Rapid Microbiological Methods and the PAT Initiative

The methods used in most microbiological test laboratories originated in the laboratories of Koch, Lister, and Pasteur. As such, they are very old, typically over a hundred years. While numerous changes have occurred in the chemistry laboratory, there have been limited improvements in methods used for microbiological testing. In the past decade, the focus of many researchers has been the study and implementation of improved methods for the isolation, early detection, characterization, and enumeration of microorganisms and their products. In common language, this translates to better methods, automated methods, miniaturized methods, and methods that require less time or expense.

All of these methods are collectively grouped into the category of rapid microbiological methods (RMM). In some compendia, these are also called alternative microbiological methods. The methods range from simple dip-stick-type tests to very complex automated systems that perform a variety of tests using a variety of techniques. The prices for these methods vary greatly, from a few dollars to hundreds of thousands of dollars. Although these methods are called rapid microbiological methods, many have their roots in other sciences, e.g., chemistry, molecular biology, biochemistry, immunology, immunochemistry, molecular electronics, and computer-aided imaging.

Rapid microbiological methods provide significant opportunities for pharmaceutical companies to obtain data that may be significantly better than that obtained via traditional methods, may be more cost effective, may provide marketing advantages, and may allow for coordinated process analytical technologies to be fully integrated within a facility.

SLOW TO ADOPT NEW METHODS

While science moved forward in developing new microbiological methods, industry was slow to accept and implement these methods. One of the greatest concerns was that regulators would not recognize these methods as superior to traditional ones. Another concern was that companies would not be allowed to change test limits based upon the test method — i.e., they would use a superior method that was likely to detect more organisms, and they would not be allowed to adjust limits to accommodate the sensitivity of the new method. A further concern was that the first company to submit a new technology for regulatory approval would have a much more difficult time obtaining approval than companies that submitted later.

REGULATORY FRAMEWORK

In recent years, a variety of documents has been issued or drafted to help the microbiologist select, purchase, implement, and regulate the submission of rapid microbiological methods. We discuss six.

Parenteral Drug Association Technical Report Number 33

The Parenteral Drug Association (PDA) was one of the first organizations to develop guidance for evaluating, implementing, and validating rapid microbiological methods. Guidance information was published as Technical Report 33 (PDA TR No 33).¹ This document was developed by a committee of individuals from industry, regulatory agencies, compendial groups, and instrument vendors. This guidance provided definitions, in microbiological terms, for validation criteria similar to the information in United States Pharmacopeia (USP) <1225> for chemistry methods.²

USP Proposed Chapter <1223> on Alternative Microbiological Methods Validation

The USP proposed a draft monograph <1223>, Validation of Alternative Microbiological Methods, which defines various validation criteria that may be used for rapid microbiological methods and defines these criteria in terms of microbiology. Additionally, the proposal identifies how to determine which criteria apply to different technologies, based on the type of testing being performed.³

GMPs for the 21st Century

FDA has initiated a program to modernize requirements for pharmaceutical manufacturing and quality. This modernization includes several objectives: encouraging early adoption of new technologies; facilitating industry application of modern quality-management technologies; encouraging implementation of risk-based approaches in critical areas; ensuring that policies for review of a submission, compliance, and facilities inspection are based on state-of-the-art technologies; and enhancing the consistency and coordination of FDA regulatory programs. This program resulted in the 2004 initiative, Pharmaceutical cGMPs [current Good Manufacturing Practices] for the 21st Century — A Risk-Based Approach.⁴

FDA's 2004 Guidance on Aseptic Processing

In 2004, the FDA published a guidance document on aseptic processing of pharmaceutical products. This document includes the following provision for the use of alternative microbiological test methods: "Other suitable microbiological test methods (e.g., rapid test methods) can be considered for environmental monitoring, in-process control testing, and finished product release testing after it is demonstrated that the methods are equivalent or better than traditional methods (e.g., USP)." This was one of the first regulatory documents that specifically recognized the potential use of alternative rapid microbiological methods.⁵

Process Analytical Technologies (PAT)

The concept of process analytical technologies (PAT) is described in the FDA's Guidance for Industry: PAT — A Framework for Innovative Pharmaceutical Development, Manufacture, and Quality Assurance. PAT is defined in this document as: "Systems for analysis and control of manufacturing processes based on timely measurements, during processing, of critical quality parameters and performance attributes of raw and in-process materials and processes to assure acceptable end product quality at the completion of the processes."⁶ Rather than relying on finished-product testing, PAT allows manufacturers to use faster, more accurate test methods capable of producing real-time or near-real-time data for process control. Traditional microbiological test methods usually cannot deliver real-time or near-real-time results, making them unsuitable for PAT applications. Nonetheless, RMMs were included as PAT applications by the FDA PAT subcommittee in October 2002, following representation from industry practitioners.

