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Biochemistry !!!! What the Hell is this!🙄

28/03/2016

Clinical Application of Molecular Biology
Molecular biological methods for the
detection and characterisation of
microorganisms have revolutionised
diagnostic microbiology and are now
part of routine specimen processing.
Polymerase chain reaction (PCR)
techniques have led the way into this
new era by allowing rapid detection of
microorganisms that were previously
difficult or impossible to detect by
traditional microbiological methods. In
addition to detection of fastidious
microorganisms, more rapid detection
by molecular methods is now possible
for pathogens of public health
importance. Molecular methods have
now progressed beyond identification to
detect antimicrobial resistance genes
and provide public health information
such as strain characterisation by
genotyping. Treatment of certain
microorganisms has been improved by
viral resistance detection and viral load
testing for the monitoring of responses
to antiviral therapies. With the advent of
multiplex PCR, real-time PCR and
improvements in efficiency through
automation, the costs of molecular
methods are decreasing such that the
role of molecular methods will further
increase. This review will focus on the
clinical utility of molecular methods
performed in the clinical microbiology
laboratory, illustrated with the many
examples of how they have changed
laboratory diagnosis and therefore the
management of infectious diseases.
Introduction
The advent of nucleic acid amplification
and detection has resulted in a change
from conventional laboratory methods
that rely on phenotypic expression of
antigens or biochemical products, to
molecular methods for the rapid
identification of a number of infectious
agents. Molecular methods have become
increasingly incorporated into the
clinical microbiology laboratory,
particularly for the detection and
characterisation of virus infections and
for the diagnosis of diseases due to
fastidious bacteria. The advantages of
rapid turn-around time and high
sensitivity and specificity are appealing
but must be matched by rigorous
validation and quality control.
Molecular detection has mostly come to
the clinical microbiology laboratory in
the form of PCR technology, initially
involving single round or nested
procedures with detection by gel
electrophoresis. However, with the
introduction of automation for the
various stages of DNA or RNA extraction,
amplification and product detection
together with real-time PCR, molecular
laboratories will continue to become
more efficient and cost-effective.
Microarray technology such as the DNA
chip will likely further increase the
utility of molecular detection in the
clinical microbiology laboratory.
This paper will provide an overview of
the clinical applications of molecular
methods for infectious diseases, these
have been summarised in Tables 1 and
2 . Applications include the discipline of
virology where it has been applied to
resistance testing, genotyping and viral
load quantification in addition to
routine viral detection. In the area of
bacteriology molecular methods have
been applied to resistance testing, the
detection of infection due to fastidious
bacteria, the more rapid detection of
serious bacterial infections compared to
conventional methods and the detection
of bacterial infection after antibiotics
have been administered. Advances into
the areas of parasitology and mycology
have also been made such as more rapid
diagnosis of fungal infection in
neutropenic patients. Other applications
such as the detection of biosecurity
agents, applications to epidemiology and
infection control together with the
potential pitfalls with molecular methods
are also discussed.
Table 1
Examples of molecular methods in use
for the diagnosis of infectious diseases
Table 2
Examples of uses for molecular methods
other than microorganism identification
in the clinical microbiology laboratory
Virology
The diagnosis of viral infections has
been hampered for many years due to
the cost, laboratory time and skilled
personnel required for the cell culture
systems used, together with the generally
low sensitivity and slow growth of many
viruses in artificial media. Serology is
often unhelpful in the early stages of
infection, specific antisera for the
serology tests can be difficult to obtain,
and the clinical detection of antibodies is
relatively insensitive for a number of
viruses. PCR technology has therefore
improved the detection of a number of
these viruses.
Herpes simplex virus (HSV) encephalitis
is a serious infection but diagnosis
previously required brain biopsy in
certain cases due to the low sensitivity of
cerebrospinal fluid (CSF) culture and
serology. PCR now allows the detection
of HSV DNA from CSF with 95%
sensitivity thus avoiding invasive brain
biopsy. Viral meningitis, commonly
caused by either enteroviruses or HSV, is
more reliably detected by PCR when
compared to culture and in a shorter
time (one versus up to five days). HSV
PCR can be multiplexed with other
pathogens responsible for meningitis.
The detection of blood borne virus
infection is also improved by both PCR
and non-PCR molecular methods. Active
hepatitis C virus (HCV) infections are
diagnosed by the presence of HCV RNA
since the detection of antibody to HCV
cannot distinguish between past and
present infection. In terms of
infectiousness only those with detectable
HCV RNA have a significant risk of
transmitting HCV by transfusion, organ
transplantation, needle-stick injury or
vertically to the child. Although
infection with the human
immunodeficiency virus (HIV) is
routinely diagnosed by serology, early
HIV infection can be detected by HIV
pro-viral DNA detection before HIV
antibodies are confirmed by Western
Blot serology. Vertical transmission of
HIV infection is also detected in the
infant using HIV pro-viral DNA
detection. The Australian Red Cross
Blood Service screens pooled samples
from all donations for HIV and HCV
using the Chiron Procleix HIV-1/HCV
transcription mediated amplification
assay, thus reducing the potentially
infectious window period from 22 and
66 days to 9 and 7 days respectively.
