead to quicker diagnosis of bacterial infections. The device, called alpha screen, is a portable, rapid, pathogen scanner that can detect as few as one bacterium.

“A rapid pathogen screener is a device capable of detecting microorganisms in near real-time, without the use of cultured colonies or a traditional microbiology lab,” PhD student Monika Weber told Security Management. Weber leads the development team.

Development of the device started as an assignment while under instruction of Prof. Mark Reed, the Harold Hodgkinson professor of engineering and applied science. The assignment was to develop a device that had marketable potential.

“We were thinking about what kind of device would be beneficial….We started asking doctors what areas of medical science could be improved,” Weber said.

Doctors told them that bacteria diagnostics would be a promising focus area because of the current time and costs of culture growth – which can sometimes take days or require separate labs. Their comments resonated with Weber, who recently became lactose intolerant because of a bacterial infection. Testing using the alpha screen is 10 to 50 times faster than the current methods—culture growth and polymerase chain reaction. The current cost of one test is estimated at $1-20 times less expensive than current testing methods. Weber says a quicker diagnosis could have prevented her condition.

Weber’s isn't the first endeavor focused on rapid bacteria detection. Researchers in the past have spent years developing similar technologies, but Weber says variations of the alpha screen are being developed to address all areas that have the need for bacteria detection.

The low cost of testing using the alpha screen will be beneficial to developing countries, Weber said—countries where a timely diagnosis could mean the difference between life and death. The device will allow doctor’s offices to make diagnoses in real-time.

For the food industry, the alpha screen has very promising applications as well, she said. “We hope that the alpha-screen technology…will help prevent disease outbreaks, like the 2011 Listeria outbreak in the U.S. and the E. coli outbreak in Germany. We anticipate that alpha-screen will have dramatic implications for domestic and international food security,” she said.

The alpha screen is still in its development stages. The basic version is a battery-powered device about the size of a coin. It can only detect one or two types of bacteria. “However, to meet the needs of prospective customers we have also designed a larger unit, which could detect simultaneously over 100 different types of bacteria,” Weber said.

The team is working to raise funds for further development and prospective commercialization. Earlier this month, they moved $20,000 closer to that goal after winning the Create the Future design contest. The contest awarded prizes to recognize research efforts in engineering focused on benefitting humanity, the environment, and the economy. More than 900 product ideas from 50 countries were entered.

Weber said she hopes the publicity for their work will pay off for the project.

“We hope to attract people and institutions that would sponsor research and investors to move the project along,” Weber said.

Weber’s isn’t the first endeavor focused on rapid bacteria detection. Researchers in the past have spent years developing similar technologies.

Wednesday, October 19, 2011

Debunking Rapid Method Myths at PDA


During the very popular Urban Myths session, I presented an overview of the myths associated with the inability of our industry to effectively implement rapid methods. Although a number of companies have successfully validated RMMs, they only represent a fraction of the industry that can benefit from using these alternative technologies. So what are the myths associated with RMMs? Here is a short list:

- Rapid methods are not accepted or understood by regulatory authorities, nor do they support QbD or PAT
- They will never replace pharmacopoeial tests
- There is little validation guidance
- RMMs offer no return on investment
- RMM use will result in exceeding specifications and action levels, which will translate to an increase in batch rejections
- Changing acceptance levels will not be allowed

During my presentation, I debunked each of these myths and many more, and provided support for rapid methods from global regulatory authorities in terms of validation, implementation and submissions. I also addressed where rapid methods can be used in contamination control programs, Quality by Design (QbD) and Process Analytical Technology (PAT) strategies. Much of what I discussed is already available for review in the Regulatory, Validation, and Return on Investment (ROI) sections at rapidmicromethods.com.

Briefly, the regulatory authorities not only accept and understand RMMs, but also have embraced and encouraged their use for a number of years. When formal changes to existing regulatory dossiers is required, both the FDA and EMA provide guidance and policies on how to accomplish this. And the Australian TGA and Japanese PMDA also support the implementation of RMMs as well.

Rapid methods can be used to support QbD and PAT strategies, including, but limited to, in-process bioburden testing, environmental monitoring, and process water and endotoxin analyses. Many companies have already validated RMMs as alternatives to compendial finished product release testing, such as USP and Ph. Eur. sterility testing (publications on the use of RMMs for finished product testing can be reviewed on our References page). And firms can and have realized significant cost savings and cost avoidances when implementing novel microbiological technologies. Finally, rapid methods can also support a comprehensive contamination control program, and when contamination arises, RMMs can be used as investigative tools, providing results much faster than traditional means.

