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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.
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
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].