Intrauterine infection of the foetus with
cytomegalovirus (CMV), rubella, and
varicella zoster virus can be detected
by PCR testing of amniocentesis fluid.
Ge***al ulceration due to HSV, usually
due to HSV type 2 infection, is now
routinely detected by PCR in many
clinical microbiology laboratories due to
its increased sensitivity over viral
culture.
Molecular detection of respiratory viral
pathogens from both upper respiratory
specimens such as nasopharyngeal
aspirates or throat swabs and lower
respiratory specimens such as sputum or
bronchoalveolar lavage fluid is cost-
effective due to the prevention of
hospitalisation, decreasing unnecessary
testing and procedures, directing specific
therapy, and reducing unnecessary
antibiotic use. Large multiplex or
tandem PCR assays testing for all the
common respiratory viruses along with
fastidious bacterial causes of pneumonia
are now feasible providing a thorough
yet cost-effective alternative to
conventional detection methods.
Uncommon yet significant respiratory
viruses such as severe acute respiratory
syndrome (SARS) coronavirus (SARS-
CoV) and influenza A/H5N1 (avian
influenza) virus can also be incorporated
into these assays thus acting as an in-
built early detection system.
During the SARS epidemic due to the
SARS-CoV, PCR testing of respiratory
specimens for other respiratory viruses
was crucial to exclude a number of
suspected cases which fulfilled the case
definition for SARS. PCR detection was
most helpful due to the ability to rapidly
screen for many respiratory viruses.
Subsequently a specific SARS CoV PCR
has been developed for the early
detection of SARS-CoV infection with a
sensitivity of 50–87% early in the
disease. Serology for SARS-CoV is up
to 100% sensitive but of limited
diagnostic value early in the disease
when the risk of transmission is
greatest.
The recent avian influenza (H5N1)
outbreaks in South East Asia and beyond
have also illustrated the need for rapid
viral diagnosis. Molecular detection
methods were developed following the
1997 Hong Kong outbreak and have
the advantage of being rapid and able to
be performed in many clinical
microbiology laboratories. Specific
serology needs live virus for the
microneutralisation assay which is
currently classed as a Biosafety Level 4
organism in Australia. Likewise direct
immunofluorescence detection requires
influenza type A/H5-specific monoclonal
antibodies.
Viruses cause more infectious diarrhoea
worldwide than bacteria and other
pathogens. The diagnosis of viral
diarrhoeal disease has improved with
the development of PCR detection. The
method of choice for microbiological
diagnosis of rotavirus from stool samples
is PCR. Norovirus, a calicivirus formerly
known as Norwalk virus and responsible
for large outbreaks both in the
community and health care facilities,
can be diagnosed by electron
microscopy, enzyme immunoassay and
PCR but PCR is the most sensitive and
rapid method. PCR is also the most
sensitive method for the diagnosis of
astroviruses and enteric adenoviruses
(serotypes 40 and 41).
Treatment Monitoring
Monitoring viral DNA or RNA loads has
become the standard of care for several
chronic viral infections. Measurement of
viral load is performed either by
competitive PCR systems, branched chain
DNA signal amplification or more
recently real-time PCR.
HIV viral load testing is an integral
component of the management of HIV
infection. It is the major tool used to
monitor the success of antiretroviral
therapy and to detect the emergence of
viral resistance, evidenced by a rise in
the viral load despite ongoing therapy.
HIV viral loads also predict progression
of disease, and give prognostic
information. Commercial tests are
available and more recently
ultrasensitive tests such as the Cobas
Amplicor HIV- 1 Monitor Ultrasensitive
Test have been released reducing the
lower limit of viral detection to 50
copies/mL.
Viral load testing is also used for the
assessment and monitoring of responses
to therapy in chronic HCV and hepatitis
B virus (HBV) infection. HCV RNA viral
loads are assessed in patients with
genotype 1 HCV infections when
monitoring for responses to combination
interferon-alpha and ribavirin therapy.
Patients who remain negative for HCV
RNA 6 months after completing
combination therapy for HCV infection
almost always remain free of the virus in
the longer term and have achieved a
sustained virological response. If the
HCV genotype 1 RNA is undetectable
after 12 weeks of therapy there is a 75%
chance of a sustained virological
response. However, even if the HCV RNA
remains detectable, a 33% chance of a
sustained virological response remains if
a 100-fold decrease in the viral load has
occurred after 12 weeks of therapy. In
HBV carriers with active liver disease
HBV DNA loads are measured not only to
assess patients regarding the need for
either interferon-alpha or lamivudine (a
DNA polymerase inhibitor) antiviral
therapy but also to monitor their
effectiveness. An increase in HBV viral
load is also used as a marker of the
emergence of lamivudine resistant viral
mutants.
Cytomegalovirus infection is a serious
infection in bone marrow and solid
organ transplant recipients together with
HIV-infected patients but detection has
been limited by the poor sensitivity of
traditional culture methods. Viral load
testing by quantitative PCR is now the
accepted standard for monitoring the
emergence of CMV infection during
immunosuppression and allows pre-
emptive therapy prior to the emergence
of clinical disease with high sensitivity
when compared to culture.
