7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 1/9
Disponible en ligne sur
www.sciencedirect.com
Médecine et maladies infectieuses 43 (2013) 322–330
General review
Broad-range PCR: Past, present, or future of bacteriology?
La PCR « universelle » : passé, présent ou futur de la bactériologie ?
A. Renvoisé , F. Brossier , W. Sougakoff , V. Jarlier , A. Aubry∗
Laboratoire de bactériologie-hygiène, faculté de médecine Pierre-et-Marie-Curie Paris-6, site Pitié-Salpêtrière, 91, boulevard de l’Hôpital, 75634 Paris cedex 13,
France
Received 6 February 2013; received in revised form 8 April 2013; accepted 17 June 2013
Available online 19 July 2013
Abstract
PCR targeting the gene encoding 16S ribosomal RNA (commonly named broad-range PCR or 16S PCR) has been used for 20 years as a
polyvalent tool to study prokaryotes. Broad-range PCR was first used as a taxonomic tool, then in clinical microbiology. We will describe the use
of broad-range PCR in clinical microbiology. The first application was identification of bacterial strains obtained by culture but whose phenotypic
or proteomic identification remained difficult or impossible. This changed bacterial taxonomy and allowed discovering many new species. The
second application of broad-rangePCR in clinicalmicrobiology is the detectionof bacterialDNAfrom clinical samples; wewill review the clinical
settings in which the technique proved useful (such as endocarditis) and those in which it did not (such as characterization of bacteria in ascites,
in cirrhotic patients). This technique allowed identifying the etiological agents for several diseases, such as Whipple disease. This review is a
synthesis of data concerning the applications, assets, and drawbacks of broad-range PCR in clinical microbiology.
© 2013 Elsevier Masson SAS. All rights reserved.
Keywords: 16S ribosomal RNA; Broad-range PCR;Molecular diagnosis
Résumé
La PCR amplifiant le gène codant pour l’ARN ribosomal 16S (plus communément appelée PCR 16S ou PCR universelle) est utilisée depuis
20 ans comme outil d’étude polyvalent des procaryotes. La PCR 16S a d’abord été utilisée comme outil d’étude taxonomique, puis son usage
s’est répandu en microbiologie clinique. Dans cette revue, nous détaillons les applications de la PCR 16S en microbiologie clinique. La première
application a été l’identification de souches bactériennes obtenues par culture, mais dont l’identification phénotypique ou protéomique est difficile
ou impossible. Cette utilisation a modifié les perspectives de la taxonomie bactérienne et a permis la découverte de nombreuses nouvelles espèces.
L’autre application de la PCR 16S enmicrobiologie clinique est la détection d’ADNbactériendirectement à partir de prélèvements cliniques ; nous
proposons une synthèse des situations cliniques dans lesquelles cette technique est utile (par exemple les endocardites) et celles dans lesquelles
elle ne l’est pas (par exemple pour les infections du liquide d’ascite chez les patients cirrhotiques). Cette technique a permis d’identifier l’agent
infectieux responsable de plusieurs pathologies (par exemple la maladie de Whipple). Cette revue fait la synthèse des données sur les indications,
avantages et inconvénients de la PCR 16S en microbiologie clinique.
© 2013 Elsevier Masson SAS. Tous droits réservés.
Mots clés : ARN ribosomal 16S ; Diagnostic moléculaire ; PCRuniverselle
∗ Corresponding author.
E-mail address: [email protected](A. Aubry).
1. Introduction
The debate on the contribution of 16S PCR can be heated
and opposes those who support its use and consider it as a
tool for broad-range identification to those who consider that its
indications are limited. This molecular technique often called
“broad-range” has rapidly become an essential element for
0399-077X/$ – see front matter© 2013 ElsevierMasson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.medmal.2013.06.003
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 2/9
A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330 323
Fig. 1. Schematic representation of 16S ribosomal RNA gene. Non-hypervariable regions are those containing conserved regions used as target sequences for
universal primers. Amplification followed by sequencing of hypervariable regions can discriminate between bacterial species. Schematic representations of short
and long amplification of the gene are presented.
Représentation schématique du gène codant pour l’ARN ribosomique 16S. Les régions non hypervariables sont celles au sein desquelles on trouve les régions
conservéesutiliséescomme séquencescibles pour les amorces« universelles». Lesrégionshypervariablessont cellesdont l’amplification et le séquencagepermettent
de différentier les espèces bactériennes. Les représentations schématiques d’un 16S long et d’un 16S court sont également figurées.
bacteriological diagnosis in hospitals. We reviewed the litera-
ture to determine the contribution of this technique to clinical
microbiology.