Table 1. Applications of Rapid Microbiological Methods

European Pharmacopoeia Proposed Chapter on RMM

PHARMEUROPA, the official forum for communicating general policies of the European Pharmacopoeia, published a draft chapter 5.1.6. "Alternative Methods for Control of Microbiological Quality" in 2004. This chapter provides an overview of some of the RMMs available and potentially applicable to pharmaceutical processes, and describes how they may be used for microbiological control of products and processes. This chapter also provides guidance regarding how to choose an appropriate methodology and how to validate the method.⁷

DOES RMM = PAT APPLICATION?

In most cases, the definition of PAT includes collecting real-time data, typically in-line, to make decisions about product quality early in the production process. Although there have been great advances in RMMs in the past few years, most methods developed to date are still conducted on the laboratory bench, off-line, i.e., samples are collected and taken to a laboratory for testing. While this may not be as advantageous as many of the chemistry applications developed, it is a significant improvement over traditional microbiological methods, because results may now be available in hours to a few days — instead of days or weeks. As a result, implementing these methods makes it possible to achieve many of the savings available from other systems.

Traditional Methods

Classical microbiological test methods frequently are divided into three general categories, based on the test function performed. These categories are: presence or absence of microorganisms (e.g., pathogen detection, absence of objectionable organisms, sterility testing), enumeration of microorganisms (e.g., bioburden testing); and identification of microorganisms. This classification answers three specific questions: "Is something there?" (presence or absence); "How much is there?" (enumeration); and "What is there?" (identification).

Table 1. (continued) Applications of Rapid Microbiological Methods

RMMs

Classification systems for rapid methods are based on how the technology works: methods that measure the growth of microorganisms; methods that determine the viability of microorganisms; methods that detect the presence or absence of cellular components or artifacts; nucleic acid methods; traditional methods combined with computer-aided imaging; and combination methods.

Growth-based Technologies. These methods are based on the measurement of biochemical or physiological parameters that reflect the growth of the microorganisms. Examples of these types of methods include: adenosine triphosphate (ATP) bioluminescence, colorimetric detection of carbon dioxide production, measurement of change in head space pressure, impedance, and biochemical assays.

Viability-based Technologies. These types of technologies do not require microorganism growth for detection. Varying methods are used to determine if the cell is viable, and if viable cells are detected, they can be enumerated. Examples of this type of technology include solid-phase and flow fluorescence cytometry.

Cellular-component or Artifact-based Technologies. These technologies look for a specific cellular component or artifact within the cell for detection or identification. Examples of these systems include: fatty acid profiles, mass spectrometry (i.e., Matrix Assisted Desorption Ionized-Time of Flight, MALDI-TOF), enzyme linked immunosorbent assay (ELISA), fluorescent probe detection, and bacterial endotoxin-limulus amebocyte lysate testing (LAL).

Nucleic-acid-based Technologies. These technologies use nucleic acid methods as the basis for operation. Examples of this type of technology include: deoxyribonucleic acid (DNA) probes, ribotyping/molecular typing, and polymerase chain reaction (PCR).

Traditional Methods with Computer-aided Imaging. This approach involves using a classical method for most of the processing of a sample, and then using imaging software to detect the growth earlier than methods requiring visual growth detection. In most cases, detection of growth using human vision typically requires growth of 10⁵ or 10⁶ cells. Computer-aided imaging can detect much lower levels of cellular growth, e.g., less than 100 cells.

Combination Methods. This term is used to describe those systems that involve more than one methodology or test to achieve a final result, e.g., a system that tells whether an organism is present and is also capable of identifying the microorganism.

TECHNOLOGY TYPES

Adenosine Tri-Phosphate (ATP) Bioluminescence

Type of Technology: Growth-based.

Premise of Technology: ATP is present in all living cells. In the presence of the substrate D-luciferin, oxygen, and magnesium ions, the enzyme luciferase will use the energy from ATP to oxidize D-luciferin and produce light. The amount of light or bioluminescence produced can be measured by sensitive luminometers, and is proportional to the amount of ATP in the sample. The emitted light is usually expressed as relative light units (RLU) rather than as direct estimates of microbial numbers. Vendors of these technologies have conducted studies to show the correlation between RLU readings and approximate number of organisms. These standard curves are used to translate the raw RLU data to more meaningful organism-quantification data. ATP bioluminescence reduces the test time required in the traditional method by approximately one-third. ATP bioluminescence can be used to screen both filterable and non-filterable samples.

Commercial Systems Available: PallCheck (Pall Life Sciences), Milliflex (Millipore Corporation), Rapiscreen (Celsis), and novaLUM (Charm). Hygiena also has a small hand-held luminometer.