Viral Genotyping and Resistance Testing
HIV genotyping for the detection of drug
resistance is the standard of care to
guide antiretroviral therapy and
complements viral load assessment.
Several databases are available such as
the Stanford reverse transcriptase and
protease database (http://
hivdb.stanford.edu ) where sequences
can be checked for resistance mutations.
Genotyping is also critical to the
management of chronic viral hepatitis.
There are six HCV genotypes
geographically distributed throughout
the world. The genotype is the single
strongest determinant for success with
combination therapy and all patients
wishing to undergo therapy firstly
undergo HCV genotyping. Those with
chronic genotype 2 or 3 HCV infections
receive 6 months of therapy with a 76%
chance of success compared to a 56%
chance of success for those with genotype
1 HCV infection receiving 12 months of
therapy.
Active chronic infections with HBV
treated with lamivudine require
surveillance for the emergence of
lamivudine resistant viral mutants.
During lamivudine monotherapy point
mutations at the active site of the
polymerase gene (YMDD variants) occur
with a frequency of 14–32% after one
year in phase III studies, and in 42%
and 52% of Asian patients after two and
three years of therapy respectively.
The emergence of lamivudine resistance
is detected by a rise in HBV viral load
and confirmed by sequencing of the
active site of the DNA polymerase
gene.
The presence of HBV pre-core mutants
may cause active liver disease despite
the absence of HBeAg, the common
marker for active hepatitis in hepatitis B
infection. This may be due to either a
premature stop codon point mutation in
the precore gene (G1896A) or a mutation
in the basal core promoter region down-
regulating HBeAg production, both of
which can only be reliably detected
genotypically.
Human papilloma virus (HPV) is now
accepted as the cause of almost all
cervical cancers and HPV genotypes are
now classified as either low or high-risk
for the causation of these cancers.
Screening for pre-neoplastic cytological
changes has traditionally been
performed by the Papanicaou (Pap)
screen, but the detection of high-risk
HPV infection is a useful adjunct. Since
HPV cannot be routinely cultured in
vitro, testing for the 15 high-risk
genotypes of HPV requires molecular
methods. Detection can be achieved by
signal amplification, such as the Digene
Hybrid Capture 2 assay which is the only
diagnostic in vitro test approved by the
Federal Drug Administration (FDA). This
assay contains specific RNA probes
directed toward the high-risk genotype
DNA sequences which are detected by an
antibody directed against the DNA-RNA
hybrids formed. Detection of the high-
risk genotypes can also be achieved by
target amplification such as multiplex
PCR, but commercial assays are not yet
available. Detection of these genotypes
by molecular analysis can help in the
assessment of equivocal Pap smears to
define those women at risk of developing
cervical cancer. Alternatively a
normal Pap smear with a negative
genetic test for the high-risk genotypes
may indicate a longer period of time
before re-testing. With the advent of a
genotype 16 HPV vaccine the role of this
testing is likely to assume more
importance.
Molecular methods have therefore gone
beyond simple detection of viral
infections to become an integral
component of the management of blood
borne virus and other viral infections.
Bacteriology
Fastidious Bacteria
Together with virology, the diagnosis of
infections due to fastidious bacteria has
benefited greatly from molecular
detection. Many of these fastidious
bacteria have public health implications
such as Mycobacterium tuberculosis ,
Chlamydia trachomatis, Neisseria
gonorrheae and Bordetella pertussis.
Non-culture-based molecular testing has
the advantage of avoiding the delays of
days to weeks for conventional culture
to allow early recognition and treatment
as a public health imperative.
Commercial assays are available for M.
tuberculosis and Mycobacterium avium
complex, C. trachomatosis, and N.
gonorrhoeae. Several nucleic acid
detection technologies are in use
including PCR, transcription based
amplification, ligase chain reaction,
strand displacement amplification and
the Qβ replicase system.
The introduction of molecular detection
for the fastidious sexually transmitted
bacteria has led to a large increase in
the proportion of laboratory confirmed
cases due to its increased sensitivity
allowing more effective contact tracing.
In the management of sexual health
traditional screening methods require
speculum examination in women and
urethral swabs in men. These require
special equipment and cause
embarrassment and discomfort, thus
reducing compliance. Molecular
detection is useful since noninvasive
specimens unsuitable for traditional
culture, such as initial stream urine and
self-collected vaginal swabs can be used.
These are more convenient and
acceptable increasing the compliance
with testing. Although molecular testing
for C. trachomatis and N. gonorrhoeae
does not allow monitoring of antibiotic
resistance or detect other sexually
transmitted diseases, urine testing has
shown equivalent sensitivity and
specificity to invasive specimens for
detection of C. trachomatis in men and
women, and for detection of N.
gonorrhoeae in men when compared to
urethral swabs. In women the sensitivity
and specificity of the PCR assay for N.
gonorrhoeae was lower for urine
compared to cervical samples, however
self-collected vaginal swabs may help in
this regard. The PCR assay for C.
trachomatis has equal sensitivity for
vaginal and cervical swabs and a
transcription mediated amplification
assay has been approved by the U.S. FDA
for testing C. trachomatis and N.
gonorrhoeae from vaginal specimens.
