Association of human beta defensin 2 serum levels and sepsis in preterm neonates

    Depósito de investigación de la Universidad de Sevilla https://idus.us.es/  Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Pediatric critical care medicine (2013 ): October 2013 DOI: 10.1097/PCC.0b013e3182975e0f Copyright: El acceso a la versión publicada del artículo puede requerir la suscripción de la revista Access to the published version may require subscription “This is a pre-copyedited, author-produced version of an article accepted for publication in [Pediatric critical care medicine]. The published version of record [ Olbrich, Peter MD1; Pavón, Antonio MD, PhD2; Rosso, Maria Luisa MD, PhD2; Molinos, Agueda MD3; de Felipe, Beatriz PhD1; Sanchez, Berta PhD4; Praena- Fernández, Juan Manuel5; Jimenez, Francisco MD, PhD2; Obando, Ignacio MD, PhD1; Neth, Olaf MD, PhD1. Association of Human Beta-Defensin-2 Serum Levels and Sepsis in Preterm Neonates*. Pediatric Critical Care Medicine 14(8): p 796- 800, October 2013. | DOI: 10.1097/PCC.0b013e3182975e0f] is available online at file:///C:/Users/user/Downloads/association_of_human_beta_defensin_2_serum_lev els.8-1.pdf and DOI: 10.1097/PCC.0b013e3182975e0f of the published article from the Journal website. Pediatric Critical Care Medicine ASSOCIATION OF HUMAN BETA DEFENSIN 2 SERUM LEVELS AND SEPSIS IN PRETERM NEONATES --Manuscript Draft-- Manuscript Number: PCCM-D-12-00390R2 Full Title: ASSOCIATION OF HUMAN BETA DEFENSIN 2 SERUM LEVELS AND SEPSIS IN PRETERM NEONATES Article Type: Online Brief Report Keywords: neonates; prematurity; sepsis; innate immunity; antimicrobial peptides; defensins Corresponding Author: Olaf Neth, M.D.; PhD Hospital Infantil Universitario Virgen del Rocio Sevilla, SPAIN Corresponding Author Secondary Information: Corresponding Author's Institution: Hospital Infantil Universitario Virgen del Rocio Corresponding Author's Secondary Institution: First Author: Peter Olbrich, M.D. First Author Secondary Information: Order of Authors: Peter Olbrich, M.D. Antonio Pavón Delgado, M.D.; PhD María Luisa Rosso, M.D.; PhD Agueda Molinos Quintana, M.D. Beatriz de Felipe, M.D.; PhD Francisco Jimenez Parrilla, M.D. Berta Sánchez Sánchez, M.D.; PhD Juan Manuel Praena-Fernandez Ignacio Obando Santaella, M.D.; PhD Olaf Neth, M.D.; PhD Order of Authors Secondary Information: Manuscript Region of Origin: SPAIN Abstract: Background: Preterm neonates are at increased risk to suffer from infections. Humanbeta-defensin-2 (HBD2) is an antimicrobial peptide, belongs to the innate immune system and has antimicrobial activity against a broad range of pathogens. Objective: To determine HBD2-levels in term and preterm neonates at birth and evaluate its impact on sepsis. Methods: Observational study of 73 neonates, determination of HBD2-levels in cord blood using ELISA technology and registration of clinical events. Results: 42 term and 31 preterm neonates were enrolled. HBD2-levels in term neonates were higher compared to preterm infants (median 1882pg/ml vs. 918pg/ml; p=0.003) and correlated with gestational age and birth weight. 7 out of 31 preterm neonates suffered from late-onset sepsis and this was associated with lower HBD2- levels (median 513 vs. 1411pg/ml; p=0.006). Conclusion: Preterm neonates show lower HBD2-levels in cord blood compared to term neonates. Low HBD2-levels in preterm neonates might be associated with an increased risk of late onset sepsis. Response to Reviewers: The Editor-in-chief Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation Patrick M. Kochanek, MD, FCCM Pediatric Critical Care Medicine Seville, 4.03.2013 Re: PCCM-D-12-00390, entitled "ASSOCIATION OF HUMAN BETA DEFENSIN 2 SERUM AND SEPSIS IN PRETERM NEONATES" Dear Editor; Thank you very much for your positive response. We are very pleased. We have carefully addressed the points made by Reviewer #2. However, we believe it to be difficult to accommodate all of his suggestions, considering the comments made previously by the remaining two Reviewers. In particularly point 6 was modified according to the suggestions made by Reviewer #1, which we share. We believe it an interesting and unique observation to find neonatal HBD2-levels to be higher in newborns than in adults. Therefore we have not adapted this specific point. However, if the editor believes this to be important we will follow your advice to remove the observation on adult HBD2- levels. Please find below a detailed and structured response to the comments made by Reviewer #2. Reviewer 2#: 1. In the