To confirm the diagnostic accuracy of candidate biomarkers in stroke-associ-
ated pneumonia (SAP), we prospectively enrolled ischemic stroke patients with
NIHSS ≥ 10 on admission from March-2016 to August-2017. Blood samples
were collected at baseline, 24 and 48 h after stroke onset. Biomarkers (MR-
proADM, suPAR, SAA) were determined by immunoassays. Regarding
biomarkers, MR-proADM at 24 h (P = 0.04) and both suPAR and SAA at 48 h
(P = 0.036 and P = 0.057) were associated with pneumonia. The combination
of SAA > 25.15 mg/dL and suPAR> 3.14 ng/mL at 48 h had 80% sensitivity
and 95.8% specificity when both biomarkers were above the cut-off. The evalu-
ated biomarkers represent promising tools to be evaluated in future large,
prospective studies on SAP. An accurate SAP diagnosis by thorax CT might
help to reduce variability in such studies

Mancha Molina, Fernando

Moniche Álvarez, Francisco

Montaner, Joan

Pardo Galiana, Blanca

Ramos Herrero, Víctor Darío

Vega Salvatierra, Ángela

Zapata Arriaza, Elena

English

idUS. Depósito de Investigación Universidad de Sevilla

BRIEF COMMUNICATIONBiomarkers predictive value for early diagnosis of Stroke-Associated PneumoniaElena Zapata-Arriaza1 , Fernando Mancha1,*, Alejandro Bustamante2, Francisco Moniche1,3, BlancaPardo-Galiana1,3, Pilar Serrano-Gotarredona4, Silvia Navarro-Herrero4, Esther Pallisa5, Julia Faura2,Angela Vega-Salvatierra1, Anna Penalba2, Irene Escudero-Martınez1,3, Vıctor Darıo Ramos-Herrero1,Leire Azurmendi6, Jean Charles Sanchez6 & Joan Montaner11Stroke Research Program, Institute of Biomedicine of Seville, IBiS/Hospital Universitario Virgen del Rocıo/CSIC, University of Seville, Seville, Spain2Neurovascular Research Laboratory. Vall d’Hebron Institute of Research (VHIR), Universitat Autonoma de Barcelona, Barcelona, Spain3Department of Neurology, Hospital Universitario Virgen del Rocıo, Seville, Spain4Radiology Department, University Hospital Virgen del Rocio, Seville, Spain5Department of Radiology, Vall d’Hebron University Hospital, Barcelona, Spain6Department of Human Protein Sciences, University of Geneva, Geneve, SwitzerlandCorrespondenceAlejandro Bustamante, NeurovascularResearch Laboratory, Vall d’Hebron Instituteof Research (VHIR), Hospital Universitari Valld’Hebron, Passeig Vall d’Hebron 119-129,08035 Barcelona, Spain. Tel: +34934894029; Fax: +34 934894015; E-mail:alejandro.bustamante@vhir.orgFunding InformationThis project was partially funded by the ISCIIIproject PI14/00971. The ITRIBiS project(Improving Translational Research Potential atthe Institute of Biomedicine of Seville) hasthe registration number REGPOT-2013-1.Cooperative Cerebrovascular DiseaseResearch Network (INVICTUS+) (RD16/0019/0015). AB is supported by a Juan Rodesresearch contract (JR16/0008) from Institutode Salud Carlos III.Received: 25 April 2019; Revised: 24 June2019; Accepted: 25 June 2019Annals of Clinical and TranslationalNeurology 2019; 6(9): 1882–1887doi: 10.1002/acn3.50849*Co-First authors.AbstractTo confirm the diagnostic accuracy of candidate biomarkers in stroke-associ-ated pneumonia (SAP), we prospectively enrolled ischemic stroke patients withNIHSS ≥ 10 on admission from March-2016 to August-2017. Blood sampleswere collected at baseline, 24 and 48 h after stroke onset. Biomarkers (MR-proADM, suPAR, SAA) were determined by immunoassays. Regardingbiomarkers, MR-proADM at 24 h (P = 0.04) and both suPAR and SAA at 48 h(P = 0.036 and P = 0.057) were associated with pneumonia. The combinationof SAA > 25.15 mg/dL and suPAR> 3.14 ng/mL at 48 h had 80% sensitivityand 95.8% specificity when both biomarkers were above the cut-off. The evalu-ated biomarkers represent promising tools to be evaluated in future large,prospective studies on SAP. An accurate SAP diagnosis by thorax CT mighthelp to reduce variability in such studies.IntroductionRespiratory tract infections occurring within 7 days ofstroke onset have been incorporated under stroke-associ-ated pneumonia (SAP) term, after adapted definitionfrom the Centre for Diseases and Control and Prevention(CDC)1 by the Pneumonia in Stroke Consensus Group(PISCES).2 However, one of the main limitations in SAPresearch is the lack of a gold-standard test for clinicaldiagnosis. Thorax high-resolution computed tomography1882 ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use anddistribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.