Download The Possibilities and limitations of nucleic acid amphfication

J. Med. Microbiol.
Vol. 46 (1W7), 188-194
1997 The Pathological Society of Great Britain and lrcland
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REVIEW ARTICLE
The Possibilities and limitations of nucleic acid
amphfication technology in diagnostic
microbiology
M. VANEECHOUTTE and J. VAN ELDERE*
Department of Clinical Chemistry, Microbiology and Immunology, Blok A, University Hospital, B 9000 Ghent
and *Laboratory of Microbiology, University Hospital St Rafael, B 3000 Leuven, Belgium
Nucleic acid amplification technology is examined from the critical viewpoint of a
clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely
recognised limitations of amplification technology include those of false-positive and
false-negative results, the difficulty of obtaining quantitative results, the problem of
using this technology for susceptibility testing, and the difficulty of detecting routinely
the wide range of possible pathogens contained in a clinical sample. On the positive side,
amplification technology brings welcome new possibilities for rapid detection of specific
pathogens in a sample, including viruses, slowly growing bacteria, fastidious o r
uncultivable bacteria, fungi and protozoa. Other possible applications include screening
normally sterile clinical samples for non-specific bacterial contamination and the use of
amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in
contrast to research and reference
facilities-routine
bacteriology laboratories will continue to rely on culture as the
preferred ‘amplification method’ for most diagnostic applications.
Introduction
This review presents a critical examination of the use
of amplification technology in microbiological diagnosis rather than a simple enumeration of its
possibilities. The technology is examined from the
viewpoint of a clinical microbiologist working in a
routine clinical laboratory to assess whether and where
amplification technology could replace existing techniques. At the same time this is an attempt to fuel a
discussion on the possibilities and limitations of DNA
technology in clinical microbiology, with a particular
einphasi s on diagnostic bacteriology.
In clinical microbiology, the possibility of detecting
the presence of organisms in clinical samples directly
is the most obvious and most thoroughly studied
application of enzymic amplification of nucleic acids
[ 1 , 21. For many microbiologists, this application holds
the promise that culture will no longer be necessary in
the future. Although, at first sight, amplification-based
specific detection is a very appealing approach in
efforts to enhance diagnostic abilities, several drawbacks limit its application in routine clinical diagnosis.
Received 16 July 1996; accepted 18 Aug. 1996.
Corresponding author: Dr M. Vaneechoutte.
It remains to be established whether, and to what
extent, nucleic acid amplification (i.e., enzymic
amplification of part(s) of the genome) will be able
to replace culture (i.e., biological amplification of the
complete genome). Other possible applications of
nucleic acid amplification, apart from species-specific
amplification for detection purposes, will also be
considered.
Limitations of nucleic acid amplification
technology for detection purposes
Practical limitations
Some technical problems of nucleic acid amplification
technology for detection purposes are recognised
widely.
Flilse-positive results. These were the most important
problem in a study in which seven laboratories were
asked to amplify Mycobacteriunz bovis strain BCG
from blinded spiked samples [3]. False-positive results
may be caused by the fact that DNA (and RNA)
amplification is highly sensitive and thus prone to
contamination. The most likely sources of contamination are other samples and products from previous
amplifications. Processing of negative samples along
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with each extraction batch can control for contamination by sample carryover, and a convenient solution to
sample carryover during extraction procedures might
be the application of automated nucleic acid extraction
techniques ( e g , GenePure 34 1; Applied Biosystems).
So far as contamination with amplification products
from previous amplifications is concerned, the need for
extensive precautions is already well-known [4, 51.
Further precautions can be taken by using uracil-Nglycosylase (UNG) to prevent contamination [4, 61.
