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Lipopolysaccharide-Binding Protein a New Biomarker for Infectious is

Lipopolysaccharide-Binding Protein a New Biomarker for Infectious is

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Published by: Rara Indriani on Apr 01, 2011
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02/05/2013

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Lipopolysaccharide-Binding Protein:A New Biomarker forInfectious Endocarditis?
Tanja Vollmer,
1
Cornelia Piper,
2
Knut Kleesiek,
1
and Jens Dreier
1*
BACKGROUND
:
Infectious endocarditis (IE) is a bacterialinfection of the endocardium. Diagnosis is based onresults obtained from echocardiography, blood cul-tures, and molecular genetic screening for bacteria andon data for inflammatory markers such as the leuko-cyte (WBC) count and the C-reactive protein (CRP)concentration. The aim of the present study was toevaluatelipopolysaccharide-bindingprotein(LBP)asasupportive biomarker for the diagnosis and therapeu-tic monitoring of IE.
METHODS
:
We measured LBP and CRP concentrationsand WBC counts in 57 IE patients at hospital admis-sion, 40 patients with noninfectious heart valve dis-eases (HVDs), and 55 healthy blood donors. The pro-gressionofthese3markersandtheinfluenceofcardiacsurgeryonthemwereevaluatedin29IEpatientsand21control patients.
RESULTS
:
Serum LBP concentrations were significantly higher in IE patients [mean (SD), 33.41 (32.10) mg/L]compared with HVD patients [6.67 (1.82) mg/L,
0.0001] and healthy control individuals [5.61 (1.20)mg/L]. The progression in the LBP concentration dur-ingtherapyofIEpatientscorrelatedwiththechangesinthe CRP concentration. The 2 markers were equally influenced by antibiotic treatment and surgicalintervention.
CONCLUSIONS
:
SerialLBPmeasurementmayprovideaneffective and useful tool for evaluating the response totherapy in IE patients. We found a strong correlationbetween LBP and CRP concentrations; LBP has a ten-dency to increase earlier in cases of reinfection.
© 2008 American Association for Clinical Chemistry 
Infectious endocarditis (IE),
3
a relatively rare diseasewith an incidence of 30 per million population
(1)
, isassociated with high rates of mortality and morbidity.Rapid diagnosis of IE is critical for effective treatment.Diagnosis is based primarily on the results of echocar-diographicexaminationsandbloodculturetests[Dukecriteria
(2)
], as well as on the results of serology testsand molecular genetic screening methods for bacteriain cases of heart valve replacement
(3, 4)
. The initialdiagnosis of IE can be difficult, however, because of nonspecific clinical symptoms, negative results inblood cultures, and ambiguous echocardiographicfindings. In this context, the availability of biomarkersis of major interest. Several studies have investigatedthe application and usefulness of important laboratory markersforIEdiagnosis
(5–12)
.Otherstudies
(13–16)
have focused on the clinical usefulness of serum C-reactive protein (CRP), a marker most frequently usedin Europe
(13)
, in IE. CRP has been assumed to beprimarily an indicator of local bacterial infections, butincreased CRP concentrations have also been strongly associated with various nonbacterial infectious dis-eases, e.g., rheumatic disorders
(17)
. CRP has beenshown to have a limited ability to distinguish betweenbacterial and viral infections
(18)
.Lipopolysaccharide-binding protein (LBP), a gly-cosylated 58-kDa protein produced predominantly inhepatocytes,hasrecentlybeendescribedasapromisingnovel diagnostic marker for the diagnosis of local bac-terial infection
(19–23)
. LBP is released constitutively into the bloodstream upon acute-phase stimulation
(24)
. It has a high affinity for lipopolysaccharide (LPS)and other bacterial-surface components
(25)
. Bindingof LPS to LBP facilitates the transportation of endo-toxin molecules to immune effector cells possessingsurfaceCD14receptorsandfurtheractivatestheproin-flammatory cascade
(24, 26)
. Association of the LPS/
1
Institut fu¨r Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszen-trum Nordrhein-Westfalen, Universita¨tsklinik der Ruhr-Universita¨t Bochum, BadOeynhausen, Germany;
2
Klinik fu¨r Kardiologie, Herz- und DiabeteszentrumNordrhein-Westfalen, Universita¨tsklinik der Ruhr-Universita¨t Bochum, BadOeynhausen, Germany.* Address correspondence to this author at: Institut fu¨r Laboratoriums- undTransfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Univer-sita¨tsklinik der Ruhr-Universita¨t Bochum, Georgstrasse 11, 32545 Bad Oeyn-hausen, Germany. Fax
49-5731-97-2307; e-mail jdreier@hdz-nrw.de.Received March 4, 2008; accepted August 26, 2008.Previously published online at DOI: 10.1373/clinchem.2008.106195
3
Nonstandard abbreviations: IE, infectious endocarditis; CRP, C-reactive protein;LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; HVD, nonin-fectious heart valve disease; WBC, leukocyte; CI, confidence interval.
Clinical Chemistry
55:2295–304 (2009)
Proteomics and Protein Markers
295
 
