Real-time PCR
as a Diagnostic Tool for Bacterial Diseases
Max Maurin
Expert Rev Mol Diagn. 2012;12(7):731-754.
Abstract and Introduction
Abstract
In recent years, quantitative real-time PCR tests have been
extensively developed in clinical microbiology laboratories for routine
diagnosis of infectious diseases, particularly bacterial diseases. This
molecular tool is well-suited for the rapid detection of bacteria directly in
clinical specimens, allowing early, sensitive and specific laboratory
confirmation of related diseases. It is particularly suitable for the diagnosis
of infections caused by fastidious growth species, and the number of these
pathogens has increased recently. This method also allows a rapid assessment of
the presence of antibiotic resistance genes or gene mutations. Although this
genetic approach is not always predictive of phenotypic resistances, in specific
situations it may help to optimize the therapeutic management of patients.
Finally, an approach combining the detection of pathogens, their mechanisms of
antibiotic resistance, their virulence factors and bacterial load in clinical
samples could lead to profound changes in the care of these infected patients.
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
Expert Commentary
PCR tests represent a major advance in the rapid diagnosis
of infectious diseases. The qPCR technology has allowed easier and more
effective implantation of molecular diagnosis in the clinical microbiology
laboratories. More recently, qPCR tests allowing rapid detection of antibiotic
resistance determinants have been commercialized. qPCR-based diagnostic
strategy has many advantages over culture, including faster diagnosis, more
rapid turn-around time, easy detection of uncultivable and fastidious
microorganisms, and the possibility to detect microbial DNA in patients already
treated with antibiotics. Conversely, culture remains essential for antibiotic
susceptibility testing and for epidemiological investigations. It is to be
noted, however, that very few commercial qPCR tests have been developed and
clinically validated for diagnosis of bacterial diseases, compared with the
broad spectrum of their potential clinical indications. On the other hand,
commercially available tests remain highly expensive, which limits their
widespread diffusion.
Five-year View
Obviously, qPCR-based diagnostic tests will continue to
expand in the coming years. However, these tests will have to meet three
requirements: adaptation of existing diagnostic tests, especially according to
the discovery of new variants of target genes of specific pathogens,
responsible for false-negative results; expansion of diagnostic applications;
and a lower cost. The development of new diagnostic applications may be slowed
because of technical limitations, but also because of low profitability for the
in vitro diagnostic industry and low cost–effectiveness, especially
for rare diseases. On the other hand, new technologies (e.g., microfluidic
devices) now allow using smaller volumes of reagents, and thus are less costly.
Ideally, in urgent clinical situations, future qPCR tests
should enable early diagnosis and rapid assessment of resistances to first-line
antibiotics, in order to significantly influence emergency treatment and
patient prognosis. These situations include bacteremia and endocarditis,
meningoencephalitis, severe pneumonia (especially nosocomial pneumonia) and neonatal
infections, among others.
An important aspect to consider is the possibility to detect
in a same clinical sample several pathogens responsible for a given syndrome or
clinical situation. These multiparameter assays will greatly improve the
management of patients suffering from clinically unspecific infectious
diseases. Tests based on multiplex qPCR are well suited for this purpose.
However, other technologies will compete with the qPCR method, including
high-throughput DNA sequencing, mass spectrometry and PCR arrays.[265]
A stepwise diagnostic approach may also be developed,
including detection of bacterial DNA (e.g., 16S rDNA), detection of DNA of the
most common pathogens and then detection of rarer etiologies. qPCR tests may
also allow evaluation of the severity of disease and patient's prognosis,
including by direct detection of bacterial genes encoding specific virulence
traits, and by quantification of the bacterial load on admission of the
patient.
Finally, qPCR tests may allow in vivo evaluation of
the antibiotic therapy, especially by rapid detection of antibiotic resistance
genes and by monitoring bacterial load during antibiotic treatment.
