Q.1 Define rapid methods & summarize the requirements to validate a rapid method.

Also
enlighten PCR used in food microbiological analysis

Rapid methods:
Rapid methods in microbiology may be categorized into those used for detecting microorganisms
and their antigens or antibodies in specimens, and for identifying and determining antimicrobial
susceptibility of microorganisms isolated in cultures. Stained or unstained wet mount
preparations are especially useful for the detection of fungi and parasites. Stained fixed
preparations are especially useful for the detection of bacteria and mycobacteria. Fluorescent
microscopy provides definitive identification of a variety of microorganisms,
including Legionella pneumophila and herpesvirus in tissue and Haemophilus influenzae.
Neisseria meningitidis, and Streptococcus pneumoniae in cerebrospinal fluid. Counter-
immunoelectrophoresis (CIE), latex agglutination (LA), and enzyme-linked immunosorbent
assay (ELISA) are techniques which are useful for detecting microbial antigens in cerebrospinal
fluid, urine, and serum, particularly in cases of partially treated bacterial meningitis and in
cryptococcal meningitis.
Rapid methods for identifying microorganisms isolated in cultures include immunological
techniques (e.g. FA, CIE, LA), kits (e.g. Micro-ID) chromatography (for anaerobic and
mycobacteria) and automated devices (e.g. MS-2, AMS). while antimicrobial susceptibility may
be rapidly determined in selected instances by direct testing of specimens, specific tests for /(-
lactamase production, and automated devices (e.g. Autobac. MS-2, AMS). Rapid techniques for
drug assay include bioassay; however, greater sensitivity and specificity are achieved with
chromatography, radio-immuno- or -enzymatic assay, and enzyme immunoassay.
The requirements to validate a rapid method:
Method validation is the process used to confirm that an analytical procedure employed for a
specific test is reliable, reproducible and suitable for its intended purpose. All analytical methods
need to be validated prior to their introduction into routine use, and this is especially true for
novel technology platforms, such as rapid microbiological methods (RMMs). Because many
RMM technologies consist of a combination of instrumentation, software, consumables and
reagents, in addition to specific detection, quantitative or identification methodologies, it is
important to develop a comprehensive and holistic approach to the validation process to ensure
that the entire RMM system is suitable for its intended use. The following sections provide an
overview of how to design a meaningful validation program in order to effectively demonstrate
that the new RMM is equivalent to, or better than, the existing method you intend to replace.
Initial Activities

Prior to purchasing and validating a RMM, there are a number of due diligence activities that
should be undertaken. These may include a thorough understanding of the scientific needs of the
RMM, the technical benefits the RMM possesses as compared with the existing method,
regulatory impact, economic advantages, and the role of the RMM supplier in terms of providing
support during the initial assessment, validation exercises, and most importantly, after the system
has been placed in service for routine use.

From a scientific perspective, it is important to understand what technical capabilities are

required, including, but not limited to, method sensitivity and specificity (e.g., detection levels
and for what types of microorganisms), sample throughput, sample type, automation, data
handling and archiving, report management, if the system needs to meet 21 CFR Part 11
expectations, and the required degree of operator training.

Proof-of-concept or feasibility testing can also be performed to determine if incompatibilities

exist between the RMM and the intended product or test sample(s). These types of studies can
also be performed in the event the RMM supplier has little or no data on testing similar product
or test materials. This can be accomplished using a rental or loaner instrument, or by sending
samples directly to the supplier for evaluation. The data obtained from these initial studies will
help with the decision to purchase the RMM and proceed with formal validation activities.

The due diligence process also involves a review of existing regulatory commitments and
whether implementing the RMM will result in significant changes that will require a formal
submission. Additionally, a financial assessment of the costs (and cost savings) associated with
the purchase, validation and implementation of the RMM should be performed.

Finally, the selection of a RMM supplier is just as important as the RMM itself, and it is
important to have a thorough understanding of the supplier’s technical capabilities and their
ability to support each phase of the validation process as well as continuing assistance once the
RMM is placed into service.
In summary, the initial assessment of a RMM should include a comprehensive scientific,
regulatory and business due diligence review, in addition to matching the appropriate technology
with the desired microbiology application. It is not uncommon in our industry to find firms that
have purchased a RMM system and spent considerable time, resources, and expense in validating
the instrumentation and method only to find, at a later date, that the technology is incompatible
with the process and/or product being evaluated, or that the sensitivity and/or specificity of the
system is not what was originally anticipated. Therefore, careful planning and fact finding during
the due diligence phase is critical to a successful RMM validation and implementation program.