Other: The majority of ATP-based systems are quantitative. The capability of these instruments can be enhanced by using the microbiology application of most probable number techniques.

Adenylate Kinase

Type of Technology: Growth-based.

Premise of Technology: Adenylate kinase is a cellular component that allows for microbial detection. The adenylate kinase released from cells reacts with ADP to form ATP. The ATP is detected using an ATP bioluminescence method. Using these technologies significantly lowers the level of detection for the system. The assay has a detection level of about 34 cells. The detection level can be further reduced (to about four cells) by extending the incubation for ATP generation time.⁸

Commercial Systems Available: Celsis has given presentations on a new system that uses this technology.

Arrays, Microarrays, Microchips

See Lab-on-a-Chip

Biochemical Assays and Physiological Reactions

Type of Technology: Cell-component-based.

Premise of Technology: Pure culture suspensions are tested with a series of biochemical substrates or subjected to analysis to generate a spectrum. The microorganisms have specific reactions to these test conditions. The results are compared to a database of expected results. These comparisons allow the user to identify the microorganism. Systems vary from manual to highly automated.

Commercial Systems Available: API Systems (bioMerieux), BIOLOG Systems (Biolog), and VITEK and VITEK2 (bioMerieux).

Other: Many of these systems require performance of a Gram stain prior to further evaluation. The accuracy of the Gram stain can significantly influence results.

Biosensors and Immunosensors

Type of Technology: Various, depending on the sensor technology used.

Premise of Technology: Immunological reagents are combined with varying sensor detection systems to produce an immunosensor. Typically, these systems are used for pathogens (including bioterrorism organisms).⁸

Commercial Systems Available: Test kits and sensors are specific to the type of application being monitored.

Changes in Headspace Pressure

Type of Technology: Growth-based.

Premise of Technology: Electronic transducers measure positive or negative pressure changes in the head space of each culture bottle. These changes are caused by microbial growth. If the growth causes significant production or consumption of gas, the samples are flagged as positive. Large quantities of samples can be placed in these instruments for testing with frequent monitoring of the head-space pressure. These systems are based on non-invasive, continuous, automated monitoring of microbial cultures.

Commercial Systems Available: BacT/Alert (bioMerieux) and ESP Microbial Detection System (AccuMed).

Colorimetric Detection of Carbon Dioxide Production

Type of Technology: Growth-based.

Premise of Technology: As microorganisms grow, they produce carbon dioxide. In this technology, the test samples are placed in culture bottles for monitoring. The samples are incubated, agitated, and monitored for the presence of microorganisms. These systems use colorimetric detection of CO₂ production from the growth of organisms. Some of the systems commercially available detect color change, flag a positive test sample, and notify the user. These systems are often considered to be non-invasive microbial detection systems and can accommodate a large number of samples. Although these systems are commonly used clinically for blood cultures, Genzyme received approval from the FDA in 2004 to use this method (with the BacT/Alert System) for sterility testing.

Commercial Systems Available: BacT/Alert (bioMerieux) and ESP Microbial Detection System (AccuMed).

Other: This technology may be useful for slow-growing organisms, e.g., mycobacteria.⁷

Concentric Arcs of Photovoltaic Detectors with Laser Scanning

Type of Technology: Combination.

Premise of Technology: The system comprises five concentric arcs of photovoltaic detectors, in an orb-like platform. The sample being evaluated is suspended in a liquid or gas inside a vial or sample collection device, which is placed near the center of the orb. A laser beam of red, solid-state composition is passed through the sample. The scattered light intensities generate a spectrum that is compared to a library of known scatter patterns, i.e., using a statistical classification algorithm. Contamination can be identified in seconds. This light-scattering pattern becomes a fingerprint-type of identification for the microorganism. The pattern includes the size of the particle, the shape of the particle, and the optical characteristics. Light patterns are evaluated at multiple angles to detect and differentiate the size of the microorganism almost instantaneously. Identification occurs within a few milliseconds after the particle passes through the beam.⁹

Commercial Systems Available: MIT System (MicroImaging Technologies, Inc.).

Other: This system was recently granted a US patent. Product claims include enumeration of microorganisms present, determination of the size of microorganisms present, identification of the microorganisms, and the ability to handle mixed cultures.⁹

Conductivity

Type of Technology: Growth-based.

Premise of Technology: This is similar to impedance methods, with measurement taken in conductance.

Commercial Systems Available: Bactometer (bioMerieux), BacTrac (Sy-Lab), RABIT (Don Whitley Scientific Ltd), and the Malthus Microbial Detection System (Malthus Diagnostics, Inc.).

Conventional Methods with Computer-Aided Imaging

Type of Technology: Growth-based.

