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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.

© 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: alexandra.aubry@upmc.fr(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

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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].

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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

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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

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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].

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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

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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.,

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