The Validation Strategy

In order to design a holistic approach to RMM validation, it is necessary to develop a

comprehensive strategy that includes qualifying not only the RMM instrumentation but the
analytical method as well. The validation plan can be comprised of a number of process steps
and these are outlined in the following sections.

Risk Assessment

Quality risk management (QRM) is an important part of science-based decision making which is
essential for the quality management of pharmaceutical manufacturing. The ICH Q9 guideline,
Quality Risk Management [1], defines QRM as a systematic process for the assessment, control,
communication and review of risk to the quality of drug product across the product lifecycle.
Similarly, the FDA Final Report for Pharmaceutical cGMPs for the 21st Century - A Risk-Based
Approach [2], states that using a scientific framework to find ways of mitigating risk while
facilitating continuous improvement and innovation in pharmaceutical manufacturing is a key
public health objective, and that a new risk-based pharmaceutical quality assessment system will
encourage the implementation of new technologies, including RMMs, to facilitate continuous
manufacturing improvements via implementation of an effective quality system.

A risk assessment should be performed prior to the start of any RMM validation activities.
Identified risks will vary depending on the RMM technology and the RMM supplier, the method
the RMM is intended to replace, the product or sample(s) for evaluation, whether the new
measurements are qualitative or quantitative and if the resulting data are significantly different
from the existing method, method variability, method robustness, pharmacopeial equivalence,
regulatory acceptance, and other attributes. Tools such as Failure Modes and Effects Analysis
(FMEA) or Hazard Analysis and Critical Control Points (HACCP) may be utilized in assessing
the potential risks when implementing the RMM.

Validation Master Plan (VMP)

A VMP should be followed which will provide the roadmap for all of the activities that will be
required to demonstrate that the RMM is validated and suitable for its intended use. The VMP
should include the overall project deliverables, the organizations or individuals that are
responsible for each phase of test execution, review and approval, and the documentation
required to satisfy the expectations of the validation strategy.

User Requirements Specifications (URS)

When choosing a RMM, the end-user must first establish the basic expectations that the system
must meet. For example, the system may have to detect and enumerate bacteria, fungi and
spores, have a sensitivity level of a single viable cell, process at least 80 samples within an 8-
hour shift, and show (at least) equivalent results to the current method. From here, the user can
develop specific requirements for the entire RMM system, including the equipment and the
analytical method, which will demonstrate that the system performs as expected. The document
that describes the functions and characteristics that the RMM system must be capable of
performing is called the URS. The requirements specified in the URS can also form the basis for
all of the validation testing requirements, test protocols and acceptance criteria.

Design Qualification (DQ)

Design Qualification (DQ) is documented verification that the proposed design of the equipment
or system is suitable for the intended purpose. Because most RMMs are commercial off-the-shelf
systems (COTS), DQ is accomplished by verifying that the supplier’s design specifications meet
the design requirements as specified in the URS. This activity can be completed prior to
purchasing the RMM system or can be incorporated into the formal VMP.

Supplier Assessment/Audit
The URS should include requirements that the RMM supplier should meet. An example is that
the supplier has an appropriate quality system for designing, manufacturing, testing and release
of equipment, software, reagents and consumables throughout the RMM life cycle. Other
requirements may include the provision of technical documentation, training, troubleshooting,
calibration and/or field service support. An assessment of whether the supplier can meet these
requirements should be conducted, either through a review of relevant documentation provided
by the supplier and/or an audit at the supplier’s manufacturing and design/development facilities.

Functional Design Specifications (FDS)

The FDS is the document that describes all of the functions and requirements for the RMM
system and what will be tested to ensure that the system performs as specified in the URS. The
FDS can be quite extensive, covering system functionality, configuration, input/outputs,
environment, utilities, architecture, interfaces, data and security. Additionally, the FDS will point
to specific test scripts where each requirement will be tested and verified against pre-established
acceptance criteria. These test scripts are normally contained within the Installation, Operational
and Performance Qualification protocols.

Requirements Traceability Matrix (RTM)

The RTM is a document that provides traceability that all the requirements listed in the FDS
have been verified and/or tested. Think of the RTM as a checklist of the validation process. The
document identifies the test script or protocol where a function or requirement will be tested,
such as the Installation, Operational and Performance Qualification (IQ, OQ and PQ,
respectively) protocols. The RTM also specifies which SOPs and other documentation that needs
to be in place in order to satisfy the criteria for meeting a specific function or requirement. The
RTM is a living document during the execution of the validation test scripts or protocols.