Sidebar
Key Issues
- Quantitative real-time PCR (qPCR) technology is a
useful diagnostic tool for early diagnosis of bacterial diseases. - qPCR is particularly adapted for diagnosis of
infections caused by fastidious and uncultivable bacterial species. - qPCR tests are more sensitive than culture in patients
who have received an antibiotic therapy before clinical sample collection. - Although qPCR tests are usually highly sensitive and
specific, false-negative and false-positive results remain major
limitations. - qPCR tests cannot accurately differentiate infection
from commensalism or chronic carriage for opportunistic pathogens and for
clinical specimens collected at nonsterile sites. - Multiplex qPCR tests may allow rapid determination of
the more frequent etiologies of a given syndrome. - The combination of various in-house and commercial qPCR
tests may allow a stepwise diagnostic approach in some specific clinical
situations. - qPCR tests may allow rapid detection of antibiotic
resistance and virulence genes, which may influence both treatment and
prognostic evaluation of infected patients. - Quantification of the bacterial load in clinical
samples may allow better evaluation of prognosis and treatment efficacy in
acutely or chronically infected patients.
＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
Introduction
This review will focus on recent advances in the development
of quantitative real-time PCR (qPCR)-based diagnostic tools allowing detection
and optional quantification of bacterial DNA in clinical specimens. The authors
will describe the existing and potential contributions of qPCR tests for
diagnosis, prognosis and treatment of diseases caused by specific bacterial
species, including assessment of the antibiotic susceptibilities of these
pathogens. Previous reviews have already addressed these issues.[1–3] The author's
goal is to describe the latest advances in this area, citing the most recent
and relevant publications, without attempting to be exhaustive. Only qPCR tests
applied to human specimens will be studied. The article aims to highlight the
many possibilities offered by these new techniques, and also the low number of
solutions marketed as compared with potential applications, as evidenced by the
large number of in-house tests developed in this area ().
Table 1. Quantitative
real-time PCR tests developed for specific pathogenic bacteria species. For
each test the qPCR technology used is specified.
Probes: TaqMan (5' nuclease probe), Dual-Hyb
(dual-hybridization probe), Beacon (molecular beacon), Scorpion (bi-labeled
fluorescent probe/primer hybrids), SYBR green (fluorescent DNA intercalating
agent).
CARDS: Community-acquired respiratory distress syndrome; HRM-qPCR: qPCR with
high resolution melt analysis; LAMP: Loop-mediated isothermal amplification;
LUX: Light upon extension PCR; MIP: Macrophage infectivity potentiator; NS: Not
specified; VRE: Vancomycin-resistant Enterococci.
Real-time PCR Technology
Although extraction of bacterial nucleic acids from clinical
samples remains of variable sensitivity, either using manual or automated
methods, currently available methods are not discussed here because they have
been presented in recent publications.[1,2] qPCR comprises both amplification and fluorescent detection
of amplified DNA target in the same step. As compared with conventional PCR, a
post-PCR step is unnecessary, which reduces the turn-around time of the
analytical process and the risk of contamination with previously amplified
nucleic acids, because there is no need for manipulation of the amplified
products after the reaction. There are two main methods for detection of
amplified qPCR products:[1,2] the first using a fluorescent dye, such as SYBR® Green,
which binds nonspecifically to double stranded DNA, where the resulting DNA–dye
complex absorbs blue light (lmax = 497 nm) and emits green light (lmax
= 520 nm) detected by the qPCR instrument; and the second using fluorescent
resonance energy transfer (FRET) probes, which bind specifically to the
amplified DNA. The term FRET probe refers to a generic mechanism in which
emission/quenching relies on the interaction between the electron-excitation
states of two fluorescent dye molecules.[4] Different FRET probes exist, including 5′-nuclease probes
(also named hydrolysis or TaqMan® probes), dual-hybridization probes (also
named LightCycler® probes), molecular beacons and scorpion probes (Figure 1). A
TaqMan probe carries both a fluorophore and a dark quencher (which absorbs
excitation energy from the fluorescent dye) at its extremities; hydrolysis of
the probe due to 5′-nuclease activity of the DNA polymerase separates the
fluorophore from the dark quencher at the time of elongation, resulting in increased
fluorescence. A dual hybridization probe consists of two DNA probes (upstream
and downstream probes), each carrying a fluorophore at the 3′ end and 5′ end,
respectively. At the time of probe hybridization, the fluorescence emitted by
the upstream probe is absorbed by the downstream probe, which emits light with
a specific wavelength detected by the qPCR instrument. A molecular beacon is a
hairpin-shaped molecule with an internally quenched fluorophore whose
fluorescence is restored when the probe binds to its target DNA. A scorpion
probe consists of a hairpin-shaped single-stranded DNA, labelled at its 5′ and
3′ ends by a fluorophore and a quencher, respectively. The probe is directly
linked to the 5′ end of a PCR primer by a blocker, which prevents the
polymerase from extending the PCR primer at the 5′ end. The DNA polymerase
extends the primer 3′ end, which allows the probe to hybridize to the newly
synthesized DNA. The distance between the fluorophore and the quencher
increases, allowing fluorescence emission detected by the real-time PCR
instrument.