(THRCT) is a useful adjunct to conventional radiographyand an accurate study to identify underlying findings instroke patients, which could serve as a guide in SAP diag-nosis. In fact, we have recently reported that this tool rep-resents an accurate imaging test for the evaluation ofclinical criteria.3Stroke alters systemic immunity, predisposing patientsto immunosuppression and infections.4 Therefore, the useof blood biomarkers associated with the systemic inflam-matory response and the immune changes would be aninteresting starting point for SAP prediction. Some sepsisbiomarkers, recently identified as promising predictors ofstroke-associated infections,5,6 represents interesting can-didates for SAP. Soluble urokinase-type plasminogen acti-vator receptor (suPAR), participates in immunologicfunctions.7 Pro-adrenomedullin (MR-proADM), seems tocorrelate with the severity of pneumonia.8 And serumamyloid A protein (SAA) with an important role inattracting leukocytes and immune cells to the sites ofdamage.9 We aimed to confirm the diagnostic accuracy ofmentioned candidate biomarkers related to the featureson THRCT.3MethodsA cohort of 45 consecutive stroke patients was includedin a monocentric, prospective, observational study fromMarch/2016-August/2017. The study was approved by ourinstitutional review committee (ID 0103-M1-14). Thepatients eligible for the study were those with ischemicstroke confirmed by CT or magnetic resonance imaging(MRI) and NIHSS ≥ 10 on patient admission. Strokeonset to inclusion was ≤24 h, and informed consentsigned by the patient or a relative was needed at inclu-sion. Mechanical ventilation pneumonia of intensive careunit transferred patients and antibiotic therapies withinlast 24 h previous to hospital admission were exclusioncriteria. Every patient underwent a comprehensive inter-view, neurological and general examination on admissionand every day after inclusion until the seventh day afteradmission by the same neurologist blinded to the patientstudy participation. Dysphagia was evaluated within thefirst 24 h with the Acute Stroke Dysphagia Screen test.10Chest X-ray (CXR) and laboratory test were run fol-lowing neurologist criteria during admission. A THRCTwith volumetric technique acquisition was performed,without contrast, between 5th and 7th day of admissionin all included patients, independently of the suspicion ofrespiratory infection. CT was performed in a 64 detectorsrows equipment. Dosage Modulation: 120–178 Kv. Colli-mation: 0.5–1 mm, according the equipment. Reconstruc-tion interval: 1 mm. PITCH could be between 0.8 and 1.Acquired data reconstruction was done with soft partsand high spatial frequency double filter. Every THRCTwas evaluated by two skilled thorax radiologists, and dis-crepancies reviewed by another expert thorax radiologist,all blinded to the clinical data. Consensus was reached incases with disagreement.In this study, SAP was diagnosed by treating physicianaccording to diagnostic categories established by thePISCES criteria.2 PISCES Probable SAP was defined bythe presence of all CDC criteria1 but in the absence ofdiagnostic changes on initial CXR and repeated CXR (orwhere CXR not undertaken), and no alternative explana-tion or diagnosis. Likewise, PISCES definite SAP wasdiagnosed when all CDC criteria met including diagnosticCXR changes on at least one CXR.Blood samples for biomarkers analysis were obtained atbaseline, 24 and 48 h after stroke onset. Then, sampleswere centrifuged at 1500g for 15 min at 4°C and serumand plasma were frozen at 80°C until biomarker mea-surement.We measured a 4-blood biomarkers panel includingSAA, MR-proADM, suPAR and CRP. MR-proADM(BRAHMS MR-proADM KRYPTORTM-ThermoFisherScientific, MA) and CRP (CardioPhase HSCRP, Siemens Healthineers Spain)were measured inplasma EDTA samples and suPAR (Quantiquine ELISAkit, R&D Systems, MA) was measured in serum. SAA1was determined in serum with the Meso Scale Discovery(MSD) Vascular Injury Panel-I ECL assay, as per manu-facturer’s instructions (MSD, Gaithersburg, MD). Allbiomarkers except MR-proADM were assessed in dupli-cate and the mean intra-assay coefficient of variation(CV) was <20%. Inter-assay variation was determinedtesting two times in each plate with a commercial internalcontrol (Human serum type AB, male, from clotted,Table 1. Baseline characteristics of the stroke patients that wereinclude in the study and received THRCT.Variable n = 41 %Male 21 51.2Age (years old) (mean  range) 75 [67–82]NIHSS on admission 20 [15–22]Time to probable SAP (PISCES) (d) (Mean [range]) 1.5 [1–2]Time to confirmed SAP on THRCT (d) (Mean [range]) 2 [1.5–3.5]Smoker 12 29.3Chronic obstructive pulmonary disease 8 19.5Dysphagia 31 75.6Urinary tract infection 5 12.1Phlebitis 1 2.4Clinical respiratory infection (PISCES Probable) 8 19.5Stroke associated pneumonia on THRCT(Bronchopneumonia)5 12.2Stroke associated chest infection on THRCT(Bronchopneumonia + Bronchitis)8 19.5ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 1883E. Zapata-Arriaza et al. Correlation with thoracic computed tomographySigma-Aldrich), and it was <20%. MR-proADM, asinstructed, was assayed by simple. Intra-assay and inter-assay CV for this method are <10% and <20%, respec-tively. Biomarker measurement was performed blinded toclinical information.Statistical analyses were conducted with StatisticalPackages for Social Sciences (SPSS), version 22. Inter-group comparisons were performed using Chi-squaredtest for categorical variables and Mann–Whitney U testfor continuous variables. Biomarkers were tested for nor-mality with the Kolgomorov-Smirnoff test. As biomarkerswere not normally distributed, comparisons on biomarkerlevels between groups were performed with non-paramet-ric tests. Intergroup differences were assessed with Mann–Whitney or Kruskal–Wallis tests. Where relevant,biomarkers were dichotomized by the cut-off with thehighest predictive accuracy for infections, which was cal-culated in receptor operating characteristic (ROC) curves.Sensitivity and specificity for every biomarker cut-off wereevaluated. A P value <0.05 was considered statistically sig-nificant. Given the small sample size and the preliminarynature of the study, P values under 0.1 were consideredas trends.ResultsOf the forty-five included patients, 3 died and one pre-sented with psychomotor agitation that precludedFigure 1. Biomarker level according to clinical diagnosis of SAP following PISCES criteria. Boxplots represent median and interquartile range inboth no SAP and probable SAP groups. Just P values <0.1 are represented. CRP, C-reactive protein; SAA, serum amyloid-A protein; MR-proADM,mid-regional proadrenomedullin; suPAR, soluble urokinase-type plasminogen activator receptor.1884 ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.Correlation with thoracic computed tomography E. Zapata-Arriaza et al.THRCT performance. PISCES probable SAP rate was17.8% (N = 8). There was no patient with PISCES defi-nite SAP according to treating physician criteria. More-over, chest radiography low quality was insufficient to acorrect interpretation of some cases to be considereddefinitive SAP. Among those mentioned 8 patients, 5(62.5%) were identified as bronchopneumonia, 2 (25%)as infectious bronchitis and 1 showed no pathology onCT. Baseline data is shown in (Table 1). According toPISCES criteria, patients with probable SAP trended tohad higher levels MR-proADM at 24 (P = 0.083) andhigher levels of suPAR at 48 h (P = 0.095). There wereno significant differences for CRP and SAA among anyvisit (Fig. 1).However, regarding THRCT findings, MR-proADM at24 h (P = 0.040) and both suPAR and SAA at 48 h(P = 0.037 and P = 0.057) were associated with pneumo-nia. At 24 h, a cut-off of MR-proADM > 1.023 nmol/Lhad 80% sensitivity and 72.2% specificity for