However, both approaches to contamination control
should be employed, as illustrated by the fact that the
laboratory with the highest number of false-positive
results in the study of Noordhoek et al. [3] used UNG
decontamination. Although, theoretically, DNA stability
may be another possible source of false-positive results
as it may lead to amplification of DNA from dead
organi sms-e. g ., after successful antibiotic treatmentthis may be a minor problem in practice, as indicated
by a study which showed that the frequency of positive
results obtained in a PCR test declined rapidly after
appropriate therapy for M. tuberculosis [7]. Amplification of more labile RNA targets [8-101 does not suffer
from this problem, but requires RNAase-free reagents
and equipment to avoid false-negative results. Finally,
low specificity of the amplification reaction can also be
a source of false-positive results [3]. Therefore,
amplification specificity should be controlled by a
second amplification with internal primers [ 111 or by
hybridisation with a specific probe [8, 9, 11-13].
Generally, commercial systems provide sufficient
specificity controls.
False-rzegative results. These may result from an
intrinsically low sensitivity of the amplification reaction. Besides optimal amplification design and thorough optimisation of the amplification conditions,
several approaches to enhance intrinsic sensitivity are
possible, such as re-amplification (nested PCR) [ 1 11,
the use of repeated sequences as the target DNA ( e g ,
IS6110 of M. tuberculosis [3, 14]), and several
ingenious hybridisation techniques [9, 10, 12, 151.
Theoretically, amplification of mRNA or rRNA [8101-which
takes advantage of the fact that each
actively replicating organism has c. 2000 rRNA
molecules compared to only one or a few rRNA
cistrons [9]-should also lead to greater sensitivity.
Another cause of false negative results can be the
presence of polymerase inhibitors in the clinical
sample. Addition to each amplification reaction of an
artificial target-with an amplification product that can
be differentiated readily from that of the real target
[16, 171-can be used as a control. Extensive DNA
purification [18, 191 is required to remove inhibitors,
although this procedure may increase the risk of
contamination as it usually comprises multiple steps.
Numerous DNA extraction and purification procedures
have been published and several systems are now
available commercially. Comparative evaluation of
189
these DNA extraction and purification techniques
may be of more interest than comparison of the
amplification methods.
The maximum sample volume of 10-50 pl for
enzymic amplification-compared
to 0.5- 10 ml for
culture-is another important reason for false negative
results. For DNA amplification reactions that start
from a maximum sample volume of 50 p l (in a 100pl total reaction volume), it can be calculated that
20 DNA target moleculks (or organisms)/ml of sample
is the detection limit with ideal DNA extraction and
amplification conditions if no concentration of the
organisms or the DNA in the total sample has been
performed. For detection of pathogens in samples with
very low initial inocula-e.g., blood and food-the
achievable sensitivity of amplification technology is
inadequate and well below that of culture. Thus, semiautomated blood culture can detect one or a few
organisms/lO ml of blood-which
is often the concentration of bacteria present during septicaemia-and
culture can detect a few organisms in contaminated
food. Concentration can be achieved by centrifugation
or precipitation, but for many clinical samples this
means that DNA from the host ( e g , leukocyte DNA
from blood samples) will also be concentrated and
may inhibit the amplification reaction. Other concentration techniques have been described that use either
immunomagnetic procedures to separate target organisms and sequences [20-241 or filtration [25]. This
last method has been shown to detect 1 cfu of Listeria
mnnocytogenes in 300 ml of milk. RNA amplification
may be a possible solution for the problem of small
sample volume, as the multiple RNA molecules
originating from a single organism may seed the
complete sample after cell lysis, increasing the chance
that the selected sample contains an amplifiable target.
Despite these efforts, amplification of targets from
clinical samples in general may have rather limited
sensitivity, in contrast to that which is often reported
from research studies. For example, with M. tuberculosis many studies report a low sensitivity for
paucibacillary specimens (i.e., specimens with
<lo4 cfu/ml). Besides the small sample volume, the
known tendency for cell clumping [26] of this microorganism provides another explanation of false negative results or low sensitivity following DNA amplification [26]. Although efforts to enhance sensitivity
differ markedly from one report to another, the overall
levels of sensitivity reached tend to be in agreement.