2. What is 16S?What is its contribution?
2.1. The bacterial ribosome
The ribosome is a ribonucleoprotein complex (made up of
proteins and RNA); it allows synthesizing proteins (also called
translation) by using mRNA as a source of information andtRNA associated with amino acids as substrates. In bacteria,
ribosomes are composed of a large sub-unit (50S) and a small
sub-unit (30S). The functional ribosome (composed of the two
sub-units assembled around the mRNA) has a molecular mass
of 2.5-megadalton and a sedimentation coefficient of 70S. The
small sub-unit is composed of 16S ribosomal RNA (encoded
by a gene of 1500 nucleotides) and of 20 proteins; it allows
“reading mRNA”. The large sub-unit is composed of 23S ribo-
somal RNA (encoded by a gene of 2900 nucleotides), of 5S
ribosomal RNA (encoded by a gene of 120 nucleotides), and
of 30 proteins; it allows synthesizing the protein correspond-
ing to the mRNA “read” by the small sub-unit. Furthermore,
various protein factors act on the ribosome at various stages of translation.
2.2. 16S rRNA
Ribosomal RNA (rRNA) 16S is the constituent RNA of the
small ribosomal sub-unit of prokaryote 30S (Fig. 1). The gene
encoding this rRNA is the “16S rRNA gene” also called ribo-
somal 16S RNA or rrs [1], present in all bacterial species in a
variablenumberof copies [2]. It is composedof 1500nucleotides
and includesnine hypervariableregions. The associationof con-
served regions and variable regions theoretically allows using
this gene to identify and detect all bacterial species.
In the1980s, itwasdemonstratedthat thephylogenicrelation-
ships among living beings could be determined by comparing
their nucleic sequences [1]. Indeed, since the 16S rRNA gene
encode for an rRNA with a constant function in evolution, it
could be used as a molecular timer to follow changes in bacte-
rial evolution. This gene was used this way in the late 1980s, as
a study tool for bacterial evolution [3], and had a major part in
the study of bacterial phylogeny and taxonomy [4].
3. Limitations of broad-range 16S PCR. Globalproblems
3.1. Cost and lack of automatization
The 16S molecular tool has some global limitations. Firstly,
thecost remains high, even though it was lowered since the tech-
niquewasfirst described[5]. Some authors suggestperforming a
“short” 16S (cf. addendum) to reduce the cost whilemaintaining
a good taxonomic value (Fig. 1) [1,6].
Furthermore, even though marketed systems such as
MicroSeqTM (Applied Biosystems) were developed [7], the
non-automatization of the technique was a limiting factor for
its global use. Nevertheless, the development of high out-put sequencing techniques could allow incorporating stages of
broad-range 16S PCR in a robotized system.
3.2. Volume of the test sample
The detection threshold for end-point PCR (such as 16S) is
weak (theoretically 1 to 5 copies of DNA), but only 1 to 5L of
the sample are used for PCR, whereas 100 to 5000 times greater
volumes are used for usual bacterial culture. The weak volume
of sample tested and PCR inhibitors may be associated to false
negative results (Fig. 2) [2].
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 3/9
324 A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330
Fig. 2. Contribution and limitations of broad-range PCR.
Intérêts et limites de la PCR 16S.
3.3. False positive results
Contaminations during gene amplifications may occur and
make it difficult to interpret results (Fig. 2). The problem of
false positives may be limited by: using “DNA free” reagents,
dedicated pipettes, cotton tip pipettes, three separate rooms for
each step (1: preparation of reagent mix, 2: DNA extraction,
and 3: amplification), nucleotides containing dUTP (deoxyuri-
dine triphosphate which is used instead of dTTP during PCR;
using uracil-DNA glycosylase in the reagent mix for the nextPCR allows degrading the DNA containing uracil, but has no
effect on“natural”DNAcontaining thymine)andwith the exper-
tise of a confirmed microbiologist. Using negative control for
each step of the protocol (extraction-amplification-sequencing)
is mandatory to detect these false positive results [8].
4. When should 16S PCR be used?
4.1. 16S PCR and bacterial identification
4.1.1. Using 16S PCR for bacterial identification of strains
isolated in culture
Bacterial identification from cultures usually relies on the
phenotypic characteristics of the bacterium: staining (Gram
for example), morphology, ability to grow on some culture
media, biochemical features detected by various techniques
on the market (APITM galleries [Biomérieux], Vitek TM sys-
tems [Biomérieux], PhoenixTM [BD biosciences], BiologTM
[Biolog], etc.). Nevertheless, some bacteria are badly identified
phenotypically for various reasons:
• small number of phenotypic characters expressed;
•
stress may have altered the phenotypic characters;
• absence of rare bacteria in the databases of systems available
on the market;
• phenotypic characters difficult to detect for some bacteria
difficult to cultivate.