Training and SOPs

Training with the RMM supplier and the proper qualification of analysts are required for the
effective execution of the testing protocols and are critical to the success of the overall validation
plan. Training may be scheduled during initial proof-of-concept or feasibility testing, either in-
house or at the supplier’s facility. SOPs that facilitate the proper execution of the RMM
instrumentation, as well as those that are required to be in place as specified in the URS and FDS
should be written and approved prior to the execution of the validation plan.

The Test Plan

The Test Plan identifies the formal testing strategy, resources, roles and responsibilities, test
procedures, test deviation handling, and required deliverables for the validation program. This is
the document that provides very specific test scripts and protocols, and their associated
acceptance criteria, which will test and verify each function and requirement as specified in the
URS and FDS. The RTM is the checklist that documents that each of the functions and
requirements have been tested and/or verified. The manner in which the functions and
requirements will be tested are specified in the IQ, OQ and PQ protocols.

Installation Qualification (IQ)

The IQ establishes that the equipment is received as designed and specified, that it is properly
and safely installed with the correct utilities in the selected environment, and that the
environment is suitable for the operation and use of the equipment. Basically, the IQ verifies that
the equipment was received and meets the design specifications for the equipment that was
ordered. The IQ can be carried out by the RMM supplier or by the end-user.

Operational Qualification (OQ)

The OQ provides documented verification that the equipment, as installed in the selected
environment, performs effectively and reproducibly as intended throughout the anticipated or
representative operational ranges, defined limits and tolerances. During this phase of the
validation test plan, the end-user may confirm their previous proof-of-concept testing and/or the
RMM supplier’s supporting data. The OQ is also the focal point for the majority of the computer
system, software and security validation activities, as well as demonstrating that the
microbiological method is appropriate for its intended application. The latter is usually
accomplished using standardized microorganism cultures and test samples/product matrices,
while demonstrating that pharmacopoeia validation criteria are met.
Computer system validation encompasses both hardware and software functionality and security,
and demonstrates that these components of the RMM system operate accurately and reliably.
Depending on the complexity of the RMM technology and the end-user’s company policies,
CSV can be quite extensive.

Functional testing for the microbiological components of the RMM can be demonstrated by
following the validation recommendations provided by the Parenteral Drug Association (PDA),
the United States Pharmacopoeia (USP) and the European Pharmacopoeia (Ph. Eur.).

The PDA provided the first true guidance on RMMs, Technical Report #33, Evaluation,
Validation and Implementation of New Microbiological Testing Methods [3]. Published in 2000,
this document provides information on validation protocol design, testing and acceptance
criteria, and installation, operational, and performance qualification strategies. Shortly thereafter,
both the USP and the Ph. Eur. published informational chapters on the same subject.

USP informational chapter <1223>,Validation of Alternative Microbiological Methods [4],

provides guidance for the validation of methods for use as alternatives to official compendial
microbiological methods. The chapter incorporates the analytical concepts from USP
<1225>,Validation of Compendial Methods, and relates these to alternative quantitative and
qualitative microbiological systems. Although there is no guidance on qualifying new microbial
identification systems, the USP has recently published a separate draft informational chapter on
this topic.

Ph. Eur. chapter 5.1.6, Alternative Methods for Control of Microbiological Quality [5], describes
alternative methods for the control of microbiological quality. A discussion of qualitative,
quantitative and identification tests and guidance for using validation criteria are provided.

A test protocol is designed to evaluate the RMM against a variety of validation criteria that are
specified in the PDA, USP and Ph. Eur. guidance documents, and the resulting data is compared
against acceptance criteria specific for the validation criteria being assessed. Standardized
microorganism cultures are used, and the concentration, number of replicates and type of
microorganisms (e.g., bacteria, fungi, stressed, dead, mixed cultures, inoculated within a test
sample) will be dependent on each validation criteria protocol and their acceptance criteria
requirements. Additionally, many of the test protocols will require the use of an appropriate
statistical model to determine if the resulting data meets the acceptance criteria as specified.

Performance Qualification (PQ)

The PQ provides confirmation that the entire RMM system performs as it is intended to by using
actual product and/or test samples in order to demonstrate equivalence to the existing or
reference method. This may include running the RMM in parallel with the current method for a
specified period of time or number of batches or samples. Test samples should be identified,
when appropriate, that are expected to contain microorganisms and some samples that are not, in
order to test the suitability of the RMM. At least three independent tests using at least three
different lots/batches of the test sample should be assessed. The actual number of batches,
replicates, sample size and/or duration of testing will be defined as a function of the application.