For example, in two studies that amplified a repetitive
sequence from M. tuberculosis in sputum samples, a
PCR sensitivity of 100% was reported in one study for
29 culture-positive samples when extracting DNA by a
simple heating protocol, performing amplification by a
single PCR and visualising the product by ethidium
bromide staining [7], while the other study [13]
reported a PCR sensitivity of 75% for 24 culturepositive samples following DNA purification with a
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M . VANEECHOUTTE A N D J. VAN ELDERE
chaotropic agent and silica, a nested PCR and
hybridisation. Similarly, limited sensitivity was reported in a comparison of two commercially available
M. tuberculosis tests; indeed, smear-negative, culturepositive samples were detected poorly [27], although it
is specifically for this kind of specimen that
mycobacteriologists seek more sensitive and reliable
diagnosis [26].
Perhaps DNA amplification sensitivity per se-i.e.,
without confounding factors such as the presence of
amplification. inhibitors-can
best be evaluated by
studies such as that in which the performance of
‘home-made’ PCR tests for detection of M. tuberculosis was compared between different laboratories [3].
The laboratories were sent samples (water, saliva or
sputum) spiked with different inocula (0-lo7) of M.
hovis strain BCG. The IS6110 insertion element was
the amplification target for all laboratories. The data
showed that only two laboratories with no falsepositive results detected 80% of the samples containing 500 cells/ml of water (most published studies
report a sensitivity of 1-2 cells/25- 100 pl reaction,
or 10-40 cells/ml). Furthermore, a sensitivity only
10-fold higher than that of microscopy-i.e.,
5 X lo3 cells/ml of sputum-was achieved reproducibly by only one laboratory without false-positive
results. From these results-and
from the fact that
so many completely different techniques are reported
to yield good results-it is clear that more comparative studies are needed to enable a thorough comparison of the value of the different methodologies.
QuantlJication problems. A further problem of nucleic
acid amplification technology is the difficulty in
obtaining quantitative results. Quantification is often
important for diagnostic work, particularly for samples
where the mere presence of an organism is not an
indication of infection, as the organism can be present
in low numbers as a commensal, or for samples (e.g.,
urine) where the number of organisms present is
used as an indication of infection. Several possible
approaches for quantification, each with its own
problems, have been reviewed by Reischl and
Kochanowski [28] but, in summary, a reproducible
quantitative amplification assay is very difficult to
construct and remains laborious and cumbersome. The
commercially available 5 ’nuclease assay (TaqmanTM
LS-SOB PCR detection system; ABI Prism 7700
Sequence Detection System; Applied BioSystems)
[29] may be a valuable alternative. However, for
bacteriological diagnosis, the capital investment is
enormous compared to the cost of culture techniques
available currently.
The current situation
As indicated by the preceding summary of some of the
possible solutions to the different technical problems of
amplification technology, scientists have been very
inventive in building in amplification controls and
preventing contamination, in rendering amplification
semi-quantitative and in enhancing sensitivity and
specificity. However, these measures often involve
additional labour, costs and complications that make
the technology even less applicable for routine
diagnostic work. Automation of amplification technology therefore becomes necessary. Perhaps of most
importance is the fact that the sensitivity of nucleic
acid amplification technology in a routine clinical
laboratory situation remains to be established by
comparative studies. It is rather surprising that even
for M. tuberculosis-which is probably the bacterium
studied most intensively with regard to nucleic acid
amplification detection methods - the current situation
is that ‘reliable detection of M. tuberculosis is still
dependent on conventional laboratory methods until the
usefulness of PCR methods has been established’ [3,
301.
Fundamental limitations of ampllJication-based
detection
Even if the preceding limitations of DNA technology
are accepted as teething troubles of a new technology
which will be resolved sooner rather than later, there
are some theoretical constraints that limit the application of this technology in the clinical microbiology
laboratory, and especially in the field of bacteriology.
Susceptibility testing. This requirement poses a fundamental problem for the introduction of current
amplification-based detection methods into diagnostic
bacteriology. Information on the susceptibility of a
pathogen is often considered to be of more clinical
importance than its correct identification. There is a
basic problem in that amplification-based detection of
all pathogens, even if this was possible (see below),
would simply mean extra work unless it was also
possible to perform culture-free susceptibility testing.