Figure 1.
Real-time PCR probes. (A) 5′ nuclease probe (TaqMan)
probe. Fluorescence occurs when the fluorophore and the quencher are separated
from each other by the nuclease activity of the DNA polymerase. (B) Dual
hybridization probes. The two probes carry a fluorophore, respectively at their
3′ and 5′ ends. At the time of probe hybridization, energy transfer occurs
between the two fluorophores 3′ ends because they are close to each other. (C)
Molecular beacon. This hairpin-shaped molecule has an internally quenched
fluorophore whose fluorescence is restored when the probe binds to its target
DNA. (D) Scorpion probe. The primer (attached to the probe by a blocker)
binds to the target DNA. After extension of the primer by DNA polymerase and
its separation from the target DNA by heating, the probe binds to the newly
synthesized DNA strand. The distance between the fluorophore and the quencher
increases, allowing fluorescence emission.
SYBR Green and dual-hybridization probes are often used for
melting point analysis at the end of the amplification. qPCR presents a number
of advantages over conventional PCR, including a faster turn-around time, a
lower risk of DNA cross contaminations and the possibility to quantify target
DNA in clinical samples. The use of qPCR and FRET probes can sometimes lead to
better sensitivity and/or specificity compared with conventional PCR,
especially when a large number of target sequences are available, allowing the
design of appropriate primers and probes. qPCR shares the same limitations as
PCR methods, including false-negative results due to inhibition of DNA
polymerase or variations in the target nucleic acid sequence among strains of a
same bacterial species, false-positive results especially because of clinical
sample contamination, and difficulties in differentiating commensa-lism from
pathogenicity for opportunistic pathogens. These limitations can be partially
controlled by proper use of negative, positive and internal controls. A
positive control (i.e., containing target DNA) will check that the DNA
amplification reaction has occurred. A negative control (DNA-free sample) will
check that no external DNA contamination has occurred. An internal process
control will test the presence of inhibitors of DNA amplification (especially
DNA polymerase). A negative qPCR result will be interpreted as unreliable in
the presence of inhibitors, and will necessitate specific procedures to remove
these inhibitors.
Diagnostic Applications for Bacterial Diseases
Panbacterial qPCR
Amplification and sequencing of the gene encoding 16S
ribosomal RNA using universal primers (panbacterial PCR) has become widely used
in clinical laboratories for identification of bacteria at the genus and
species levels, either after their isolation in culture or directly from
clinical samples.[5] Panbacterial qPCR tests for direct detection of bacterial
DNA from clinical samples have also been developed.[5–7] This method
allows rapid and wide-spectrum detection of bacteria, especially in patients
with negative cultures because of previous administration of antibiotics. It is
often successful, provided that a single bacterial species is present in the
clinical sample and the bacterial load is high enough to be detected. When
multiple bacterial species are present in a clinical sample, a mix of 16S
ribosomal DNA (rDNA) sequences will be obtained, and identification of
individual species usually necessitates specific techniques (DNA cloning,
high-throughput sequencing, ESI mass spectrometry, and so on) that are not yet
available in most clinical laboratories. It is to be noted, however, that commercial
software potentially allowing specific identification of mixed 16S rDNA
sequences is currently sold.[8] Almost all clinical samples can be tested by this method,
but those contaminated with a rich and varied commensal flora are not suitable.
