research article advantages and limitations of direct pcr … · 2019. 7. 30. · research article...

Research ArticleAdvantages and Limitations of Direct PCRAmplification of Bacterial 16S-rDNA from ResectedHeart Tissue or Swabs Followed by Direct Sequencing forDiagnosing Infective Endocarditis A RetrospectiveAnalysis in the Routine Clinical Setting

Daniela Maneg1 Janina Sponsel1 Iris Muumlller1 Benedikt Lohr1

John Penders2 Katharina Madlener3 and Klaus-Peter Hunfeld1

1 Institute of Laboratory Medicine Microbiology and Infection Control The Northwest Medical Centre60488 Frankfurt am Main Germany2CAPHRI School for Public Health and Primary Care Department of Medical MicrobiologyMaastricht University Medical Center 6200 MDMaastricht Netherlands3Kerckhoff Klinik 61231 Bad Nauheim Germany

Correspondence should be addressed to Daniela Maneg manegdanielakhnwde

Received 30 December 2015 Revised 24 February 2016 Accepted 3 March 2016

Academic Editor Kazuhiko Nakano

Copyright copy 2016 Daniela Maneg et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Infective endocarditis (IE) is a life-threatening disease that is associated with highmorbidity andmortality Its long-term prognosisstrongly depends on a timely and optimized antibiotic treatment Therefore identification of the causative pathogen is crucial andcurrently based on blood cultures followed by characterization and susceptibility testing of the isolateHowever antibiotic treatmentstarting prior to blood sampling or IE caused by fastidious or intracellular microorganismsmay cause negative culture results Herewe investigate the additional diagnostic value of broad-range PCR in combinationwith direct sequencing on resected heart tissue orswabs in patients with tissue or swab culture-negative IE in a routine clinical setting Sensitivity specificity and positive and negativepredictive values of broad-range PCR fromdiagnosticmaterial in our patients were 333 769 909 and 143 respectivelyWeidentified a total of 20 patients (215) with tissue or culture-negative IE who profited by the additional application of broad-rangePCR We conclude that broad-range PCR on resected heart tissue or swabs is an important complementary diagnostic approachIt should be seen as an indispensable new tool for both the therapeutic and diagnostic management of culture-negative IE and wethus propose its possible inclusion in Dukersquos diagnostic classification scheme

1 Introduction

In spite of improvements in the diagnostic and treatmentinfective endocarditis (IE) remains a serious life-threateningdisease [1ndash4] with an unchanged mortality rate up to 30and an incidence of 2 to 6 per 100000 individuals per yearover the last 30 years [5] The stagnation in mortality andincidence can be ascribed to the progressively evolving pre-disposing factors [6] leading to a change in the epidemiologyand etiology of IE thereby resulting in worsening of the

individual course of IE [1 5 6] Particularly noteworthy isthe general increase of reported health care associated IE[7] with a potentially higher proportion of microorganismswith a potentially complex resistance pattern Especiallyfor S aureus bloodstream infections IE is referred to asmost frequent complication and occurs in 40 of patientswithout heart disease [8] Still the primary sites of infec-tion are native heart valves but individuals with implantedmechanical devices such as valvular prostheses pacemakersor implantable defibrillators are increasingly involved [9ndash11]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7923874 10 pageshttpdxdoiorg10115520167923874

2 BioMed Research International

Early diagnosis of IE and rapid identification of thecausative pathogen are crucial for providing a specific andefficient antibiotic therapy and improvement of the patient [512ndash15] Generally in IE patients blood cultures are frequentlypositive [16] Nevertheless such cultures can remain negativein 25 to 31 of all cases of IE depending on causativeagent and the clinical circumstances [2 17 18] Blood culturestypically remain negative due to antibiotic therapy prioror concurrent to blood sampling (sim50 of cases) due toinfection by fastidious organisms such as nutritionally variantstreptococci fastidious Gram-negative bacilli of the HACEKgroup (Aggregatibacter aphrophilus former Haemophilusaphrophilus and H paraphrophilus Aggregatibacter actino-mycetemcomitans formerActinobacillus actinomycetemcomi-tans Cardiobacterium hominis Eikenella corrodens Kingellakingae) Brucella spp (in endemic areas) and fungi or due toinfections by intracellular bacteria such as the zoonotic agentsCoxiella burnetii and Bartonella and Tropheryma whipplei[5] In such cases negative cultures may significantly delaydiagnosis and effective treatment which possibly negativelyimpacts on the outcome of the patient [17]

These obvious drawbacks of conventional techniquesclearly impair the current criteria used for clinical diagnosisof IE and call for adjunctive diagnostic methods increas-ing the sensitivity for more rapid pathogen recovery andidentification This is why in such cases of blood culture-negative or clinically questionable IE culture-independentnew molecular diagnostic techniques such as broad-rangepolymerase chain reaction (PCR) with subsequent DNA-sequencing fromheart valve specimenhave gained increasingattention and were shown to be useful in the detection andclassification of bacterial DNA in several recent diagnosticstudies [19ndash28] Besides detecting organisms that are missedby culture-dependent methods PCR-based methods alsoallow exact identification of the causative organism and theidentification of new hitherto unknown pathogens causing IEeven if the patient is already under antibiotic treatment [29]These possibly beneficial features of broad-range PCR for thediagnosis in such cases have even led to the proposition ofPCR positivity as a major diagnostic criterion for IE [14 1930] However further research is clearly required until PCR-based methods become accepted as routine diagnostic tool[2 9]

The aim of the present study was to investigate theadditional diagnostic value of broad-range PCR targeting the16S ribosomal DNA in heart tissue or swabs in patients withconfirmed suspected or possible IE in a routine clinicaldiagnostic setting We hypothesized that the use of broad-range PCR amplification followed by direct sequencingmightincrease the number of identified causative pathogens in caseswith culture-negative IE and in antibiotically pretreated indi-viduals thereby possibly improving the rapidity of diagnosisin such patients also in the routine diagnostic setting

2 Material and Methods

21 StudyDesign and Patients For our retrospective observa-tional study we identified all patients admitted to KerckhoffClinic in Bad Nauheim (Germany) between 2011 and 2014

(Figure 1)This cohort was then subjected to further in-depthcomputer-assisted analysis identifying all patients admittedwith a diagnosis of suspected or definite infective endocardi-tis encoded by the ICD-10-GM (International Classificationof Diseases 10th Revision German Modification) for ldquoacuteand subacute endocarditisrdquo (I33-) and undergoing cardio-surgical treatment We also looked for all patients subjectedto diagnostic testing after admission to Kerckhoff Clinic bybroad-range PCR amplification and direct sequencing fromcardiosurgical specimens as performed at the Institute ofLaboratory Medicine Microbiology and Infection Control atthe NorthwestMedical Centre in FrankfurtMain (Germany)during the same time period In combining both selectorsall patients were identified with both an encoded diagnosisof ldquoacute and subacute endocarditisrdquo and a diagnostic testresult of broad-range PCR and direct sequencing fromcardiosurgical specimens for further investigation (Figure 1)Medical charts of all patients of this study group werethen retrospectively reviewed and patients were includedin the study if both the microbiological findings or broad-range PCR and the corresponding tissue or swab culture ofcardiosurgical specimens were available