However, for the following reasons, culture seems to be
essential for susceptibility testing. First, different
primer sets are needed to cover all possible resistance
mechanisms, as the encoding genes are scattered all
over the genome. Moreover, the genetic basis of
resistance to a single class of antibiotics may be very
diverse. Thus, p-lactam resistance can be a consequence of P-lactamase production, altered penicillinbinding proteins, porin alterations resulting in reduced
membrane permeability, or other mechanisms. Multiplex PCRs - in which up to three or four sets of
primers are used simultaneously-provide only a very
partial and tedious solution. On the other hand, the
Kirby-Bauer diffusion antibiogram-despite its many
shortcomings [3 11-enables rapid screening on a single
plate of genetically unrelated resistance mechanisms
towards antibiotics of different classes. Second, defective or constitutively suppressed resistance genes may
be amplified. Finally, the simultaneous demonstration
of the presence of a particular resistance mechanism
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NUCLEIC ACID AMPLIFICATION TECHNOLOGY
and pathogen in a clinical sample is not definite proof
of the presence of a resistant pathogen, as similar
resistance mechanisms can be found in commensals.
Thus, even genotypic susceptibility testing should start
ideally from isolated, cultured organisms. Even if a
DNA-based non-culture method could be developed for
the detection of all known resistance mechanisms, this
would allow detection of only the known (i.e.,
sequenced) mechanisms, while newly evolving resistance mechanisms-which are recognised readily on a
diffusion antibiogram by an experienced microbiologist-would escape attention. In general, it can be said
that nucleic acid amplification technology is very
useful for gaining an insight of molecular mechanisms
of resistance, and that it can be used by specialised
reference laboratories for large-scale monitoring of
certain resistance mechanisms, but that it has little
future in the daily work of a routine clinical
bacteriology laboratory.
Spec$c detection. This is the application of DNA
amplification thought currently to be of most use in
microbiological diagnosis. Species-specific primers are
used for amplification (or species-specific probes for
hybridisation). Thus the method is limited to detection
of a single species or genus. However, most clinical
samples may contain a wide range of possible
pathogens. Specific detection of all these pathogens
would require the use of different primer sets (and
probes), which would be highly impracticable. Again,
multiplex PCRs [32] or amplification with a single pair
of primers that yields products with different lengths
according to the species [33] are only partial solutions.
Furthermore, in contrast to virology and parasitology,
the current culture alternative used in routine bacteriology is relatively fast and less labour-intensive, as
most pathogens can be grown easily and quickly on a
limited number of growth media, leading in some
instances to an immediate preliminary identification. It
is also relevant that culture technology itself is
continuing to improve. Thus, for M. tuberculosis, the
recent major advances in reduced detection time and
enhanced sensitivity have been achieved by combining
non-radioactive detection methods with automated
liquid culture techniques (Bactec, Becton Dickinson;
BacT/Alert, Organon Teknika). The last few years have
also seen the development of semi-automated liquid
culture systems that permit identification and susceptibility testing of several pathogens in <24 h (VITEK,
bioMeri eux; Micr oScan, Baxter) .
19 1
gical agent(s) directly from clinical samples; (b)
identification of cultured organism(s) to species level;
(c) assessment of the antibiotic susceptibility of
isolated organisms; and-to a lesser extent and mainly
in hospital laboratories-(d)
differentiation between
strains of the same species in order to trace nosocomial
outbreaks (i.e., strain identification or typing).
Detection of micro-organisms
Specific detection. As discussed previously, although
amplification for specific detection purposes has
limited practical applicability for covering the whole
array of possible pathogens, it brings welcome new
possibilities for detection of viruses, slowly growing
bacteria (e.g., Mycobacterium spp.), fastidious or noncultivable bacterial pathogens (e.g., Borrelia,
Chlamydia, Ehrlichia, Mycoplasma, Tropheryma [34],
certain fungi [4] and protozoa [35]. These are all
organisms for which the current alternatives-culture
or serology-are
slower than DNA amplification
technology, but just as labour intensive, or for which
no alternatives are available. Consequently several
companies (GenProbe, Roche, Abbott, Organon Teknika, Becton Dickinson, Genetrak, Chiron, etc.) have
either marketed or are about to market amplificationbased kits for specific detection of some viruses (HIV,
HBV, HCV, HSV, etc.) and selected bacterial species
( M . tuberculosis, Chlamydia, etc.). Amplification for
specific detection purposes can also be applied
routinely in reference laboratories that process a large
number of samples while searching for only one or a
few species (e.g., screening for Salmonella or Listeria
spp. in food quality control or for M, tuberculosis in
clinical samples).