first paragraph of results - serum and plasma results are compared and the serum levels were deemed higher despite a non-significant p value. These data could be referred to as "no differences were seen between serum and plasma levels and the addition of aprotinin did not influence HBD2 levels. Alternativley, the entire paragraph could be removed. Study to study variability of absolute mediator levels is common. Add 1. As correctly mentioned by the reviewer plasma and serum levels, determined in the cord blood of each neonate, did not differ significantly (p=0.068), which is likely due to the small sample size (n=4) tested. Nevertheless we believe this information to be important, not only to clarify differences between our results and previously published data, but also to avoid their misinterpretation. Furthermore, it might facilitate comparative research studies in the future. However, if the editor feels this information of minor importance for this article we will remove the paragraph, rather than modify it. 2. Additional demographics such as organisms present and source could be included in Table 1b. Add 2. We have followed the suggestion of the reviewer and have included the data in Table 1b (highlighted in red). 3. Mentioning that HBD2 does not correlate with other markers of inflammation is enough - there is no need to include the info about the specific cases on p 10-11. Add 3. We have included the information about inflammatory markers in the article following the comments made by Reviewer #2: “Demonstrating other predictors of inflammation/sepsis (CRP, neutrophil count, etc) simultaneously with HBD2 would strengthen the argument that b defensins are important markers of inflammation/sepsis in neonates”. In the modified manuscript we maintain the sentence: “No consistent correlation was observed between neutrophil count, C-reactive protein (CRP) and HBD2-levels (data not shown)”. As suggested by Reviewer #2 the additional information about newborns with extreme high and extreme low HBD2- levels has now been removed: “However, one patient with chorioamnionitis suffered from early-onset K.pneumonia sepsis, had raised CRP (34.5mg/L), very high HBD2-levels (10649pg/ml) and died at 72 hours of life. In contrast, two patients with normal neutrophil counts and CRP but Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation extremely low HBD2-levels at birth (98 and 54pg/ml respectively) suffered from LOS, the former from S.warnerii, followed by S.epidermidis and finally died at 40 days of age from K.pneumonia sepsis”. 4. No relationship between type of delivery and HBD2 levels is mentioned in the text but does not appear in Table 1b - showing CS values vs NVD values with a p value would answer this question more clearly. Add 4. We agree with the Reviewer and have therefore added the data to table 1b (highlighted in red). 5. At the bottom of page 10, I assume the AUC, sensitivity and specificity etc values are for HBD2 and LOS - is that correct? Add 5. We thank the Reviewer for this comment. We have now clarified this paragraph: “AUC-calculations for the variables HBD2 and LOS, confirmed the predictive ability of the model (AUC: 0.85 (CI 0.68-1, p=0.005); sensitivity 71.4%; specificity: 95.8%; PPV 83.3%; NPV 92%; Youden´s-Index: 0.7)”. 6. I still don't see the relevance of comparing preterm, term, and adult values. The comparison of term to preterm is sufficient to demonstrate a potential deficiency espec with the data showing that levels are lower in preterm infants with LOS. Comparing preterms and adults doesn't support any concept put forth in the paper. As highlighted by Reviewer #1 it is a unique finding to detect levels of the innate immune system higher in neonates than in adults. This might be explained by the importance of this antimicrobial peptide in the first weeks of life as a primordial defence mechanism, raising therefore the possibility that low levels of HBD2 at birth might increase the susceptibility of newborns (especially preterms) to suffer from invasive infections. As mentioned in the letter we feel this information to be an important part of the conclusions of our article. Provided the acceptance of the editor we would like to maintain this in the manuscript. We hope that we have addressed the above comments satisfactorily for the editor as well as for Reviewer #2. Yours sincerely, Olaf Neth, MD MRCPCH Department of Paediatric Infectious Diseases and Immunology Hospital Universitario Virgen del Rocio, Sevilla 41013, Spain Fax: 0034-955012921 E-mail: [email protected] Section/Category: Immunology Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation The Editor-in-chief Patrick M. Kochanek, MD, FCCM Pediatric Critical Care Medicine Seville, 4.03.2013 Re: PCCM-D-12-00390, entitled "ASSOCIATION OF HUMAN BETA DEFENSIN 2 SERUM AND SEPSIS IN PRETERM NEONATES" Dear Editor; Thank you very much for your positive response. We are very pleased. We have carefully addressed the points made by Reviewer #2. However, we believe it to be difficult to accommodate all of his suggestions, considering the comments made previously by the remaining two Reviewers. In particularly point 6 was modified according to the suggestions made by Reviewer #1, which we share. We believe it an interesting and unique observation to find neonatal HBD2-levels to be higher in newborns than in adults. Therefore we have not adapted this specific point. However, if the editor believes this to be important we will follow your advice to remove the observation on adult HBD2- levels. Please find below a detailed and structured response to the comments made by Reviewer #2. Reviewer 2#: 1. In the first paragraph of results - serum and plasma results are compared and the serum levels were deemed higher despite a non-significant p value. These data could be referred to as "no differences were seen between serum and plasma levels and the addition of aprotinin did not influence HBD2 levels. Alternativley, the entire paragraph could be removed. Study to study variability of absolute mediator levels is common. Add 1. As correctly mentioned by the reviewer plasma and serum levels, determined in the cord blood of each neonate, did not differ significantly (p=0.068), which is likely due to the small sample size (n=4) tested. Nevertheless we believe this information to be important, not only to clarify differences between our results and previously published data, but also to avoid their misinterpretation. Furthermore, it might facilitate comparative research studies in the future. However, if the editor feels this information of minor importance for this article we will remove the paragraph, rather than modify it. 2. Additional demographics such as organisms present and source could be included in Table 1b. Add 2. We have followed the suggestion of the reviewer and have included the data in Table 1b (highlighted in red). Cover Letter 3. Mentioning that HBD2 does not correlate with other markers of inflammation is enough - there is no need to include the info about the specific cases on p 10-11. Add 3. We have included the information about inflammatory markers in the article following the comments made by Reviewer #2: “Demonstrating other predictors of inflammation/sepsis (CRP, neutrophil count, etc) simultaneously with HBD2 would strengthen the argument that b defensins are important markers of inflammation/sepsis in neonates”. In the modified manuscript we maintain the sentence: “No consistent correlation was observed between neutrophil count, C-reactive protein (CRP) and HBD2-levels (data not shown)”. As suggested by Reviewer #2 the additional information about newborns with extreme high and extreme low HBD2- levels has now been removed: “However, one patient with chorioamnionitis suffered from early-onset K.pneumonia sepsis, had raised CRP (34.5mg/L), very high HBD2-levels (10649pg/ml) and died at 72 hours of life. In contrast, two patients with normal neutrophil counts and CRP but extremely low HBD2-levels at birth (98 and 54pg/ml respectively) suffered from LOS, the former from S.warnerii, followed by S.epidermidis and finally died at 40 days of age from K.pneumonia sepsis”. 4. No relationship between type of delivery and HBD2 levels is mentioned in the text but does not appear in Table 1b - showing CS values vs NVD values with a p value would answer this question more clearly. Add 4. We agree with the Reviewer and have therefore added the data to table 1b (highlighted in red). 5. At the bottom of page 10, I assume the AUC, sensitivity and specificity etc values are for HBD2 and LOS - is that correct? Add 5. We thank the Reviewer for this comment. We have now clarified this paragraph: “AUC-calculations for the variables HBD2 and LOS, confirmed the predictive ability of the model (AUC: 0.85 (CI 0.68-1, p=0.005); sensitivity 71.4%; specificity: 95.8%; PPV 83.3%; NPV 92%; Youden´s-Index: 0.7)”. 