In these cases, amplificationand sequencingof the 16S rRNA
gene followed by the comparison of the obtained sequencewith
databases have proved their value for bacterial identification
[9]. This is a broad-range method, accurate and reliable, theinter-operator variability of which is limited compared to usual
techniques [2].
The authors of two important studies measured the perfor-
mance of 16S PCR for the identification of clinical isolates not
identified by conventional methods. Kiratisin et al. reported that
isolates not identified by conventional techniqueshad been iden-
tified by a long 16S PCR (cf. addendum) at the species level for
74% of strains compared to 83.1% for Mignard et al. who had
used a short 16S (cf. addendum); at the genus level for 21% of
strains compared to 15.8% forMignard et al.; and that 1 to 5%of
strains could definitely not be identified by Kiratisin et al. com-
pared to1%byMignard et al. [5,10]. Furthermore, 16SPCRwas
especially effective for the identification of Gram-positive aero-bicbacilli. For 136 clinical strains of Gram-positivebacilli badly
identified by conventional techniques (only 52.2%of strains had
been identifiedat the genus level by usual techniques),Bosshard
et al. reported that 16S PCR had identified 65.4% of strains at
the species level (defined by similarity≥ 99%) and 31.6%more
at the genus level (defined by similarity ranging from 95 to 99%)
[11].
Nevertheless, 16SPCRisweaklyeffective as an identification
tool for some species when the variations of sequences are too
small amongspecies to allowdiscriminating.This is the case, for
example, for: some streptococci (Streptococcus mitis and Strep-
tococcus pneumoniae), some enterobacteria ( Escherichia coli
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 4/9
A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330 325
and Shigella spp.), or some Bacillus spp. ( Bacillus cereus and
B. anthracis) [2,4,10,12]. In these cases, other genes can be
used to discriminate among these species, such as rpoB (which
encodes the sub-unit beta of RNA polymerase), or other target
genes including variable sequences surrounded by conserved
sequences such as: recA, tuf , gyrA, and gyrB [12].
Finally, some species have several copies of the 16S rRNA
gene. These copies may present with variations, which has for
consequence to generate sequences containing ambiguousness
[2]. Furthermore, some authors have reported a certain degree of
“micro-heterogeneity”among species; this corresponds to intra-
species variations inferior to 0.5% and to different genotypes of
sub-species [1]. But these small variations would have a weak
impact on routine hospital practice [10].
4.1.2. Contribution of 16S PCR for the description of new
species
Defining the concept of bacterial species is a delicate point;
the correspondence between a strain and a previously described
species is based on phenotypic and genetic similarity [13]. Thecurrent methods used to define prokaryote species do not allow
covering the diversity found in nature and bacterial taxonomy
is influenced by breakthroughs in the genetics of populations,
ecology, genomics, and by the facility with which data may be
obtained [13]. Traditionally, a bacterial species is defined from
DNA-DNA hybridization, which is a complex and costly tech-
nique, less and less frequently used [14]. The genetic definition
of a species is quantifiable taking into account the kinetics of
DNA-DNA recombination. A species is then defined geneti-
cally as a group of strains, which have DNA-DNA relationships
resulting in:
• a rate of DNA-DNA hybridization greater or equal to 70%;
• thermic stability of hybrids less or equal to 5 ◦C [4].
The Stackebrandt committee determined that any descrip-
tion of a new species should include a complete sequence of
the gene encoding for 16S rRNA [14]. Nevertheless, there is
no clearly determined threshold value for rates of similarity,
beyondwhich the scientific community agrees to define the rank
of species [4]. Indeed, if a close similarity (≥ 97%) between two
16S sequences does not allow systematically determining that
two strains belong to the same species, the contrary holds true:
similarity between two 16S sequences less than 97% allows
determining that the corresponding strains belong to differentspecies [4,13,15].When 16S rRNAsequencespresentmore than
97%of homology, theStackebrandtcommittee recommends that
the study of DNA-DNA hybridization rate as well as thermic
stability of hybrids remain the reference to define a genomic
bacterial species [14].
The routine use of 16S PCR for bacterial identification in a
laboratory has allowed discovering new species or new bacte-
rial genera. Numerous strains with a rate of genetic similarity
inferior to 97%were thus discovered thanks to the globalization
and the availability of the molecular tool. Broad-range PCR has
imposed itself as a means of discoveringnew taxa. For example,
Drancourt et al. reported discovering 11 new bacterial species
among 1404 isolates for which usual techniques had not been
contributive [15]. Rates of similarity greater than 99% had been
chosen to assign a strain to a previously described species, in
this study; 97 to 99% to assign it at the genus level; and less
than 97% to consider it as a new species. Thus, 16S PCR is tool
of great importance for the exploration of bacterial diversity
[4,15].