Universal detection. This application involves the
detection of any micro-organism in normally sterile
samples. The presence of highly conserved genomic
regions throughout the eubacterial kingdom [36] or
within some eukaryote groups (e.g., fungi [37]) enables
the design of ‘universal’ primers that enable amplification of all members of a particular group. Such primers
can be used to screen for the presence of ( e g ) bacteria
in normally sterile clinical samples such as blood,
cerebrospinal fluid and tissue samples. This approach
has been used to detect non-cultivable species [34].
However, limited sensitivity may be a problem (see
above).
Culture-based applications of ampllJication
Possible applications of amplification
technology in diagnostic microbiology
Given the limitations of nucleic acid-based technology,
it is worth considering how this versatile technology
could be applied usefully to the different tasks of a
clinical microbiology laboratory. The main potential
applications include: (a) detection of possible aetiolo-
Susceptibility testing. The limitations outlined previously mean that amplification-based susceptibility
testing may be applicable only for slowly growing
bacteria. Susceptibility testing of potential multipledrug-resistant isolates of M. tuberculosis is the most
obvious application [38, 391. DNA technology (hybridisation or amplification) may also indicate latent
resistance by detection of non-expressed resistance
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M. VANEECHOUTTE AND J. VAN ELDERE
genes (e.g., some mecA gene positive strains of
Staphylococcus aweus which are methicillin-sensitive
until challenged in vivo [40]). This is an example of
how culture-based susceptibility testing can sometimes
yield false-negative results that are resolved by DNA
technology. However, it should be noted that both of
these applications of amplification technology for
susceptibility testing still start from cultured organisms.
Identification. Since there still seems to be a need for
viable organisms in pure culture (see above), the many
problems of phenotypic identification could be solved
by the use of amplification-based techniques for
determining species identity of cultured organisms.
Several PCR-based DNA fingerprinting methods have
been claimed to produce species-specific fingerprints,
including rDNA RFLP analysis [41, 421, rDNA SSCP
analysis [43], tRNA inter-repeat length polymorphism
analysis [44] and rDNA spacer length polymorphism
analysis [45]. These PCR-based DNA fingerprinting
techniques have several advantages over the phenotypic
identification methods used currently. They may be
faster (automatable) and more reliable, they may enable
finer discrimination (allowing differentiation between
genomic species), and they may replace the different
biochemical techniques and identification schemes by a
single universally applicable approach. They also have
several advantages compared to approaches based on
specific amplification and hybridisation as: (a) they are
independent of sequence information so that new
species can be added to a databank as new patterns,
obviating the need to develop new primers or probes;
(b) they are universally applicable, which means that
all species can be amplified with the same set of
primers; and (c) they are fast, requiring only simple
DNA extraction, amplification, restriction and electrophoresis techniques, but no hybridisation. These
techniques are currently being evaluated and developed,
and fingerprint databanks have yet to be constructed.
All of these fingerprinting techniques are indirect
means for sequence polymorphism analysis. The
reason why the most direct and informative approach
of DNA sequencing-although strongly advocated by
some groups [8, 461-is not applicable currently as a
routine technique is, of course, the inherent high
workload. However, several improvements and novel
approaches are being developed. First, biochemical
sequencing, based on strand termination by dideoxynucleotide incorporation, has developed rapidly since
the availability of thermostable polymerases because
sufficient product for direct sequencing, together with
isolation of the target DNA, can be obtained without
the need of cloning and because starting from much
lower amounts of target (e.g., by means of cycle
sequencing) becomes possible. Analysis of the sequence reaction products has been automated by the
use of fluorescent DNA fragment electrophoresis
equipment (e.g., ABI 377 DNA sequencer, Applied
BioSystems; ALF Express DNA sequencer, Pharmacia
Biotech; ABI Prism 3 10 capillary electrophoresis
apparatus, Applied BioSystems), sometimes referred
to by the somewhat misleading term of ‘automated
sequencer’. This equipment has also been shown to be
useful for DNA fingerprint analysis [47, 481.