In summary, most if not all RMM myths are not true, the regulatory authorities want to see RMMs implemented and encourage their use, RMMs are directly aligned with the future state of pharmaceutical manufacturing, QbD, PAT and continuous process and product improvement, there is validation guidance available, and the cost of implementation can be a good investment.

Tuesday, October 18, 2011

Rapid Methods Session 2


The first speaker during this session was Claudio D. Denoya, PhD, Adjunct Professor Department of Molecular and Cell Biology, University of Connecticut. Dr. Denoya discussed the current microbiology curricula taught within academic institutions and the role pharmaceutical science and modern microbiological methods should play in these programs. The focus was to understand whether microbiology students and the courses they take are appropriate in preparing individuals for a career in microbiology within the pharmaceutical industry.

When reviewing the microbiology curriculum, many courses focus on biochemistry and very few will cover the application of microbiology concepts in industry, especially pharmaceuticals. Additionally, one of the areas that are absent in the academic curricula is how to assess, validate and implement alternative, molecular and rapid microbiological methods. Because alternative methods will play an important role in the future of contamination monitoring and control, and QC microbiology activities, it is critical that these concepts be included in academic courses, especially since this enhanced knowledge and associated skill sets will be the expectation for pharma microbiologists who will want to move up the technical career ladder.

As a case study, Dr. Denoya stated that out of 65 graduates from a scientific masters program in applied genomics (at the University of Connecticut), 63 of them obtained careers in their first choice within the pharmaceutical industry. The remaining students returned to school for advanced degrees.

The next speaker was Rudolf Gilmanshin, PhD, Vice President, Advanced Platform Research, Pathogenetix, who presented on a single-molecule technology for broadband detection and identification of bacteria. The technology is based on genome sequence scanning, or GSS. Generally, genomic DNA is obtained from a sample under evaluation, and is tagged, linearized and detected in a microfluidic chip. The DNA travels through areas of focused laser light (within the microchip) and the responses are compared against an internal database.

Long fragments of DNA are required from the sample (60-350 kb or 20-115 um), must be double stranded and free of nicks, be free floating in solution, be able to hybridize with the fluorescent tags, and be of high purity. Following lysis of the sample organisms, preparation occurs in about 3 hours in an automated washing and purification instrument. The fragment is then stretched within the microfluidics chip, and will then pass through separate lasers, which will excite the fluorescent tags. Organism concentration for use in the system is between 10^7-10^9 cells.

Restriction enzymes and the tags generate fluorescent signatures used for fingerprinting. Because of the uniqueness of underlying genomic sequences, hybridized tags generate the unique signals. The system is extremely sensitive, with a detection level of 0.1% sensitivity against the internal database. Beta systems will be placed in hospital clinical labs in Q4, 2012.

The last presentation in this session was made by Alessio Fantuzzi, PhD, Microbiological Project Supervisor, and Michele Bosi, Quality Control Manager, Chiesi Pharmaceutical. They presented a case study in development and qualification of an alternative method for the release of non-sterile and sterile products (e.g., sterility testing).

For sterility testing, they perform the compendial sterility test; however, they have added an additional reading phase during the test incubation period using the ATP bioluminescent Pallchek system. The use of the Pallcheck system represents a qualitative assessment of the growth of organisms if they were present in the original sample. To demonstrate equivalence against the compendial methods, they followed the validation guidance as specified in USP 1223 and Ph. Eur. 5.1.6.

For non-sterile product the incubation period lasts for 24 hours, which is the time it takes for the system to detect the number of organisms that is at a threshold level higher than background noise. For sterile product, after a 48-hour incubation period, they filter the media and assess the media for microorganisms using the Pallchek system. To support this rapid sterility test, they also utilize an enhanced environmental monitoring and risk assessment program, and the total sterility testing program has been reduced from 14 days to 8 days.

The success of these validation programs have prompted their company to implement rapid methods ofr all of their future products/formulations.

Rapid Methods Session 1 at the PDA Global Microbiology Conference


This is the first of two rapid method sessions at the PDA Micro Conference. The first speaker was Michele J. Storrs-Mabilat, PhD, Global Scientific Partnerships Manager, Industrial Microbiology Division, bioMerieux, Inc. She presented information on a novel rapid and automated prototype system for the microbiological monitoring of sterile pharmaceutical environments. The Midass system was introduced, and Midass is an acronym for microbial detection in air system for space. This technology was originally developed for use by astronauts on route to and from Mars, where the air in the space capsule will recirculate for a period of up to 3 years, and there will be a need to assess the microbiological state of the capsule environment during the journey.