pneumonia.At 48 h, both SAA > 25.15 mg/dL (60% sensitivity,85.7% specificity) and suPAR > 3.14 ng/mL (80% sensi-tivity, 57.1% specificity) were associated with infections.The combination of SAA > 25.15 mg/dL andsuPAR > 3.14 ng/mL at 48 h had 80% sensitivity and95.8% specificity when both biomarkers were above thecut-off, and 60% sensitivity and 94.1% specificity whenone of them was above the cut-off (Fig. 2).DiscussionOur results provide various key findings, about the mostcommon complication after stroke. Employment ofTHRCT in SAP, could reduce diagnostic discrepancies inpatients with clinical suspicion and low quality chestradiography. From the evaluated biomarkers, MR-proADM seems to be the earliest predictor of SAP, whenrespiratory infection is yet not suspected. The combina-tion of SAA/suPAR improves the predictive value, but atlater time-points.The poor predictive value of previously SAP studiedbiomarkers11 and the limited specificity of validatedpneumonia predictive scores12 induce uncertainty in SAPprediction and diagnosis. This leads to a late start of theantibiotic treatment, with the unfortunate consequencesassociated, or an overuse of antibiotics in patients withouta clear diagnosis, in order to avoid these fatal outcomes,increasing antibiotic resistance in cases without real SAP.Therefore, the identification of an early reliable biomar-ker, could guide respiratory infection management instroke patient.Figure 2. Biomarker level according to radiological diagnosis of SAP using thoracic HRCT findings. Boxplots represent median and interquartilerange in normal, bronchitis and pneumonia groups. Just P values < 0.1 are represented. CRP, C-reactive protein; SAA, serum amyloid-A protein;MR-proADM, mid-regional proadrenomedullin; suPAR, soluble urokinase-type plasminogen activator receptor.ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 1885E. Zapata-Arriaza et al. Correlation with thoracic computed tomographyOur previous study showed for the first time THRCTradiological findings of stroke associated respiratory infec-tions,3 allowing us to conduct this confirmatory study totest the diagnostic accuracy of candidate biomarkersrelated to both clinical and radiological diagnosis. In ourcohort, regarding clinical criteria, patients with probableSAP had higher levels of all evaluated biomarkers acrossthe time-points, but without statistical significance. How-ever, when SAP was diagnosed according to THRCT find-ings, we found that MR-proADM at 24 h and bothsuPAR and SAA at 48 h were significantly associated withpneumonia. These results may suggest a correlationbetween the acute-phase response and development of aninfection, which would expand biomarkers scope of usebecause of their predictive value in SAP patients, con-firmed by THRCT.According to our results, it is interesting to highlightthe insufficient association of tested biomarkers with SAPwhen they are explored regarding clinical criteria. Suchclinical criteria have shown high sensitivity rates (87.5%)when respiratory infection suspicion is confirmed withTHTCT.3 However, high false positive rate of clinical cri-teria leads to discrepancies in biomarkers-SAP relation-ship that clearly improves when compared with THRCTfindings. In fact, with the use of THRCT we would havebeen able to give a diagnosis of definitive SAP instead ofprobable SAP in seven out of the eight patients with clini-cal suspicion. Such results are consistent with low accu-racy of clinical criteria; showing reliable results due toTHRCT performance in this study.Finally, for post-stroke infections prediction, to definebiomarker´s cut-points in patients with SAP would helpin preventive strategies, especially in early stages. In ourcohort, the combination of SAA > 25.15 mg/dL andsuPAR > 3.14 ng/mL at 48 h showed 80% sensitivity and95.8% specificity. Given most SAP cases appears withinfirst seven days after stroke onset,2 the availability ofdescribed biomarkers, even after 48 h, could help in earlydetection of respiratory infection in stroke patients, withlow rates of false