Finally, the main problem is the threshold from which a rate
of similarity would allow assigning the studied sequence to a
species, or to a genus, is not clearly determined. Several thresh-
oldshave been suggested.Most taxonomists accept thresholdsof
97% for the genus and 99% for species [2]. Nevertheless, some
authors suggest using a threshold of 99.5% for species, whereas
for others, it seems impossible to determine a single threshold
for all the bacterial world [6]. Recommendations for the inter-
pretation by bacterial “categories” were recently suggested by
the Clinical and Laboratory Standard Institute (CLSI), but their
prohibitive costs restrict their availability to routine laboratories
[12].
It should be noted that the rates of similarity obtained dif-fer, whether a “short 16S” or a “long 16S” is used, but also
depending on the program used, and the parameters used for a
given program (cf. addendum) [1]. It should also be kept in mind
that the final interpretation of a bacterial identification result
based on 16S rRNA, should obviously also take into account the
phenotypic characteristics.
4.2. 16S PCR and bacterial detection from clinical samples
4.2.1. Global features
Even though 16S PCR has limitations, it is an alternativemethod, independent of culture, used for bacterial detection
directly from clinical samples [2]. The advantage of 16S PCR
compared to usual bacterial culture was clearly demonstrated in
the following circumstances: (1) detection of bacteria difficult
to grow (mycobacteria and other bacteria containing mycolic
acids, intracellularbacteria such asCoxiella burnetii, Bartonella
spp., Ehrlichia spp., Anaplasma spp., Francisella tularensis,
Mycoplasma spp.) and (2) detection of bacteria the usual culture
of which is made impossible because of, among other reasons,
a previous antibiotic treatment (Fig. 2) [16]. But, as Rampini
et al. have very well demonstrated, the contribution of this tech-
nique depends almost exclusively on the value of criteria used
to select patients suspects of infection, as is illustrated in Fig. 3.It should be specified that 16S PCR used for bacterial detection
never allows strain culture and thus prevents obtaining an antibi-
ogram,which is a limitation for the therapeutic management of
patients. It should also be noted that detection of bacterial DNA
(by amplification of all or a part of the gene) in a sample, if it
is positive, should be followed by bacterial identification with
sequencing of all or a part of the 16S gene (which is similar to
identification of strains mentioned in Section 4.1).
Furthermore, the scope of 16S PCR is not theoretically
restricted to samples from normally sterile sites and sites of
mono-microbial infections, because cloning and pyrosequenc-
ing techniques allow identifying various microorganisms from
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 5/9
326 A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330
Fig. 3. It is essentialto selectpatientsfor whom a diagnostic test is contributive (thus improving pre-test probability). Defining criteria fora biological test: sensitivity,
specificity, negative predictive value (NPV), and positive predictive value (PPV). This is a previously reported example, with a sensitivity of 93% and a specificity
of 98% for broad-range PCR applied in a population with a prevalence of meningitis at 44% [17]. Broad-range PCR is first performed on cerebrospinal fluid (CSF)
for any patient. This corresponds to a low prevalence of meningitis. Secondly, broad-range PCR is performed on selected samples for patients highly suspect of
meningitis. This corresponds to a high prevalence of the disease. We observe that negative and positive predictive values vary according to the incidence of the
disease. Thus, it is essential to select patients highlysuspect of infection (whatever the type of infection) to adequately use broad-range PCRin a targeted population.
This allows improving the pre-test diagnostic probability and consequently the positive predictive value of broad-range PCR.
Illustration de l’intérêt de déterminer les patients pour lesquels un test diagnostique a un intérêt (augmenter la probabilité pré-test). Définitions des critères d’un
test biologique: sensibilité, spécificité, valeur prédictive positive (VPP) et valeur prédictive négative (VPN). On prend ici l’exemple d’une publication rapportant
une sensibilité de 93% et une spécificité de 98% pour une PCR 16Sappliquée dans une population où la prévalence de laméningite était de 44% [17]. On applique
la PCR16S dans une première situation où la PCR16S est effectuée sur des prélèvements de liquides céphalorachidiens (LCR) tout-venants. On se place donc dansune situation où la prévalence des infectionsméningées est faible. On applique ensuite le test dans une seconde situation où seuls les prélèvements de LCRsuspects
de méningite sont soumis à une PCR 16S. On se place alors dans une situation où la prévalence des méningites est élevée. On observe que les valeurs prédictives
positives et négatives du test varient selon que le test est appliqué à une population à faible (situation 1) ou à haute incidence (situation 2) de la maladie. Il est donc
indispensable de sélectionner les patients suspects d’infection (cela quel que soit le cadre nosologique) afin d’utiliser la PCR16S à bon escient dansune population
ciblée. Cela permet d’améliorer la probabilité diagnostique pré-test et par conséquent la valeur prédictive positive de la PCR16S.
the same sample [18]. Nevertheless, such a strategy, long and
costly, is difficult to apply in the routine activity of a laboratory.