In remote areas, molecular methods have
the advantage of being performed on
dry swabs with little degradation of the
DNA during transit compared to the
difficulties of transporting samples in
specialised transport medium to
preserve viability. In addition, molecular
methods can test for multiple ge***al
pathogens such as C. trachomatis, N.
gonorrhoeae, the Donovanosis agent and
the ge***al mycoplasmata from the same
swab.
Mycobacteriology has been aided by the
introduction of molecular methods.
However, it is important to note that
molecular detection of M. tuberculosis is
one of the few examples where
conventional culture remains more
sensitive. This is possibly due to the
difficulty in releasing the DNA from the
bacterial cells during the extraction
process. Despite this limitation,
molecular detection of M. tuberculosis
has a definite role as it allows
confirmation of acid-fast bacilli seen on
microscopy with up to 98% sensitivity in
pulmonary tuberculosis within a day
compared to two weeks or more by
culture. Specimens that are smear-
negative have a much lower chance of
molecular confirmation, with reported
sensitivities as low as 40%. In
addition to direct detection from clinical
specimens, molecular methods can
confirm a positive culture within a day
compared to approximately four weeks
using phenotypic methods. This has
shortened the time for laboratory
confirmation of suspected tuberculosis
even for smear-negative but culture-
positive cases.
Mycobacteriology has also advanced
through the use of molecular methods for
the speciation of the many
nontuberculous mycobacterial species.
Phenotypic methods are slow and the
limited number of tests available is
inadequate to differentiate between the
large number of species. Genetic
sequencing of the 16S rRNA gene has
simplified this process in many
laboratories. However, some species
such as the rapid grower group cannot
be distinguished by 16S rRNA gene
sequencing alone, and require a multi-
gene approach incorporating the hsp65 ,
rpoB and sod genes.
Due to its significantly enhanced
sensitivity, PCR has replaced direct
fluorescent-antibody and culture as the
“gold standard” method for detection of
B. pertussis early in the disease
process. In one pertussis school
outbreak using nasopharyngeal
aspirates, PCR detected 48% of clinical
cases compared to 5% confirmed by
culture. For this pathogen of public
health significance, a combination of
PCR detection early in disease and
serology for suspected cases late in the
disease process is used for maximal case
ascertainment. Other fastidious
respiratory pathogens that can be
rapidly diagnosed by molecular means
include Legionella spp., Mycoplasma
pneumoniae and Chlamydia pneumoniae .
Some bacteria can only be detected by
molecular means as culture is either
extremely difficult or impossible for the
routine microbiology laboratory, or
represents a significant occupational
risk to the laboratory personnel.
Whipple’s disease is a rare but
ultimately lethal infection due to
Tropheryma whipplei which could
previously only be diagnosed by
characteristic histopathology and
electron microscopy, often from post-
mortem material. PCR now allows
diagnosis of neuro-Whipple’s disease
and endocarditis by the detection of T.
whipplei from noninvasive specimens.
Other examples where molecular
diagnosis can help in the diagnosis of
difficult or uncultivable bacteria include
cat scratch disease due to Bartonella
henselae , Q fever due to Coxiella burnetii ,
and male urethritis due to Mycoplasma
ge***alium. A more detailed discussion
on the molecular methods for the
diagnosis of fastidious bacteria can be
found in Fenollar and Raoult.
Rapid Bacterial Diagnosis
Meningococcal disease can have
devastating consequences and requires
early diagnosis for correct antibiotic
therapy as well as early provision of
chemoprophylaxis for close contacts. PCR
methods can now provide same-day
detection from sterile site specimens
with a superior speed and sensitivity to
culture, and when combined with
culture and other laboratory methods,
PCR maximises the laboratory
confirmation of clinically suspected
cases. Genoserogrouping for serogroup
B and C N. meningitidis strains helps
with decisions regarding vaccination
and can be combined with N.
meningitidis detection. In our laboratory
as well as others, combined N.
meningitidis detection and
genoserogrouping is routinely
performed on clinical specimens from
suspected cases.
Rapid detection of the other common
bacterial causes of meningitis has also
been developed. N. meningitidis with
Streptococcus pneumoniae and
Haemophilus influenzae type B account
for 90% of cases of bacterial meningitis
and multiplex PCR methods have been
developed for their detection.
Antibiotic Resistance
Following on from the success of
molecular methods for the detection of
several bacterial infections, genotypic
detection of antibiotic resistance is
appealing due to the avoidance of
problems such as variable phenotypic
resistance expression. Applying rapid
and reliable genotypic detection to
bacteria with infection control
implications such as methicillin-resistant
Staphylococcus aureus (MRSA) and
vancomycin-resistant enterococci (VRE)
is of great potential benefit. The
discrimination of MRSA from other S.
aureus is confirmed by the detection of
the mecA gene responsible for this
resistance. The detection of the mecA
gene can be multiplexed with the nuc
gene to allow rapid molecular detection
of S. aureus and confirmation of MRSA
from positive blood culture bottles.
This is important to provide information
regarding antibiotic selection as early as
possible since mortality rates are higher
with MRSA infection compared to
methicillin-sensitive S. aureus .