Examples of possible applications include detection of bacteria responsible for
meningitis, endocarditis, arthritis, osteitis and deep abscesses and
suppurations.[5–7,9,10] Panbacterial qPCR has also been used for quantification of
the bacterial flora, for example, in blood,[11] intraocular samples[12] or chronic wounds.[13]
qPCR for Easy-to-Grow
Bacteria
Staphylococcus Aureus.S. aureus is responsible
for a wide range of infections in humans including skin and soft tissue
infections, arthritis, osteitis, deep abscesses, pneumonia, endocarditis,
urinary tract infections, enteritis and bacteremia.[14,15] In addition,
methicillin-resistant S. aureus (MRSA) is a major nosocomial pathogen.[14,15] In healthy
carriers, S. aureus is found primarily in the nasal cavity. Culture remains the
reference method for the screening and clinical diagnosis of S. aureus/MRSA carriage
and infection.[15] qPCR tests may be useful for rapid detection of MRSA nasal
carriage.[16–21] The cost–effectiveness of this strategy has been debated,
and a targeted rather than universal screening is warranted in hospitals with
an MRSA incidence lower than 5% of admitted patients.[22] qPCR tests may
also be useful for diagnostic purposes.[14] In-house and commercial qPCR tests have been developed for
rapid detection of S. aureus/MRSA in clinical samples and blood cultures.[21,23,24]
Streptococcus Pneumoniae.S. pneumoniae is
primarily responsible for respiratory tract infections (e.g., pneumonia),
meningitis and bacteremia. The natural reservoir of this bacterium is the
oropharynx of humans. Therefore, detection of S.
pneumoniae using culture or qPCR from
respiratory samples may correspond to infection or contamination by the
commensal flora. Quantitative sputum cultures are used to tentatively
differentiate infection from contamination, and authors have tried to do so
using qPCR.[25]S. pneumoniae
DNA can also be detected by qPCR in blood samples or in positive blood cultures
in patients with bacteremia.[26–30] qPCR is currently used on a routine basis mainly for rapid
detection of S. pneumoniae DNA in cerebrospinal fluid in patients with meningitis,[31] and in sputum
samples in patients with pneumonia.[26,28] Among gene targets, the lytA and Spn9802 genes have been shown to allow more specific detection of S. pneumoniae
than the ply gene.[29,30] qPCR tests targeting the cpsA gene (capsule protein) also allow determination of S. pneumoniae
serotype.[26]
Streptococcus Agalactiae.S. agalactiae is
responsible for skin and soft tissue infections, urinary tract infections,
bacteremia and occasionally severe neonatal infections.[32] Culture
remains the reference diagnostic method for most S. agalactiae
infections. The prevention of neonatal infections is currently based on the
systematic screening of pregnant women for S. agalactiae vaginal carriage (usually by culturing a vaginal
swab collected during the eighth month of pregnancy) and antibiotic treatment
in those with a positive screening test at the time of delivery.[32] However,
approximately 15–20% of parturient women have unknown S. agalactiae
carriage status. A number of qPCR tests have been evaluated for rapid per
partum detection of S. agalactiae in vaginal and rectal samples of the parturient woman.[33–37] These tests
are well-adapted in this situation, allowing rapid and sensitive detection of S. agalactiae,
and more appropriate decisions concerning administration of antibiotic prophylaxis.
Streptococcus Pyogenes.S. pyogenes is
the primary bacterial cause of pharyngitis.[38] Culturing S.
pyogenes from throat samples remains the
reference method, allowing evaluation of antibiotic resistances, especially in
patients allergic to b-lactams or in case of treatment failure. Many tests
allowing rapid detection of S.
pyogenes antigens in throat samples are now
available.[38] These tests display high sensitivities and specificities
(>95%) compared with culture,[38] and although more expensive, they are more suitable for a
quick decision regarding the need for antibiotic therapy.[38] Most
guidelines still recommend obtaining a throat culture in patients with severe
pharyngitis and a negative antigen test because of higher sensitivity of the
former technique.[38] qPCR tests for rapid detection of S. pyogenes
DNA in throat samples have been developed.[39,40] Their sensitivity is higher than that of culture. However,
these tests remain costly and they cannot be used as point-of-care tests, nor
for evaluation of antibiotic resistances.
Listeria Monocytogenes.L. monocytogenes
primarily causes bacteremia, meningoencephalitis, enteritis and maternofetal
infections. This bacterium can be grown from various clinical samples,
including blood, cerebrospinal fluid, placenta and fetal or neonatal specimens.