4.2.2. Cardiovascular infections
The example of endocarditis with negative blood cultures
illustrates the contribution of 16S PCR for the diagnostic and
therapeutic management of patients. Indeed, the positivity of bloodcultures ispart of Duke’s criteria for the diagnosisof infec-
tious endocarditis, but in 2.5 to 31% of cases, blood cultures
remain negative [19]. Furthermore, Greub et al. reported that
the culture of cardiac valves has a weak sensitivity (13%) and is
not more contributive to the diagnosis than 16S PCR [20], usual
culture of cardiac valves also being associated to numerous false
positives (soiled cultures). For more than 10 years, the contri-
bution of 16S PCR on cardiac valves of patients undergoing
surgery for infectious endocarditis was largely described in the
literature [20–25]. For example, Greub et al. reported that PCR
had allowed obtaining an etiological diagnosis for 23% of endo-
carditis with negative blood cultures [20]. The authors of these
studies showed that 16S PCRwas especially contributive when:
(i) the patients had received previous antibiotic therapy, and (ii)
wheninfectious endocarditiswasdue toa fastidiousbacteriumor
to streptococci [19]. For example, Podglajen et al. had obtained
four diagnoses of endocarditis due to Bartonella sp. with PCR,
out of six cases of endocarditiswith negative blood cultures [23].
This also allows improving the post-surgical therapeutic man-agement of patients [22–24]. It was also suggested to integrate
the molecular approach to Duke’s criteria for the diagnosis of
infectious endocarditis, but this has not been taken into account
yet. In any case, 16S PCR isno longer an isolated diagnostic tool
and is currently part of a multimodal diagnostic strategy (sero-
logical, molecular, and histopathological) for endocarditis with
negative blood cultures [19]. Fournier et al. used this strategy
to identify a bacterium in 62.7% of cases of endocarditis with
negativebloodcultures,with57.3%of C. burnetii, 19.2%of Bar-
tonella sp., 4% of Tropherymawhipplei, 0.4% of Legionella sp.,
0.4% of mycobacteria, 0.2% of Mycoplasma hominis, 0.2% of
Gemella morbillorum, and 0.2% of Abiotrophia defectiva [19].
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 6/9
A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330 327
But 16S PCR on cardiac valves may be associated to false
positives. The contaminations of PCR have already been men-
tioned(in13 to 20% of cases [20]), and it was also demonstrated
that, for infectious endocarditis, bacterial DNA could persist
long after antibiotic treatment, especially for Bartonella spp.
and Streptococcus spp., making difficult the interpretation of
molecular results in case of a new episode of infectious endo-
carditis [26]. Conversely, false negatives may also occur in case
of previous antibiotic therapy, when inhibitors are present in
the sample or when a “bad” aliquot of the valve is selected for
the PCR (when the selected aliquot does not correspond to the
infectious micro-focus) [20,23,25].
But 16S PCR performed on whole blood in non-operated
patients and thus without any cardiac tissue for analysis is
not contributive for endocarditis with negative blood cultures,
because its sensitivity is too weak for the etiological diagnosis
of infectious endocarditis [19].
Finally, for endocarditis with positive blood cultures, 16S
PCR on cardiac valves allows confirming the diagnosis. There
may nevertheless be discordance between blood cultures and16S PCR when the delay between the initiation of antibi-
otic therapy and surgery is too important; thus for 30 patients
with endocarditis with positive blood cultures, Podglajen et al.
obtained four cases of negative PCR with an average duration
of antibiotic therapy of 34.5 days whereas for the 26 cases with
a positive PCR, the average duration of antibiotic therapy was
24.6 days [23].
4.2.3. Neuromeningeal infections
The diagnosis of neuromeningeal infections, and more
especially of meningitis, is particularly important becauseman-
agement of these severe infections isan emergency. Broad-range16S PCR has proved contributive in this context, in specific
situations: when patients have been given a previous antibiotic
therapy, and/or when the microscopic examination is negative,
and/or when usual culture remains negative [27–29]. For other
authors, PCRallows ruling out the diagnosis of meningitis when
molecular technique is used on “routine” samples [30]. But for
thespecificcase of postoperativemeningitis, PCRdoesnot allow
performing any complementarymicrobiological diagnosis com-
pared to culture and thus would not change the management of
aseptic postoperativemeningitis [31].
Theuse of 16S PCR for the diagnosis of community-acquired
or postoperative meningitis is not clearly defined and according
to authors, there are important differences in sensitivity for 16SPCRcompared to culture; the great variability of protocols used
and of extraction techniques may account for these differences
[29]. Furthermore, the prevalence of the diseasevaries according
to study results, which implies that the test characteristics are
not comparable (Fig. 3).