22 Definition of Diagnosed IE For the purpose of this studypatients of the study group were divided into three differentcategories (ldquodefiniterdquo ldquopossiblerdquo and ldquono evidence forrdquo IEFigure 1) according to somemodification ofDukersquos criteria forthe diagnosis of IE [31] and including all available data fromthe medical charts at Kerckhoff Clinic as follows definite IEa typical valve histopathology (considered as gold standard)in addition to 2 or more findings indicating IE in any ofthe following diagnostic procedures is present echocardio-graphy preoperative BC (preBC) and typical intraoperativemacroscopic aspect possible IE there is negative valvehistopathology or evidence for IE resulting from one of theabove-mentioned diagnostic procedures only but there is apositive result upon PCR andor tissue or swab culture fromvalve tissue after surgery no evidence for IE only one ofthe above described procedures provided evidence for IE butPCR conventional culture fromvalve tissue after surgery andhistopathology proved negative (Figure 1)

23 Surgical Material Tissue specimens or swabs fromintraoperatively excised cardiac tissue were taken duringcardiosurgical treatment at Kerckhoff Clinic in Bad Nauheimaccording to local standard surgical and diagnostic proce-dures and specimens for microbiological analysis were thendelivered to the Institute of Laboratory Medicine Microbiol-ogy and Infection Control at the Northwest Medical Centrein FrankfurtMain (Germany) for microbiological analysisperformed under standard conditions

24MolecularMethods Bacterial DNAwas isolated from theintraoperative excised specimens by means of the MagNAPure Compact System (Roche Diagnostics Mannheim Ger-many) For the amplification of the 16S-rDNA with aGeneAmp PCR System 2700 or a Thermal Cycler 2720(Applied Biosystems Darmstadt Germany) the universal51015840 primer 285 (gag agt ttg atc ctg gct cag position 9ndash29

BioMed Research International 3

Cohort of patients admitted to the Kerckhoff Clinic between

January 2011 and April 2014

Patients encoded withICD ldquoacute and subacute

endocarditisrdquo

Patients whose cardiosurgicalspecimens were analyzed by

broad-range PCR

Patients with diagnosis encoded by ICD ldquoacute andsubacute endocarditisrdquo and PCR diagnostic findings of

cardiosurgical specimen

Definite Possible No evidenceN = 10N = 4N = 100

N = 114

N = 122N = 210

N = 40190

Classification of diagnosis of IE according to clinicaland laboratory findings

Figure 1 Overview of the assortment of the study group Workflow scheme for the assortment of patients according to the criteria describedin detail in Section 2

within gene according to 16S-rDNA of Escherichia coli) andthe universal 31015840 primer 243r (acg agc tga cga cag cca tgE coli position 1073ndash1054) (both primers synthesised byTiB Molbiol Berlin Germany) were used [32] From visibleUV-bands PCR products were purified with the QIAquickPCR Purification Kit (50250) (QIAGEN Hilden Germany)Sequencing was carried out using a BigDye Terminator v1or alternatively using a BigDye Terminator v31 (AppliedBiosystems Darmstadt Germany) according to the manu-facturerrsquos instructions Analysis of the sequencing productswas carried out using ABI 3500 Dx Sequencer (AppliedBiosystems Darmstadt Germany) For the identification

of the pathogens the resulting sequences were then com-pared with reference sequences obtained from the NCBIdatabase using BLAST (Basic Local Alignment Search Toolhttpblastncbinlmnihgov)

3 Results

31 Assortment of Patients In our observational retrospectivestudy we identified a total of 40190 patients admitted toKerckhoff Clinic in Bad Nauheim between January 2011 andApril 2014 A total of 114 patients with suspected or diagnosedIE who underwent cardiosurgical treatment during this time


Table 1 Distribution of types of valves infected within the studygroup

Type of valve infected (119873 = 113lowast)Native Prosthetic Cardiac devices

Aortic 37 28Mitral 34 5Tricuspidal 2 1

6119873 () 73 (646) 34 (301) 6 (53)

104 patients FM ratio 1 3 mean age 660 plusmn 126 yearslowastHeart valves of patients with two infected heart valves are countedseparately

period were included in our study One hundred patients haddefinite IE and 4 patients had possible IE according to ourabove defined criteria For 10 patients there was no evidencefor the clinically suspected diagnosis of IE The patients withdefinite or possible IE (119899 = 104) were included in our furtheranalysis whereas the 10 patients with no definite evidence forIE were excluded and used for the computation of sensitivityspecificity and positive and negative predictive values onlyThe incidence of IE in our cohort of admitted hospitalpatients was found to be 259 per 100000 patientsyear

32 Description of Patients and Surgical Material Out of 104selected patients with possible or definite IE 75weremen and29 were women (FM ratio sim1 3) with a mean age of 660 plusmn126 years The primary sites of infection were native heartvalves (646) with 37 aortic (507) 34 mitral (466) and2 tricuspid (27) valves 301 were prosthetic valves (aorticvalve 119899 = 28 824 mitral valve 119899 = 5 147 and tricuspidvalve 119899 = 1 29) whereas 53 showed infected cardiacdevices (119899 = 6) (Table 1) Nine of the 104 patients sufferedfrom two infected heart valves or an infected valve togetherwith an infected cardiac device (aorticmitral valve 119899 = 7prosthetic aorticmitral valve 119899 = 1 mitral valvecardiacdevice 119899 = 1) according to the diagnosis documented in themedical charts (Table 1)

33 Comparison of PCR Results with Clinical Findings Bac-terial broad-range PCR amplification was positive in 40samples (385) obtained from 104 patients with possibleor definite diagnosis of IE In 36 of 100 patients (360)PCR results showed analytical agreement with the clinicallydefined diagnosis of definite IE In 4 patients with possibleIE that lacked clinical diagnostic evidence broad-range PCRfrom valve tissue yielded a positive result supporting theclinical suspicion (Table 2 Figure 2)

In 33 of the 40 patients with positive PCR amplification(825) sequencing of the amplicons was possible up to thespecies level and identified mainly streptococci and entero-cocci (both 119899 = 9 272) followed by staphylococci (119899 = 8242) and sporadically occurring pathogens (119899 = 7 212)Themajority of the streptococcal sequences were identified asstreptococci of the viridans group (119899 = 7 778) for exampleS mitis and S gordonii Among the staphylococci S aureus

was identified most frequently (119899 = 4 50) Among thesporadically occurring bacteria Aerococcus urinae (119899 = 1)Anoxybacillus spp Bartonella quintana (119899 = 1) Gemellahaemolysans (119899 = 1) Granulicatella adiacens (119899 = 1) andPropionibacterium acnes (119899 = 2) were found (Table 3)

In the remaining 7 (175) of the 40 patients with positivebroad-range PCR sequencing was not possible due to amixture of DNA belonging to different organisms (119899 = 4571) or due to low DNA concentrations (119899 = 3 429) inthe sample At least 33 of the 40 PCR-positive patients weretreated with antibiotics preoperatively for the remaining 7patients these data were not available

In 64 of 100 specimens PCR amplification was negativedespite the clinically confirmed diagnosis of IE in thosepatients At least 60 of these 64 negative tested patients(938) received antibiotics before surgery For the 10 patientswithout clinical evidence for IE PCR amplification wasnegative and further confirmed the absence of IE (Table 2)

34 Comparison of PCR Results with Preoperative BloodCultures Data of preBC were available for 82 of the 104patients (788) among which 71 were positive (866) and11 were negative (134) Staphylococci were detected mostfrequently with 355 (119899 = 27) followed by streptococci (119899 =24 316) enterococci (119899 = 13 171) and other (119899 = 12158) On species level 630 were Staphylococcus aureus(119899 = 17) one of which was methicillin-resistant S aureus(MRSA) 667 of the streptococci (119899 = 16) were representedby streptococci of the viridans group for a detailed overviewsee Figure 2