Likewise, detection of VRE is more
sensitive and rapid using DNA-based
amplification techniques. Commercial
kits are now becoming available for
MRSA and VRE detection using real-time
PCR instrumentation which will further
improve the speed of detection.
Extended spectrum β-lactamases (ESBL)
are found in Escherichia coli and
Klebsiella pneumoniae and are readily
transmitted on plasmids and
transposons. ESBL-containing bacteria
can spread rapidly in health care
facilities to cause wound infections,
urinary tract infections and septicaemia.
Their detection requires special
laboratory tests since routine antibiotic
susceptibility testing may not detect
strains carrying the resistance gene.
Although most clinical microbiology
laboratories currently use phenotypic
methods to detect ESBL, molecular
detection of these point mutations at the
active site of the β-lactamase gene can
confirm the ESBL and allow the
characterisation of the type of ESBL for
monitoring of its spread through health
care facilities around the world.
Multi-drug resistant tuberculosis
(defined as the presence of both
rifampicin and isoniazid resistance) is a
serious problem in many parts of the
world such as Eastern Europe.
Traditional methods for detecting
rifampicin and isoniazid resistance
require additional incubation from
culture, delaying the diagnosis and
increasing the risk of transmission of
resistant disease in the community. A
multiplex PCR for the sequencing of rpo B
and hsp 65 gene targets can facilitate
same day detection of the majority of
multi-drug resistant strains but
reliable resistance testing will require
multi-gene and whole of gene
sequencing better suited to
microarray technology.
Mycology and Parasitology
Although not as frequently applied to
eukaryotic infections, in a number of
clinical circumstances molecular testing
can be helpful. Pneumocystis jiroveci (a
fungus previously called Pneumocystis
carinii) can cause a severe pneumonia in
HIV-infected patients and other
immunosuppressed patients but
detection is limited to microscopy of
respiratory tract specimens. Microscopy
for the detection of P. jiroveci usually
involves methenamine silver staining of
tissue specimens or calcofluor white
staining of induced sputum or
bronchoalveolar lavage specimens.
Immunofluorescence is more sensitive
than these stains but is more expensive
and needs specialised facilities.
Sensitivity remains an issue, however,
especially in HIV-non-infected patients
such that the more sensitive PCR can be
very useful. The specificity of PCR is
limited, however, as this organism is a
ubiquitous commensal and can be
detected by PCR in the absence of
pneumonia. Another mycological
example is the use of 18S rRNA gene PCR
to detect Aspergillus spp. infection in
neutropenic haematology patients. This
disease is notoriously difficult to
diagnose due to the poor sensitivity of
culture early in disease and the
difficulty in obtaining histopathological
specimens in those with reduced platelet
counts. Early treatment is essential for
the best outcomes resulting in empiric
use of costly and toxic antifungal
therapy. When performed frequently
Aspergillus PCR can reduce the time
required for a specific diagnosis,
however, its exact role to improve the
management and therefore the outcome
of this devastating disease is still unclear.
Parasitological diagnosis is aided by
molecular methods since most parasites
are not cultured in routine laboratory
settings and therefore diagnosis relies
mostly on the relatively less sensitive
microscopy or serology. Toxoplasma
gondii can be detected by PCR from
amniocentesis fluid to confirm foetal
infection and from CSF to diagnose
toxoplasma encephalitis. Microscopy
remains the mainstay of malaria
diagnosis but Plasmodium spp. PCR,
because of its superior sensitivity
compared to microscopy, can diagnose
malaria in those with negative thick and
thin blood films due to administration of
chemoprophylaxis or partial immunity.
Plasmodium species PCR can also detect
mixed infections that can be difficult to
discern microscopically.
Broad-range PCR
Unlike specific PCR testing where a
particular organism is being sought, the
use of broad-range PCR for the diagnosis
of infectious diseases is more of a fishing
expedition. Primers complementary to a
conserved region of a gene are used,
such as the 16S rRNA bacterial gene or
the 18S rRNA gene of fungi. Any
amplified product is usually sequenced
and compared to more than 9,000
sequences from different organisms in
Internet databases. There are several
comprehensive databases such as
GenBank (www.ncbi.nlm.nih.gov/
Genbank ), EMBL Data Library
(www.ebi.ac.uk/embl ), and the DNA Data
Bank of Japan (www.ddbj.nig.ac.jp ) with
daily data exchange between them, and
more specialised high quality databases
such as RIDOM (www.ridom-rdna.de/ )
for bacterial rDNA sequences used for
mycobacterial speciation. Broad-range
PCR using 16S rRNA sequences is
appealing as it can, in theory, detect
bacteria in any sterile site specimen such
as blood or cerebrospinal fluid, in other
words a “molecular petri dish.” In fact
this method was used to identify B.
henselae in bacillary angiomatosis and T.
whipplei as the bacterium associated with
Whipple’s disease. A good example of
its potential use in diagnostic medicine
is for the aetiological diagnosis of
infective endocarditis. Antibiotic
regimens for the therapy of this serious
disease rely on the identification of the
microbiological aetiology which can be
problematic when conventional blood
cultures are negative due to the prior
administration of antibiotics. Broad-
range PCR can be performed on the
excised heart valves and vegetations or
peripheral blood to reveal a diagnosis
that would otherwise be missed. Broad-
range PCR has also been applied to the
diagnosis of bacterial meningitis.