16SPCRused formoleculardetection on cerebro-spinalfluid
samples has limitations common to other uses of 16S PCR; fur-
thermore, this is a biological sample containing PCR inhibitors
(proteins or other agents), making it a limiting element [29].
Finally, 16S PCR used on samples from cerebral abscesses
has proved contributive when amplification is followed by
cloning of PCR products; in this case, the technique allows
identifyingmorebacteria thanculture [18].But suchanapproach
is difficult to apply in the daily activity of a laboratory.
4.2.4. Bone and joint infections
The diagnosis of bone and joint infections (osteitis,
osteomyelitis, arthritis, or infections on material) is usually
made by bacterial culture. Nevertheless, culture results may
also be falsely positive (contamination of samples by cuta-
neous flora) but also falsely negative (because of a previous
antibiotic therapy or an infection due to fastidious bacteria)
[32]. Thus, Fenollar et al. analyzed 525 bone and joint sam-
ples and found 89 samples positive with PCR and culture,
nine samples positive with culture and negative with PCR, and
16 samples negative with culture and positive with PCR [32].
The falsely negative results with PCR could have been due a
bad quality of extraction while falsely negative results with
culture were associated, in this study, to previous antibiotic
therapy in seven out of 16 cases, to fastidious bacterium infec-
tions in twoout of 16 cases,whereas in the seven remainingcases
16S PCR detectedmost frequently streptococci and enterococci[32]. Broad-range 16S PCR could improve the diagnostic man-
agement but the results of studies are still too preliminary and
contradictory for 16S PCR to be used in the routine diagnosis of
bone and joint infections. The molecular tool allows detecting
new pathogens and allows improving the diagnosis of bone and
joint infections due to Kingella kingae, S. pneumoniae, Strepto-
coccus agalactiae, Enterococcus faecalis, Mycoplasma spp. and
anaerobicbacteria [32,33]. Likewise, in caseof spondylodiscitis,
16S PCR allowed making a diagnosis on 44% of the samples
that were negative in culture, the negativity being most often
related to fastidious bacteria [34]. Finally, 16S PCR associated
with cloning allowed improving the diagnosis of polymicro-bial bone and joint infections [32]. Nevertheless, the diagnostic
value of 16S PCR for bone and joint infections varies according
to authors; this may be due to the variation of population panels
studied (Fig. 3) [32,35,36].
Furthermore, the absence of associatedantibiogrammay be a
limitation for therapeutic management. This limitation is espe-
cially a problem when dealing with bone and joint infections
on material, in which the causative germs have greatly variable
antibiotic susceptibility profiles, and for which microbiological
documentation is recommended. Even if the use of 16S PCR
remains to be defined for bone and joint infections, it seems
that it allows clarifying unreliable culture results but that it
should be kept for specificcases, such as infectionswith negativeculture butwith clinical and biological arguments suggesting the
infection [32].
4.2.5. Other nosological settings
There are many studies reporting the use of 16S PCR in
various contexts, nevertheless, it has proved truly contributive
only in some indications (salpingitis, postoperative and post-
traumatic uveitis, pericarditis), whereas for other indications it
should notbe used (pacemaker infections or chronic pneumonia
in patients presenting with cystic fibrosis [37]).
Thus, for the salpingitis, 16S PCR allows identifying new
bacteria difficult to grow in culture [38]. The authors of a
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 7/9
328 A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330
Fig. 4. Strategic contribution of mass spectrometry compared to molecular biology (including broad-range PCR) in clinical bacteriology.
Place stratégique de la spectrométrie de masse par rapport à la biologie moléculaire (dont la PCR 16S) en bactériologie clinique.
study on uveitis analyzed 1520 samples according to a global
molecular strategy using 16S PCR and other molecular tools;
they concluded that 16S PCR lacked sensitivity and was not
contributive for the detection of intracellular bacteria, but was
recommended to improve the etiological diagnosis of post-
operative and post-traumatic uveitis [39]. The authors of a
study on the molecular and non-molecular analysis of peri-
cardial fluids, in a context of pericarditis, demonstrated that16S PCR was useful in case of previous antibiotic therapy
[40].
But, in case of ascitic fluid infections in cirrhotic patients,
bacterial DNA may be detected in patients not meeting criteria
of ascitic fluid infections and for whom the diagnosis could be
episodes of bacterial translocation [41]. For some authors, there
could be a continuum between bacterial colonization and the
spontaneous primary peritonitis; the presence of bacterial DNA
in ascitic fluid could be associated to an increased risk of devel-
oping an infection of ascitic fluid [42]. In this case, 16S PCR
could allow defining a category of patients “at risk” in whom
the bacterial population would have been identified, thus allow-
ing shortening the delay before therapeutic management [42];nevertheless, the contribution of such a strategy remains to be
demonstrated.
