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Review Article

Clinical Applications of Molecular Biology for Infectious Diseases

David J Speers
Division of Clinical Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Hospital Avenue,
Nedlands, WA 6009, Australia
For correspondence: Dr David Speers e-mail: david.speers@health.wa.gov.au

Abstract
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 This paper will provide an overview of the clinical
resulted in a change from conventional laboratory methods applications of molecular methods for infectious diseases,
that rely on phenotypic expression of antigens or biochemical these have been summarised in Tables 1 and 2. Applications
products, to molecular methods for the rapid identification of a include the discipline of virology where it has been applied to
number of infectious agents. Molecular methods have become resistance testing, genotyping and viral load quantification in
increasingly incorporated into the clinical microbiology addition to routine viral detection. In the area of bacteriology
laboratory, particularly for the detection and characterisation molecular methods have been applied to resistance testing,
of virus infections and for the diagnosis of diseases due to the detection of infection due to fastidious bacteria, the more
fastidious bacteria. The advantages of rapid turn-around time rapid detection of serious bacterial infections compared to
and high sensitivity and specificity are appealing but must be conventional methods and the detection of bacterial infection
matched by rigorous validation and quality control. after antibiotics have been administered. Advances into the
areas of parasitology and mycology have also been made such
Molecular detection has mostly come to the clinical as more rapid diagnosis of fungal infection in neutropenic
microbiology laboratory in the form of PCR technology, patients. Other applications such as the detection of biosecurity
initially involving single round or nested procedures agents, applications to epidemiology and infection control
with detection by gel electrophoresis. However, with the together with the potential pitfalls with molecular methods
introduction of automation for the various stages of DNA or are also discussed.
RNA extraction, amplification and product detection together
with real-time PCR, molecular laboratories will continue Virology
to become more efficient and cost-effective. Microarray The diagnosis of viral infections has been hampered for many
technology such as the DNA chip will likely further increase years due to the cost, laboratory time and skilled personnel
the utility of molecular detection in the clinical microbiology required for the cell culture systems used, together with the
laboratory. generally low sensitivity and slow growth of many viruses

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Table 1. Examples of molecular methods in use for the diagnosis of infectious diseases*

Discipline Examples

Virology Herpes simplex virus, cytomegalovirus, Epstein-Barr virus,


varicella zoster virus, human herpes virus type 6, 7, 8
respiratory viruses (such as influenza virus, respiratory syncytial virus,
parainfluenza virus, adenovirus, rhinovirus)
#
SARS-CoV, avian influenza virus
@
HIV, hepatitis B, hepatitis C
human papilloma virus, enterovirus
orf virus, mulloscum contagiosum
rotavirus, norovirus, enteric adenoviruses

Bacteriology C. trachomatis, N. gonorrhoeae, B. pertussis, M. tuberculosis, nontuberculous


mycobacteria, T. whipplei, B. henselae, genital mycoplasmata, C. burnettii
M. pneumoniae, C. pneumoniae, Legionella spp., N. meningitidis, S. pneumoniae

Parasitology Plasmodium spp., T. gondii

Mycology P. jiroveci, Aspergillus spp.

* the complement of molecular diagnostic tests available varies between laboratories


#
severe acute respiratory syndrome-coronavirus
@
human immunodeficiency virus

in artificial media. Serology is often unhelpful in the early The detection of blood borne virus infection is also improved by
stages of infection, specific antisera for the serology tests can both PCR and non-PCR molecular methods. Active hepatitis
be difficult to obtain, and the clinical detection of antibodies is C virus (HCV) infections are diagnosed by the presence of
relatively insensitive for a number of viruses. PCR technology HCV RNA since the detection of antibody to HCV cannot
has therefore improved the detection of a number of these distinguish between past and present infection. In terms of
viruses. infectiousness only those with detectable HCV RNA have
a significant risk of transmitting HCV by transfusion, organ
Herpes simplex virus (HSV) encephalitis is a serious infection transplantation, needle-stick injury or vertically to the child.5
but diagnosis previously required brain biopsy in certain Although infection with the human immunodeficiency virus
cases due to the low sensitivity of cerebrospinal fluid (CSF) (HIV) is routinely diagnosed by serology, early HIV infection
culture and serology.1 PCR now allows the detection of HSV can be detected by HIV pro-viral DNA detection before HIV
DNA from CSF with 95% sensitivity2 thus avoiding invasive antibodies are confirmed by Western Blot serology.6 Vertical
brain biopsy. Viral meningitis, commonly caused by either transmission of HIV infection is also detected in the infant
enteroviruses or HSV, is more reliably detected by PCR when using HIV pro-viral DNA detection.7 The Australian Red Cross
compared to culture3 and in a shorter time (one versus up to Blood Service screens pooled samples from all donations
five days). HSV PCR can be multiplexed with other pathogens for HIV and HCV using the Chiron Procleix HIV-1/HCV
responsible for meningitis.4 transcription mediated amplification assay, thus reducing the
potentially infectious window period from 22 and 66 days to
9 and 7 days respectively.8

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Table 2. Examples of uses for molecular methods other than microorganism identification in the clinical microbiology
laboratory*

Test Examples

Viral load monitoring Cytomegalovirus, Epstein-Barr virus


hepatitis B, hepatitis C, @HIV

Viral genotyping @
HIV, hepatitis B, hepatitis C, human papillomavirus

Bacterial resistance detection MRSA, ^VRE, ESBL containing E. coli, K. pneumoniae


#

M. tuberculosis

Bacterial genotyping M. tuberculosis, N. meningitidis

Broad-range PCR Infective endocarditis, bacterial meningitis

* the complement of molecular tests available varies between laboratories


#
methicillin resistant S. aureus
^ vancomycin resistant enterococci

extended spectrum beta-lactamase
@
human immunodeficiency virus

Intrauterine infection of the foetus with cytomegalovirus (SARS) coronavirus (SARS-CoV) and influenza A/H5N1
(CMV),9 rubella,10 and varicella zoster virus11 can be detected (avian influenza) virus can also be incorporated into these
by PCR testing of amniocentesis fluid. Genital ulceration due assays thus acting as an in-built early detection system.
to HSV, usually due to HSV type 2 infection, is now routinely
detected by PCR in many clinical microbiology laboratories During the SARS epidemic due to the SARS-CoV, PCR
due to its increased sensitivity over viral culture. testing of respiratory specimens for other respiratory viruses
was crucial to exclude a number of suspected cases which
Molecular detection of respiratory viral pathogens from both fulfilled the case definition for SARS. PCR detection was
upper respiratory specimens such as nasopharyngeal aspirates most helpful due to the ability to rapidly screen for many
or throat swabs and lower respiratory specimens such as respiratory viruses. Subsequently a specific SARS CoV PCR
sputum or bronchoalveolar lavage fluid is cost-effective due has been developed for the early detection of SARS-CoV
to the prevention of hospitalisation, decreasing unnecessary infection with a sensitivity of 50-87% early in the disease.13
testing and procedures, directing specific therapy, and reducing Serology for SARS-CoV is up to 100% sensitive but of
unnecessary antibiotic use.12 Large multiplex or tandem limited diagnostic value early in the disease when the risk of
PCR assays testing for all the common respiratory viruses transmission is greatest.14
along with fastidious bacterial causes of pneumonia are now
feasible providing a thorough yet cost-effective alternative to The recent avian influenza (H5N1) outbreaks in South East
conventional detection methods. Uncommon yet significant Asia and beyond have also illustrated the need for rapid viral
respiratory viruses such as severe acute respiratory syndrome diagnosis. Molecular detection methods were developed