More recently a spectacular use of
broad-range PCR was the identification
of the novel virological cause of SARS.
Broad-based primers were used to detect
unknown viruses in specimens from
SARS clinical cases. The sequences
showed homology to the coronavirus
genus, supported by other laboratory
results that resulted in a specific SARS
CoV PCR within weeks of the first report
of the disease.
A major drawback of broad-range PCR is
the risk of amplifying DNA that may be
contaminating the specimen or the PCR
reagents themselves, especially the Taq
DNA polymerases, resulting in false
positive results. Also the accuracy of
the data available through public
databases is difficult to assess and is
dependent on the quality of the
sequences deposited, a critical factor
when comparing an unknown sequence.
It is possible that the matching sequence
is either inaccurate or is shared by
another organism for which data is not
currently available.
Public Health Aspects
Since rapid and reliable aetiological
diagnosis underpins the effective
management of contagious diseases,
molecular diagnostics have an important
role. The outbreak of SARS CoV
illustrated the importance of ruling out
other respiratory viruses such as
influenza to facilitate the early
identification and quarantine of
suspected cases of SARS. This proved
effective in controlling the outbreak
even though a specific diagnostic test
was not available during most of the
outbreak. Now several PCR-based
diagnostic kits are available and we will
be much better equipped for early
virological diagnosis should the SARS
CoV re-emerge. Diarrhoeal viruses such
as noroviruses, which spread rapidly
through health care facilities and
residential care facilities, can now be
rapidly diagnosed to facilitate case
isolation. The infectiousness of those
with blood borne viruses is also
determined predominantly by molecular
testing. The ability of health care
workers who have been infected with
hepatitis B and C to perform exposure-
prone procedures such as surgery is
determined by PCR testing such that
workers with detectable hepatitis B DNA
or hepatitis C RNA may be restricted
from performing such procedures.
The management of bacterial infections
of public health significance is also
improved by molecular methods. Early
diagnosis of B. pertussis , M. tuberculosis ,
N. meningitidis is important for the early
prevention of transmission, an aim that
is best achieved by a combination of
conventional and molecular testing.
The advent of molecular epidemiology,
which allows the tracking of pathogens
based on genotyping of the involved
strains from outbreaks, has
revolutionised how outbreaks are
investigated and managed. The problem
with strain differentiation using
phenotypic methods in bacterial
outbreaks, such as meningococcal
disease, is the variable expression of the
phenotypic markers. Other methods like
multilocus enzyme electrophoresis are
very labour-intensive and unable to be
performed in many laboratories.
Multilocus sequence typing (MLST)
avoids these problems since every strain
can be typed unambiguously. MLST
involves the comparison of nucleotide
sequences from internal fragments of a
number of housekeeping genes.
Sequences obtained are submitted to
websites such as http://mlst.zoo.ox.ac.uk/
for N. meningitidis to give an allelic
profile which can be compared to
existing clones from anywhere in the
world. This system avoids the problems
of interlaboratory interpretation
encountered with other genotyping
methods yet can be used to investigate
local outbreaks even in some cases
where a viable culture is not obtained.
This has been applied to N. meningitidis
and S. pneumoniae which has helped
map the spread of virulent clones around
the world. Similarly genotyping of M.
tuberculosis using different methods
such as mycobacterial interspersed
repetitive unit genotyping is
recommended for the evaluation of
community and health care facility
tuberculosis outbreaks.
In the future it is likely that point-of-
care molecular tests will be available
allowing reliable results with rapid
turnaround time in the field.
Biosecurity
Biological warfare agents such as
Bacillus anthracis , variola major virus
(smallpox), Clostridium botulinum and
Yersinia pestis (plague) are problematic
since they may be invisible, may cause
no ill-effect for several days, and are
communicable with very small amounts
affecting many people. For example, 10g
of anthrax spores could kill as many
people as a ton of the nerve agent
sarin. The rapidity at which such an
incident could escalate mandates rapid,
reliable and sensitive detection methods.
Real-time PCR methods fulfil these
criteria best as conventional methods
either lack discriminatory power, are
slow, or require highly trained
personnel. However, PCR systems
require the release of DNA from spores,
which can be difficult to achieve without
inhibiting the PCR process. Spore
disruption and PCR can now be achieved
in 15 minutes using newly developed
hardware. Battery-powered portable
machines using TaqMan real-time PCR
are being developed with processing
times of only 30 minutes.
In addition microarray technologies
have a great deal of potential in this area
but are restricted by the sample
pretreatment required for such
microfluidic devices. These problems are
likely to be overcome as new
technologies are developed.
Limitations of Molecular Methods
Despite significant advantages of
molecular diagnostics it cannot yet
replace conventional methods for a
range of infectious diseases since many
common tests performed in the clinical
microbiology laboratory are rapid and
inexpensive. Advances in conventional
technologies have resulted in many rapid
antigen tests requiring only minutes for
results and the modern automated
culture systems allow relatively rapid
identification and susceptibility testing.