Other breakthroughs may come from unexpected
directions. The possibility of developing microchips
coated with an array of oligonucleotides of known
sequence at well-determined locations [49, 501 may
allow sequence determination of a target based on the
combination of the hybridisation events of the target
with different probes. Other applications of bioelectronics [5 I] include coupling oligonucleotides to a
fibreoptic biosensor [52] to enable rapid analysis of
hybridisation events. Developments in microscopy
might even allow a DNA sequence to be literally
‘read’ through direct recognition of the morphology of
the nucleotides [53]. As amplification usually yields a
homogeneous mixture of molecules, this approach
might not be too difficult. However, although these
developments might turn sequence determination into
a rapid technique that may supplant the indirect
sequence analysis techniques of DNA fingerprinting
and classical hybridisation, it should be emphasised
that the possible availability of these rapid sequencing
techniques does not alleviate the limitations of specific
DNA amplification technology outlined above.
Tvping. The techniques of arbitrarily-primed PCR or
RAPD [54, 551, rRNA spacer length polymorphism
analysis (‘PCR-ribotyping’) [56, 571, inter-repeat
spacer length polymorphism analysis [5 81, tandem
repeat number polymorphism analysis [59, 601 and
gene restriction analysis (PCR-RFLP) [6 I , 621 are
examples of the numerous DNA amplification-based
possibilities for differentiating between strains of a
single species [63]. The major advantage of these
simple and rapid PCR-based techniques is that they
bring genotypic epidemiological analysis within the
reach of most routine diagnostic laboratories, so that
typing becomes an effective tool for dealing with
nosocomial outbreaks. Currently, most phenotypic
typing methods (e.g., serotyping, phage typing, etc.)
and other genotypic typing methods (e.g., chromosomal
DNA restriction analysis with low frequency restriction
enzymes, ribotyping) are slow or are carried out in
reference laboratories because they are complicated or
need special reagents. As a consequence, too much
time is often lost for the typing results to be used
effectively in tracing the source of nosocomial outbreaks. Rapid sequencing techniques might also enable
strain differentiation and could eventually replace these
indirect means of sequence polymorphism analysis.
Conclusions
In summary, from the critical viewpoint of this review,
the application of amplification methods for species-
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specific detection is usefully limited to viruses, to
poorly or non-cultivable bacteria, or to reference
laboratories that concentrate on detecting a single
pathogen. This is because of the many technical
difficulties of this technology and the continuing need
for cultured bacteria to perform susceptibility testing,
and because the non-universality and lack of standardisation of amplification-based detection make it
impracticable for routine laboratories. Susceptibility
testing based on DNA technology is applicable for
large-scale studies of the epidemiology of particular
resistance mechanisms and for obtaining a molecular
insight into resistance mechanisms, but will probably
remain limited in the clinical bacteriology laboratory to
organisms for which culture is slow or impossible.
Therefore, it can be predicted that routine bacteriology
will also continue to rely on culture for detection of
most pathogens. Several culture-dependent, but broadly
applicable, nucleic acid-based amplification techniques
are being developed for discrimination of different
strains within a species and for differentiation between
species, although rapid sequence determination might
conceivably replace the indirect approaches of hybridisation and DNA fingerprinting in the future.
Thus, the overall conclusion of this review is that
provided culture is not unduly slow-as is the case for
most known pathogenic bacteria-it
remains the
preferred ‘nucleic acid amplification method’ for
diagnostic microbiology, in part because it yields a
complete organism and genome instead of one or a
few DNA fragments. Therefore, it seems unlikely that
the achievements of Watson, Crick, Mullis and
Faloona will replace those of Pasteur in our routine
bacteriology laboratories in the immediate future.
Rather, microbiologists will continue to take the best
of both approaches.
The authors thank Anne-Mie Vandamme for critical reading of the
manuscript.
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