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following the 1997 Hong Kong outbreak15 and have the a 100-fold decrease in the viral load has occurred after 12
advantage of being rapid and able to be performed in many weeks of therapy.20 In HBV carriers with active liver disease
clinical microbiology laboratories. Specific serology needs HBV DNA loads are measured not only to assess patients
live virus for the microneutralisation assay which is currently regarding the need for either interferon-alpha or lamivudine
classed as a Biosafety Level 4 organism in Australia. Likewise (a DNA polymerase inhibitor) antiviral therapy but also to
direct immunofluorescence detection requires influenza type monitor their effectiveness. An increase in HBV viral load is
A/H5-specific monoclonal antibodies.16 also used as a marker of the emergence of lamivudine resistant
viral mutants.21
Viruses cause more infectious diarrhoea worldwide than
bacteria and other pathogens. The diagnosis of viral diarrhoeal Cytomegalovirus infection is a serious infection in bone
disease has improved with the development of PCR detection. marrow and solid organ transplant recipients together with
The method of choice for microbiological diagnosis of HIV-infected patients but detection has been limited by the
rotavirus from stool samples is PCR. Norovirus, a calicivirus poor sensitivity of traditional culture methods.22 Viral load
formerly known as Norwalk virus and responsible for large testing by quantitative PCR is now the accepted standard
outbreaks both in the community and health care facilities, can for monitoring the emergence of CMV infection during
be diagnosed by electron microscopy, enzyme immunoassay immunosuppression and allows pre-emptive therapy prior to
and PCR but PCR is the most sensitive and rapid method. the emergence of clinical disease with high sensitivity when
PCR is also the most sensitive method for the diagnosis of compared to culture.23
astroviruses and enteric adenoviruses (serotypes 40 and
41).17 Viral Genotyping and Resistance Testing
HIV genotyping for the detection of drug resistance is
Treatment Monitoring the standard of care to guide antiretroviral therapy and
Monitoring viral DNA or RNA loads has become the standard complements viral load assessment. Several databases are
of care for several chronic viral infections. Measurement of available such as the Stanford reverse transcriptase and
viral load is performed either by competitive PCR systems, protease database (http://hivdb.stanford.edu) where sequences
branched chain DNA signal amplification or more recently can be checked for resistance mutations.
real-time PCR.
Genotyping is also critical to the management of chronic
HIV viral load testing is an integral component of the viral hepatitis. There are six HCV genotypes geographically
management of HIV infection. It is the major tool used to distributed throughout the world. The genotype is the single
monitor the success of antiretroviral therapy and to detect strongest determinant for success with combination therapy
the emergence of viral resistance, evidenced by a rise in and all patients wishing to undergo therapy firstly undergo
the viral load despite ongoing therapy. HIV viral loads HCV genotyping. Those with chronic genotype 2 or 3 HCV
also predict progression of disease, and give prognostic infections receive 6 months of therapy with a 76% chance
information.18 Commercial tests are available and more of success compared to a 56% chance of success for those
recently ultrasensitive tests such as the Cobas Amplicor HIV- with genotype 1 HCV infection receiving 12 months of
1 Monitor Ultrasensitive Test have been released reducing the therapy.20
lower limit of viral detection to 50 copies/mL.19
Active chronic infections with HBV treated with lamivudine
Viral load testing is also used for the assessment and require surveillance for the emergence of lamivudine resistant
monitoring of responses to therapy in chronic HCV and viral mutants. During lamivudine monotherapy point
hepatitis B virus (HBV) infection. HCV RNA viral loads are mutations at the active site of the polymerase gene (YMDD
assessed in patients with genotype 1 HCV infections when variants) occur with a frequency of 14-32% after one year in
monitoring for responses to combination interferon-alpha phase III studies, and in 42% and 52% of Asian patients after
and ribavirin therapy. Patients who remain negative for HCV two and three years of therapy respectively.24 The emergence
RNA 6 months after completing combination therapy for HCV of lamivudine resistance is detected by a rise in HBV viral
infection almost always remain free of the virus in the longer load and confirmed by sequencing of the active site of the
term and have achieved a sustained virological response. If DNA polymerase gene.25
the HCV genotype 1 RNA is undetectable after 12 weeks
of therapy there is a 75% chance of a sustained virological The presence of HBV pre-core mutants may cause active liver
response. However, even if the HCV RNA remains detectable, disease despite the absence of HBeAg, the common marker
a 33% chance of a sustained virological response remains if for active hepatitis in hepatitis B infection. This may be due