A qPCR test was recently developed for rapid detection of L. monocytogenes
DNA in the cerebrospinal fluid of patients with meningoencephalitis.[41]
Neisseria Meningitidis.N. meningitidis is
a commensal of the oropharynx in humans and is primarily responsible for
bacteremia, meningitis and purpura fulminans.[42] Detection of N. meningitidis
oropharyngeal carriage is not performed on a routine basis, whereas diagnosis
of bacteremia, meningitis and purpura fulminans is usually based on isolation
of bacteria from blood, cerebrospinal fluid or less frequently from other
clinical specimens (e.g., skin lesion biopsies). qPCR may be useful for early
detection of N. meningitidis DNA in these clinical samples, especially in patients with
very severe infections (e.g., purpura fulminans).[43–46] Its
sensitivity is greater than that of culture in patients who have received
antibiotics before clinical sample collection, a common situation for N. meningitidis
infections. False-negative results have been reported, however, due to nucleic
acid polymorphism in the ctrA gene.[47,48] Finally, specific qPCR tests also allow determination of
the involved serogroup,[44,45] thereby guiding vaccine prophylaxis in healthy persons who
have been in contact with the patient. Vaccines are available to prevent
infections caused by serogroups A, C, Y and W135 strains, but not yet for
serogroup B strains, which are most prevalent in many developed countries.
Haemophilus Influenzae.H. influenzae is
primarily involved in upper and lower respiratory tract infections (e.g.,
otitis, sinusitis, epiglottitis, bronchitis and pneumonia), meningitis and
bacteremia.[49] Infections caused by H.
influenzae serotype b (Hib) predominate and
are the more severe, but the systematic use of specific anti-Hib vaccines in
infants has dramatically reduced the incidence of these infections in developed
countries. Diagnosis relies on culture of this pathogen from various clinical
specimens. However, qPCR tests have been developed for rapid detection of H. influenzae
DNA, especially in the cerebrospinal fluid and sputum samples from patients
with meningitis or pneumonia, respectively.[50,51] Some qPCR tests also allow determination of the capsular
type (a to f) of H. influenzae strains.[52] Wang et al. recently developed a new qPCR test allowing better
detection of non-b H. influenzae-related infections, a significant advantage in
Hib-vaccinated populations where other capsular types may now be more
frequently encountered.[50]
Shiga Toxin-producing Escherichia Coli. In
recent years, outbreaks of severe enteritis cases caused by Shiga toxin-producing
E. coli (STEC) have been described worldwide, with a variable
percentage of patients experiencing severe hemolytic and uremic syndrome.[53,54] The involved E. coli strains may be
of different pathotypes (enterohemorragic, enteroaggregative) and genotypes,
with probable variable virulence potential. However, their most significant
virulence factor is the production of the Shiga like toxins (also named
Vero-toxins) encoded by the stx1 and stx2 genes. Because STEC are difficult to isolate from the rich
and varied intestinal flora and display only few and inconstant phenotypic
traits (e.g., sorbitol-negative 'O157' E.
coli strains), qPCR tests targeting stx1 and stx2 in stool
samples are currently the most efficient diagnostic techniques, allowing a
rapid, sensitive and specific diagnosis of STEC-related infections.[55–60] The
sensitivity of qPCR tests may be further increased by testing stool specimens
that have been previously enriched by short-time culture.[56]
qPCR for Slow-Growing,
Fastidious-Growing & Uncultivable Bacteria
Mycobacterium Species.