LBP complex with HDL inactivates LPS, thereby pro-tecting against LPS toxicity 
(24)
.The aims of this study were to evaluate (
a
) theclinicalusefulnessofLBPasasupportivebiomarkerforIE diagnosis, (
b
) the applicability of LBP for monitor-ing the response to antibiotic therapy, and (
) the in-fluence of cardiac surgery on the progression of LBPvalues in both IE patients and patients with noninfec-tious heart valve disease (HVD). The use of LBP as amarker was compared with the frequently used endo-carditis markers CRP and the leukocyte (WBC) count.
MaterialsandMethods
PATIENTS AND CONTROLS
Between May 2005 and November 2007, serum sam-ples were obtained from 57 patients with definite IEdiagnosed according to the Duke criteria. The caus-ative pathogens were identified with standard blood-culture methods or broad-range PCR of the gene forbacterial 16S ribosomal DNA, as described previously 
(27)
. Patients with immunosuppression, cardiogenicshock, or septic shock were excluded. Three of the pa-tients had experienced 2 independent episodes of IE.Patients were subdivided into 4 groups according tothe duration of antibiotic treatment [0–10 days (n
38), 11–20 days (n
7), 21–30 days (n
6), and
30days (n
9)]. Serum samples were also collected from40 control HVD patients before surgical intervention.HVDs consisted of noninfectious and nonrheumaticvalve insufficiencies or stenosis. We measured serumLBP values in 55 healthy blood donors to confirm thepreviously determined reference interval of the com-mercially available LBP assay.To examine the progression in LBP and CRP con-centrations and the WBC count, we collected serumsamples and EDTA-anticoagulated blood samplesfrom 29 IE patients and 21 control patients with HVDduring their admission at our hospital. Samples werecollecteddaily.Thestudyprotocolwasapprovedbytheinstitutional review board, and all patients gave theirinformed consent.
MEASUREMENT OF CRP, LBP, AND THE WBC COUNT
Serum samples were collected in serum Monovettes(Sarstedt). The samples were clotted, centrifuged at4000
for10min,andstoredat
70 °Cwithin2daysof collection.Sampleswerestoredatthistemperatureun-til assayed.Serum CRP was measured with the ultrasensitivelatex immunoassay CRP
Vario
(Abbott Diagnostics),and the total LBP concentration in serum samples wasmeasured with a commercially available chemilumi-nescence assay (IMMULITE LBP, Diagnostics Prod-uctsCorporation/SiemensHealthcareDiagnostics)ac-cordingtothemanufacturer’sinstructions.Allsamplesweretestedinrandomorderwithcontrolsandcalibra-tion curves incorporated into each run. Blood sampleswere collected in EDTA Monovettes (Sarstedt), andWBCs were counted directly with the Cell-Dyn 3500(Abbott Diagnostics).
STATISTICAL ANALYSIS
All data are presented as the mean (SD). The Mann–Whitney 
-test and the Student
-test were used forstatistical analyses as appropriate, and the assumptionofagaussiandistributionwastestedinalldatasetswiththe Kolmogorov–Smirnov test. We used Pearson andSpearmancorrelationcoefficientstoassesscorrelationsbetweenvariables.
values
0.05wereconsideredsta-tistically significant. GraphPad Prism 4.0 software(GraphPad Software) was used for statistical analyses.
Results
Table 1 summarizes the clinical characteristics of thepatients and the control cohorts. LBP was measured inserum samples obtained from 57 patients with 60 epi-sodes of IE, a control group of 40 HVD patients, and asecond control group of 55 healthy blood donors. Weconfirmed the manufacturer’s LBP reference intervalin our healthy control population. Fig. 1A presentsscatter plots of LBP concentration for the 3 cohorts.Serum LBP concentrations were increased in 58 epi-sodes of IE, and the increases were independent of thedurationofantibiotictreatment.In2episodes,theLBPconcentration was within the reference interval. LBPconcentrations were significantly higher in IE patientsthan in the 2 control groups [IE patients, 33.41 (32.10)mg/L; HVD patients, 6.67 (1.82) mg/L; healthy blooddonor control group, 5.61 (1.20) mg/L] (see Fig. 1A).The diagnostic sensitivity was 96.7% [95% confidenceinterval (CI), 92.2%–100.0%], and the diagnosticspecificity was 87.5% (95% CI, 77.3%–97.7%) with re-specttotheHVDpatientsand94.5%(95%CI,88.5%–100.0%)withrespecttothehealthycontrols.ThemeanLBPconcentrationoftheIEpatientsampleswas83.2%higher than the mean concentrations of the controlgroups.With respect to the duration of antibiotic treat-ment, LBP concentrations were considerably lower af-ter11–20daysand
30daysoftreatment[14.56(5.29)mg/L and 15.71 (8.05) mg/L, respectively], comparedwith treatment for 0–10 days [41.97 (37.20) mg/L](Fig. 1A, inset). Comparatively high LBP concentra-tionswereobservedafter21–30daysofantibiotictreat-ment[25.45(7.30)mg/L].Theserumconcentrationof CRP in patients upon hospital admission showed atrend similar to that of LBP, with IE patients havingsignificantly higher concentrations than the individu-
296
Clinical Chemistry
55:2 (2009)
 