Of the 71 positive preBC 27 were confirmed by positivebroad-range PCR amplification results (380) However in14 cases (52) the pathogens identified differed between bothtechniques on genus andor species level (see Table 3) Inone patient where preBC identified two species of bacteria(E coli and Streptococcus mitis) the presence of more thanone species was confirmed by broad-range PCR whichidentified a DNA mixture in the sample In the remaining44 of the 71 blood culture-positive cases (620) broad-range PCR was negative 43 of these patients with bloodculture-positive but PCR-negative results (977) receivedpreoperative antibiotics Only 1 of the 11 blood culture-negative patients (91) had a positive PCR result In theremaining 10 patients with blood culture-negative resultsPCR results were consistent All of these patients were treatedwith antibiotics preoperatively (Figure 2)

35 Comparison of PCR Results with Results of Tissue or SwabCulture Broad-range PCR was positive in 40 of 104 patientswith definite or possible IE (385) but at least 33 patients ofthose PCR-positive IE received antibiotics preoperatively Forthe remaining 7 patients data about preoperative antibiotictreatment were not available (Table 2)

In 11 patients broad-range PCR was performed excep-tionally before having obtained a negative result of tissueor swab culture upon special clinical request Seven ofthe culture-positive cases (636) were concordant with apositive broad-range PCR whereas 4 cases (364) showeda negative PCR result Among the cases with both a positive


Table 2 Overview of PCR results and preoperative antibiotic treatment Crude numbers refer to number of patients numbers in parenthesesindicate the proportion of valve tissue samples versus swabs

Tissue samplesswabs1

Number of patients PCRPositive Negative

Total 114 40 (2812) 74 (4232)Preoperative antibiotic treatment 101 33 lowastND 7 68 lowastND 4

IEDefinite 100 36 (2511) 64 (3529)Possible 4 4 (31) 0

No evidence 10 0 10 (73)lowastND no data available for 2 patients a preoperative antibiotic treatment could be excluded by medical chart1Positive tissueswab cultures were available for 11 specimens that were exceptionally analyzed using broad-range PCR before a negative culture result wasobtainedThese data were described but not used for the computation of sensitivity specificity and positive and negative predictive values of broad-range PCR

preBC pos

preBC neg

No information available

Information of preBCdefinitepossible IE PCR results

Staphylococcusthereof S aureus 63

Streptococcusthereof viridans group 667

Enterococcus

Other

Additional information orconfirmation

(for more details see Table 3)

lowastThe detected amplicon could not be sequenced due to detection of more than one organism or low DNA content

Add-on n = 9

Add-on n = 1

pos n = 12

pos n = 1

pos n = 27

neg n = 44

neg n = 10

neg n = 10

No sequencing lowastn = 4

n = 33


Concordance n = 9

Discordance n = 14

n = 104n = 104

n = 22

n = 11

n = 71

MRSA 37

Figure 2 Comparative presentation of PCR results with preoperative blood culture Bars represent on a percentage basis the distributionof preoperative blood culture results (preBC) correlated with the results of PCR followed by direct sequencing Blue shaded bars representnegative results or no available information Red shaded bars represent a positive result in blood culture or PCR amplification In the columnon the right hand side further information is provided The results of direct sequencing are classified according to either a concordance ordiscordance in comparison to the results of blood culture or as add-on information in case of a negative or not available result An in-depthdetailed comparison is additionally shown in Table 3

culture and PCR result the identified pathogens differedamong both techniques in 2 cases where broad-range PCRidentified Streptococcus gordonii and Streptococcus gallolyti-cus whereas tissue or swab culture revealed Staphylococcusepidermidis and Staphylococcus pasteuri suggesting possiblecontamination of culture (Table 4) Nevertheless these datawere described only but not used for the computation ofsensitivity specificity and positive and negative predictivevalues of broad-range PCR

Of the 33 culture-negative cases (Table 5) the identifi-cation of the causative pathogens by broad-range PCR waspossible in 26 specimens (787) and revealed predominantlystreptococci [119899 = 7 212] followed by enterococci and

staphylococci [both 119899 = 6 182] For 7 specimens [212]sequencing of the PCR products could not identify theetiology of the pathogens due to the presence of a DNAmixture or too low DNA concentrations in the sample

36 Sensitivity Specificity and Positive andNegative PredictiveValue of Broad-Range PCR For the computation of thesensitivity specificity and positive and negative predictivevalues of broad-range PCR performed on tissue or swabspecimens in tissue or swab culture-negative patients allpatients of the study (with definite possible and no evidencefor IE) with a negative tissue or swab culture were included(119899 = 103)


Table3Overviewof

speciesidentificatio

nob

tained

with

PCRandsequ

encing

metho

dsandcomparis

onto

ther

esultsof

preoperativ

eblood

cultu

res(preB

C)

PCRpo

sitive119899=40species

identifi

catio

n119899=33

lowast

Con

cordance119899=9

(PCR

preBClowastlowast)

119899

Disc

ordance119899=14(ongenu

sand

orspecies

level)

Additio

nalinformation119899=10

(nopreB

Cdataavailable)

119899

PCR

Resultof

preB

Clowastlowast

Enterococcus

faecalis

5En

terococcus

faecalis

Streptococcusgordonii

Enterococcus

faecalis

1Staphylococcus

aureus

1En

terococcus

faecium

Enterococcus

faecalis

Staphylococcus

aureus

2Streptococcusa

galactiae

1Staphylococcus

aureus

Staphylococcus

auric

ularis

Staphylococcus

epidermidis

1Ae

rococcus

urinae

1Staphylococcus

epidermidis

Streptococcuso

ralis

Staphylococcus

lugdun

ensis

1Gem

ellahaem

olysan

s1

Streptococcusgallolyticus

subsppaste

urianu

sStreptococcusa

lactolyticu

sStreptococcusm

itis

1Streptococcusgordonii

Streptococcusp

neum

oniae

Streptococcusp

neum

oniae


Streptococcusv

iridans

Streptococcussanguinis

1Streptococcusm

itis

Streptococcusintermedius

Anoxybacillus

spp

1Ba

rtonellaquintana

Staphylococcus

epidermidis

Propionibacterium

acnes

1Gr

anulica

tella

adiacens

Streptococcusv

iridans

Streptococcuso

ralis

Streptococcusv

iridans

Enterococcus

faecium

Gram-positive

cocci

Staphylococcus

epidermidis

Gram-positive

cocci

Propionibacterium

acnes

Negativep

reBC

lowastIn

7patie

ntsw

ithpo

sitiveP

CRam

plicon

dire

ctsequ

encing

faileddu

etoab

acteria

lmixture119899=4or

lowDNAconcentrations119899=3

lowastlowastRe

sults

ofpreB

Cweretaken

andanalyzed

inho

spita

lsof

prim

arycare

accordingto

localstand

ardmicrobiologicallabo

ratory

procedures


Table 4 Comparison of PCR with results of tissue or swab culture For a small patient group (119899 = 11) results of both methods (PCRamplification and culture of swab or tissue) were exceptionally gained upon special clinical requestThe direct comparison is provided in thistable these data were not used for further computation of sensitivity specificity and positive and negative predictive values of broad-rangePCR

Result Culture PCR

Positive in both methodsconcordance on species level119899 = 5

3 times Enterococcus faecalisStaphylococcus epidermidisStaphylococcus lugdunensis

Positive but discordant119899 = 2

Staphylococcus epidermidis Streptococcus gordoniiStaphylococcus pasteuri Streptococcus gallolyticus