A statistical analysis of the resulting data should be conducted in order to demonstrate

equivalency between the two methods. There is the possibility that the RMM may recover a
higher number of microorganisms as some technologies are not dependant on the growth of
microorganisms, and cells that are physiologically stressed and/or damaged may not be detected
on conventional, growth-based media.

Furthermore, the RMM may report the detection of viable microorganisms as a completely
different measurement than colony forming units (CFUs), such as fluorescent units, relative light
units or genetic copy number. In this case, a strategy should be developed for determining the
correlation of the new data with the existing method’s data, and whether there may be an impact
to existing acceptance levels or in-process/product specifications. This strategy should be
incorporated into the validation plan, and if necessary, within specific testing protocols.

Finally, when conducting equivalency testing using test samples that will not normally contain
microorganisms, as would be expected during sterility testing, other strategies for demonstrating
equivalency may need to be developed. For example, standardized cultures may be diluted to a
theoretical level of less than 1 viable cell. The resulting suspension is then inoculated into the
test material such that a proportion of the inoculated samples will contain a microorganism and
the remaining proportion will not. The two methods may be determined to be equivalent if the
proportion of positive to negative results is not statistically different.

PCR is a method for synthesising multiple copies of (amplifying) a specific piece of DNA. For
PCR to take place four basic components are required:

 A DNA template containing the target sequence that is to be amplified. For pathogen
detection this sequence must be highly specific to the organism concerned, often a single
gene, such as a virulence gene.
 Primers – a pair of short single-stranded DNA sections, which are exactly complementary
for specific parts of the target sequence.
 A heat-stable DNA-polymerase enzyme, usually Taq polymerase from a thermophilic
bacterium, which catalyses the reaction.
 Free nucleotides that are used as the building blocks for multiple copies of the DNA
template.

The first stage in the PCR process is to raise the temperature to about 90-95oC. This causes the
double stranded DNA to denature, or melt, into single strands.

The temperature is then reduced to about 50-65oC to allow the two primers to bind, or anneal, at
specific points on the single-stranded DNA of the target sequence.

Finally, the temperature is raised to 70-74oC and the DNA-polymerase enzyme catalyses the
duplication of the target sequence, starting at the annealed primers on each single strand, in a
process known as extension. This results in two double-stranded DNA fragments that are
identical copies of the original target sequence.

The temperature cycling process is then repeated a number of times, typically 30-40, creating a
theoretical doubling of the number of copies of the target sequence at each cycle. This gives an
exponential increase in target DNA concentration and produces sufficient DNA for reliable
detection from a single target sequence in a few hours.

PCR Kits for Food Microbiology

There are two broad types of detection method:

End-point PCR detection takes place, as the name suggests, when the amplification process is
complete. Typically, agarose gel electrophoresis, followed by staining with fluorescent ethidium
bromide is used to detect the amplified DNA fragments. This method is time consuming and not
sensitive enough to measure the accumulated DNA copies accurately, so can only give a
qualitative result.

Most food testing applications employ a method known as real-time PCR detection – combining
the amplification and detection stages of the process so that amplification is monitored
continuously during the exponential phase. Real-time detection is more accurate and the result
can also be quantified.

Real-time PCR detection methods The simplest method is to use intercalating fluorescent dyes,
such as SYBR Green. These fluoresce only when bound to double-stranded DNA and the
increase in fluorescence can be measured at each cycle. Unfortunately, these dyes bind to all
double-stranded DNA present, including any non-specific PCR products. This makes it difficult
to quantify the result accurately. The problem can be overcome to some extent, but only by
adding an extra stage at the end of the PCR process.

A second, more accurate and reliable method is to use fluorescent reporter probes. This method
utilises an additional primer, the probe, which also binds specifically to the target DNA sequence
during annealing. Probes have a fluorescent reporter dye at one end and a quencher dye, which
inhibits fluorescence, at the other. During the extension stage the probe is broken apart by the
DNA-polymerase and begins to fluoresce more strongly. The fluorescence emitted can be
measured at each cycle and increases in proportion to the number of target sequence copies
produced. To quantify the assay, the cycle at which the fluorescence intensity rises above the
background level is recorded for each test sample and for a set of standards run at the same time.
A standard curve can then be constructed. The amount of target DNA present in the sample can
be calculated from the standard curve. Since the probe only binds specifically to the target DNA,
non-specific PCR products are not detected.