For the pharma industry, the Midass system is a complete system for monitoring surfaces, personnel and air. The system utilizes a peppermill-type collection device for air sampling, cellular lysis and nucleic acid purification. A separate NASBA card, which contains primers and probes/beacons, is used to amplify the purified rRNA targets. NASBA is used instead of conventional DNA/PCR amplification because RNA is a better predictor of cellular viability, is not susceptible to contamination by extraneous DNA, and the amplification reaction is carried out at a single temperature instead of multiple temperatures as is required by PCR. Amplification takes place in 60-90 minutes, and the system will detect both bacteria and fungi. The time to result is 3 hours, but there is an opportunity to reduce this time in the future. A table-top instrument is used to process the peppermill and the amplification card.

Total viable counts are obtained not in the form of colony forming units (cfu), but in gene copies or genomic equivalents (Geqs). Sensitivity is estimated at 1 cfu (or 1 Geq) per cubic meter of air or per 25 square cm for fungi, and 20 cfu (20 Geqs) per 25 square cm for bacteria (work is still underway to determine the sensitivity for bacteria in air). Initial testing shows encouraging equivalence between a cfu and a Geq. Finally, the system is considered to be non-destructive, where the purified nucleic acid material may be stored for further analysis, such as microbial identification.

The second speaker was Gene Zhang, PhD, Principal Scientist, Bayer Healthcare Pharmaceutical, who presented a Case Study on Validating a Microbial ID System to Meet the New Regulatory Requirements for Part 11.

There are a number of microbial identification rapid methods systems available and many are operating via computerized systems. The pharma industry is now expected to ensure that the data management capabilities and electronic records for these types of systems meet Part 11 compliance. In fact, FDA warning letters have included reference to computer systems that have not been validated against the expectations to Part 11 requirements. Dr. Zhang reviewed how a firm can meet these requirements and used a rapid nucleic acid amplification identification system based on 16S rRNA sequencing as an example.

USP Update on Rapid Micro Methods and Chapter 1223


Radhakrishna Tirumalai and James Akers both provided updates to the USP Microbiology Expert Committee activities. Of note, the following rapid method topics were discussions.

During last year’s PDA Micro meeting, the USP stated that they were going to update/revise the existing USP 1223 informational chapter and provide additional guidance with respect to the use of alternative micro methods. First, we must be reminded that the use of RMMs as a replacement for existing methods is nothing new, as the USP provides guidance on the validation of alternative methods. More importantly, USP micro methods are intended to be referee tests (i.e., adjudicative) for the analysis of monograph products and compendial articles, and they were not intended to be QC release assays or in-process tests. Actually, USP referee tests were not intended to be used as QC assays without modification, and this modification is the responsibility of the method user.

USP <1223>, the informational chapter that provides guidance on validating alternative or rapid methods, is under revision. Some of the changes from the current version may include enhanced guidance on method selection and qualification, and more specific content than what is currently provided. Additionally, the committee is concerned that RMM implementation is being held up because users are too concerned at arriving at a perfect definition of method equivalence. Therefore, we may see some changes in how the USP recommends how to determine whether an alternative method is as good (or better) than a compendial method that is current in use.

Next, the expert committee is now exploring the development of a new referee sterility test; however, the referee method cannot be sourced from a single, patented technology. The committee will initially y will focus on biologics (cytotherapy/regenerative medicine products) and radiopharmaceuticals. And they will get support from the USP biologics committee as well as scientists from CBER (please see my August blog posts for additional guidance form the FDA on the use of RMMs for sterility testing).

Finally, the committee plans on providing validation-useful information for the development and validation of HPLC methods for antibiotic assays for products where micro methods are still being employed.

Monday, October 17, 2011

Dr. Fung Discusses a 30 Year Review of Rapid Methods in the Food Industry


Welcome to the 6th Annual Global Conference on Pharmaceutical Microbiology! There are a record number of attendees this year, so the conference is sure to provide excellent opportunities for interaction with microbiologists from across the industry. Over the next few days, I will be blogging on presentations related to rapid and alternative microbiological methods.

The opening keynote address is being presented by a world-renowned microbiologist and subject matter expert in rapid methods for the food industry, Dr. Daniel Y.C. Fung. Dr. Fung is Industry Professor, Food and Science, at Kansas State University. His presentation focused on Global Developments of Rapid Methods and Automation in Microbiology: A Thirty Year Review and Predictions into the Future.