Culture positive but PCR amplificationnegative119899 = 4

2 times Enterococcus faecium negStaphylococcus aureus neg

Coagulase-negative staphylococci neg

Table 5 Frequencies and etiology of causative pathogens identifiedby broad-range PCR in tissue or swab culture-negative IE patients(119899 = 33)

Causative pathogens identifiedby PCR in culture-negative IE 119899

Aerococcus urinae 1 30Anoxybacillus spp 1 30Bartonella quintana 1 30Enterococci 6 182Gemella haemolysans 1 30Granulicatella adiacens 1 30Propionibacterium acnes 2 61Staphylococcus aureus 4 121Staphylococcus epidermidis 2 61Streptococci 7 212No sequencing possible 7 212Total 33 100

Sensitivity and specificity of the broad-range PCR per-formed in cardiac tissue or swab specimens in tissue or swabculture-negative IE were 333 (238ndash441 95 interval)and 769 (462ndash947) respectively whereas the positiveand negative predictive values of the broad-range PCRwere 909 (756ndash980) and 143 (71ndash247) respectively(Table 6)

4 Discussion

Despite advances in diagnostic and therapeutic proce-dures infective endocarditis remains a serious [1ndash3] life-threatening disease where long-term prognosis stronglydepends on an immediate and optimized antibiotic treat-ment Therefore besides an early establishment of the diag-nosis the rapid and correct identification of the causativemicroorganism is particularly important for the patientrsquosprognosis [13 33] Currently blood cultures remain the diag-nosticmainstay for identification and species-specific charac-terization of IE-associated pathogens also because they offerthe possibility of antimicrobial susceptibility testing for target

Table 6 Calculation of sensitivity specificity and positive andnegative predictive values Calculation of sensitivity specificity andpositive and negative predictive values is based on a dataset of 119899 =103 The gold standard is defined according to the criteria outlinedin Definition of Diagnosed IE (Section 2)

Diagnosis+ minus

PCR

+ 30 3 909 Positivepredictive value

minus 60 10 143 Negativepredictive value

333 769Sensitivity Specificity

directed antimicrobial treatment If antibiotic treatmenthowever is started prior to or concurrent to blood samplingor if IE is caused by slow-growing fastidious or intracellularmicroorganisms blood cultures may yield negative results [218] which may lead to inadequate treatment and worseningof the patient Here molecular methods such as broad-range PCR amplification followed by direct sequencing ofbacterial DNA performed in excised heart valve tissue holdpromise in revealing the etiology of culture-negative IE Thebroader diagnostic value of PCR for routine microbiologicaldiagnostics however needs further evaluation until thesetechniques may become more generally accepted [13] In thisretrospective study we therefore investigated the possibleadditional value of broad-range PCR amplification followedby direct sequencing performed in resected heart tissue orswabs for diagnosing IE in 104 patients with suspected ordiagnosed IE in a routine clinical setting

The incidence of IE in our cohort was found to be 26 per1000 patients per year which is much higher than reportedin the general population (sim2ndash6 per 100000 individualsper year) [5] due to the fact that we focused on patientsadmitted to a specialized clinic for cardiac surgery Thepatients included in our study still reflect the changingepidemiology of IE described in the literature [5] with ahigh mean age of 660 years Also men were affected moreoften than women (sim3 1) and besides native heart valves


(646) we observed an increased number of infectionsof prosthetic valves (301) and cardiac devices (53) asthe primary site of infection [11] The number of infectedprosthetic valves in our study is higher than described inrecent investigations [5] This may again be explained by theselection of our study population from individuals admittedfor cardiac surgery which is indicated in case of progressiveprosthetic valve endocarditis [34] Relating to the microbialspectrum all microorganisms identified corresponded tocommonly known pathogens causing IE [16] except for oneisolate of Anoxybacillus spp detected by broad-range PCRand two identified by blood culture only (S alactolyticus andS auricularis)

Considering mainly clinical findings as the gold standardfor diagnosing IE the sensitivity and specificity of broad-range PCR performed in resected heart valve tissue orswabs to diagnose IE in heart valve tissue or swab culture-negative IE were 333 and 769 respectively whereasthe positive and negative predictive values were 909 and143 respectively In our study broad-range PCR testingwas only applied when tissue or swab culture from resectedheart tissue remained sterile Thus for the evaluation of theaccuracy of broad-range PCR in diagnosing IE only patientswith tissue or swab culture-negative IE were consideredThis is probably why values of specificity sensitivity andpositive and negative predictive values were somewhat lowerthan those reported in other studies (ranges sensitivity 412to 960 specificity 953ndash100 positive predictive value984ndash100 and negative predictive value 262ndash885) [1419 24 25 35] Additionally when comparing such valuesit should be kept in mind that not all of these studieswere performed in a routine clinical setting In contrastto classical prospective study conditions routine applicationmay impair the elaborateness of preanalytical conditions andthe management of practical procedures such as samplingand handling of the resected material which can negativelyimpact on the outcome In this study broad-range PCR wasperformed not only on resected heart tissue as the preferredanalyticalmaterial but also on clinical swabs taken fromhearttissue that are known for a lower recovery rate Moreoverthe type of tissue biopsied may be crucial with vegetationsmore likely than heart valves to yield a positive PCR product[36 37] Also differences in study design method appliedand prolonged antibiotic treatment periods before surgerymay explain our relatively lower broad-range PCR sensitivitycompared to other studies In fact at least 938 of patientswith negative broad-range PCR received antibiotics beforesurgery Broad-range PCR test results can turn negative morequickly than histopathological or gross evidence of IE [38]which can lead to discrepancies between PCR and otherdiagnostic methods when applying nucleic amplificationtechniques Nevertheless the high positive predictive valueof PCR in our study shows that broad-range PCR on resectedheart tissue or swabs is a reliable method to diagnose IEin culture-negative patients as in 909 of all patients withpositive test results it further confirmed the presence of IEwhereas the low negative predictive value of 143 indicatesthat a negative PCR result apparently cannot exclude IE

For 4 patients in whom the diagnosis of IE could notbe established by clinical evidence alone broad-range PCRprovided the etiologic diagnosis of IE thereby confirmingthe suspicion of IE If broad-range PCR had been primarilyaccepted as a specific diagnostic criterion as proposed bysome [19 21 24] these cases of ldquopossible IErdquo would havebeen reclassified as ldquodefinite IErdquo which would have increasedthe sensitivity and positive predictive value of PCR in ourinvestigation

The majority of the patients with definite and possible IEof whom data of preBC were available (788) had a positiveblood culture (866) In only 380 of these patients broad-range PCRwas positive In one-third of these patients (119899 = 9)with a positive blood culture and a broad-range PCR themicroorganisms identified differed among both techniques inboth the species and the genera In two of these cases bloodculture revealed S alactolyticus and S auricularis whichare not reported in literature to cause IE whereas broad-range PCR identified microorganisms (S gallolyticus and Saureus resp) consistent withmicroorganisms causing IE [39]pointing to possible misidentification or even contaminationof blood cultures in these cases In two other cases broad-range PCR identified fastidious bacteria such as Granuli-catella adiacens and the intracellular organism Bartonellaquintanawhich can be easily missed by conventional culture-based methods [13 36 40] In the latter case conventionalculture revealed S epidermidis which can be clearly assumedto be a contamination in the clinical context For 2 casesin which blood culture revealed the genus only broad-range PCR identified the species of the bacteria and therebyspecified the etiology of the causative microorganisms Allthese cases represent a possible diagnostic ldquoadd-onrdquo of PCRdiagnostics as the molecular identification of the causativepathogen by broad-range PCR is regarded more accuratelythan by blood culture and biochemical identification [41]Furthermore broad-range PCR from valve tissue is known tobemore specific in the etiologic diagnosis of IE and thereforemay also lead to better tailored treatment regimens after IEand cardiac surgery