16S PCR performed on blood, serum, or plasma samples
theoretically allows detecting bacteria that may be cultivated or
not. The technique performed on EDTA-treated whole blood
could allow targeting a broader bacterial spectrum, while serum
or plasma samples contain less inhibitors [43]. It was reported
as contributive in case of neonatal sepsis and for the detection of
“non-cultivable”bacteria such as Mycoplasmaspp.,Ureaplasma
spp., or Treponema pallidum [43].
16S PCR performed on pacemakers of symptomatic patients
shows that the presence of bacterial DNA is not a proof of
pacemaker infection [44]; 16S PCR should not be used in this
case.
4.3. 16S PCR and evidence of new diseases
Molecular detection in atypical cases without any prior
microbiological diagnostic orientation remains the prerogative
of broad-range 16S PCR. Thus, 16S PCR allowed linking Bar-tonella henselae or T.whipplei to bacillary angiomatosis and
Whipple’s disease, respectively [2,45]. The association between
the detection of a bacterial sequence and an infectious disease
was in this case guided by histopathological data. When this
is not the case, the causality between the detection of bacterial
DNA in a human sample and an infectious diseasemay be more
difficult to establish [2].
5. Is 16S PCR inferior to mass spectrometry?
For a few years, mass spectrometry, a new tool for
“broad-range” bacterial identification, has been available for
microbiologists (Fig. 4). It allows to identify rapidly and at alow cost most strains cultivated in a medical bacteriology labo-
ratory (Fig. 4) [46]. It has reduced the use of molecular biology,
but the latter remains the gold standard for rare bacteria or bacte-
ria badly identified by mass spectrometry (check below), as
reported in a recent study, in which less than half of strains
identified by 16S PCR were identified by mass spectrometry
[47]. There are indeed some limitations to identification, for
example for streptococci of the viridans group, pneumococci,
anaerobic bacteria, but also for bacteria of the HACEK group,
Shigella spp., and some strictly aerobic bacteria [48]. It should
be noted that for some limitations of identification such as those
encountered with streptococci, pneumococci, or Shigella spp.,
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 8/9
7/25/2019 PCR past, future.pdf
http://slidepdf.com/reader/full/pcr-past-futurepdf 9/9
330 A. Renvoisé et al. / Médecine et maladies infectieuses 43 (2013) 322–330
[23] Podglajen I, Bellery F, Poyart C, Coudol P, Buu-Hoi A, Bruneval P, et al.
Comparative molecular and microbiologic diagnosis of bacterial endo-
carditis. Emerg Infect Dis 2003;9:1543–7.
[24] Lang S, Watkin RW, Lambert PA, Bonser RS, Littler WA, Elliott TS.
Evaluation of PCR in the molecular diagnosis of endocarditis. J Infect
2004;48:269–75.
[25] Vondracek M, Sartipy U, Aufwerber E, Julander I, Lindblom D, West-
ling K. 16S rDNA sequencing of valve tissue improves microbiological
diagnosis in surgically treated patients with infective endocarditis. J Infect2011;62:472–8.
[26] Rovery C, Greub G, Lepidi H, Casalta JP, Habib G, Collart F, et al. PCR
detection of bacteria on cardiac valves of patients with treated bacterial
endocarditis. J ClinMicrobiol 2005;43:163–7.
[27] SchuurmanT, de Boer RF, Kooistra-Smid AM, van Zwet AA.Prospective
study of use of PCRamplification and sequencing of 16S ribosomal DNA
from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical
setting. J ClinMicrobiol 2004;42:734–40.
[28] Welinder-OlssonC,DotevallL,HogevikH, JungneliusR,TrollforsB,Wahl
M, et al. Comparison of broad-range bacterial PCR and culture of cere-
brospinal fluid for diagnosis of community-acquired bacterial meningitis.
ClinMicrobiol Infect 2007;13:879–86.
[29] Srinivasan L, Pisapia JM, Shah SS, Halpern CH, Harris MC. Can Broad-
range 16S ribosomal ribonucleic acid gene polymerase chain reactions
improve the diagnosis of bacterial meningitis? A systematic review and
meta-analysis. Ann Emerg Med 2012;60:609–20.
[30] Saravolatz LD, Manzor O, VanderVelde N, Pawlak J, Belian B. Broad-
range bacterial polymerase chain reaction for early detection of bacterial
meningitis. Clin Infect Dis 2003;36:40–5.