Rapid methods and automation in microbiology is a dynamic area of technological advancement sustaining a stream of emerging technologies. Rapid microbial methods continue to offer unique opportunities for improving product quality assurance and economy of quality control and manufacturing operations. Almost ten years ago, improvements in microbial isolation, rapid detection, characterization, and enumeration lead to his prediction “…companies that aren’t converting to rapid methods won’t be in business in 10 years…”

Dr. Fung reviewed the use of rapid methods within the food and medical sectors since the 1960’s. Methods have included modifications of traditional, growth-based procedures using conventional medium, including a double tube agar method Dr. Fung developed himself. In this procedure, growing C. perfringens was able to be viewed within a few hours. And over the years, more automated systems were being introduced. For example, impedance microbiology procedures have been around for more than 30 years, as well as methods for the detection of ATP.

Immunological dip-sticks then came on the market, which provided results on the presence of food-borne pathogens in as early as 10 minutes. Today, we can utilize a wide variety of molecular and nucleic amplification systems, including automated, real-time PCR, as well as novel biosensors, microarrays and nanosensors.

Within the food processing sector, it was projected that more than 740 million micro tests were performed in 2008 by more than 40,000 food processing plants, and it is estimated that the worldwide market for micro testing is more than $2 billion. And the market for food microbiology testing continues to grow, year over year. For example, the rate of growth of micro testing from 2008 to 2010 was more than 6%. But Dr. Fung also stated that the use of rapid methods can also provide considerable cost savings, depending on the method being utilized.

The take home message from Dr. Fung’s keynote is that the number of microbiology assays associated with the monitoring of food will continue to increase, especially in light of recent contamination events, and that rapid technologies will play a very important role in protecting the world’s food supplies.

When asked what the pharmaceutical industry can learn from the food industry (in terms of the adoption of rapid methods), Dr. Fung stated that the expectations for microbiological safety is much higher in the pharmaceutical industry than in the food industry, and that we can benefit greatly from the implementation of rapid methods. Interestingly, the food industry looks up to the pharma industry for guidance on excellence in microbiology testing. Their perception is that we pharma microbiologists strive for perfection, and that we are always looking at ways to implement new technologies. Unfortunately (from my point of view), our industry has been extremely slow to adopt rapid methods for a number of reasons, and that the food industry is actually well ahead of where we are today. This will be a topic of discussion during my rapid methods presentation tomorrow afternoon.

Saturday, October 15, 2011

Live Blogging from the PDA Microbiology Conference


As I did last year, I will be blogging live from the PDA 6th Annual Global Conference on Pharmaceutical Microbiology. Here are the following rapid micro method presentations that I will be attending on Monday and Tuesday:

Keynote Address: Global Developments of Rapid Methods and Automation in Microbiology: A Thirty Year Review and Predictions into the Future. Daniel Y.C. Fung, PhD, Industry Professor, Food and Science, Kansas State University

A Rapid and Automated Prototype System for the Microbiological Monitoring of Sterile Pharmaceutical Environments. Michele J. Storrs-Mabilat, PhD, Global Scientific Partnerships Manager, Industrial Microbiology Division, bioMerieux, Inc.

Advanced Microbiological Systems—A Case Study on Validating a Microbial ID System to Meet the New Regulatory Requirements for Part 11. Gene Zhang, PhD, Principal Scientist, Bayer Healthcare Pharmaceutical

The Benefits of Developing an In-house Disinfectants Qualification Process: Understanding, Responsiveness, and Control. Eric Myers, Senior Supervisor - QC Microbiology, Pfizer, Inc., Claudio D. Denoya, PhD, Adjunct Professor, Department of Molecular and Cell Biology University of Connecticut

Single-Molecule Technology for Broadband Detection and Identification of Bacteria. Rudolf Gilmanshin, PhD, Vice President, Advanced Platform Research, Pathogenetix

Rapid Micro Project in Chiesi Pharmaceuticals Group: Development and Qualification of Alternative Method for the Release of Non-sterile and Sterile Products. Alessio Fantuzzi, PhD, Microbiological Project Supervisor, Chiesi Pharmaceutical, Michele Bosi, Quality Control Manager, Chiesi Pharmaceutical

Debunking the Myth’s Surrounding Rapid Microbiological Methods and Their Impact on Pharmaceutical Manufacturing and the Quality of Medicinal Products. Michael J. Miller, PhD, President, Microbiology Consultants, LLC.