Unlike bacterial culture, which can
detect a large number of cultivable
bacteria without initially knowing the
specific organism responsible, all PCR
tests except broad-range PCR can only
detect the organism whose DNA is
complementary to the primers used.
Therefore to cover a similar breadth of
possible organisms would require the
introduction of inexpensive and simple
microarray technologies that are not
yet available.
False Positive and False Negative Results
Another problem restricting the
application of molecular techniques to
routine diagnosis is that of false positive
and false negative results. To avoid false
positive results due to laboratory
contamination relatively large
laboratory areas are required for
physical separation of reagent
preparation, specimen preparation and
product detection areas together with a
high level of staff training and skill.
Amplicon laboratory contamination can
be reduced by ultraviolet light
irradiation of reagents and chemical
inactivation of surface contamination
with sodium hypochlorite. Amplicons can
be destroyed by the use of dUTP to
replace dTTP for amplification then
uracil N -glycosylase (UNG) treatment of
preassembled starting reactions to
destroy the dUTP-containing amplicons.
Intersample contamination can be
reduced by the use of disposable
equipment and cotton filter tips, and
using disposable personal protective
equipment such as caps, gowns and
gloves. Even with scrupulous technique
problems can be encountered, especially
with broad-range PCR due to the
presence of foreign DNA in the PCR
reagents. It is therefore crucial that
appropriate negative controls are
included in every PCR run to detect any
contamination. The advent of real-time
PCR has reduced this risk due to single
tube PCR reaction and detection systems.
Poor primer design can also lead to
erroneously positive results. Primers
may be poorly designed such that
incidental amplification of
microorganisms other than those sought
occurs. Also primers are designed based
on the known sequences available
through international databases but
organisms or sequences yet to be
discovered can subsequently reduce the
specificity of the PCR.
False negative results may also be a
problem. Some organisms such as
mycobacteria are difficult to extract DNA
from, reducing the sensitivity of the
PCR. Substances in some clinical
specimens such as sputum and faeces
can degrade the DNA and RNA and other
specimens may contain substances such
as polysaccharides, haem and
therapeutic drugs that inhibit the PCR
enzymes. It is therefore important to
include inhibitor checks for each
specimen to ensure a negative PCR
reaction is not actually an inhibited
reaction. This can be done by
incorporation of an internal
amplification control to check for both
inhibitors and successful DNA extraction.
This can be achieved by the addition of
non-human pathogen DNA, for example
equine herpesvirus as used in our
laboratory, to the extraction buffer and
its subsequent detection from the
extracted sample by PCR using
complementary primers. The amount
of spiked DNA is titrated to be as
sensitive as possible yet allow regular
detection in non-inhibitory specimens.
Alternatively the human β-globin gene
can be detected by PCR following sample
extraction without the need for spiking
the reagents with foreign DNA. A
problem with this method is that the
amount of human DNA in each specimen
cannot be controlled resulting in an
inhibitor check of varying sensitivity.
The addition of non-human pathogen
RNA, such as bacteriophage RNA, can be
used as an inhibitor and extraction
control for reverse transcription PCR. If
inhibitors are detected, they may be
overcome by dilution of the DNA extract
or treatment of the DNA extract with
products such as GeneReleaser before
PCR, or by including PCR facilitators such
as bovine serum albumin in the PCR
step. If such manoeuvres fail to
overcome the inhibition the sample must
be reported as inhibitory and a repeat
sample requested.
Lack of Uniformity in Molecular Testing
Molecular diagnosis is also complicated
by the vast array of in-house PCR tests
used in different laboratories.
Commercial tests are available for a
number of common and important
infectious diseases such as HIV, hepatitis
C and B, C. trachomatis, and N.
gonorrhoeae but many infectious
diseases are unlikely to have a
commercial PCR test developed due to
their rarity. Differences in primer
selection (different genes or different
sequences within genes), amplification
format such as single round, nested or
real-time PCR or one of the other nucleic
amplification methods, and product
detection methods such as ethidium
bromide gel electrophoresis, DNA probes
or sequencing make comparisons for
sensitivity and specificity difficult.
Differentiation between Infection and Disease
Since the presence of nucleic acid does
not necessarily mean the presence of
viable organisms a problem with
interpretation of PCR results can emerge
that does not occur with culture. For
some infections such as invasive
meningococcal disease the presence of
meningococcal DNA from a sterile site
has a very high positive predictive value.
However, the detection of P. jiroveci in
suspected P*P may have only a 50%
positive predictive value in
immunosuppressed patients since it may
colonise as well as cause disease.
Likewise herpes viruses such as Epstein-
Barr virus (EBV), CMV and HSV are
intermittently shed following primary
infection without causing disease. Less
sensitive detection methods such as
culture have a higher specificity but
quantitative PCR may be helpful in this
regard since higher viral loads are
usually more specific for disease.
Quantification in viral infections such as
HIV, CMV, EBV, HBV and HCV is well
established for assessing disease severity
or monitoring response to treatment.
In CMV disease viral load testing can
monitor either increases of viral loads to
threshold levels or rates of viral load
increase to improve the positive
predictive value for clinical CMV disease.
Another approach is to detect RNA
species that are usually degraded within
minutes of cell death to indicate
pathogen viability and replication.