[31] Zarrouk V, Leflon-Guibout V, Robineaux S, Kalamarides M, Nicolas-
Chanoine MH, Sterkers O, et al. Broad-range 16S rRNA PCR with
cerebrospinal fluid may be unreliable for management of postoperative
aseptic meningitis. J ClinMicrobiol 2010;48:3331–3.
[32] Fenollar F, Roux V, Stein A, Drancourt M, Raoult D. Analysis of 525
samples to determine the usefulness of PCRamplification and sequencing
of the 16S rRNA gene for diagnosis of bone and joint infections. J Clin
Microbiol 2006;44:1018–28.
[33] FenollarF,LevyPY,RaoultD.Usefulness of broad-rangePCRfor thediag-
nosis of osteoarticular infections. Curr Opin Rheumatol 2008;20:463–70.
[34] Fuursted K, Arpi M, Lindblad BE, Pedersen LN. Broad-range PCR as a
supplement to culture for detection of bacterial pathogens in patients with
a clinically diagnosed spinal infection. Scand J Infect Dis 2008;40:772–7.
[35] Marin M, Garcia-Lechuz JM, Alonso P, Villanueva M, Alcala L, Gimeno
M,et al.Roleof universal16S rRNAgenePCRandsequencingindiagnosis
of prosthetic joint infection. J Clin Microbiol 2012;50:583–9.
[36] Ince A, Rupp J, Frommelt L, Katzer A, Gille J, Lohr JF. Is “aseptic” loos-
ening of theprosthetic cupafter total hipreplacementdue to nonculturable
bacterial pathogens in patients with low-grade infection? Clin Infect Dis
2004;39:1599–603.
[37] Rudkjobing VB, Thomsen TR, Alhede M, Kragh KN, Nielsen PH,
Johansen UR, et al. True microbiota involved in chronic lung infection
of cystic fibrosis patients found by culturing and16S rRNA gene analysis.
J ClinMicrobiol 2011;49:4352–5.
[38] Hebb JK,CohenCR, AsteteSG, Bukusi EA,TottenPA.Detection of novel
organisms associated with salpingitis, by use of 16S rDNA polymerase
chain reaction. J Infect Dis 2004;190:2109–20.[39] Drancourt M, Berger P, Terrada C, Bodaghi B, Conrath J, Raoult D, et al.
High prevalence of fastidious bacteria in 1520 cases of uveitis of unknown
etiology. Medicine (Baltimore) 2008;87:167–76.
[40] Levy PY, Fournier PE, Charrel R, Metras D, Habib G, Raoult D. Molec-
ular analysis of pericardial fluid: a 7-year experience. Eur Heart J
2006;27:1942–6.
[41] Such J, Frances R, Munoz C, Zapater P, Casellas JA, Cifuentes A, et al.
Detectionand identificationof bacterialDNAin patientswithcirrhosisand
culture-negative, nonneutrocytic ascites. Hepatology 2002;36:135–41.
[42] Rogers GB, Russell LE, Preston PG, Marsh P, Collins JE, Saunders J,
et al. Characterisation of bacteria in ascites–reporting the potential of
culture-independent, molecular analysis. Eur J Clin Microbiol Infect Dis
2010;29:533–41.
[43] ManciniN, CarlettiS,GhidoliN, CicheroP,BurioniR, ClementiM. Theera
of molecular and other non-culture-based methods in diagnosis of sepsis.
ClinMicrobiol Rev 2010;23:235–51.
[44] PichlmaierM,Marwitz V, KuhnC, NiehausM,KleinG,Bara C,et al.High
prevalence of asymptomatic bacterial colonization of rhythmmanagement
devices. Europace 2008;10:1067–72.
[45] Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent
of bacillary angiomatosis. An approach to the identification of uncultured
pathogens. N Engl J Med1990;323:1573–80.
[46] Seng P, Drancourt M, Gouriet F, La SB, Fournier PE, Rolain JM, et al.
Ongoing revolution in bacteriology: routine identification of bacteria by
matrix-assisted laser desorption ionization time-of-flightmass spectrome-
try. Clin Infect Dis 2009;49:543–51.
[47] Bizzini A,JatonK,RomoD, BilleJ, Prod’homG,GreubG.Matrix-assisted
laser desorption ionization-time of flight mass spectrometry as an alterna-
tive to 16S rRNA genesequencing for identificationof difficult-to-identify
bacterial strains. J ClinMicrobiol 2011;49:693–6.
[48] Croxatto A, Prod’hom G, Greub G. Applications of MALDI-TOF mass
spectrometry in clinical diagnostic microbiology. FEMS Microbiol Rev
2012;36:380–407.
[49] Clifford RJ, Milillo M, Prestwood J, Quintero R, Zurawski DV, Kwak
YI, et al. Detection of bacterial 16S rRNA and identification of four
clinically important bacteria by real-time PCR. PLoS One 2012;7:
e48558.