In the majority of blood culture-positive cases broad-range PCR remained negative (620) which can be ascribedeither to prolonged preoperative antibiotic treatment as 977of these patients received antibiotics before surgery or topossible inadequate intraoperative specimen collection Thisis why several fractions of the valve tissue should best becollected for broad-range PCR [22 36] and swabs shouldbe avoided In one case out of 11 blood culture-negative IEcases (91) broad-range PCR provided the sole etiologicdiagnosis of IE Although in the routine clinical setting thesensitivity of PCR testing was somewhat lower than expectedour study confirms the usefulness of PCR amplification andsequencing of the bacterial 16S-rDNA as an important add-on technology in the analysis of resected heart valve tissueof swabs in cases with tissue or swab culture-negative IE asdescribed previously [19 21 24 28] Our study identified atotal of 20 patients (215) who possibly profited from theapplication of broad-range PCR in case of heart tissue orswab culture-negative IE by the following (i) reclassifying aldquopossible IErdquo to ldquodefinite IErdquo considering broad-range PCR


as a criterion for IE (150) (ii) providing the sole etiologicidentification of the causative pathogen (250) (iii) correc-tion of genera and species of probably misidentified causativepathogens resulting from preBC diagnostics (350) (iv)adding the correct species identification specification to thepathogen identified by preBC (200) and (v) excluding pos-sible contaminants resulting from preBC diagnostics (50)

5 Conclusion

Novel perspectives on the management of IE are neededto decrease the rate of residual deaths by accelerating theprocesses of diagnosis risk stratification and facilitating theindividualised adaption of antimicrobial therapy In view ofour results we thus consider broad-range PCR on resectedheart valve tissue as a helpful supplementary tool in patientswith culture-negative or questionable IE Further researchinto the ldquoadded valuerdquo of broad-range PCR in the diagnosis ofIE compared to conventional diagnostics in routine clinicalsettings as well as a better standardization and furtherimprovements of PCR technology including increased sen-sitivity and specificity are necessary before this techniquecan be finally included in Dukersquos classification schemeNevertheless culture-dependent methods will remain themainstay in the diagnosis of IE as they offer the possibility ofantimicrobial susceptibility testing of the causative pathogen

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

DanielaManeg and Janina Sponsel contributed equally to thiswork

References

[1] B Zaloudıkova E Nemcova J Pol et al ldquoValue of PCRin surgically treated patients with staphylococcal infectiveendocarditis a 4-year retrospective studyrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 31 no 6 pp1187ndash1194 2012

[2] G Habib B Hoen P Tornos et al ldquoGuidelines on the preven-tion diagnosis and treatment of infective endocarditis (newversion 2009) the Task Force on the Prevention Diagnosis andTreatment of Infective Endocarditis of the European Societyof Cardiology (ESC) Endorsed by the European Society ofClinical Microbiology and Infectious Diseases (ESCMID) andthe International Society of Chemotherapy (ISC) for Infectionand Cancerrdquo European Heart Journal vol 30 no 19 pp 2369ndash2413 2009

[3] F Thuny D Grisoli J Cautela A Riberi D Raoult and GHabib ldquoInfective endocarditis prevention diagnosis andman-agementrdquo Canadian Journal of Cardiology vol 30 no 9 pp1046ndash1057 2014

[4] M Wolff B Mourvillier R Sonneville and J-F Timsit ldquoMypaper 10 years later infective endocarditis in the intensive careunitrdquo Intensive Care Medicine vol 40 no 12 pp 1843ndash18522014

[5] Y-A Que and P Moreillon ldquoInfective endocarditisrdquo NatureReviews Cardiology vol 8 no 6 pp 322ndash336 2011

[6] PMoreillon and Y-A Que ldquoInfective endocarditisrdquoTheLancetvol 363 no 9403 pp 139ndash149 2004

[7] D H Bor S Woolhandler R Nardin J Brusch and D UHimmelstein ldquoInfective endocarditis in the US 1998ndash2009 anationwide studyrdquo PLoS ONE vol 8 no 3 Article ID e600332013

[8] V Le Moing F Alla T Doco-Lecompte et al ldquoStaphylococcusaureus bloodstream infection and endocarditismdasha prospectivecohort studyrdquo PLoS ONE vol 10 no 5 Article ID e01273852015

[9] N Westphal B Plicht and C Naber ldquoInfective endocarditismdashprophylaxis diagnostic criteria and treatmentrdquo DeutschesArzteblatt International vol 106 no 28-29 pp 481ndash489 2009

[10] C H Cabell E Abrutyn and A W Karchmer ldquoCardiologypatient page Bacterial endocarditis the disease treatment andpreventionrdquo Circulation vol 107 no 20 pp e185ndashe187 2003

[11] HW Ann J Y Ahn Y D Jeon et al ldquoIncidence of and risk fac-tors for infectious complications in patients with cardiac deviceimplantationrdquo International Journal of Infectious Diseases vol36 pp e9ndashe14 2015

[12] C K Naber A Bauhofer M Block et al ldquoS2-leitlinie zur diag-nostik und therapie der infektiosen endokarditisrdquoZeitschrift furKardiologie vol 93 no 12 pp 1005ndash1021 2004

[13] A Moter MMusci and D Schmiedel ldquoMolecular methods fordiagnosis of infective endocarditisrdquo Current Infectious DiseaseReports vol 12 no 4 pp 244ndash252 2010

[14] M Marın P Munoz M Sanchez et al ldquoMolecular diagnosisof infective endocarditis by real-time broad-range polymerasechain reaction (PCR) and sequencing directly from heart valvetissuerdquoMedicine vol 86 no 4 pp 195ndash202 2007

[15] G Marsch P Orszag B Mashaqi C Kuehn and A HaverichldquoAntibiotic therapy following polymerase chain reaction diag-nosis of infective endocarditis a single centre experiencerdquoInteractive Cardiovascular and Thoracic Surgery vol 20 no 5pp 589ndash593 2015

[16] G Martinez and K Valchanov ldquoInfective endocarditis Contin-uing Education inAnaesthesiardquoCritical Careamp Pain vol 12 no3 pp 134ndash139 2012

[17] P Tornos T Gonzalez-Alujas FThuny andGHabib ldquoInfectiveendocarditis the European viewpointrdquo Current Problems inCardiology vol 36 no 5 pp 175ndash222 2011

[18] H Kanamori R Kakuta H Yano et al ldquoA case of culture-negative endocarditis due to Streptococcus tigurinusrdquo Journal ofInfection and Chemotherapy vol 21 no 2 pp 138ndash140 2015

[19] P P Bosshard A Kronenberg R Zbinden C Ruef E CBottger andM Altwegg ldquoEtiologic diagnosis of infective endo-carditis by broad-range polymerase chain reaction a 3-yearexperiencerdquo Clinical Infectious Diseases vol 37 no 2 pp 167ndash172 2003

[20] D Goldenberger A Kunzli P Vogt R Zbinden and MAltwegg ldquoMolecular diagnosis of bacterial endocarditis bybroad-range PCR amplification and direct sequencingrdquo Journalof Clinical Microbiology vol 35 no 11 pp 2733ndash2739 1997

[21] V Gauduchon L Chalabreysse J Etienne et al ldquoMoleculardiagnosis of infective endocarditis by PCR amplification anddirect sequencing of DNA from valve tissuerdquo Journal of ClinicalMicrobiology vol 41 no 2 pp 763ndash766 2003