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to either a premature stop codon point mutation in the pre- The introduction of molecular detection for the fastidious
core gene (G1896A) or a mutation in the basal core promoter sexually transmitted bacteria has led to a large increase in the
region down-regulating HBeAg production, both of which proportion of laboratory confirmed cases due to its increased
can only be reliably detected genotypically.26 sensitivity allowing more effective contact tracing. In the
management of sexual health traditional screening methods
Human papilloma virus (HPV) is now accepted as the cause require speculum examination in women and urethral
of almost all cervical cancers and HPV genotypes are now swabs in men. These require special equipment and cause
classified as either low or high-risk for the causation of these embarrassment and discomfort, thus reducing compliance.
cancers. Screening for pre-neoplastic cytological changes has Molecular detection is useful since noninvasive specimens
traditionally been performed by the Papanicaou (Pap) screen, unsuitable for traditional culture, such as initial stream urine
but the detection of high-risk HPV infection is a useful adjunct. and self-collected vaginal swabs can be used. These are
Since HPV cannot be routinely cultured in vitro, testing for the more convenient and acceptable increasing the compliance
15 high-risk genotypes of HPV requires molecular methods. with testing. Although molecular testing for C. trachomatis
Detection can be achieved by signal amplification, such as the and N. gonorrhoeae does not allow monitoring of antibiotic
Digene Hybrid Capture 2 assay which is the only diagnostic in resistance or detect other sexually transmitted diseases, urine
vitro test approved by the Federal Drug Administration (FDA). testing has shown equivalent sensitivity and specificity to
This assay contains specific RNA probes directed toward the invasive specimens for detection of C. trachomatis in men
high-risk genotype DNA sequences which are detected by an and women, and for detection of N. gonorrhoeae in men when
antibody directed against the DNA-RNA hybrids formed.27 compared to urethral swabs. In women the sensitivity and
Detection of the high-risk genotypes can also be achieved by specificity of the PCR assay for N. gonorrhoeae was lower for
target amplification such as multiplex PCR, but commercial urine compared to cervical samples, however self-collected
assays are not yet available. Detection of these genotypes by vaginal swabs may help in this regard. The PCR assay for
molecular analysis can help in the assessment of equivocal C. trachomatis has equal sensitivity for vaginal and cervical
Pap smears to define those women at risk of developing swabs and a transcription mediated amplification assay has
cervical cancer.28 Alternatively a normal Pap smear with a been approved by the U.S. FDA for testing C. trachomatis and
negative genetic test for the high-risk genotypes may indicate N. gonorrhoeae from vaginal specimens.31 In remote areas,
a longer period of time before re-testing.29 With the advent of molecular methods have the advantage of being performed
a genotype 16 HPV vaccine the role of this testing is likely to on dry swabs with little degradation of the DNA during
assume more importance. transit compared to the difficulties of transporting samples in
specialised transport medium to preserve viability. In addition,
Molecular methods have therefore gone beyond simple molecular methods can test for multiple genital pathogens
detection of viral infections to become an integral component such as C. trachomatis, N. gonorrhoeae, the Donovanosis
of the management of blood borne virus and other viral agent and the genital mycoplasmata from the same swab.
infections.
Mycobacteriology has been aided by the introduction of
Bacteriology molecular methods. However, it is important to note that
Fastidious Bacteria molecular detection of M. tuberculosis is one of the few
Together with virology, the diagnosis of infections due to examples where conventional culture remains more sensitive.
fastidious bacteria has benefited greatly from molecular This is possibly due to the difficulty in releasing the DNA
detection. Many of these fastidious bacteria have public health from the bacterial cells during the extraction process. Despite
implications such as Mycobacterium tuberculosis, Chlamydia this limitation, molecular detection of M. tuberculosis has
trachomatis, Neisseria gonorrheae and Bordetella pertussis. a definite role as it allows confirmation of acid-fast bacilli
Non-culture-based molecular testing has the advantage of seen on microscopy with up to 98% sensitivity in pulmonary
avoiding the delays of days to weeks for conventional culture tuberculosis within a day compared to two weeks or more
to allow early recognition and treatment as a public health by culture. Specimens that are smear-negative have a much
imperative. Commercial assays are available for M. tuberculosis lower chance of molecular confirmation, with reported
and Mycobacterium avium complex, C. trachomatosis, and sensitivities as low as 40%.32 In addition to direct detection
N. gonorrhoeae. Several nucleic acid detection technologies from clinical specimens, molecular methods can confirm a
are in use including PCR, transcription based amplification, positive culture within a day compared to approximately four
ligase chain reaction, strand displacement amplification and weeks using phenotypic methods. This has shortened the time
the Qβ replicase system.30 for laboratory confirmation of suspected tuberculosis even for
smear-negative but culture-positive cases.

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Mycobacteriology has also advanced through the use vaccination and can be combined with N. meningitidis
of molecular methods for the speciation of the many detection. In our laboratory as well as others,40 combined
nontuberculous mycobacterial species. Phenotypic methods N. meningitidis detection and genoserogrouping is routinely
are slow and the limited number of tests available is inadequate performed on clinical specimens from suspected cases.
to differentiate between the large number of species. Genetic
sequencing of the 16S rRNA gene has simplified this process Rapid detection of the other common bacterial causes of
in many laboratories.33 However, some species such as the meningitis has also been developed. N. meningitidis with
rapid grower group cannot be distinguished by 16S rRNA Streptococcus pneumoniae and Haemophilus influenzae
gene sequencing alone, and require a multi-gene approach type B account for 90% of cases of bacterial meningitis
incorporating the hsp65, rpoB and sod genes.34 and multiplex PCR methods have been developed for their
detection.41
Due to its significantly enhanced sensitivity, PCR has
replaced direct fluorescent-antibody and culture as the “gold Antibiotic Resistance
standard” method for detection of B. pertussis early in the Following on from the success of molecular methods for the
disease process.35 In one pertussis school outbreak using detection of several bacterial infections, genotypic detection
nasopharyngeal aspirates, PCR detected 48% of clinical cases of antibiotic resistance is appealing due to the avoidance of
compared to 5% confirmed by culture.36 For this pathogen of problems such as variable phenotypic resistance expression.
public health significance, a combination of PCR detection Applying rapid and reliable genotypic detection to bacteria
early in disease and serology for suspected cases late in the with infection control implications such as methicillin-
disease process is used for maximal case ascertainment. Other resistant Staphylococcus aureus (MRSA) and vancomycin-
fastidious respiratory pathogens that can be rapidly diagnosed resistant enterococci (VRE) is of great potential benefit. The
by molecular means include Legionella spp., Mycoplasma discrimination of MRSA from other S. aureus is confirmed by
pneumoniae and Chlamydia pneumoniae. the detection of the mecA gene responsible for this resistance.
The detection of the mecA gene can be multiplexed with the
Some bacteria can only be detected by molecular means nuc gene to allow rapid molecular detection of S. aureus and
as culture is either extremely difficult or impossible confirmation of MRSA from positive blood culture bottles.42
for the routine microbiology laboratory, or represents a This is important to provide information regarding antibiotic
significant occupational risk to the laboratory personnel. selection as early as possible since mortality rates are higher
Whipple’s disease is a rare but ultimately lethal infection with MRSA infection compared to methicillin-sensitive
due to Tropheryma whipplei which could previously only S. aureus.43 Likewise, detection of VRE is more sensitive
be diagnosed by characteristic histopathology and electron and rapid using DNA-based amplification techniques.44
microscopy, often from post-mortem material. PCR now Commercial kits are now becoming available for MRSA and
allows diagnosis of neuro-Whipple’s disease and endocarditis VRE detection using real-time PCR instrumentation which
by the detection of T. whipplei from noninvasive specimens.37 will further improve the speed of detection.
Other examples where molecular diagnosis can help in the
diagnosis of difficult or uncultivable bacteria include cat Extended spectrum β-lactamases (ESBL) are found in
scratch disease due to Bartonella henselae, Q fever due to Escherichia coli and Klebsiella pneumoniae and are readily
Coxiella burnetii, and male urethritis due to Mycoplasma transmitted on plasmids and transposons. ESBL-containing
genitalium. A more detailed discussion on the molecular bacteria can spread rapidly in health care facilities to cause
methods for the diagnosis of fastidious bacteria can be found wound infections, urinary tract infections and septicaemia.
in Fenollar and Raoult.38 Their detection requires special laboratory tests since routine
antibiotic susceptibility testing may not detect strains carrying
Rapid Bacterial Diagnosis the resistance gene. Although most clinical microbiology
Meningococcal disease can have devastating consequences laboratories currently use phenotypic methods to detect
and requires early diagnosis for correct antibiotic therapy as ESBL, molecular detection of these point mutations at the
well as early provision of chemoprophylaxis for close contacts. active site of the β-lactamase gene can confirm the ESBL and
PCR methods can now provide same-day detection from allow the characterisation of the type of ESBL for monitoring
sterile site specimens with a superior speed and sensitivity to of its spread through health care facilities around the world.
culture, and when combined with culture and other laboratory
methods, PCR maximises the laboratory confirmation of Multi-drug resistant tuberculosis (defined as the presence of
clinically suspected cases.39 Genoserogrouping for serogroup both rifampicin and isoniazid resistance) is a serious problem
B and C N. meningitidis strains helps with decisions regarding in many parts of the world such as Eastern Europe. Traditional