Sunday, September 18, 2011

Significant Updates to our Regulatory Pages on rapidmicromethods.com


We have very recently updated all of our rapidmicromethods.com Regulatory Pages to better reflect the most current perspectives, expectations and guidance from the U.S. FDA, EMA, Australian TGA, Japanese PMDA, ICH, as well as added an expanded page that discusses the regulatory impact of changing acceptance levels and microbial specifications. Please visit our Regulatory starting page at http://rapidmicromethods.com/files/regulatory.html. Links to each section can be found at the top of this page.

Look for more revisions to our website in the upcoming month, especially in the area of available technologies and implementation strategies. Our Blog and Newsletter will announce when our updates are effective (you can sign up for our FREE monthly newsletter at http://rapidmicromethods.com/newsletter/).

Sunday, September 11, 2011

Nature New Focus of USF Lab Targeting Biological Terror


There's some very interesting research being conducted (literally) right in my own back yard. The University of South Florida's (USF) Center for Biological Defense is focused on the emerging infections cooked up by Mother Nature herself. Here is an overview of what's happening at the Tampa campus:

Some people were skeptical when the Center for Biological Defense opened at the University of South Florida 11 years ago. There had been only one biological attack in U.S. history – a Salmonella poisoning at an Oregon salad bar that sickened 751 people. Within two years, the fledgling lab was Florida's bioterror research and detection center, with up to 100 samples of potentially deadly white powder arriving for testing every day. Soon it would get $4 million from the U.S. Department of Defense.

Today, $67 million later, the USF Center for Biological Defense is focused on a different kind of threat – the emerging infections cooked up by Mother Nature, whom lab supervisor Andrew Cannons called "a much better terrorist than man could ever be."

Judging by the five security doors a visitor must pass through to reach one of the labs, it's work that requires a good deal of protection. USF is trying to be ready for "what's around the corner," Cannons said, be it a deadly new virus, antibiotic-resistant organism or food-borne illness. And the grim truth, Cannons said, is that the operation might not exist if not for the anthrax letter attacks that closely followed the Sept. 11 attacks on the World Trade Center towers.

Before most people had even heard of anthrax or the antibiotic Cipro, Phil Amuso, of the Florida Department of Health, and then-USF research vice president George Newkome sketched out the biological defense center on a Dunkin' Donuts napkin. Biological dangers weren't at the top of people's minds, but the threats were out there, Amuso said. "We were looking for ways DOH could work with the university" to deal with them if they did emerge.

In 2000, U.S. Rep. C.W. Bill Young, a Republican from Seminole, helped USF get about $1 million to work in partnership with the Department of Health, drawing on research from the University of Florida, University of West Florida and Florida Atlantic University. "I saw a growing threat from people around the world who were anti-American and I was concerned about someone who might try to poison our water supplier or damage air filtration systems," Young said.

"After 9/11, the threats became far more serious and far more specific."

About a week after the attacks, anthrax spores spilled out of letters delivered to two U.S. senators and several media offices, including the National Inquirer in Lantana, Florida. Five people eventually died.

The following week, on Sept. 26, USF announced the center had been awarded $4 million to build a system to prepare public health workers to deal with bioterrorism attacks. The next year it announced a $9 million Department of Defense grant. "It went up and up and up," Cannons said.

The center focused heavily on helping emergency responders gather samples, creating a streamlined process that ensured the sample would be preserved untainted and workers would be protected. Then they developed a fast way to figure out whether any substance was dangerous. The new test could identify anthrax spores in about 15 minutes. That led to rapid new methods for genetically identifying the "bugs" and finding out if they had been seen elsewhere and would respond to antibiotics or not.

Along the way, the center amassed "a unique collection of bacteria that no one else has," Cannons said. It includes about 1,500 strains, "most of them not nasty." The lab has a federal biosafety level of 3; the highest is 4. Level 3 includes bacteria and viruses that can be deadly but are treatable. It follows strict Centers for Disease Control safety rules, Cannons said.

The center keeps a low profile in a state Department of Health building on the Tampa campus of USF. It doesn't hide its presence, but it doesn't advertise it, either. Everything except beyond the lobby is behind one or more locked doors. No photography of the lab equipment is allowed. Cannons didn't explain why, other than to say it was the DOH's rule. "It's not all that exciting anyway," he said.

The biological defense center is quiet these days. Its reduced to pre-Sept. 11 levels. But its mission hasn't changed, Cannons said. It still trains emergency workers on how to handle biological threats. Since it opened, it has trained more than 25,000 people from around the country, said Harry Glenn, spokesman for Rep. Young.