Most mycobacterial species pathogenic for humans are slow-growing
microorganisms.[61,62] Also, the traditional methods for their phenotypic
identification and antibiotic susceptibility testing are fastidious and
time-consuming. The modern diagnostic strategy of mycobacterial diseases
includes rapid culture in liquid media (e.g., Middlebrook) using automated
systems, direct detection in clinical samples using nucleic acid amplification
methods, molecular identification of Mycobacterium spp. from positive cultures or directly from clinical
samples, and direct detection of antibiotic resistance genes.[61] qPCR methods
have been developed to detect either nontuberculous mycobacteria (NTM)[63–66] or tuberculous
mycobacteria.[67–71] Because Mycobacteriumleprae (the agent of leprosy) is an uncultivable microorganism,
reverse transcriptase qPCR tests have been developed for simultaneous detection
and determination of the viability of this bacterium.[72] qPCR tests for
diagnosis of tuberculosis display sensitivities close to that of culture
methods for respiratory samples,[67,68,70,71] but their usefulness has also been demonstrated for
extrapulmonary specimens (e.g., cerebrospinal fluid, pleural fluid, synovial
fluid, lymph node biopsies).[69,70] These tests are more sensitive for smear-positive than for
smear-negative samples.[69,71] In-house qPCR tests targeting multicopy DNA such as the
IS6110 showed increased sensitivity.[69,73,74] Commercial qPCR tests may not allow identification of all Mycobacterium
species, especially for NTM. A number of in-house qPCR tests have been
developed for species identification of isolated strains, but few have been
evaluated directly on clinical samples.[66]
Chlamydia Trachomatis & Neisseria Gonorrhoeae.C. trachomatis[75] and N.
gonorrhoeae[76] are two main etiological agents of sexually transmitted
infections (STIs). Both organisms may cause obstetrical complications and
infection in the neonate. Culture of N.
gonorrhoeae is usually obtained using enriched
selective media and allows testing for acquired resistances to antibiotics,
especially to β-lactams, tetracyclines and fluoroquinolones.[76]C.
trachomatis isolation necessitates eukaryotic
cell systems because of its strict intracellular lifestyle.[75] Serological
techniques for C. trachomatis infections lack specificity, and these techniques are not
useful for N. gonorrhoeae.[75,76] Patients suffering STIs are often coinfected with both
pathogens. PCR-based techniques are now considered reference diagnostic methods
for C. trachomatis infections, and a number of commercial qPCR tests now allow
rapid, sensitive and specific detection of both C. trachomatis
and N. gonorrhoeae in urogenital samples.[77–81] qPCR tests should be able to detect the new variant of C. trachomatis
with a deletion in the cryptic plasmid.[82]
Chlamydophila (Chlamydia) Pneumoniae & Chlamydophila
(Chlamydia) Psittaci. These two
species are strict intracellular bacteria responsible for pneumonia.[83,84]C.
pneumoniae is responsible for approximately
10% of community-acquired pneumonia, and 5% of bronchitis and sinusitis
worldwide, in both adults and children.[83,85]C. psittaci is
the agent of psittacosis, a zoonosis occurring in specific endemic areas,
especially in persons in contact with infected poultry and in psittacine
owners.[84] Culturing of these pathogens in cell systems is tedious,
poorly sensitive and even a risk for the laboratory personnel (C. psittaci).[83,84] Serological
techniques based on detection of anti-lipopolysaccharide antibodies are not
species-specific and only allow a retrospective diagnosis.[83,84] Because C. pneumoniae
infections are common, serum residual antibody titers are found in many
patients. Compared with serological methods, PCR-based techniques are useful
for early diagnosis of Chlamydophila-related infections.[85] Target sequences include a portion of the 16S rDNA,[86,87] the 16S–23S interspacer
sequence,[87] outer membrane proteins OmpA[88,89] and OmpB,[90] or the incA and envB genes,
respectively encoding inclusion membrane protein A[91] or a
cysteine-rich protein.[92] qPCR tests display sensitivities of 10 inclusion forming
units (IFU)[89,92] to 0.1–1 IFU.[86,93] A number of commercial or in-house qPCR tests allow rapid
detection of C. pneumoniae in respiratory samples from pneumonia patients.[85,86,93–95]
However, these tests cannot differentiate chronic oropharyngeal carriage and
acute respiratory infection.[95] qPCR tests have also been used for detection of C. pneumoniae in
cerebrospinal fluid and vascular tissues in order to tentatively correlate
chronic infection with this pathogen and neurological diseases (e.g., multiple
sclerosis) or cardiac diseases (e.g., coronary artery disease[88,90]). Home-made
qPCR tests have been developed for specific detection of C. psittaci in
respiratory samples.[87,89,91,92]
(本文太長。欲讀全文者可留言，或查詢 google)
沒有留言:
張貼留言