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methods for detecting rifampicin and isoniazid resistance Broad-range PCR


require additional incubation from culture, delaying the Unlike specific PCR testing where a particular organism is
diagnosis and increasing the risk of transmission of resistant being sought, the use of broad-range PCR for the diagnosis
disease in the community. A multiplex PCR for the sequencing of infectious diseases is more of a fishing expedition. Primers
of rpoB and hsp65 gene targets can facilitate same day complementary to a conserved region of a gene are used, such
detection of the majority of multi-drug resistant strains33 but as the 16S rRNA bacterial gene or the 18S rRNA gene of fungi.
reliable resistance testing will require multi-gene and whole Any amplified product is usually sequenced and compared to
of gene sequencing45 better suited to microarray technology. more than 9,000 sequences from different organisms in Internet
databases.51 There are several comprehensive databases such
Mycology and Parasitology as GenBank (www.ncbi.nlm.nih.gov/Genbank), EMBL Data
Although not as frequently applied to eukaryotic infections, Library (www.ebi.ac.uk/embl), and the DNA Data Bank
in a number of clinical circumstances molecular testing can of Japan (www.ddbj.nig.ac.jp) with daily data exchange
be helpful. Pneumocystis jiroveci (a fungus previously called between them, and more specialised high quality databases
Pneumocystis carinii) can cause a severe pneumonia in such as RIDOM (www.ridom-rdna.de/) for bacterial rDNA
HIV-infected patients and other immunosuppressed patients sequences used for mycobacterial speciation. Broad-range
but detection is limited to microscopy of respiratory tract PCR using 16S rRNA sequences is appealing as it can, in
specimens. Microscopy for the detection of P. jiroveci usually theory, detect bacteria in any sterile site specimen such as
involves methenamine silver staining of tissue specimens or blood or cerebrospinal fluid, in other words a “molecular petri
calcofluor white staining of induced sputum or bronchoalveolar dish.” In fact this method was used to identify B. henselae
lavage specimens. Immunofluorescence is more sensitive in bacillary angiomatosis and T. whipplei as the bacterium
than these stains but is more expensive and needs specialised associated with Whipple’s disease.52 A good example of its
facilities. Sensitivity remains an issue, however, especially potential use in diagnostic medicine is for the aetiological
in HIV-non-infected patients such that the more sensitive diagnosis of infective endocarditis.53 Antibiotic regimens for
PCR can be very useful.46 The specificity of PCR is limited, the therapy of this serious disease rely on the identification
however, as this organism is a ubiquitous commensal and can of the microbiological aetiology which can be problematic
be detected by PCR in the absence of pneumonia.47 Another when conventional blood cultures are negative due to the
mycological example is the use of 18S rRNA gene PCR to prior administration of antibiotics. Broad-range PCR can be
detect Aspergillus spp. infection in neutropenic haematology performed on the excised heart valves and vegetations or
patients. This disease is notoriously difficult to diagnose peripheral blood to reveal a diagnosis that would otherwise
due to the poor sensitivity of culture early in disease and the be missed. Broad-range PCR has also been applied to the
difficulty in obtaining histopathological specimens in those diagnosis of bacterial meningitis.54 More recently a spectacular
with reduced platelet counts. Early treatment is essential for use of broad-range PCR was the identification of the novel
the best outcomes resulting in empiric use of costly and toxic virological cause of SARS. Broad-based primers were used
antifungal therapy. When performed frequently Aspergillus to detect unknown viruses in specimens from SARS clinical
PCR can reduce the time required for a specific diagnosis,48 cases. The sequences showed homology to the coronavirus
however, its exact role to improve the management and genus, supported by other laboratory results that resulted in
therefore the outcome of this devastating disease is still a specific SARS CoV PCR within weeks of the first report of
unclear. the disease.55

Parasitological diagnosis is aided by molecular methods since A major drawback of broad-range PCR is the risk of amplifying
most parasites are not cultured in routine laboratory settings DNA that may be contaminating the specimen or the PCR
and therefore diagnosis relies mostly on the relatively less reagents themselves, especially the Taq DNA polymerases,
sensitive microscopy or serology. Toxoplasma gondii can be resulting in false positive results.56 Also the accuracy of the
detected by PCR from amniocentesis fluid to confirm foetal data available through public databases is difficult to assess
infection49 and from CSF to diagnose toxoplasma encephalitis. and is dependent on the quality of the sequences deposited,
Microscopy remains the mainstay of malaria diagnosis but a critical factor when comparing an unknown sequence. It is
Plasmodium spp. PCR, because of its superior sensitivity possible that the matching sequence is either inaccurate or is
compared to microscopy, can diagnose malaria in those with shared by another organism for which data is not currently
negative thick and thin blood films due to administration of available.
chemoprophylaxis or partial immunity. Plasmodium species
PCR can also detect mixed infections that can be difficult to
discern microscopically.50

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Speers DJ

Public Health Aspects as mycobacterial interspersed repetitive unit genotyping is


