Ojchd.000536

Ana Sanjurjo Rivo1
and Javier de la Fuente Aguado2
*
1
Department of Internal Medicine, Unit of Infectious Diseases, Spain
2
Department of Internal Medicine, Povisa Hospital, Spain
*Corresponding author: Javier de la Fuente Aguado, Internal Medicine Department. GTEI SEMI, Povisa Hospital, Vigo, Spain,
Email:
Submission: June 08, 2018; Published: July 18, 2018
Role of New Microbiological Techniques in the
Diagnosis of Infective Endocarditis
Introduction
The polymerase chain reaction (PCR) is the most commonly
used. A useful target-gene is the 16S ribosomal RNA (rRNA), which
is composed of highly conserved regions and variable regions that
allow for design of either broad-range PCR primers or genus-or
species-specific primers Sequencing of the 16S rRNA gene allows
culture-independent phylogenetic classification of many bacteria,
and to date is the bacterial gene with the most available sequences
in public databases.
PCR-based techniques in blood samples from patients
with IE
The application of molecular techniques directly in whole
blood samples offers the possibility of identifying the responsible
etiological agent in a short period of time. There are few studies that
use these techniques on patients with IE. In most cases, patients
with sepsis were evaluated using the standard blood culture as a
comparator, whose sensitivity is not adequate to be considered
the gold standard [9]. This extraordinarily sensitive method has a
theoretical detection threshold of only 1 to 10 microorganisms. Its
main limitations are false positive results: risk of attributing an IE to
the detection of contaminating microorganisms and its inability to
detect the real viability of the isolations. There are commercialized
techniques that allow the detection of various bacteria and fungi
in direct blood samples in real time. In this regard, Casalta et al.
[9], compare the sensitivity of the blood culture to a commercially
available wide-range polymerase chain reaction (PCR) assay
[(SeptiFast (Roche Diagnostics)] for the detection in blood of
19 bacterial species and six fungal species in 63 patients with
infectious endocarditis (IE).
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Copyright © All rights are reserved by Javier de la Fuente Aguado.
Volume 2 - Issue - 2
Open Journal of
Cardiology & Heart Diseases
C CRIMSON PUBLISHERS
Wings to the Research
ISSN 2578-0204
Abstract
Infective endocarditis is a life-threatening disease caused by bacterial infection of the endothelium and cardiac valves, either native or prosthetic.
In the present work the role of the new microbiological techniques (techniques of detection and amplification of the subunit 16 ribosomal sRNA by
means of the chain reaction of the polymerase in blood or tissue, fluorescent in situ hybridization, and matrix-assisted laser is reviewed desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the diagnosis of infective endocarditis. Infective endocarditis (IE) is a life-threatening
disease caused by septic vegetations and inflammatory foci on the surface of the endothelium and the valves. Although most of endocarditis cases are
community-acquired, healthcare-associated endocarditis is increasing and now accounts for approximately one third of IE. Risk factors include the
presence of a prosthetic heart valve, structural or congenital heart disease, intravenous drug use and a recent history of invasive procedures. Diagnosis is
made using the Duke criteria, which include clinical, laboratory and echocardiographic findings. IE associates high morbidity and a mortality rate close
to 25% in the year following diagnosis, thus early diagnosis and adequate treatment is fundamental in its evolution [1].