Its Advanced Biosensors Lab continues to refine ways to recognize and identify dangerous organisms. Much of the staff's time is spent testing detection and decontamination equipment developed by others. The center is part of a $1.3 million Defense Department grant to Mote Marine Laboratory in Sarasota to test whether the mucus sharks use to fight infection could help wounded soldiers.

In addition to its work on emerging infections, the center has also begun working with the state Department of Agriculture to develop a food-testing program. "We're trying to be ready," Cannon said. "You can't really know what's coming." That's the nature of these threats. "But you can respond as soon as it happens."

Wednesday, September 7, 2011

Optical Biosensor for Continuous Rapid Detection of Health Threats


Researchers from Stratophase, a firm out of Southampton, United Kingdom, just published a paper in journal Biosensors and Bioelectronics, describing the technology inside their SpectroSens chip, a new optical micro device designed to rapidly detect pathogens and biochemicals. The chip can be loaded into a robust device to simultaneously identify 16 different potential health threats like anthrax and ricin toxin.

The chip works with light that reflects in different wavelengths in different situations. The reflectors, which are called Bragg gratings, will reflect one wavelength and let all other wavelengths pass through unaffected. The specific color it reflects can be correlated with a location on the chip. Interactions between target antigens in the test sample and respective immobilized antibodies on the chip result in localized changes in the refractive-index. This increases the wavelength of the reflected light which can be detected. With one chip it is possible to multiplex sixteen different biological agents like spores, viruses and toxins in real-time. You can either load samples manually or, when continuous monitoring is necessary, the cartridge can be combined with air sampling technology.

The company tested the device first on harmless biological agents like Bacillus atrophaeus (BG) spores, Escherichia coli, MS2 viruses and albumin protein. Thereafter, they tested the method on organisms like Bacillus anthracis (BA) spores, Vaccinia viruses (heat-killed) and ricin toxin. The soluble protein antigens seem to give a higher and earlier response than the larger bacterial and viral antigens, but in the end all were picked up by the detector.

The disposable microchips and robust device are compact and easy to transport, which makes them ideal to do rapid on-site monitoring. The system can be used in security and defense operations, but also in regular in-field medical diagnostics for human and veterinary health.

The paper is in press but is currently available for online purchase through Science Direct.

Optical microchip array biosensor for multiplexed detection of bio-hazardous agents.

D. Bhatta, A. Michel, M. Marti Villalba, G.D. Emmerson, I.J.G. Sparrow, E.A. Perkins, M.B. McDonnell, R.W. Ely and G.A. Cartwright. Biosensors and Bioelectronics. Volume 28, Issue 2. 2011.

Abstract

An optical waveguide array biosensor suitable for rapid detection of multiple bio-hazardous agents is presented. SpectroSens™ optical microchip sensors contain multiple spatially-separated waveguide channels with integral high-precision Bragg gratings sensitive to changes in refractive-index; selective surface-functionalisation of discrete sensing channels with different antibodies as bio-recognition elements enables selective multi-analyte biological detection. Interactions between target antigens in the test sample and respective surface-immobilised antibodies result in localised changes in refractive-index; the biosensor response manifests as increases in wavelength of light reflected from specific sensing channels. Multiplexed, label-free detection of 8 different biological agents, encompassing bacterial spores, vegetative cells, viruses and proteinaceous toxins has been demonstrated in real-time. Selective detection of Bacillus atrophaeus (BG) spores, Escherichia coli cells, MS2 viruses and ovalbumin (OVA) protein (simulant bio-hazardous agents) was first demonstrated as proof-of-concept; subsequently, detection of Bacillus anthracis (BA) spores (UM23CL2 strain), Franciscella tularensis (FT) cells (live vaccine strain), Vaccinia viruses (heat-killed) and ricin toxin (bio-hazardous agents) was proven. Two optical microchip sensors, each comprising 8 sensing channels were packaged into a single disposable cartridge allowing simultaneous 16-channel data acquisition. The specific antibody deposition sequence used in this study enabled detection of either 4 simulants or 4 bio-hazardous agents using a single consumable. The final device, a culmination of the multidisciplinary convergence of the fields of biology, chemistry, optoelectronics and microfluidics, is man-portable and inherently robust. The performance characteristics of the SpectroSens™ technology platform highlight its potential for exploitation as a ‘detect to warn/treat’ biodetector in security and defence operations.