als in the 2 control groups [IE patients, 101.90 (81.67)mg/L; HVD patients, 2.31 (2.19) mg/L; healthy blooddonor control group, 1.11 (1.18) mg/L] (see Fig. 1B).Likewise, WBC counts were higher in IE patients[10.54
10
9
/L(5.51
10
9
/L)]thanintheHVDgroup[6.94
10
9
/L (1.55
10
9
/L)] and the healthy controlgroup [7.37
10
9
/L (1.65
10
9
/L)] (Fig. 1C). Thediagnostic sensitivities were 95.0% (95% CI, 89.5%–100.0%) for CRP and 26.7% (95% CI, 15.5%–37.9%)for the WBC count. The diagnostic specificity for CRPwas 87.5% (95% CI, 77.3%–97.7%) with respect to theHVD patients and 100% (95% CI, 93.5%–100%) withrespect to the healthy controls. Although the WBCcount had the lowest diagnostic sensitivity (26.7%), ithad a high diagnostic specificity [100% (95% CI,91.2%–100%)withrespecttoHVDpatientsand96.3%(95% CI 91.3%–100.0%) with respect to the healthy controls]. Combining the LBP and CRP markers pro-duced a diagnostic sensitivity of 95.8% (95% CI,92.2%–99.4%) and a specificity of 87.5% (95% CI,80.3%–94.7%) with respect to HVD patients and97.3% (95% CI, 94.3%–100.0%) with respect to thehealthy controls.LBPandCRPvaluesweresignificantlyhigher(
0.0014, and
0.0009, respectively) in the HVD con-trol group than in the healthy blood donor controlgroup, although all observed LBP concentrations werewithin the reference interval. In contrast, the WBCcounts in the 2 control groups were not significantly different. Comparisons of LBP and CRP concentra-tions and the WBC count according to the infectingmicroorganisms showed no differences in mean valuesfor any of the variables tested (data not shown). Theexamined markers had equivalent positive predictivevalues (LBP, 87.9%; CRP, 91.9%; WBC count, 88.9%)comparedwiththeaverageofthe2controlgroups.Thenegative predictive values were 97.8% (LBP), 96.8%(CRP),and67.9%(WBCcount).Acorrelationanalysisof LBP, CRP, and WBC values for IE patients showed astrong correlation between LBP and CRP concentra-tions (Spearman rank correlation coefficient
0.70;
0.0001) and a moderate correlation between theLBP concentration and the WBC count (
0.33;
0.001). We found weak correlations between the CRPconcentrationandtheWBCcount(
0.26;
0.05).No correlations between the 3 variables were found inthe HVD patients or the healthy blood donors.Fig. 2A presents an example of the progression inLBPandCRPconcentrationsandtheWBCcountdur-inganuncomplicatedepisodeofIE.Thetemporalpro-gression in the concentration of LBP strictly followedthatofCRP.BothLBPandCRPshowedincreasedcon-centrations upon hospital admission and a prompt de-crease after the administration of adequate antibiotictreatment. CRP and LBP concentrations increasedwithinthefirst48haftersurgicalinterventionandthendecreased continuously toward reference values. WBCcounts persisted within the upper portion of the refer-ence interval during the entire IE episode, except forsurgical intervention, which caused a moderate in-crease. This profile is typical and has been observed inmost analyzed episodes of uncomplicated IE. Fig. 2Bsummarizes the progression in LBP and CRP concen-trations and the WBC count in a patient with a notice-ably low LBP response and typical progression in theCRP concentration and the WBC count during an un-complicated IE episode. Even surgical interventioncausedonlya2-foldincreaseintheLBPconcentration.Fig. 2C summarizes the progression in a case of a com-
Table 1.
Demographic characteristics ofthe patients.
Variable
IE patientsAge, years
a
Male (n
42) 64.67 (14.05), 21102Female (n
15) 66.00 (15.35), 4189Affected valves, %Native 70Prosthetic 30Causative microorganism, no. of patients
Staphylococcus aureus 
17Coagulase-negative staphylococci 10
Streptococcus 
spp. 12
Enterococcus 
spp. 12Others 6Culture-negative endocarditis 2Surgical intervention, no. of episodesYes 47No 13HVD patientsAge, yearsMale (n
19) 61.63 (16.36), 2982Female (n
21) 73.19 (7.32), 5986Affected valves, %Native 100Prosthetic 0Healthy blood donorsAge, yearsMale (n
28) 36.75 (10.51), 1955Female (n
27) 37.96 (13.30), 1964
a
Age data are presented as the median (SD), range.
LBP in Infectious EndocarditisClinical Chemistry
55:2 (2009)
297

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