6. I still don't see the relevance of comparing preterm, term, and adult values. The comparison of term to preterm is sufficient to demonstrate a potential deficiency espec with the data showing that levels are lower in preterm infants with LOS. Comparing preterms and adults doesn't support any concept put forth in the paper. As highlighted by Reviewer #1 it is a unique finding to detect levels of the innate immune system higher in neonates than in adults. This might be explained by the importance of this antimicrobial peptide in the first weeks of life as a primordial defence mechanism, raising therefore the possibility that low levels of HBD2 at birth might increase the susceptibility of newborns (especially preterms) to suffer from invasive infections. As mentioned in the letter we feel this information to be an important part of the conclusions of our article. Provided the acceptance of the editor we would like to maintain this in the manuscript. We hope that we have addressed the above comments satisfactorily for the editor as well as for Reviewer #2. Yours sincerely, Olaf Neth, MD MRCPCH Department of Paediatric Infectious Diseases and Immunology Hospital Universitario Virgen del Rocio, Sevilla 41013, Spain Fax: 0034-955012921 E-mail: [email protected] 4 ASSOCIATION OF HUMAN BETA DEFENSIN 2 SERUM LEVELS AND SEPSIS IN PRETERM NEONATES Authors, highest academic degrees and affiliations: Peter Olbrich, M.D.1; Antonio Pavón, M.D., Ph.D.2; Maria Luisa Rosso, M.D., Ph.D2; Agueda Molinos, M.D.3, Beatriz de Felipe, Ph.D.1, Berta Sanchez, Ph.D.4, Juan Manuel Praena-Fernández5, Francisco Jimenez, M.D., PhD2; Ignacio Obando, M.D., Ph.D.1 and Olaf Neth, M.D., Ph.D.1 1Department of Paediatric Infectious Diseases and Immunodeficiency; Hospital Infantil Universitario Virgen del Rocio, Seville, Spain. 2Department of Neonatology and Neonatal Intensive Care Unit; Hospital Infantil Universitario Virgen del Rocio, Seville, Spain. 3Department of Paediatric Haematology; Hospital Infantil Universitario Virgen del Rocio, Seville, Spain. 4Department of Immunology; Hospital Universitario Virgen del Rocio, Seville, Spain. 5Statistics, Methodology and Research Evaluation Unit, Andalusian Public Foundation for Health Research Management in Seville, Seville, Spain. Corresponding author: Olaf Neth Department of Paediatric Infectious Diseases and Immunodeficiency Hospital Infantil Universitario Virgen del Rocio, Av Manuel Siurot s/n, 41013 Sevilla, Spain (T) 0034955012991; e-mail: [email protected] The authors have not disclosed any potential conflicts of interest *Manuscript (All Manuscript Text Pages in MS Word format, including References and Figure Legends) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 Abstract: Background: Preterm neonates are at increased risk to suffer from infections. Human-beta-defensin-2 (HBD2) is an antimicrobial peptide, belongs to the innate immune system and has antimicrobial activity against a broad range of pathogens. Objective: To determine HBD2-levels in term and preterm neonates at birth and evaluate its impact on sepsis. Methods: Observational study of 73 neonates, determination of HBD2-levels in cord blood using ELISA technology and registration of clinical events. Results: 42 term and 31 preterm neonates were enrolled. HBD2-levels in term neonates were higher compared to preterm infants (median 1882pg/ml vs. 918pg/ml; p=0.003) and correlated with gestational age and birth weight. 7 out of 31 preterm neonates suffered from late-onset sepsis and this was associated with lower HBD2-levels (median 513 vs. 1411pg/ml; p=0.006). Conclusion: Preterm neonates show lower HBD2-levels in cord blood compared to term neonates. Low HBD2-levels in preterm neonates might be associated with an increased risk of late onset sepsis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12 It remains to be clarified whether very low as well as very high HBD2-levels may be detrimental for the clinical outcome. All studies were performed in blood serum and measured overall higher HBD2- levels, compared to published data [8] where plasma was used. Indeed, HBD2- levels from control blood samples were analyzed using plasma as well as serum and found to be higher in serum. This observation might be explained by the fact that HBD2 are highly positively charged peptides, which bind to negatively charged proteins in plasma and serum, resulting in a reduction of free circulating HBD2. Furthermore, numerous coagulation factors in plasma are serine proteases, which may affect HBD2-levels. Finally differences of published HBD2-levels might be due to the use of different laboratory techniques. Neither the addition of a protease inhibitor to serum samples nor its analysis