Premise of Technology: Many conventional methods for processing samples can be used for testing, and using advanced image-analysis software can significantly reduce the incubation and enumeration time required. Commercial software programs are available that can be customized for use with current methods. With this technology, images are collected using a charge-coupled device camera. The collected images are digitized on a computer, using image processing software that has programming capabilities (alternatively, some systems collect the data directly with a digital camera); the digitized picture is processed to detect colonies present, and the separated colonies are counted. Depending on the system used, one can obtain movie-like presentations of colony growth, overgrowth, and confluence, if present. These systems use either a fluorescent staining procedure, cellular epifluorescence, or autofluorescence as a method of cellular detection. This technology can be used as a series of commercially available products used together to achieve the desired enumeration, or as a combined industrialized (automated) system that performs these tasks.

Commercial Systems Available: COVASIAM¹⁰ (University of Mexico) and Growth Direct (Genomic Profiling Systems).

Direct Epifluorescent Filter Technique (DEFT)

Type of Technology: Viability-based.

Premise of Technology: In the direct epifluorescent filter technique (DEFT), samples are filtered and stained using a fluorescent viability indicator. Acridine orange was used originally, but more recent applications use 4',6-diamidino-2-phenylindole. Epifluorescence microscopy detects fluorescing microorganisms. The sensitivity of the technique depends on the volume filtered and the number of fields viewed under the microscope. Micro-colony formation can further enhance the accuracy of the technique and the detection of cell viability. Automated and semi-automated systems have been developed to speed up the process and increase the accuracy of the technique.

Commercial Systems Available: Microscopes with epifluorescence capabilities.

Other: The ability to differentiate between fluorescing organisms and auto-fluorescing particles can be a concern. The robustness of the test can be affected by the distribution of the microorganisms on the membrane. This methodology is best suited for low viscosity fluids, although it may be possible to use pre-filtration to allow testing of other solutions.⁷

Endospore Detection

Type of Technology: Cell-component-based.

Premise of Technology: A major component of the spore case is calcium dipicolinate (Ca[dpa]). Dipicolinate anions are present only in bacterial endospores. Ca (dpa) and dipicolinate anions (dpa^2- ), when dissolved, are not photoluminescent. It has been shown that terbium (Tb³⁺ ) is able to complex with dpa^2- , forming a photoluminescence complex. The procedure for detecting spores (US patent issued to the US Army Research Laboratory) involves the following steps:¹¹

• Mixing: Terbium chloride (TbCl₃) is added to the aqueous-based test solution (potentially including endospores). It may be desirable also to add aluminum chloride (AlCl₃), as it has been shown to bind to phosphates that may interfere with the results obtained. Buffers can compete with the dipicolinate for the Tb³⁺ , thereby reducing the expected photoluminescence. Tris buffer was found to be a good choice for the solution.

• Filtration: A 0.22-µm filter is used to remove excess residual bacterial particles from the terbium-treated solution as the solution is passed from the mixing container to a cuvette containing [Tb(dpa)]⁺ .

• Analysis: The sample is exposed to an ultraviolet (UV) source and excited. The corresponding emission is perceived by a detector and is used to generate an emission spectrum (UV-visible spectrophotometer used with wavelength showing range of 250-300 nm).

Commercial Systems Available: The equipment needed to perform this procedure is commercially available as individual instruments.

Other: Insoluble items may be photoluminescent, and there may be light loss due to the presence of insoluble particulate matter.

Enzyme-Linked Immunosorbent Assay (ELISA)

Type of Technology: Cell-component-based.

Premise of Technology: In the ELISA technique, an antigen-antibody reaction detects unique microorganisms or cellular components.

Commercial Systems Available: VIDAS and Mini-VIDAS (bioMerieux), Tecra Salmonella ELISA (International Bioproducts), and Salmonella Tek ELISA (Organon Teknika).

Other: This technology has been used for many years in clinical labs.

Fatty Acid Profiles (Fatty Acid Methyl Esters [FAMEs])

Type of Technology: Cell-component-based.

Premise of Technology: Fatty acids are present in microorganisms. The fatty acid composition typically is homogeneous within different taxonomic groups. Isolates are grown on standard media and selected for testing. The testing procedure includes saponification of fatty acids, methylation, and extraction, to produce fatty acid methyl esters (FAMEs). The FAMEs are measured using gas chromatography, and the measurements are compared to a library of known organisms.⁷

Commercial Systems Available: Sherlock Microbial Identification System (MIDI).

Other: The methods used for growth of microorganisms (media and incubation) should be standardized. Gas chromotographs should be calibrated frequently.

Fluorescent Detection of Carbon Dioxide

Type of Technology: Growth-based.