Most IE, regardless of valve type, native or prosthetic, are due to gram-positive cocci. Currently Staphylococcus aureus is the microorganism most
frequently involved, causes 25% to 30% of cases, followed by viridans group Streptococci (20-25%), coagulase negative Staphylococci (11%) and
Enterococcus faecalis 10% [2]. Gram-negative bacilli make up 5% of cases and include organisms from the HACEK (Haemophilus, Aggregatibacter,
Cardiobacterium, Eikenella, and Kingella species) group, Enterobacteriaceae and nonfermenting Gram-negative bacilli. Finally, there is a series of
microorganisms difficult to grow in conventional media that can cause IE, the most common being Coxiella Burnetii, Bartonella spp and Tropheryma
whipplei and rare cases of IE caused by fungi [3,4]. With traditional microbiological methods, up to one third of IE remain without an etiological
diagnosis [5,6]. This is usually due to two factors: the prior taking of antibiotics, which causes a lower yield of blood cultures, and the local prevalence of
fastidious microorganisms that are difficult to isolate with conventional microbiological techniques or uncultivable or challenging to cultivate organisms
in routine methods [7]. In recent years, various identification techniques have been developed, both in blood cultures and valvular tissue, which entail
shorter times and greater diagnostic precision [8], thus reducing the percentage of blood culture negative IE (BCNE)

Open J Cardiol Heart Dis Copyright © Javier de la Fuente Aguado
2/4
How to cite this article: Ana S R, Javier de la F A.Role of New Microbiological Techniques in the Diagnosis of Infective Endocarditis. Open J Cardiol Heart
Dis. 2(2). OJCHD.000536.2018. DOI: 10.31031/OJCHD.2018.02.000536
Theyprovedthatitwasnotmoresensitivefornormalorganisms,
although in 20 patients presenting IE with negative blood cultures
caused by previous antibiotic therapy, the trial detected three
cases with Streptococcus gallolyticus, Staphylococcus aureus and
Enterococcus faecalis, respectively. However, the test does not
include the organisms of the HACEK group. The analytical specificity
of the test was 100%. More recently, Marco et al. [10] publishes a
review on the different molecular methods currently marketed
to make a direct diagnosis using blood samples in patients with
septicaemia [11]. It concludes that, although these techniques
have the potential to reduce the necessary time to reach a final
microbiological diagnosis, it does not replace the one obtained with
the usual blood cultures. Both methods are currently considered
complementary and studies are needed to assess the possible
impact they may have on the management of these patients.
In addition, other aspects such as the number of samples to be
analysed, their frequency or the interpretation of the results from
a clinical and microbiological point of view should be considered.
PCR-based techniques for the diagnosis of IE in cardiac
valvular tissue
PCR on valvular tissue may offer a higher sensitivity than
that obtained in blood cultures, due to the greater abundance
of bacterial DNA in the valvular tissue than in blood. For this
reason, the application of PCR to explanted valves may increase
the sensitivity of the Duke criteria in establishing the diagnosis
of definite IE. Moter et al. [11] in 2010 issues a compilation of
studies that investigate the impact of PCR-based methods using
heart valve samples for the microbiological diagnosis of IE. The
results were contrasted with the data obtained by routine blood
culture procedures. A total of 481 IE cases from 10 studies were
reviewed, including 110 (22.9%) of BCNE cases. In the BCNE group,
PCR identified microorganisms in 47 cases, which correspond to
42.7% of BCNE and 9.8% of the total cases investigated. All these
studies recommended the application of molecular techniques for
the microbiological diagnosis of BCNE and possible IE. However, it
should be emphasized that the data from these studies are complex
and require careful interpretation. There are several limitations:
1. The results are influenced by differences in regional
epidemiology and the inclusion criteria in the different studies.
2. The differences between laboratories and the lack
of standardization in PCR-based methods complicate the
interpretation of the results.
3. The identification of false positive results. It has been
proven that bacterial DNA can persist in cardiac valves under
antibiotic therapy and after IE is cured. The viability of these
microorganisms is not clear, and the detection of bacterial DNA
should be interpreted with caution.