at different time points did influence HBD2-levels. This is important as it may facilitate clinical studies in the future. In summary, in this study HBD2-serum levels were lower in preterm neonates compared to term infants, but not different from adult controls, suggesting a relative deficiency at birth. Low HBD2-levels might be an independent risk factor for the susceptibility to infections in this risk group. The value of HBD2- measurements at birth and its impact on clinical outcome remains to be determined. References: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 1. Vergnano S, Menson E, Kennea N, et al. Neonatal infections in England: the NeonIN surveillance network. Arch Dis Child Fetal Neonatal Ed. 2011;96:F9-F14. 2. Wynn JL, Levy O. Role of innate host defenses in susceptibility to earlyonset neonatal sepsis. Clin Perinatol. 2010;37:307-337. 3. Underwood MA, Bevins CL. Defensin-barbed innate immunity: clinical associations in the pediatric population. Pediatrics. 2010;125:1237-1247. 4. Stoll BJ, Hansen N, Fanaroff AA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network. Pediatrics. 2002;110:285–291 5. Strunk T, Doherty D, Richmond P, et al. Reduced levels of antimicrobial proteins and peptides in human cord blood plasma. Arch Dis Child Fetal Neonatal Ed 2009;94:F230-231. 6. Dzwonek AB, Neth OW, Thiébaut R, et al. The role of mannose-binding lectin in susceptibility to infection in preterm neonates. Pediatr Res. 2008;63:680-685. 7. Starner TD, Agerberth B, Gudmundsson GH, McCray PB Jr. J Immunolo. 2005; 174:1608-15. 8. Book M, Chen Q, Lehmann LE, et al. Inducibility of the endogenous antibiotic peptide beta-defensin 2 is impaired in patients with severe sepsis. Crit Care. 2007;11:R19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 Figure legend Figure 1a: HBD2-serum levels in preterm, term neonates and healthy adults Figure 1b: HBD2-serum levels with and without late-onset sepsis. Two patients with early-onset sepsis have been excluded from this analysis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Figure Click here to download high resolution image 11 Table 1a: Demographic and clinical details of the neonatal cohort Preterm neonates (n=31) Term neonates (n=42) P value Gestational age (weeks) Median 30 (IQR 29-31) Median 39 (IQR 38-40) < 0.001 Birth weight (g) Mean 1298 (SD 338.4) Mean 3247 (SD 378.2) < 0.001 Gender - Female; n (%) - Male; n (%) 11 (35.5%) 20 (64.5%) 28 (66.7%) 14 (33.3%) 0.008 Maternal age (y) 32 (29-34) 30.5 (22.7-35.2) 0.151 Type of Delivery - NVD; n (%) - Instrumental; n (%) - CS; n (%) 9 (29%) 0 (0.0%) 22 (71%) 27 (64.3%) 10 (23.8%) 5 (11.9%) < 0.001 Chorioamnionitis - Yes 16 (48.4%) 9 (21.4%) 0.007 y, years; NVD, normal vaginal delivery; CS, caesarean section Table 12 Table 1b: HBD2 levels in relation to demographic and clinical data Patients n=73 Median HBD2-Levels in pg/ml (IQR 25-75) p value Gestational age (weeks) - Preterm neonates (28-32) - Term neonates (37-41) 31 42 918 (556-2331) 1882 (1204-3364) p= 0.003 Birth weight <1500g ≥1500g 20 53 880 (524-2645) 1748 (956-2969) p= 0.038 Gender - Female - Male 39 34 1639 (691-2968) 1661 (842-2579) p= 0.812 Mode of Delivery - Preterm neonates o CS o NVD - Term neonates o CS o NVDa 22 9 5 37 1020 (546-2385) 707 (488-2423) 1602 (762-3588) 1888 (1257-3398) p= 0.862 p= 0.522 Corioamnionitis - Yes - No 25 48 1639 (869-3591) 1662 (740-2775) p= 0.754 Term neonates with CA Term neonates without CA Preterm neonates with CA Preterm neonates without CA 9 33 16 15 2106 (1639-7187) 1813 (1146-2971) 1046 (578-2213) 655 (248-2293) p= 0.244 p= 0.268 Preterm neonates with proven LOSb Preterm neonates without proven LOS and/or EOSc 7 22 513 (98-557) 1411 (609-2579) p=0.006 Table 12 CS, caesarean section, NVD, normal vaginal delivery, CA, chorioamnionitis; LOS, late-onset sepsis; EOS, early-onset sepsis a 10 of 37 vaginal deliveries were instrumental. b S. epidermidis (n=5); K.pneumonia (n=2); S.warnerii (n=1), E.faecalis (n=1). Whilst one patient suffered from 3 LOS episodes there were no cases of meningitis, line infections or nosocomial pneumonias. c Two preterm neonates suffering from EOS were excluded from this analysis Delgado Click here to download Copyright and Disclosure Form: PCCM-D-12-Neth-2012 Delgado.pdf Felipe Click here to download Copyright and Disclosure Form: PCCM-D-12-Neth-2012 Felipe.pdf Neth Click here to download Copyright and Disclosure Form: PCCM-D-12-Neth-2012 Neth.pdf Santaella Click here to download Copyright and Disclosure Form: PCCM-D-12-Neth-2012 Santaella.pdf