Premise of Technology: This technology allows continuous monitoring for contamination using a fluorescent carbon dioxide system. A pH-sensitive, fluorescent CO₂ sensor is poured into the bottom of each container. A series of computer algorithms assess an increased rate of change and a sustained increase in CO₂ production.¹²

Commercial Systems Available: Bactec (Becton Dickinson and Company).

Other: Commercial formulations of all microbial growth media may not be available.

Flow Cytometry (Fluorescence)

Type of Technology: Viability-based.

Premise of Technology: Using flow cytometry, microorganisms are labeled in solution with a non-fluorescent marker. The marker is taken up into the cell and cleaved by intracellular enzymatic activity to produce a fluorescing substrate. The labeled sample is automatically injected into a quartz flow cell, which passes each microorganism individually through a laser excitation beam for detection. The staining and detection mechanisms are similar to solid-phase cytometry. Flow cytometry detects organisms in solution and not in the solid phase, allowing for non-filterable solutions to be tested. Typically, results are obtained within 1.5 to 2 hours, although the limit of detection is approximately 100 cfu per mL.

Commercial Systems Available: D-Count (AES Chemunex) and RBD3000 (AATI).

Other: Systems developed for the pharmaceutical sector range from simple manual systems with single-test capability to highly automated units with high test throughput potential.

Fluorescent Probe Detection

Type of Technology: Combination, cell-component-based, nucleic-acid-based.

Premise of Technology: Nucleic acid probes are designed to bind to specific target sites on or in cells. The probes contain a molecule that is capable of fluorescing when stimulated by an energy source such as a laser. (See Nucleic Acid Probes)

Commercial Systems Available: RBD3000 (AATI).

Other: Some systems have restrictions on the sample size allowed.

Fourier Transform Infrared Spectroscopy (FTIR)

Type of Technology: Cell-component-based.

Premise of Technology: Fourier transform infrared spectroscopy can be used to generate an infrared spectrum of microorganisms. The patterns generated are stable across taxonomic groups. The patterns are compared to a database of spectra of known microorganisms.

Commercial Systems Available: A variety of systems is commercially available.

Other: Standardization is a critical performance aspect, e.g., using isolates grown on standard media using standard incubation conditions.

Gram Stains

Type of Technology: Cell-component-based.

Premise of Technology: This technology uses a single solution, without fixatives or washes. Results are obtained in a few minutes. Syto-9 stain and red-fluorescent hexidium iodide nucleic-acid stain are used. The method can be used with mixed cultures. Using the LIVE Bac Light Bacterial Gram Stain Kit, Gram-positive organisms stain a reddish-orange, and Gram-negative organisms stain green. The fluorescent stains can be viewed and assessed using a fluorescent microscope (with a standard fluorecein long-pass optical filter set) or using flow cytometry. The reagents have been designed to show low background (intrinsic) stain. Dead cells do not show a predicted staining pattern. There are also procedures specified for use with DEFT. A second staining kit — ViaGram Red⁺ Bacterial Gram Stain and Viability Kit — is similar to the kit described above, but it uses two stains and three colors, so that viable and non-viable cells can be readily detected in addition to knowing the Gram reaction. Plasma membrane integrity is used as the distinguishing factor of live bacterial cells. Intact membranes are detected with a blue stain, while damaged membranes stain green. The red stain identifies Gram-positive bacteria.¹³

Commercial Systems Available: LIVE Bac Light Bacterial Gram Stain Kit and ViaGram Red⁺ Bacterial Gram Stain.

Immunological Methods

Type of Technology: Cell-component-based.

Premise of Technology: One can use an antigen-antibody reaction to detect unique microorganisms or cellular components.

Commercial Systems Available: Pathogen detection kits are available for various types of pathogen. ELISA is available.

Other: Immunological methods are useful for pathogen detection, and they may also be used for identification. In some cases, the systems may not distinguish whether the detected cells are viable.⁷

Impedance (Electrochemical) Methods

Type of Technology: Growth-based.

Premise of Technology: Growing microorganisms metabolize large complex constituents, such as proteins and carbohydrates, and convert them to smaller charged by-products such as amino acids, carbon dioxide, and acids. These smaller by-products of metabolism build up and eventually change the electrical conducting properties of the supporting growth medium. When an alternating current is applied across electrodes to this growth media, a change in impedance can be observed. Impedance is the resistance to the flow of an alternating current through a conducting material. Microbial detection systems based on impedance technology are classified in two types of systems: direct and indirect impedance. Direct impedance systems work by detecting changes in electrical conductivity of growth media when an a.c. current is passed across two electrodes. Indirect impedance systems detect carbon dioxide produced by metabolizing organisms, via the use of chemical sinks such as potassium hydroxide. As the carbon dioxide is ionized, changes in impedance result. There is no direct contact between the electrodes and the microorganisms under investigation. When microorganisms multiply, a detection threshold is reached, above which an electrical signal is detected by both types of systems. Generally, this detection limit is approximately 10⁶ cfu/mL for many microbial species. The lower the initial population, the longer the time taken to reach the detection threshold.