[22] I Podglajen F Bellery C Poyart et al ldquoComparative molecularand microbiologic diagnosis of bacterial endocarditisrdquo Emerg-ing Infectious Diseases vol 9 no 12 pp 1543ndash1547 2003


[23] S Lang R WWatkin P A Lambert R S Bonser W A LittlerandT S J Elliott ldquoEvaluation of PCR in themolecular diagnosisof endocarditisrdquoThe Journal of Infection vol 48 no 3 pp 269ndash275 2004

[24] C Breitkopf D Hammel H H Scheld G Peters and KBecker ldquoImpact of a molecular approach to improve themicrobiological diagnosis of infective heart valve endocarditisrdquoCirculation vol 111 no 11 pp 1415ndash1421 2005

[25] G Greub H Lepidi C Rovery et al ldquoDiagnosis of infectiousendocarditis in patients undergoing valve surgeryrdquo The Ameri-can Journal of Medicine vol 118 no 3 pp 230ndash238 2005

[26] P Kotilainen M Heiro J Jalava et al ldquoAetiological diagnosisof infective endocarditis by direct amplification of rRNA genesfrom surgically removed valve tissue An 11-year experience ina Finnish teaching hospitalrdquo Annals of Medicine vol 38 no 4pp 263ndash273 2006

[27] P Pathipati T Menon N Kumar T Francis P Sekar andK M Cherian ldquoUsefulness of 16S rDNA sequencing for thediagnosis of infective endocarditis caused by Corynebacteriumdiphtheriaerdquo Journal of Medical Microbiology vol 61 no 8 pp1159ndash1161 2012

[28] P-E Fournier F Thuny H Richet et al ldquoComprehensivediagnostic strategy for blood culture-negative endocarditis aprospective study of 819 new casesrdquo Clinical Infectious Diseasesvol 51 no 2 pp 131ndash140 2010

[29] G E Madico and P A Rice ldquo16S-ribosomal DNA to diag-nose culture-negative endocarditisrdquo Current Infectious DiseaseReports vol 10 no 4 pp 280ndash286 2008

[30] P Tattevin G Watt M Revest C Arvieux and P E FournierldquoUpdate on blood culture-negative endocarditisrdquo Medecine etMaladies Infectieuses vol 45 no 1-2 pp 1ndash8 2015

[31] J S Li D J Sexton N Mick et al ldquoProposed modificationsto the Duke criteria for the diagnosis of infective endocarditisrdquoClinical Infectious Diseases vol 30 no 4 pp 633ndash638 2000

[32] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[33] W-L Cheng C W Li M C Li N Y Lee C C Lee and W CKo ldquoSalmonella infective endocarditisrdquo Journal of MicrobiologyImmunology and Infection 2015

[34] M Grijalva R Horvath M Dendis J Erny and J BenedıkldquoMolecular diagnosis of culture negative infective endocarditisclinical validation in a group of surgically treated patientsrdquoHeart vol 89 no 3 pp 263ndash268 2003

[35] M Voldstedlund L N Pedersen U Baandrup K E Klaaborgand K Fuursted ldquoBroad-range PCR and sequencing in routinediagnosis of infective endocarditisrdquo APMIS vol 116 no 3 pp190ndash198 2008

[36] Y T Chin RHasan andAQamruddin ldquo16S rRNAPCR for thediagnosis of culture-negative Bartonella quintana endocarditisthe importance of sample typerdquo Indian Journal of MedicalMicrobiology vol 33 no 1 pp 185ndash186 2015

[37] H Lepidi J-P Casalta P-E Fournier G Habib F Collart andD Raoult ldquoQuantitative histological examination of biopros-thetic heart valvesrdquo Clinical Infectious Diseases vol 42 no 5pp 590ndash596 2006

[38] P A Rice and G E Madico ldquoPolymerase chain reaction todiagnose infective endocarditis will it replace blood culturesrdquoCirculation vol 111 no 11 pp 1352ndash1354 2005

[39] L Mauri J A de Lemos and P T OrsquoGara ldquoInfective endocardi-tisrdquo Current Problems in Cardiology vol 26 no 9 pp 562ndash6102001

[40] S Edouard C Nabet H Lepidi P-E Fournier and D RaoultldquoBartonella a common cause of endocarditis a report on 106cases and reviewrdquo Journal of Clinical Microbiology vol 53 no 3pp 824ndash829 2015

[41] D Harmsen and H Karch ldquo16S rDNA for diagnosingpathogens a living treerdquo ASM News vol 70 no 1 pp 19ndash242004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Early diagnosis of IE and rapid identification of thecausative pathogen are crucial for providing a specific andefficient antibiotic therapy and improvement of the patient [512ndash15] Generally in IE patients blood cultures are frequentlypositive [16] Nevertheless such cultures can remain negativein 25 to 31 of all cases of IE depending on causativeagent and the clinical circumstances [2 17 18] Blood culturestypically remain negative due to antibiotic therapy prioror concurrent to blood sampling (sim50 of cases) due toinfection by fastidious organisms such as nutritionally variantstreptococci fastidious Gram-negative bacilli of the HACEKgroup (Aggregatibacter aphrophilus former Haemophilusaphrophilus and H paraphrophilus Aggregatibacter actino-mycetemcomitans formerActinobacillus actinomycetemcomi-tans Cardiobacterium hominis Eikenella corrodens Kingellakingae) Brucella spp (in endemic areas) and fungi or due toinfections by intracellular bacteria such as the zoonotic agentsCoxiella burnetii and Bartonella and Tropheryma whipplei[5] In such cases negative cultures may significantly delaydiagnosis and effective treatment which possibly negativelyimpacts on the outcome of the patient [17]

These obvious drawbacks of conventional techniquesclearly impair the current criteria used for clinical diagnosisof IE and call for adjunctive diagnostic methods increas-ing the sensitivity for more rapid pathogen recovery andidentification This is why in such cases of blood culture-negative or clinically questionable IE culture-independentnew molecular diagnostic techniques such as broad-rangepolymerase chain reaction (PCR) with subsequent DNA-sequencing fromheart valve specimenhave gained increasingattention and were shown to be useful in the detection andclassification of bacterial DNA in several recent diagnosticstudies [19ndash28] Besides detecting organisms that are missedby culture-dependent methods PCR-based methods alsoallow exact identification of the causative organism and theidentification of new hitherto unknown pathogens causing IEeven if the patient is already under antibiotic treatment [29]These possibly beneficial features of broad-range PCR for thediagnosis in such cases have even led to the proposition ofPCR positivity as a major diagnostic criterion for IE [14 1930] However further research is clearly required until PCR-based methods become accepted as routine diagnostic tool[2 9]

The aim of the present study was to investigate theadditional diagnostic value of broad-range PCR targeting the16S ribosomal DNA in heart tissue or swabs in patients withconfirmed suspected or possible IE in a routine clinicaldiagnostic setting We hypothesized that the use of broad-range PCR amplification followed by direct sequencingmightincrease the number of identified causative pathogens in caseswith culture-negative IE and in antibiotically pretreated indi-viduals thereby possibly improving the rapidity of diagnosisin such patients also in the routine diagnostic setting

2 Material and Methods

21 StudyDesign and Patients For our retrospective observa-tional study we identified all patients admitted to KerckhoffClinic in Bad Nauheim (Germany) between 2011 and 2014