Since rapid and reliable aetiological diagnosis underpins recommended for the evaluation of community and health
the effective management of contagious diseases, molecular care facility tuberculosis outbreaks.
diagnostics have an important role. The outbreak of SARS
CoV illustrated the importance of ruling out other respiratory In the future it is likely that point-of-care molecular tests will
viruses such as influenza to facilitate the early identification be available allowing reliable results with rapid turnaround
and quarantine of suspected cases of SARS. This proved time in the field.
effective in controlling the outbreak even though a specific
diagnostic test was not available during most of the outbreak. Biosecurity
Now several PCR-based diagnostic kits are available and we Biological warfare agents such as Bacillus anthracis, variola
will be much better equipped for early virological diagnosis major virus (smallpox), Clostridium botulinum and Yersinia
should the SARS CoV re-emerge. Diarrhoeal viruses such as pestis (plague) are problematic since they may be invisible,
noroviruses, which spread rapidly through health care facilities may cause no ill-effect for several days, and are communicable
and residential care facilities, can now be rapidly diagnosed to with very small amounts affecting many people. For example,
facilitate case isolation. The infectiousness of those with blood 10g of anthrax spores could kill as many people as a ton of
borne viruses is also determined predominantly by molecular the nerve agent sarin.59 The rapidity at which such an incident
testing. The ability of health care workers who have been could escalate mandates rapid, reliable and sensitive detection
infected with hepatitis B and C to perform exposure-prone methods. Real-time PCR methods fulfil these criteria best as
procedures such as surgery is determined by PCR testing such conventional methods either lack discriminatory power, are
that workers with detectable hepatitis B DNA or hepatitis C slow, or require highly trained personnel. However, PCR
RNA may be restricted from performing such procedures. systems require the release of DNA from spores, which can
be difficult to achieve without inhibiting the PCR process.
The management of bacterial infections of public health Spore disruption and PCR can now be achieved in 15 minutes
significance is also improved by molecular methods. Early using newly developed hardware.60 Battery-powered portable
diagnosis of B. pertussis, M. tuberculosis, N. meningitidis machines using TaqMan real-time PCR are being developed
is important for the early prevention of transmission, an aim with processing times of only 30 minutes.59
that is best achieved by a combination of conventional and
molecular testing. In addition microarray technologies have a great deal
of potential in this area but are restricted by the sample
The advent of molecular epidemiology, which allows the pretreatment required for such microfluidic devices. These
tracking of pathogens based on genotyping of the involved problems are likely to be overcome as new technologies are
strains from outbreaks, has revolutionised how outbreaks developed.61
are investigated and managed. The problem with strain
differentiation using phenotypic methods in bacterial Limitations of Molecular Methods
outbreaks, such as meningococcal disease, is the variable Despite significant advantages of molecular diagnostics
expression of the phenotypic markers. Other methods like it cannot yet replace conventional methods for a range of
multilocus enzyme electrophoresis are very labour-intensive infectious diseases since many common tests performed in the
and unable to be performed in many laboratories. Multilocus clinical microbiology laboratory are rapid and inexpensive.
sequence typing (MLST) avoids these problems since every Advances in conventional technologies have resulted in
strain can be typed unambiguously. MLST involves the many rapid antigen tests requiring only minutes for results
comparison of nucleotide sequences from internal fragments and the modern automated culture systems allow relatively
of a number of housekeeping genes. Sequences obtained rapid identification and susceptibility testing. Unlike bacterial
are submitted to websites such as http://mlst.zoo.ox.ac.uk/ culture, which can detect a large number of cultivable bacteria
for N. meningitidis to give an allelic profile which can be without initially knowing the specific organism responsible,
compared to existing clones from anywhere in the world. This all PCR tests except broad-range PCR can only detect the
system avoids the problems of interlaboratory interpretation organism whose DNA is complementary to the primers used.
encountered with other genotyping methods yet can be used Therefore to cover a similar breadth of possible organisms
to investigate local outbreaks57 even in some cases where a would require the introduction of inexpensive and simple
viable culture is not obtained. This has been applied to N. microarray technologies30 that are not yet available.
meningitidis and S. pneumoniae58 which has helped map
the spread of virulent clones around the world. Similarly False Positive and False Negative Results
genotyping of M. tuberculosis using different methods such Another problem restricting the application of molecular

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techniques to routine diagnosis is that of false positive and cannot be controlled resulting in an inhibitor check of varying
false negative results. To avoid false positive results due to sensitivity. The addition of non-human pathogen RNA,
laboratory contamination relatively large laboratory areas such as bacteriophage RNA, can be used as an inhibitor and
are required for physical separation of reagent preparation, extraction control for reverse transcription PCR. If inhibitors
specimen preparation and product detection areas together are detected, they may be overcome by dilution of the DNA
with a high level of staff training and skill. Amplicon extract or treatment of the DNA extract with products such as
laboratory contamination can be reduced by ultraviolet GeneReleaser before PCR, or by including PCR facilitators
light irradiation of reagents and chemical inactivation of such as bovine serum albumin in the PCR step.65 If such
surface contamination with sodium hypochlorite. Amplicons manoeuvres fail to overcome the inhibition the sample must
can be destroyed by the use of dUTP to replace dTTP for be reported as inhibitory and a repeat sample requested.
amplification then uracil N-glycosylase (UNG) treatment
of preassembled starting reactions to destroy the dUTP- Lack of Uniformity in Molecular Testing
containing amplicons. Intersample contamination can be Molecular diagnosis is also complicated by the vast array of
reduced by the use of disposable equipment and cotton filter in-house PCR tests used in different laboratories. Commercial
tips, and using disposable personal protective equipment such tests are available for a number of common and important
as caps, gowns and gloves. Even with scrupulous technique infectious diseases such as HIV, hepatitis C and B, C.
problems can be encountered, especially with broad-range trachomatis, and N. gonorrhoeae but many infectious diseases
PCR due to the presence of foreign DNA in the PCR reagents. are unlikely to have a commercial PCR test developed due to
It is therefore crucial that appropriate negative controls are their rarity. Differences in primer selection (different genes or
included in every PCR run to detect any contamination. The different sequences within genes), amplification format such
advent of real-time PCR has reduced this risk due to single as single round, nested or real-time PCR or one of the other
tube PCR reaction and detection systems. nucleic amplification methods, and product detection methods
such as ethidium bromide gel electrophoresis, DNA probes or
Poor primer design can also lead to erroneously positive sequencing make comparisons for sensitivity and specificity
results. Primers may be poorly designed such that incidental difficult.
amplification of microorganisms other than those sought
occurs. Also primers are designed based on the known Differentiation between Infection and Disease
sequences available through international databases but Since the presence of nucleic acid does not necessarily mean
organisms or sequences yet to be discovered can subsequently the presence of viable organisms a problem with interpretation
reduce the specificity of the PCR.62 of PCR results can emerge that does not occur with culture.
For some infections such as invasive meningococcal disease
False negative results may also be a problem. Some organisms the presence of meningococcal DNA from a sterile site has a
such as mycobacteria are difficult to extract DNA from, very high positive predictive value. However, the detection
reducing the sensitivity of the PCR.63 Substances in some of P. jiroveci in suspected PCP may have only a 50% positive
clinical specimens such as sputum and faeces can degrade the predictive value in immunosuppressed patients since it
DNA and RNA and other specimens may contain substances may colonise as well as cause disease. Likewise herpes
such as polysaccharides, haem and therapeutic drugs that viruses such as Epstein-Barr virus (EBV), CMV and HSV
inhibit the PCR enzymes.1 It is therefore important to include are intermittently shed following primary infection without
inhibitor checks for each specimen to ensure a negative PCR causing disease. Less sensitive detection methods such as
reaction is not actually an inhibited reaction. This can be done culture have a higher specificity but quantitative PCR may
by incorporation of an internal amplification control to check be helpful in this regard since higher viral loads are usually
for both inhibitors and successful DNA extraction. This can more specific for disease. Quantification in viral infections
be achieved by the addition of non-human pathogen DNA, such as HIV, CMV, EBV, HBV and HCV is well established
for example equine herpesvirus as used in our laboratory, to for assessing disease severity or monitoring response to
the extraction buffer and its subsequent detection from the treatment.30 In CMV disease viral load testing can monitor
extracted sample by PCR using complementary primers.64 The either increases of viral loads to threshold levels or rates of
amount of spiked DNA is titrated to be as sensitive as possible viral load increase to improve the positive predictive value
yet allow regular detection in non-inhibitory specimens. for clinical CMV disease. Another approach is to detect RNA
Alternatively the human β-globin gene can be detected species that are usually degraded within minutes of cell death
by PCR following sample extraction without the need for to indicate pathogen viability and replication.66
spiking the reagents with foreign DNA.30 A problem with this
method is that the amount of human DNA in each specimen

Clin Biochem Rev Vol 27 February 2006 I 47


BadrickDJ
Speers et al.