negative results.Therefore, these new cut-offs should now be tested inlarger cohorts, since prospectively validating a cut-off is anecessary and relevant approach in the biomarkersresearch field that we do not usually do systematically.The main limitation of the study is that our results arebased on a single cohort and our findings need to be vali-dated in an independent and larger cohort before anintervention based on these results may be recommended.In addition, the small sample size and the low rate ofevents in both clinical and radiological diagnosis pre-vented us to perform logistic regression analysis to adjustby potential confounders, such as stroke severity or dys-phagia. However, we consider that given laboratory testare compared for the first time with THRCT findings,this adds important strength to our study.In conclusion, among explored biomarkers, MR-proADM would have an important role in SAP predic-tion, due to its early elevation in stroke patients sufferingrespiratory infections later on. In addition, the combina-tion of SAA/suPAR improves the predictive value, espe-cially adding specificity, but at later time-points. Furtherprospective studies should be developed to assess the pre-dictive value of these results. An accurate SAP diagnosisby thorax CT might help to reduce diagnostic uncertaintyin such studies.AcknowledgmentsTo the Pneumonia in Stroke Consensus (PISCES) Groupand specially to Craig Smith and Andreas Meisel forinteresting conversations on the use of chest CT andother biomarkers for the diagnosis of post stroke pneu-monia.Conflict of InterestNone.References1. Horan T, Andrus M, Dudeck M. CDC/NHSN surveillancedefinition of health care-associated infection criteria forspecific types infections in acute care settings. Am J InfectControl 2008;36:309–332.2. Smith CJ, Kishore AK, Vail A, et al. Diagnosis of stroke-associated pneumonia: recommendations from thepneumonia in stroke consensus group. Stroke2015;46:2335–462340.3. Zapata-Arriaza E, Serrano-Gotarredona P, Navarro-Herrero S, et al. Chest CT findings and validation ofclinical criteria of stroke associated pneumonia. J Stroke2019;21. (Epub ahead of print).4. Meisel C, Schwab JM, Prass K, et al. Central nervoussystem injury-induced immune deficiency syndrome. NatRev Neurosci 2005;6:775–786.5. Bustamante A, Garcıa-Berrocoso T, Penalba A, et al. Sepsisbiomarkers reprofiling to predict stroke-associatedinfections. J Neuroimmunol 2017;15:19–23.6. Azurmendi L, Lapierre-Fetaud V, Schneider J, et al.Proteomic discovery and verification of serum amyloidA as a predictor marker of patients at risk of post-stroke infection: a pilot study. Clin Proteomics2017;14:27.7. Thunø M, Macho B, Eugen-Olsen J. SuPAR: the molecularcrystal ball. Dis Markers 2009;27:157–172.8. Schuetz P, Christ-Crain M, Muller B. Biomarkers toimprove diagnostic and prognostic accuracy in1886 ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.Correlation with thoracic computed tomography E. Zapata-Arriaza et al.systemic infections. Curr Opin Crit Care 2007;13:578–585.9. Gabay C, Kushner I. Acute-phase proteins and othersystemic responses to inflammation. N Engl J Med1999;340:448–454.10. Edmiaston J, Connor LT, Loehr L, Nassief A. Validationof a dysphagia screening tool in acute stroke patients. AmJ Crit Care 2010;19:357–364.11. Fluri F, Morgenthaler NG, Mueller B, et al. Copeptin,procalcitonin and routine inflammatory markers-predictors of infection after stroke. PLoS ONE 2012;7:e48309.12. Zapata-Arriaza E, Moniche F, Blanca PG, et al. Externalvalidation of the ISAN, A2DS2, and AIS-APS scores forpredicting stroke-associated pneumonia. J StrokeCerebrovasc Dis 2018;27:673–676.ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 1887E. Zapata-Arriaza et al. Correlation with thoracic computed tomography

Biomarkers predictive value for early diagnosis of Stroke- Associated Pneumonia.

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Biomarkers predictive value for early diagnosis of Stroke- Associated Pneumonia.

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