(Figure 1)This cohort was then subjected to further in-depthcomputer-assisted analysis identifying all patients admittedwith a diagnosis of suspected or definite infective endocardi-tis encoded by the ICD-10-GM (International Classificationof Diseases 10th Revision German Modification) for ldquoacuteand subacute endocarditisrdquo (I33-) and undergoing cardio-surgical treatment We also looked for all patients subjectedto diagnostic testing after admission to Kerckhoff Clinic bybroad-range PCR amplification and direct sequencing fromcardiosurgical specimens as performed at the Institute ofLaboratory Medicine Microbiology and Infection Control atthe NorthwestMedical Centre in FrankfurtMain (Germany)during the same time period In combining both selectorsall patients were identified with both an encoded diagnosisof ldquoacute and subacute endocarditisrdquo and a diagnostic testresult of broad-range PCR and direct sequencing fromcardiosurgical specimens for further investigation (Figure 1)Medical charts of all patients of this study group werethen retrospectively reviewed and patients were includedin the study if both the microbiological findings or broad-range PCR and the corresponding tissue or swab culture ofcardiosurgical specimens were available

22 Definition of Diagnosed IE For the purpose of this studypatients of the study group were divided into three differentcategories (ldquodefiniterdquo ldquopossiblerdquo and ldquono evidence forrdquo IEFigure 1) according to somemodification ofDukersquos criteria forthe diagnosis of IE [31] and including all available data fromthe medical charts at Kerckhoff Clinic as follows definite IEa typical valve histopathology (considered as gold standard)in addition to 2 or more findings indicating IE in any ofthe following diagnostic procedures is present echocardio-graphy preoperative BC (preBC) and typical intraoperativemacroscopic aspect possible IE there is negative valvehistopathology or evidence for IE resulting from one of theabove-mentioned diagnostic procedures only but there is apositive result upon PCR andor tissue or swab culture fromvalve tissue after surgery no evidence for IE only one ofthe above described procedures provided evidence for IE butPCR conventional culture fromvalve tissue after surgery andhistopathology proved negative (Figure 1)

23 Surgical Material Tissue specimens or swabs fromintraoperatively excised cardiac tissue were taken duringcardiosurgical treatment at Kerckhoff Clinic in Bad Nauheimaccording to local standard surgical and diagnostic proce-dures and specimens for microbiological analysis were thendelivered to the Institute of Laboratory Medicine Microbiol-ogy and Infection Control at the Northwest Medical Centrein FrankfurtMain (Germany) for microbiological analysisperformed under standard conditions

24MolecularMethods Bacterial DNAwas isolated from theintraoperative excised specimens by means of the MagNAPure Compact System (Roche Diagnostics Mannheim Ger-many) For the amplification of the 16S-rDNA with aGeneAmp PCR System 2700 or a Thermal Cycler 2720(Applied Biosystems Darmstadt Germany) the universal51015840 primer 285 (gag agt ttg atc ctg gct cag position 9ndash29





endocarditisrdquo


broad-range PCR




N = 114

N = 122N = 210

N = 40190





3 Results






6119873 () 73 (646) 34 (301) 6 (53)




















preBC pos

preBC neg





Enterococcus

Other




Add-on n = 9

Add-on n = 1

pos n = 12

pos n = 1

pos n = 27

neg n = 44

neg n = 10

neg n = 10


n = 33


Concordance n = 9

Discordance n = 14

n = 104n = 104

n = 22

n = 11

n = 71

MRSA 37







Table3Overviewof


nob

tained

with

PCRandsequ

encing

metho

dsandcomparis

onto

ther

esultsof

preoperativ

eblood

cultu

res(preB

C)

PCRpo


identifi

catio

n119899=33

lowast

Con

cordance119899=9

(PCR

preBClowastlowast)

119899

Disc


sand

orspecies

level)

Additio


(nopreB

Cdataavailable)

119899

PCR

Resultof

preB

Clowastlowast

Enterococcus

faecalis

5En

terococcus

faecalis


Enterococcus

faecalis

1Staphylococcus

aureus

1En

terococcus

faecium

Enterococcus

faecalis

Staphylococcus

aureus

2Streptococcusa

galactiae

1Staphylococcus

aureus

Staphylococcus

auric

ularis

Staphylococcus

epidermidis

1Ae

rococcus

urinae

1Staphylococcus

epidermidis

Streptococcuso

ralis

Staphylococcus

lugdun

ensis

1Gem

ellahaem

olysan

s1


subsppaste

urianu

sStreptococcusa

lactolyticu

sStreptococcusm

itis


Streptococcusp

neum

oniae

Streptococcusp

neum

oniae


Streptococcusv

iridans


1Streptococcusm

itis


Anoxybacillus

spp

1Ba

rtonellaquintana

Staphylococcus

epidermidis

Propionibacterium

acnes

1Gr

anulica

tella

adiacens

Streptococcusv

iridans

Streptococcuso

ralis

Streptococcusv

iridans

Enterococcus

faecium

Gram-positive

cocci

Staphylococcus

epidermidis

Gram-positive

cocci

Propionibacterium

acnes

Negativep

reBC

lowastIn

7patie

ntsw

ithpo

sitiveP

CRam

plicon

dire

ctsequ

encing

faileddu

etoab

acteria

lmixture119899=4or


lowastlowastRe

sults

ofpreB

Cweretaken

andanalyzed

inho

spita

lsof

prim

arycare

accordingto

localstand


ratory

procedures



Result Culture PCR












4 Discussion



Diagnosis+ minus

PCR














5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





endocarditisrdquo


broad-range PCR




N = 114

N = 122N = 210

N = 40190





3 Results






6119873 () 73 (646) 34 (301) 6 (53)




















preBC pos

preBC neg





Enterococcus

Other




Add-on n = 9

Add-on n = 1

pos n = 12

pos n = 1

pos n = 27

neg n = 44

neg n = 10

neg n = 10


n = 33


Concordance n = 9

Discordance n = 14

n = 104n = 104

n = 22

n = 11

n = 71

MRSA 37







Table3Overviewof


nob

tained

with

PCRandsequ

encing

metho

dsandcomparis

onto

ther

esultsof

preoperativ

eblood

cultu

res(preB

C)

PCRpo


identifi

catio

n119899=33

lowast

Con

cordance119899=9

(PCR

preBClowastlowast)

119899

Disc


sand

orspecies

level)

Additio


(nopreB

Cdataavailable)

119899

PCR

Resultof

preB

Clowastlowast

Enterococcus

faecalis

5En

terococcus

faecalis


Enterococcus

faecalis

1Staphylococcus

aureus

1En

terococcus

faecium

Enterococcus

faecalis

Staphylococcus

aureus

2Streptococcusa

galactiae

1Staphylococcus

aureus

Staphylococcus

auric

ularis

Staphylococcus

epidermidis

1Ae

rococcus

urinae

1Staphylococcus

epidermidis

Streptococcuso

ralis

Staphylococcus

lugdun

ensis

1Gem

ellahaem

olysan

s1


subsppaste

urianu

sStreptococcusa

lactolyticu

sStreptococcusm

itis


Streptococcusp

neum

oniae

Streptococcusp

neum

oniae


Streptococcusv

iridans


1Streptococcusm

itis


Anoxybacillus

spp

1Ba

rtonellaquintana

Staphylococcus

epidermidis

Propionibacterium

acnes

1Gr

anulica

tella

adiacens

Streptococcusv

iridans

Streptococcuso

ralis

Streptococcusv

iridans

Enterococcus

faecium

Gram-positive

cocci

Staphylococcus

epidermidis

Gram-positive

cocci

Propionibacterium

acnes

Negativep

reBC

lowastIn

7patie

ntsw

ithpo

sitiveP

CRam

plicon

dire

ctsequ

encing

faileddu

etoab

acteria

lmixture119899=4or


lowastlowastRe

sults

ofpreB

Cweretaken

andanalyzed

inho

spita

lsof

prim

arycare

accordingto

localstand


ratory

procedures



Result Culture PCR












4 Discussion



Diagnosis+ minus

PCR














5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





6119873 () 73 (646) 34 (301) 6 (53)




















preBC pos

preBC neg





Enterococcus

Other




Add-on n = 9

Add-on n = 1

pos n = 12

pos n = 1

pos n = 27

neg n = 44

neg n = 10

neg n = 10


n = 33


Concordance n = 9

Discordance n = 14

n = 104n = 104

n = 22

n = 11

n = 71

MRSA 37







Table3Overviewof


nob

tained

with

PCRandsequ

encing

metho

dsandcomparis

onto

ther

esultsof

preoperativ

eblood

cultu

res(preB

C)