Future Directions of Molecular Technology factor identification, pathogen response to drugs, and vaccine
PCR coupled with sequencing has become a powerful tool for development. However, these companies will need to compete
the identification of previously unknown pathogens and the with the multiplex real-time PCR kits becoming commercially
epidemiological investigation of new and emerging infectious available such as those produced by Prodesse for the detection
diseases. Molecular methods have helped reveal that over of the common respiratory pathogens.
30 species of bacteria can form uncultivable forms under
unfavourable environmental conditions.67 This now means that Economic pressures will force the development of more
Koch’s postulates cannot be applied to investigate the validity automated and less expensive test procedures similar to those
of certain microorganisms in the causation of disease, such in clinical chemistry laboratories. Nucleic acid extraction
as T. whipplei as the cause of Whipple’s disease. Molecular and purification and the manual loading of the isolated
technology has gone beyond the simple identification of nucleic acids and master mixes into the PCR reaction
causative organisms for infectious diseases and either now or vessels remain the most labour-intensive parts of molecular
in the near future will be pivotal to the study of the evolution technology. However, new technology has been developed to
of pathogens, the maintenance of infective cycles in nature, perform these tasks in the form of automated extraction and
the investigation of causes and mechanisms of new pathogens, purification systems and pipetting robots, respectively. One
the mechanisms of susceptibility of different host groups and of the first automated extraction systems developed was the
the development of DNA and RNA banking of genes encoding COBAS AmpliPrep from Roche.70 This system uses specific
pathogenic factors. This will be achieved with new molecular biotinylated oligonucleotide probes that capture released
methods such as microarray, microchips, in situ PCR and DNA which is then attached to streptavidin-coated magnetic
automation of molecular procedures. beads. Roche have since released the MagNA Pure LC System
which is well suited to the diagnostic laboratory. This system
Microarray and gene chip assays, first published in 1991, have can process up to 32 samples in 60 minutes and has positive
the advantages of miniaturisation and automated construction pressure pipetting, built-in UV decontamination and HEPA-
using industrial robots together with sensitivity and rapid filtration to avoid cross-contamination. However, reduced
reading of large amounts of detailed genetic information. In efficiency of extraction compared to manual extraction
fact up to 106 different probes per cm2 can be attached to specific methods resulting in lower PCR sensitivity may be a problem.71
sites on the microarray platform68 of either nylon membrane A number of other automated extraction systems are now
or glass slide. Microarrays can identify simultaneously a available, such as the QIAGEN BioRobot EZ1 and M48/9604
range of pathogens for particular diseases such as infective systems, the Abbott m1000 system, the ABI PRISM™ 6100
diarrhoea, pneumonia or meningitis as well as genetic Nucleic Acid PrepStation and 6700 Automated Nucleic Acid
markers of virulence and antibiotic susceptibility. One of the Workstation, and the Corbett Robotics X-tractor Gene.™
first applications of this technology was in the field of HIV, in There are therefore a number of systems available offering
which an array was used to detect protease gene resistance.69 a range of purchase costs, sample capacities and processing
Microarrays can be used for complete genome sequencing, times. Automated fluid handling systems such as the Corbett
an example being the development of an array to sequence CAS-1200™ Automated DNA Sample Setup allow automated
the SARS virus following the 2003 outbreak. In addition their PCR setup, including reagent preparation, dilution series and
role in the detection and characterisation of biosecurity agents sample pipetting.
is rapidly progressing,59 an example being the sequencing
of hundreds of different variola major strains by the U.S. If the performance characteristics of these systems are found
Centers for Disease Control and Prevention. However, its use to be acceptable, the molecular diagnostic laboratory will be
at this stage is mainly research-based and is currently very able to analyse more samples with higher throughput in an
expensive.1 Issues of reproducibility must be addressed as economic fashion and require less highly trained personnel.
the technology is highly sensitive and processing conditions This will allow the clinical microbiology laboratory to answer
must be standardised and followed rigidly. Also, because more questions routinely by molecular methods than just the
multiple data points are generated from each array, computer detection and quantification of microorganisms.
algorithms are needed to analyse the data.69 Commercially,
initial efforts focussed on applications of microarrays for use As with all new technologies new questions arise which can
in detecting drug resistance and mycobacterial identification limit the clinical utility of the test. For example how long
but the biotechnology companies are now assessing the market should we expect DNA to persist after recovery or treatment
for molecular diagnosis using microarrays in the infectious and in what body fluids or tissues will they persist, how can
diseases laboratories. For example, Affymetrix produce we distinguish between colonisation and active infection, and
GeneChip microarrays for pathogen identification, virulence is the detection of DNA from microorganisms from so-called
sterile sites a normal variant?