Commercial Systems Available: Bactometer (bioMerieux), BacTrac (Sy-Lab), RABIT (Don Whitley Scientific Ltd), and the Malthus Microbial Detection System (Malthus Diagnostics, Inc.).

Lab-on-a-Chip (LOC), Arrays, Microarrays, and Microchips

Type of Technology: Combination.

Premise of Technology: An array is defined as an orderly arrangement of data. For example, a microtiter well plate can be considered an array of rows and columns of data. Arrays of data have been used in many microbiology applications to perform a single test or series of tests. Microchips are manufactured using microminiaturization technologies, such as photolithography, hot embossing, reactive ion etching, and microinjection molding. Each microchip is much like a miniature laboratory, and some scientists refer to these as "lab on a chip" devices. The chips have become miniature analyzers, i.e., small versions of analytical instrumentation. Reagents used with these chips have been manufactured utilizing micro-fabrication technologies, e.g., micro-dispensing, ink-jet printing. Typical microbiological reagents include oligonucleotides, proteins, and DNA. One application of this technology is the antibody dot or microspot assay. A small amount of antibody, typically 10-100 mL, is placed on the bottom surface of a plastic well. This antibody dot is used as the capture antibody in a microimmunoassay.⁸ This technology can be used for a variety of applications, e.g., microbial virulence factors, antimicrobial resistance and identification, and bacterial discrimination.^14-16

Commercial Systems Available: Various sources are available for these technologies. Others choose to "build" a chip to meet specific laboratory requirements.

Other: Many of these technologies are very expensive. As more commercial systems become available, the costs may be reduced.

Limulus Amebocyte Lysate Endotoxin (LAL) Testing

Type of Technology: Cell-component-based.

Premise of Technology: Amebocyte lysate recovered from horseshoe crabs (Limulus) has similarities with blood coagulation in humans. This similarity allows this reagent to be used to detect the presence of bacterial endotoxins. Quantitation of the endotoxin present requires the use of standards that are appropriately certified or licensed. Three different methods are available: gel clot, kinetic turbidometric, and chromagenic. The gel clot method is an endpoint determination of the amount of endotoxin present. Several different dilutions are evaluated as part of the test to determine the lowest concentration of endotoxin at which a clot forms. This type of test can require a large test sample to perform the various dilutions. The kinetic-turbidometric test allows for faster handling and smaller sample sizes. The chromogenic test is another variation of this method.

Commercial Systems Available: Pyrogent Gel Clot (BioWhittaker), Pyrotell (Associates of Cape Cod), BioTek, and handheld unit (Charles Rivers Endosafe).

Other: This technology has been available for many years as a replacement for the rabbit pyrogen test. Many systems are available, and they have gained widespread regulatory acceptance. Several characteristics, such as pH composition and ions present, can affect results.

Mass Spectrometry (Matrix-Assisted Laser Desorption-Time Of Flight, MALDI-TOF)

Type of Technology: Cell-component-based.

Premise of Technology: When microbial isolates are heated in a vacuum, the gaseous breakdown products can be analyzed using mass spectrometry. A spectrum can be generated and compared to a database of known organisms for identification. When subjected to intense ionization (MALDI-TOF), intact cells will release charged particles in distinct patterns. These patterns can be compared to a database of known microorganisms.

Commercial Systems Available: MALDI-TOF (Kratos Analytical Systems) and Voyager (Perspective Biosystem).

Other: This technology has been used for microbial identifications. The size of the database is important in evaluating the effectiveness of the system.

Microcalorimetry

Type of Technology: Viability-based, but requires catabolic activity.

Premise of Technology: The process of microbial catabolism results in heat that can be measured by micro-calorimetry. The sample is placed in a sealed ampoule with media inside a calorimeter. The instrumentation can be used to establish growth curves. When high levels of contamination are present, one may need to use flow calorimetry.⁷

Commercial Systems Available: A variety of systems is available.

Other: This process cannot be used to determine if a single contaminant is present, nor can it be used on samples with mixed contaminants.⁷

Nucleic Acid Probes

Type of Technology: Nucleic-acid-based.

Premise of Technology: Data available from nucleic acid sequencing are used to select a desired nucleic acid. The desired nucleic acids are extracted, immobilized to a solid phase, and hybridized to a labeled probe. Alternatively, the extracted nucleic acids can be labeled and hybridized to an immobilized probe.¹⁷

Commercial Systems Available: The systems available depend on the type of outcome desired. They include Gene-Trak Systems (Gene-Trak) and Gene-Probe Systems (Gene-Probe).