PCRpo


identifi

catio

n119899=33

lowast

Con

cordance119899=9

(PCR

preBClowastlowast)

119899

Disc


sand

orspecies

level)

Additio


(nopreB

Cdataavailable)

119899

PCR

Resultof

preB

Clowastlowast

Enterococcus

faecalis

5En

terococcus

faecalis


Enterococcus

faecalis

1Staphylococcus

aureus

1En

terococcus

faecium

Enterococcus

faecalis

Staphylococcus

aureus

2Streptococcusa

galactiae

1Staphylococcus

aureus

Staphylococcus

auric

ularis

Staphylococcus

epidermidis

1Ae

rococcus

urinae

1Staphylococcus

epidermidis

Streptococcuso

ralis

Staphylococcus

lugdun

ensis

1Gem

ellahaem

olysan

s1


subsppaste

urianu

sStreptococcusa

lactolyticu

sStreptococcusm

itis


Streptococcusp

neum

oniae

Streptococcusp

neum

oniae


Streptococcusv

iridans


1Streptococcusm

itis


Anoxybacillus

spp

1Ba

rtonellaquintana

Staphylococcus

epidermidis

Propionibacterium

acnes

1Gr

anulica

tella

adiacens

Streptococcusv

iridans

Streptococcuso

ralis

Streptococcusv

iridans

Enterococcus

faecium

Gram-positive

cocci

Staphylococcus

epidermidis

Gram-positive

cocci

Propionibacterium

acnes

Negativep

reBC

lowastIn

7patie

ntsw

ithpo

sitiveP

CRam

plicon

dire

ctsequ

encing

faileddu

etoab

acteria

lmixture119899=4or


lowastlowastRe

sults

ofpreB

Cweretaken

andanalyzed

inho

spita

lsof

prim

arycare

accordingto

localstand


ratory

procedures



Result Culture PCR












4 Discussion



Diagnosis+ minus

PCR














5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








preBC pos

preBC neg





Enterococcus

Other




Add-on n = 9

Add-on n = 1

pos n = 12

pos n = 1

pos n = 27

neg n = 44

neg n = 10

neg n = 10


n = 33


Concordance n = 9

Discordance n = 14

n = 104n = 104

n = 22

n = 11

n = 71

MRSA 37







Table3Overviewof


nob

tained

with

PCRandsequ

encing

metho

dsandcomparis

onto

ther

esultsof

preoperativ

eblood

cultu

res(preB

C)

PCRpo


identifi

catio

n119899=33

lowast

Con

cordance119899=9

(PCR

preBClowastlowast)

119899

Disc


sand

orspecies

level)

Additio


(nopreB

Cdataavailable)

119899

PCR

Resultof

preB

Clowastlowast

Enterococcus

faecalis

5En

terococcus

faecalis


Enterococcus

faecalis

1Staphylococcus

aureus

1En

terococcus

faecium

Enterococcus

faecalis

Staphylococcus

aureus

2Streptococcusa

galactiae

1Staphylococcus

aureus

Staphylococcus

auric

ularis

Staphylococcus

epidermidis

1Ae

rococcus

urinae

1Staphylococcus

epidermidis

Streptococcuso

ralis

Staphylococcus

lugdun

ensis

1Gem

ellahaem

olysan

s1


subsppaste

urianu

sStreptococcusa

lactolyticu

sStreptococcusm

itis


Streptococcusp

neum

oniae

Streptococcusp

neum

oniae


Streptococcusv

iridans


1Streptococcusm

itis


Anoxybacillus

spp

1Ba

rtonellaquintana

Staphylococcus

epidermidis

Propionibacterium

acnes

1Gr

anulica

tella

adiacens

Streptococcusv

iridans

Streptococcuso

ralis

Streptococcusv

iridans

Enterococcus

faecium

Gram-positive

cocci

Staphylococcus

epidermidis

Gram-positive

cocci

Propionibacterium

acnes

Negativep

reBC

lowastIn

7patie

ntsw

ithpo

sitiveP

CRam

plicon

dire

ctsequ

encing

faileddu

etoab

acteria

lmixture119899=4or


lowastlowastRe

sults

ofpreB

Cweretaken

andanalyzed

inho

spita

lsof

prim

arycare

accordingto

localstand


ratory

procedures



Result Culture PCR












4 Discussion



Diagnosis+ minus

PCR














5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Overviewof


nob

tained

with

PCRandsequ

encing

metho

dsandcomparis

onto

ther

esultsof

preoperativ

eblood

cultu

res(preB

C)

PCRpo


identifi

catio

n119899=33

lowast

Con

cordance119899=9

(PCR

preBClowastlowast)

119899

Disc


sand

orspecies

level)

Additio


(nopreB

Cdataavailable)

119899

PCR

Resultof

preB

Clowastlowast

Enterococcus

faecalis

5En

terococcus

faecalis


Enterococcus

faecalis

1Staphylococcus

aureus

1En

terococcus

faecium

Enterococcus

faecalis

Staphylococcus

aureus

2Streptococcusa

galactiae

1Staphylococcus

aureus

Staphylococcus

auric

ularis

Staphylococcus

epidermidis

1Ae

rococcus

urinae

1Staphylococcus

epidermidis

Streptococcuso

ralis

Staphylococcus

lugdun

ensis

1Gem

ellahaem

olysan

s1


subsppaste

urianu

sStreptococcusa

lactolyticu

sStreptococcusm

itis


Streptococcusp

neum

oniae

Streptococcusp

neum

oniae


Streptococcusv

iridans


1Streptococcusm

itis


Anoxybacillus

spp

1Ba

rtonellaquintana

Staphylococcus

epidermidis

Propionibacterium

acnes

1Gr

anulica

tella

adiacens

Streptococcusv

iridans

Streptococcuso

ralis

Streptococcusv

iridans

Enterococcus

faecium

Gram-positive

cocci

Staphylococcus

epidermidis

Gram-positive

cocci

Propionibacterium

acnes

Negativep

reBC

lowastIn

7patie

ntsw

ithpo

sitiveP

CRam

plicon

dire

ctsequ

encing

faileddu

etoab

acteria

lmixture119899=4or


lowastlowastRe

sults

ofpreB

Cweretaken

andanalyzed

inho

spita

lsof

prim

arycare

accordingto

localstand


ratory

procedures



Result Culture PCR












4 Discussion



Diagnosis+ minus

PCR














5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Result Culture PCR












4 Discussion



Diagnosis+ minus

PCR














5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusion


Competing Interests




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article advantages and limitations of direct pcr … · 2019. 7. 30. · research article...

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