Many of the commercially available PCR-based food microbiology applications use this type of
detection. Probe-based detection also allows for more than one target DNA sequence to be
assayed in the same sample, by using specific probes equipped with different coloured dyes. For
example, some Campylobacter detection kits allow for several species of the pathogen to be
detected and quantified in the same sample.

Commercial real-time PCR assays for food microbiology applications generally employ a high
degree of automation to minimise the number of operations involved and reduce the risk of
contamination. The reaction usually takes place inside a combined thermocycler/fluorescence
detection instrument and uses pre-prepared reagents, often in dried tablet form, and validated
controls. Typically, identical run conditions are used for all pathogens so that several assays can
be run simultaneously. The thermocycling and detection processes are controlled by dedicated
software that also calculates and interprets the results.

Typical Laboratory Work Flow A typical pathogen detection assay is performed as follows.
  Stage 1. Sample enrichment 25g of sample added to 225ml of enrichment broth and
incubated overnight. Enrichment times can be as little as 8 hours but 16-24 hours is more
normal.
 Stage 2. Sample preparation Collect enriched samples and lyse cells to extract target
DNA. Add extracted DNA to PCR tubes/wells containing PCR assay reagents. Time 1-3
hours
 Stage 3. DNA amplification and analysis Place samples in automated
thermocycler/analyser and start PCR. Dedicated software manages cycling and detection
and calculates results. Time 2-3 hours

Total time for assay normally 20-30 hours but can be as little as 12 hours depending on the
assay.

Benefits and Costs

The main advantage for PCR systems over other methods is in time saving, both in the total time
from sampling to result and in the technician time needed to set up and run the assay. For
example, a typical immunoassay-based pathogen detection method will often require a secondary
enrichment stage before low numbers of cells can be detected and can take up to 48 hours to
achieve a clear result. The high degree of automation built into PCR systems means that staff
time and training is effectively minimised.

PCR has also been shown to have a very high specificity and generally requires fewer repeat
tests than most other rapid methods, leading to further time saving. Many commercially
available kits have also achieved AFNOR and/or AOAC validation.

PCR systems are flexible, allowing for several pathogens to be assayed in a single run (multiplex
PCR). Real-time PCR assays can also be made quantitative without adding additional stages or
operations to the assay.

Costs Capital costs for automated PCR systems are relatively high compared to those for some
other rapid methods.Consumables costs are also high by comparison to rapid culture based
techniques. However, there are considerable cost benefits in terms of reduced technician time
and training. There is a clear cost benefit in rapid test results allowing faster HACCP verification
and positive release of finished food products. A recent study carried out at the German Federal
Institute for Risk Assessment (BfR) concluded that, taking all costs into account, real-time PCR
could be significantly less expensive than conventional ISO culture methods for the enumeration
of salmonellae in food samples.

Future developments Real time PCR systems have sufficient flexibility to allow the rapid
development of new assays for emerging pathogens and for specific spoilage organisms. The
facility to conduct tests for more than one gene in the same sample well is also likely to be
further exploited in the future. This has the potential to reduce costs by allowing more efficient
use of instrument capacity and reagents.
Starter Cultures
Starter cultures are those microorganisms that are used in the production of cultured dairy products
such as yogurt and cheese. The natural microflora of the milk is either inefficient, uncontrollable, and
unpredictable, or is destroyed altogether by the heat treatments given to the milk. A starter culture
can provide particular characteristics in a more controlled and predictable fermentation. The primary
function of lactic starters is the production of lactic acid from lactose. Other functions of starter
cultures may include the following:

 flavour, aroma, and alcohol production

 proteolytic and lipolytic activities
 inhibition of undesirable organisms

There are two groups of lactic starter cultures:

1. simple or defined: single strain, or more than one in which the number is known
2. mixed or compound: more than one strain each providing its own specific characteristics

Starter cultures may be categorized as mesophilic, for example:

 Lactococcus lactis subsp. cremoris

 L. delbrueckii subsp. lactis
 L. lactis subsp. lactis biovar diacetylactis
 Leuconostoc mesenteroides subsp. cremoris

or thermophilic:

 Streptococcus salivarius subsp. thermophilus (S.thermophilus)

 Lactobacillus delbrueckii subsp. bulgaricus
 L. delbrueckii subsp. lactis
 L. casei
 L. helveticus
 L. plantarum

Mixtures of mesophilic and thermophilic microorganisms can also be used as in the production of
some cheeses.