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Infectious Diseases

Although molecular methods have already replaced a number Infectious Diseases 2002. p 45-50.
of traditional methods in the virology laboratory, until they 12. Henrickson KJ. Cost-effective use of rapid diagnostic
can quickly and inexpensively analyse many genetic markers techniques in the treatment and prevention of viral
to determine aetiology and susceptibility, conventional culture respiratory infections. Pediatr Ann 2005;34:24-31.
and susceptibility testing using traditional methods will be 13. Ng EK, Hui DS, Chan KC, et al. Quantitative analysis
required for some time to come. and prognostic implication of SARS coronavirus RNA
in the plasma and serum of patients with severe acute
Competing interests: None declared respiratory syndrome. Clin Chem 2003;49:1976-80.
14. Ho PL, Chau PH, Yip PSF, et al. A prediction rule for
References clinical diagnosis of severe acute respiratory syndrome.
Eur Respir J 2005;26:474-9.
1. Gilbert GL, James GS, Sintchenko V. Culture shock. 15. Yuen KY, Chan PK, Peiris M, et al. Clinical features and
Molecular methods for diagnosis of infectious diseases. rapid viral diagnosis of human disease associated with
Med J Aust 1999;171:536-40. avian influenza A H5N1 virus. Lancet 1998;351:467-
71.
2. Lakeman FD, Whitley RJ. Diagnosis of herpes simplex
encephalitis: application of polymerase chain reaction 16. World Health Organisation. Recommended laboratory
to cerebrospinal fluid from brain-biopsied patients and tests to identify avian influenza A virus in specimens
correlation with disease. National Institute of Allergy from humans. http://www.who.int/csr/disease/avian_
and Infectious Diseases Collaborative Antiviral Study influenza/guidelines/labtests/en/index.html. Accessed
Group. J Infect Dis 1995;171:857-63. October 17, 2005.
3. van Vliet KE, Glimaker M, Lebon P, et al. Multicenter 17. Clark B, McKendrick M. Review of viral gastroenteritis.
evaluation of the Amplicor Enterovirus PCR test Curr Opin Infect Dis 2004;17:461-9.
with cerebrospinal fluid from patients with aseptic 18. Katzenstein TL. Molecular biological assessment
meningitis. The European Union Concerted Action on methods and understanding the course of the HIV
Viral Meningitis and Encephalitis. J Clin Microbiol infection. APMIS 2003;114(S):1-37.
1998;36:2652-7. 19. Berger A, Scherzed L, Sturmer M, Preiser W, Doerr
4. Read SJ, Jeffery KJM, Bangham CRM. Aseptic HW, Rabenau HF. Comparative evaluation of the
meningitis and encephalitis: the role of PCR in the Cobas Amplicor HIV-1 Monitor Ultrasensitive Test, the
diagnostic laboratory. J Clin Microbiol 1997;35:691-6. new Cobas AmpliPrep/Cobas Amplicor HIV-1 Monitor
5. Dore GJ, Kaldor JM, McCaughan GW. Systematic Ultrasensitive Test and the Versant HIV RNA 3.0 assays
review of role of polymerase chain reaction in defining for quantitation of HIV-1 RNA in plasma samples. J
infectiousness among people infected with hepatitis C Clin Virol 2005;33:43-51.
virus. BMJ 1997;315:333-7. 20. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon
6. Dax EM. The window period and HIV tests. ASHM alpha-2a plus ribavirin for chronic hepatitis C virus
Journal Club 2004;13:9-11. infection. N Engl J Med 2002;347:975-82.
7. Luzuriaga K, Sullivan JL. Pathogenesis of vertical 21. Thomson EC, Main J. Advances in hepatitis B and C.
HIV-1 infection: implications for intervention and Curr Opin Infect Dis 2004;17:449-59.
management. Pediatr Ann 1994;23:159-66. 22. Long CM, Drew L, Miner R, Jekic-McMullen D,
8. Seed CR, Cheng A, Ismay SL, et al. Assessing the Impraim C, Kao SY. Detection of cytomegalovirus
accuracy of three viral risk models in predicting the in plasma and cerebrospinal fluid specimens from
outcome of implementing HIV and HCV NAT donor human immunodeficiency virus-infected patients by
screening in Australia and the implications for future the AMPLICOR CMV test. J Clin Microbiol 1998;36:
HBV NAT. Transfusion 2002;42:1365-72. 2434-8.
9. Palasanthiran P, Jones C, Garland S. Cytomegalovirus. 23. Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker
In: Management of Perinatal Infections. Palasanthiran AF, Griffiths PD. Application of viral-load kinetics to
P, Starr M, Jones C editors. Australasian Society for identify patients who develop cytomegalovirus disease
Infectious Diseases 2002. p 1-4. after transplantation. Lancet 2000;355:2032-6.
10. Nourse C. Rubella. In: Management of Perinatal Infections. 24. Dixon JS, Boehme RE. Lamivudine for the treatment
Palasanthiran P, Starr M, Jones C editors. Australasian of chronic hepatitis B. Acta Gastroenterol Belg
Society for Infectious Diseases 2002. p 33-5. 2000;63:348-58.
11. Heuchan A, Isaacs D. Varicella zoster virus. In: 25. Allen MI, Deslauriers M, Andrews CW, et al.
Management of Perinatal Infections. Palasanthiran Identification and characterization of mutations in
P, Starr M, Jones C editors. Australasian Society for hepatitis B virus resistant to lamivudine. Lamivudine

Clin Biochem Rev Vol 27 February 2006 I 49


BadrickDJ
Speers et al.