Future Directions of Molecular Technology
PCR coupled with sequencing has become
a powerful tool for the identification of
previously unknown pathogens and the
epidemiological investigation of new and
emerging infectious diseases. Molecular
methods have helped reveal that over 30
species of bacteria can form uncultivable
forms under unfavourable
environmental conditions. This now
means that Koch’s postulates cannot be
applied to investigate the validity of
certain microorganisms in the causation
of disease, such as T. whipplei as the
cause of Whipple’s disease. Molecular
technology has gone beyond the simple
identification of causative organisms for
infectious diseases and either now or in
the near future will be pivotal to the
study of the evolution of pathogens, the
maintenance of infective cycles in
nature, the investigation of causes and
mechanisms of new pathogens, the
mechanisms of susceptibility of different
host groups and the development of DNA
and RNA banking of genes encoding
pathogenic factors. This will be achieved
with new molecular methods such as
microarray, microchips, in situ PCR and
automation of molecular procedures.
Microarray and gene chip assays, first
published in 1991, have the advantages
of miniaturisation and automated
construction using industrial robots
together with sensitivity and rapid
reading of large amounts of detailed
genetic information. In fact up to 10
different probes per cm can be
attached to specific sites on the
microarray platform of either nylon
membrane or glass slide. Microarrays
can identify simultaneously a range of
pathogens for particular diseases such as
infective diarrhoea, pneumonia or
meningitis as well as genetic markers of
virulence and antibiotic susceptibility.
One of the first applications of this
technology was in the field of HIV, in
which an array was used to detect
protease gene resistance. Microarrays
can be used for complete genome
sequencing, an example being the
development of an array to sequence the
SARS virus following the 2003 outbreak.
In addition their role in the detection
and characterisation of biosecurity
agents is rapidly progressing, an
example being the sequencing of
hundreds of different variola major
strains by the U.S. Centers for Disease
Control and Prevention. However, its use
at this stage is mainly research-based
and is currently very expensive. Issues
of reproducibility must be addressed as
the technology is highly sensitive and
processing conditions must be
standardised and followed rigidly. Also,
because multiple data points are
generated from each array, computer
algorithms are needed to analyse the
data. Commercially, initial efforts
focussed on applications of microarrays
for use in detecting drug resistance and
mycobacterial identification but the
biotechnology companies are now
assessing the market for molecular
diagnosis using microarrays in the
infectious diseases laboratories. For
example, Affymetrix produce
GeneChip microarrays for pathogen
identification, virulence factor
identification, pathogen response to
drugs, and vaccine development.
However, these companies will need to
compete with the multiplex real-time PCR
kits becoming commercially available
such as those produced by Prodesse for
the detection of the common respiratory
pathogens.
Economic pressures will force the
development of more automated and less
expensive test procedures similar to
those in clinical chemistry laboratories.
Nucleic acid extraction and purification
and the manual loading of the isolated
nucleic acids and master mixes into the
PCR reaction vessels remain the most
labour-intensive parts of molecular
technology. However, new technology
has been developed to perform these
tasks in the form of automated
extraction and purification systems and
pipetting robots, respectively. One of the
first automated extraction systems
developed was the COBAS AmpliPrep™
from Roche. This system uses specific
biotinylated oligonucleotide probes that
capture released DNA which is then
attached to streptavidin-coated magnetic
beads. Roche have since released the
MagNA Pure LC System which is well
suited to the diagnostic laboratory. This
system can process up to 32 samples in
60 minutes and has positive pressure
pipetting, built-in UV decontamination
and HEPA- filtration to avoid cross-
contamination. However, reduced
efficiency of extraction compared to
manual extraction methods resulting in
lower PCR sensitivity may be a
problem. A number of other
automated extraction systems are now
available, such as the QIAGEN BioRobot
EZ1 and M48/9604 systems, the Abbott
m 1000 system, the ABI PRISM™ 6100
Nucleic Acid PrepStation and 6700
Automated Nucleic Acid Workstation,
and the Corbett Robotics X-tractor
Gene.™ There are therefore a number of
systems available offering a range of
purchase costs, sample capacities and
processing times. Automated fluid
handling systems such as the Corbett
CAS-1200™ Automated DNA Sample
Setup allow automated PCR setup,
including reagent preparation, dilution
series and sample pipetting.
If the performance characteristics of
these systems are found to be acceptable,
the molecular diagnostic laboratory will
be able to analyse more samples with
higher throughput in an economic
fashion and require less highly trained
personnel. This will allow the clinical
microbiology laboratory to answer more
questions routinely by molecular
methods than just the detection and
quantification of microorganisms.
As with all new technologies new
questions arise which can limit the
clinical utility of the test. For example
how long should we expect DNA to
persist after recovery or treatment and
in what body fluids or tissues will they
persist, how can we distinguish between
colonisation and active infection, and is
the detection of DNA from
microorganisms from so-called sterile
sites a normal variant?
Although molecular methods have
already replaced a number of traditional
methods in the virology laboratory, until
they can quickly and inexpensively
analyse many genetic markers to
determine aetiology and susceptibility,
conventional culture and susceptibility
testing using traditional methods will be
required for some time to come.