Other: This technology is frequently used for characterization or identification of microorganisms, and for pathogen detection.

Polymerase Chain Reaction

Type of Technology: Nucleic-acid-based.

Premise of Technology: PCR makes copies of nucleic acid fragments. Nucleic acid fragments are amplified using polymerization techniques. The fragment of interest is heat-denatured; the reaction vessel is then cooled, and the polymerase begins to create the complementary strand. Another denaturation step, followed by another polymerization step, doubles the amount of DNA. Several iterations of this process produce a massive amount of DNA. A variety of PCR methods may be used: reverse transcripterase PCR, nucleic acid sequence-based amplification, or transcription-mediated amplification.

Commercial Systems Available: BAX Microbial Identification System (Qualicon) and Probelia System (BioControl Systems).

Other: This technology is widely used in other sciences, such as anthropology and forensics.

Raman Spectroscopy

Type of Technology: Cell-component-based.

Premise of Technology: A Raman spectrophotometer can generate a spectrum unique to the microorganism. The spectra are stable across taxonomic groups. The patterns are compared to a database of spectra of known microorganisms.

Commercial Systems Available: Raman spectrophotometer.

Other: Studies performed in clinical settings indicated that identifications could be made from cultures incubated for approximately five hours. The test is non-destructive.

Ribotyping - Molecular Typing

Type of Technology: Nucleic-acid-based.

Premise of Technology: This technology uses restriction fragment length polymorphisms (RFLPs) of nucleic acids from bacterial genomes. The size-separated RFLPs are hybridized to a ribosomal ribonucleic acid (RNA) probe. A chemiluminescent substrate is applied. A camera is used to convert the luminescing RFLPs to digital information. The digital information is captured and the data extracted. A pattern is generated and compared to a database of known patterns for identification. The ribotype is a stable epidemiological marker and provides definitive taxonomic information.

Commercial Systems Available: MicroSeq 16S rDNA Bacterial Identification System (Applied Biosystems) and Riboprinter (DuPont Qualicon).

Other: Molecular typing is considered the "gold standard" for identifying microorganisms.

Solid-Phase Cytometry

Type of Technology: Viability-based.

Premise of Technology: Solid-phase cytometry uses membrane filtration to separate potential microbial contaminants from filterable samples before labeling the captured cells with a universal viability substrate. Once within the cytoplasm of metabolically active microorganisms, the non-fluorescent substrate is enzymatically cleaved to release free fluorochrome by a ubiquitous hydrolytic enzyme esterase. Only the viable microorganisms with membrane integrity retain the marker used in the assay. A laser-based detector then automatically scans the membrane, and the number of fluorescently labeled cells is immediately reported. Solid-phase cytometry eliminates the need for cell multiplication. Sensitivities to the single cell level are possible, independent of the volume of sample filtered. In addition to vegetative cells, the technique also can detect spores (bacterial and fungal), stressed organisms, and fastidious organisms. Near real-time results are obtained, typically within two to five hours of sample preparation. Solid-phase cytometry was accepted for pharmaceutical-grade water testing by the FDA in February 2004, and in the United Kingdom in 2000.

Commercial Systems Available: ScanRDI (AES-Chemunex).

Other: Several articles have been published on the topic of viable but not culturable microorganisms. Using a viability-based technology may require changes to existing limits or levels.

Turbidimetry

Type of Technology: Growth-based.

Premise of Technology: As microorganisms grow, one can detect changes in the opacity of the growth medium. Optical density measurements can detect differences in opacity at specified wavelengths, using a spectrophotometer (usually in the range of 420-615 nm). Another version of this methodology uses microtitre plate readers with continuous detectors, to detect organism growth earlier.⁷ A common use for this type of test is to determine microbiological suspension or inoculum sizes.

RMM APPLICATIONS

Table 1 provides a table of some of the ways rapid microbiological methods can be applied in a pharmaceutical environment.

When evaluating a system, one should consider a variety of factors, such as the following:

• Type of technology considered and the type of microbiological test being performed

• Cost for purchase of the initial system

• Cost per test on an ongoing basis

• System's ability to handle the type of products manufactured, e.g., filterability, sample size, detection limits appropriate for the test

• System throughput

• Level of automation required and available

DISCLAIMER

There are reports of thousands of systems that are in some stage of development for use in place of traditional microbiological methods. This article introduces some of the technologies available. Inclusion or exclusion of available methods is not meant to confer credibility, endorsement, or acceptance of some methods over other methods.

ACKNOWLEDGEMENTS

Special thanks to Casey Costello and Vicky Strong for their aid in compiling this information.