Clinical Investigation Group. Hepatology 1998;27: based identification and serogroup prediction of Neisseria
1670-7. meningitidis. J Clin Microbiol 2000;38:855-7.
26. Papatheodoridis GV, Hadziyannis SJ. Diagnosis and 41. Tzanakaki G, Tsopanomichalou M, Kesanopoulos K, et
management of pre-core mutant chronic hepatitis B. J al. Simultaneous single-tube PCR assay for the detection
Viral Hepat 2001;8:311-21. of Neisseria meningitidis, Haemophilus influenzae type
27. Hubbard RA. Human papillomavirus testing methods. b and Streptococcus pneumoniae. Clin Microbiol Infect
Arch Pathol Lab Med 2003;127:940-5. 2005;11:386-90.
28. Brestovac B, Harnett GB, Smith DW, Frost F, Shellam 42. Louie L, Goodfellow J, Mathieu P, Glatt A, Louie M,
GR. Multiplex PCR (MNP) assay for the detection of Simor AE. Rapid detection of methicillin-resistant
15 high risk genotypes of human papillomavirus. J Clin staphylococci from blood culture bottles by using
Virol 2005;33:116-22. a multiplex PCR assay J Clin Microbiol 2002;40:
2786-90.
29. Cuzick J. Role of HPV testing in clinical practice. Virus
Res 2002;89:263-9. 43. Cockerill FR. Rapid Detection of Pathogens and
Antimicrobial Resistance in Intensive Care Patients
30. Yang S, Rothman RE. PCR-based diagnostics for
Using Nucleic Acid-Based Techniques. Scand J Clin
infectious diseases: uses, limitations, and future
Lab Invest 2003;63(S239):34-46.
applications in acute-care settings. Lancet Infect Dis
2004;4:337-48. 44. Paule SM, Trick WE, Tenover FC, et al. Comparison
of PCR assay to culture for surveillance detection of
31. Cook RL, Hutchison SL, Ostergaard L, Braithwaite RS,
vancomycin-resistant enterococci. J Clin Microbiol
Ness RB. Systematic review: noninvasive testing for
2003;41:4805-7.
Chlamydia trachomatis and Neisseria gonorrhoeae. Ann
Intern Med 2005;142:914-25. 45. Jenkins C. Rifampicin resistance in tuberculosis outbreak,
London, England. Emerg Infect Dis 2005;11:931-4.
32. Bergmann JS, Woods GL. Clinical evaluation of the
Roche AMPLICOR PCR Mycobacterium tuberculosis 46. Helweg-Larsen J, Jensen JS, Benfield T, Svendsen UG,
test for detection of M. tuberculosis in respiratory Lundgren JD, Lundgren B. Diagnostic use of PCR for
specimens. J Clin Microbiol 1996;34:1083-5. detection of Pneumocystis carinii in oral wash samples.
J Clin Microbiol 1998;36:2068-72.
33. Kapur V, Li LL, Hamrick MR, et al. Rapid
Mycobacterium species assignment and unambiguous 47. Helweg-Larsen J, Jensen JS, Dohn B, Benfield TL,
identification of mutations associated with antimicrobial Svendsen UG, Lundgren B. Detection of Pneumocystis
resistance in Mycobacterium tuberculosis by automated DNA in samples from patients suspected of bacterial
DNA sequencing. Arch Pathol Lab Med 1995;119: pneumonia-a case control study. BMC Infect Dis
131-8. 2002;25:28.
34. Devulder G, Perouse de Montclos M, Flandrois JP. A 48. Williamson ECM, Leeming JP, Palmer HM, et al.
multigene approach to phylogenetic analysis using Diagnosis of invasive aspergillosis in bone marrow
the genus Mycobacterium as a model. Int J Syst Evol transplant recipients by polymerase chain reaction. Br J
Microbiol 2005;55:293-302. Haematol 2000;108:132-9.
35. Cockerill FR, Smith TF. Response of the clinical 49. Gilbert L. Toxoplasmosis. In: Management of Perinatal
microbiology laboratory to emerging (new) and Infections. Palasanthiran P, Starr M, Jones C editors.
reemerging infectious diseases. J Clin Microbiol Australasian Society for Infectious Diseases;2002.
2004;42:2359-65. p 39-41.
36. He Q, Mertsola J, Soini H, Skurnik M, Ruuskanen 50. Speers DJ, Ryan S, Harnett G, Chidlow G. Diagnosis of
O, Viljanen MK. Comparison of PCR with culture malaria aided by polymerase chain reaction in two cases
and EIA for diagnosis of pertussis. J Clin Microbiol with low-level parasitaemia. Intern Med J 2003;33:
1993;31:642-5. 613-5.
37. Dutly F, Altwegg M. Whipple’s disease and “Tropheryma 51. Fredricks DN, Relman DA. Application of PCR to
whippelii”. Clin Microbiol Rev 2001;14:561-83. the diagnosis of infectious diseases. Clin Infect Dis
1999;29:475-88.
38. Fenollar F, Raoult D. Molecular genetic methods for
the diagnosis of fastidious microorganisms. APMIS 52. Relman DA, Schmidt TM, MacDermott RP.
2004;112:785-807. Identification of the uncultured bacillus of Whipple’s
disease. N Engl J Med 1992;327:293-301.
39. Carrol ED, Thomson AP, Riordan FA, et al.
Increasing microbiological confirmation and changing 53. Hryniewiecki T, Gzyl A, Augustynowicz E, Rawczynska-
epidemiology of meningococcal disease on Merseyside, Englert I. Development of broad-range polymerase
England. Clin Microbiol Infect 2000;6:259-62. chain reaction (PCR) bacterial identification in diagnosis
of infective endocarditis. J Heart Valve Dis 2002;11:
40. Taha, MK. Simultaneous approach for nonculture PCR-
870-4.

50 I Clin Biochem Rev Vol 27 February 2006


Infectious Diseases

54. Schuurman T, de Boer RF, Kooistra-Smid AM, van Zwet infectious diseases: the past, the present and the future.
AA. Prospective study of use of PCR amplification and Pediatr Infect Dis J 2002;21:605-12.
sequencing of 16S rDNA from cerebrospinal fluid for 69. Roberson BH, Nicholson KA. New microbiology tools
diagnosis of bacterial meningitis in a clinical setting. J for public health and their implications. Annu Rev
Clin Microbiol 2004;42:734-40. Public Health 2005;26:281-302.
55. CDC. Outbreak of severe acute respiratory syndrome- 70. Jungkind D. Automation of laboratory testing for
worldwide. MMWR Morb Mortal Wkly Rep infectious diseases using the PCR – our past, our present,
2003;52:226-8. our future. J Clin Virol 2001;20:1-6.
56. Corless CE, Guiver M, Borrow R, Edwards-Jones V, 71. Schuurman T, van Breda A, de Boer R, et al. Reduced
Kaczmarski EB, Fox AJ. Contamination and sensitivity sensitivity due to impaired DNA recovery with the
issues with a real-time universal 16S rRNA PCR. J Clin MagNA Pure LC toal nucleic acid isolation kit. J Clin
Microbiol 2000;38:1747-52. Microbiol 2005;43:4616-22.
57. Clarke SC, Diggle MA, Edwards GF. Semiautomation
of multilocus sequence typing for the characterization
of clinical isolates of Neisseria meningitidis. J Clin
Microbiol 2001;39:3066-71.
58. Maiden MC Bygraves JA, Feil E, et al. Multilocus
sequence typing: a portable approach to the identification
of clones within populations of pathogenic organisms.
Proc Natl Acad Sci USA 1998;95:3140-5.
59. Ivnitski D, O’Neil DJ, Gattuso A, Schlicht R, Calidonna
M, Fisher R. Nucleic acid approaches for detection
and identification of biological warfare and infectious
disease agents. Biotechniques 2003;35:862-9.
60. Belgrader P, Hansford D, Kovacs GT, et al. A
minisonicator to rapidly disrupt bacterial spores for
DNA analysis. Anal Chem 1999;71:4232-6.
61. Peruski LF, Peruski AH. Rapid diagnostic assays in
the genomic biology era: detection and identification
of infectious disease and biological weapon agents.
Biotechniques 2003;35:840-6.
62. McHugh TD, Ramsay AR, James EA, Mognie R,
Gillespie SH. Pitfalls of PCR: misdiagnosis of cerebral
nocardia infection. Lancet 1995;346:1436.
63. Roth A, Schaberg T, Mauch H. Molecular diagnosis
of tuberculosis: current clinical validity and future
perspectives. Eur Respir J 1997;10:1877-91.
64. Druce J, Catton M, Chibo D, et al. Utility of a multiplex
PCR assay for detecting herpesvirus DNA in clinical
samples. J Clin Microbiol 2002;40:1728-32.
65. Jiang J, Alderisio KA, Singh A, Xiao L. Development
of procedures for direct extraction of Cryptosporidium
DNA from water concentrates and for relief of PCR
inhibitors. Appl Environ Microbiol 2005;71:1135-41.
66. Birch L, Dawson CE, Cornett JH, et al. A comparison of
nucleic acid amplification techniques for the assessment
of bacterial viablitiy. Lett Appl Microbiol 2001;33:
296-301.
67. Tarasevich IV, Shaginyan IA, Mediannikov OY.
Problems and perspectives of molecular epidemiology
of infectious diseases. Ann N Y Acad Sci 2003;990:
751-6.
68. Nissen MD, Sloots TP. Rapid diagnosis in pediatric

Clin Biochem Rev Vol 27 February 2006 I 51