In recent years, to improve the sensitivity and minimize the
limitations of these molecular methods, various studies have been
carried out; Brinkman CL et al. [12], evaluated the combined use
of the mass spectrometry technique and a wide-spectrum PCR in
83 paraffin-fixed heart valves of subjects with endocarditis who
had positive valve disease and/or blood cultures. In 55% of cases,
there was concordant microbiology at the level of genus/species or
group of organisms, 11% positivity with discordant microbiology
and 34% without detection. It also detected all the antimicrobial
resistance encoded by mec A or van A/B and identified a case
of Tropheryma whipplei endocarditis previously unrecognized
[13]. The association of both techniques has the potential to
reduce the response time compared to the standard culture and
in addition to the detection and classification of organisms, the
selected antimicrobial resistance is also defined. All several studies
conducted with different commercial systems of real-time broad-
spectrum PCR that compare the results with conventional valve
cultures show a significant difference in the rate of detection
of pathogens in favour of the molecular methods, with a high
specificity (77% to 100%) and high PPV 100% and NPV (85-90%)
[8,14-16].
In order to improve the efficacy of broad-spectrum PCR
techniques, new modalities are being used, such as the use of a
multiple PCR associated with a nested one, which demonstrates
its effectiveness particularly in cases involving a limited number
of bacteria, in BCNE and other contexts that involved bacterial
species that were not susceptible to identification by phenotypic
research [17]. When considering molecular testing of cardiac
valves, it should be noted that organism-specific PCR assays often
demonstrate superior sensitivity compared to wide-range PCR
[18]. For the diagnosis of Bartonella endocarditis, the Bartonella
specific PCR applied to the heart valve tissue was positive in 92%
(48/52) of the cases, while the broad-spectrum PCR identified
Bartonella in the valvular tissue in only 60% (21/35) of the cases
[19]. The group led by Didier Raoult demonstrates this in a study
conducted in 283 patients with BCNE by adding a specific PCR to the
systematic PCR test already used in patients with BCNE, increasing
the diagnostic efficiency by 24.3%, mainly by detecting enterococci
and streptococci that had not been detected by other diagnostic
methods, but also agents that required specific management such
as Mycoplasma hominis and Tropheryma whipplei [20].
Diagnosis Based on Fluorescent in situ Hybridization
Fluorescence in situ hybridization (FISH) is a molecular
technique that uses fluorescently labelled probes to detect
RNA or DNA. Fluorescent probes are applied to fixed samples,
either smears or sections of tissue on slides. They penetrate in
morphologically intact microorganisms and hybridize specifically
at their target sites on highly abundant ribosomes. Therefore,
FISH allows the independent identification of the culture and the
simultaneous visualization of bacteria. Although FISH techniques
could potentially be useful to improve the diagnostic yield in EIs,
there are not enough contrasted studies that allow this technique to
be incorporated into routine clinical practice. Mallmann et al. [20]
published the only pilot study in which, with the FISH technique,
he detected bacteria in 26 out of the 54 heart valves and allowed a
successful diagnosis of infective endocarditis in five out of 13 cases
with negative blood cultures. Isolated studies conducted in patients
with IE by Coxiella burnetii also recommend this technique to
improve diagnostic yield [21-23]

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Mass spectrometry: MALDI-TOF
Massspectrometry,matrix-assistedlaserdesorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS), is a technique
used in the identification of microorganisms by creating a spectrum
based on the protein profile, which is unique to a given species.
Numerous studies have shown in recent years the advantages
of the use of MALDI-TOF for bacterial identification [24,25].
Several papers that analyze the profitability of this technique in
blood cultures [26,27], but not specifically in IE have also been
published. As a matter of fact, articles published by IE that use
the MALDI TOF technique for diagnosis always refer to isolated
cases in which an unusual pathogen is involved and difficult to
diagnose with traditional culture techniques [28-30]. Although
MALDI-TOF is undoubtedly a very promising technique that can
contribute to substantially reduce the time of diagnosis in IE and
as a consequence to a more adequate management, it is neither
widely available nor is its use in these infections standardized at the
moment. The application of all techniques alone or in combination
will contribute in the immediate future to reducing the number of
BCNE, increasing the diagnostic accuracy of the Duke criteria and
will possibly have a positive impact on the morbidity and mortality
associated with this life-threatening condition.
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