Jeanne Moldenhauer is a senior quality assurance and regulatory affairs professional and pharma consultant at Vectech Pharmaceutical Consultants, Inc., 24543 Indoplex Circle, Farmington Hills, MI, 48335, 248.478.5820, fax: 248.442.0060, jeannemoldenhauer@yahoo.com

REFERENCES

1. Parenteral Drug Association. Technical Report 33: Evaluation, validation and implementation of new microbiological testing methods. J Pharm Sci Technol. 2000; 54(suppl):TR33.

2. <1225> Validation of compendial methods (2nd supp to USP 27). Pharmacopeial Forum. 2003; 29.

3. Proposed Chapter <1223> Validation of alternative microbiological methods. Pharmacopeial Forum. 2003; 29:256-264.

4. US Food and Drug Administration. Pharmaceutical cGMPs for the 21st Century — A Risk-Based Approach: Final Report, Fall 2005. Rockville, MD: Department of Health and Human Services; 2004. Available at: http://www.fda.gov/cder/gmp/gmp2004/GMP_finalreport2004.htm

5. US Food and Drug Administration. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice. Rockville, MD: Office of Training and Communication; 2004. Available at: http://www.fda.gov/cber/gdlns/steraseptic.htm.

6. US Food and Drug Administration. Guidance for Industry: PAT — A Framework for Innovative Pharmaceutical Development, Manufacture, and Quality Assurance. Rockville, MD: Office of Training and Communication; 2004. Available at: http://www.fda.gov/cder/guidance/6419fnl.htm.

7. 5.1.6.: Alternative Methods for Control of Microbiological Quality. Pharmeuropa. 2004; 16:555-565.

8. Kricka LJ. New Technologies for Microbiological Assays. In: Easter MC, ed. Rapid Microbiological Methods in the Pharmaceutical Industry. Washington, DC: Interpharm/CRC; 2003:233-248.

9. DeSorbo MA. Rapid contamination detection technology patent granted. CleanRooms. August 2002. Available at: http://cr.pennnet.com/Articles/Article_Display.cfm?Section=Archives&Subsection=Display&ARTICLE_ID=150543&KEYWORD=%22patent%20granted%22.

10. Corkidi G, Trejo M, Nieto-Sotelo J. Automated Colony Counting Using Image-Processing Techniques. In: Olson WP, ed. Rapid Analytical Microbiology: The Chemistry and Physics of Microbial Identification. Bethesda, MD and Godalming Surrey, UK: Parenteral Drug Association and Davis Horwood International Publishing; 2003.

11. Rosen DL, Fell Jr NF, Pellegrino PM. Spectroscopic Detection of Bacterial Endospores Using Terbium Cation Reagent. In: Olson WP, ed. Rapid Analytical Microbiology: The Chemistry and Physics of Microbial Identification. Bethesda, MD and Godalming Surrey, UK: Parenteral Drug Association and Davis Horwood International Publishing; 2003:229-240.

12. Meszaros A. Alternative Technologies for Sterility Testing. In: Easter MC, ed. Rapid Microbiological Methods in the Pharmaceutical Industry. Washington, DC: Interpharm/CRC; 2003:179-185.

13. Tools for microbiology. BioProbes. 2003;43:11-13. Available at: http://probes.invitrogen.com/lit/bioprobes43/4.pdf.

14. Chizhikov V, Rasooly A, Chumakov K, and Levy DD. Microarray analysis of microbial virulence factors. Applied and Environmental Microbiology. 2001; 67:3258-3263.

15. Westin L, Miller C, Vollmer D, et al. Antimicrobial resistance and bacterial identification utilizing a microelectronic chip array. J of Clin Microbiology. 2001; 39:1097-1104.

16. Busit E, Bordoni R, Castiglioni B, et al. Bacterial Discrimination by Means of a Universal Array Approach Mediated by LDR (Ligase Detection Reaction). BMCMicrobiology. 2002; 2:27. Available at: www.biomedcentral.com/1471-2180/2/27.

17. Rudi K. Application of Nucleic Acid Probes for Analyses of Microbial Communities. In: Olson WP, ed. Rapid Analytical Microbiology: The Chemistry and Physics of Microbial Identification. Bethesda, MD and Godalming Surrey, UK: Parenteral Drug Association and Davis Horwood International Publishing; 2003:13-40.

ADDITIONAL RESOURCES

Additional resources are available as microbiologists gain information on rapid microbiology. These include the following:

• www.fda.gov has guidance documents on PAT, information on presentations, and other data.

• www.rapidmicrobiology.com includes information on vendors, technologies, press releases, etc.

• Rapid Microbiology User's Group (RMUG), with information available at www.vectech.com has resources and support information for seminars and newsletters.

• Pharmaceutical Microbiology Forum (PMF), www.microbiol.org, is for pharmaceutical microbiologists. It includes an e-mail discussion group, virtual library, and information relevant to microbiologists.