Original Article
Preterm Infants Can Mount Appropriate C-Reactive Protein Responses to Early Onset Sepsis Sanket D. Shah, MD1 Ajay J. Talati, MD1,2 Mohamad T. Elabiad, MD, MS1 Ramasubbareddy Dhanireddy, MD1,2 Massroor Pourcyrous, MD1,2,3 1 Department of Pediatrics, University of Tennessee Health Science
Center, Memphis, Tennessee 2 Department of Obstetrics and Gynecology, University of Tennessee Health Science Center, Memphis, Tennessee 3 Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee
Address for correspondence Massroor Pourcyrous, MD, Division of Neonatology, Department of Pediatrics, College of Medicine, 853 Jefferson Avenue, 2nd floor, Memphis, TN 38163 (e-mail: [email protected]).
Abstract
Keywords
► ► ► ►
CRP early onset sepsis GBS CONS
Objective This study aims to evaluate whether infants born at 32 weeks’ gestational age (GA) can mount C-reactive protein (CRP) responses during early onset bacterial sepsis that are comparable to infants born at > 32 weeks’ GA. Methods Retrospectively (2003–2012) infants with a positive bacterial culture during the first 72 hours of life were identified and grouped into two categories based on their GA: 32 weeks (group A) and > 32 weeks (group B). Results Group A included 25 and group B included 122 infants. Both groups responded similarly to sepsis with an increase in CRP (p ¼ 0.59). Each group had a significant change in intragroup CRP levels over time (p < 0.0001). However, in both groups, the degree of this change was at the same rate over time (p ¼ 0.74). Conclusion CRP responses to bacterial sepsis during the first 72 hours of life in infants born at 32 weeks’ GA are comparable to infants born at > 32 weeks’ GA.
Neonatal sepsis is a leading cause of mortality and morbidity in preterm infants.1 There still remains a difficulty in diagnosing neonatal sepsis because of the nonspecific signs of infection. Although blood culture is considered as the “gold standard” for the diagnosis of sepsis, it can be falsely positive because of the contamination of blood samples. Conversely, false-negative blood cultures are not uncommon and are usually because of the small volume of blood sample, an intermittent presence and low density of circulating pathogens, and antepartum antibiotic administration.2–4 Among the ancillary tests, C-reactive protein (CRP) is the most frequently studied and used laboratory test to screen for neonatal infection.5,6 Serial CRP measurements have high sensitivity and negative predictive value in neonatal sepsis.3,7–10 Fetuses are capable of producing CRP as early as 4 to 5 weeks of gestational age (GA), and CRP does not cross the placenta. Therefore, an abnormal level is an indication of
neonatal origin.11–15 Compared with other infection markers, measurement of CRP is simple, reliable, cost effective, and with a quick turn-around time.5 Recently, a few investigators had expressed concern for delayed or diminished CRP responses to early onset sepsis (EOS) in premature infants.16,17 We hypothesized that in EOS, premature infants can mount CRP responses that are similar to those of more mature infants. We thus conducted a retrospective chart review with an objective to compare CRP responses to EOS between infants born 32 weeks’ GA and those born > 32 weeks’ GA.
received January 7, 2015 accepted after revision May 13, 2015
Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
Methods and Design Study Population This retrospective chart review was performed at the neonatal intensive care unit (NICU) at the Regional One Health, in Memphis, TN, between January 2003 and December 2012.
DOI http://dx.doi.org/ 10.1055/s-0035-1555127. ISSN 0735-1631.
Downloaded by: University of Pittsburgh. Copyrighted material.
Am J Perinatol
This study was approved by the Institutional Review Board at the University of Tennessee Health Science Center in Memphis, TN. Infants with a positive bacterial culture during the first 72 hours of life were identified through our perinatal database and were grouped into two categories based on their birth GA at 32 weeks (group A) and > 32 weeks (group B). On the basis of our unit guidelines, when sepsis work-up was required, blood culture was obtained before starting antibiotic therapy. CRP levels were measured every 12 hours for three times and then every 24 hours if values remained elevated. Common indications for sepsis work-up included hyperthermia/hypothermia, meconium aspiration, respiratory distress, apnea/bradycardia, or abnormal glucose homeostasis. Sepsis work-up was also initiated because of the obstetrical risk factors, that is, intra- or postpartum fever, prolonged rupture of membranes, chorioamnionitis, premature onset of labor, urinary tract infection, or group B streptococcus (GBS) colonization without appropriate treatment. An “illness severity scoring” is not routinely calculated in our unit. EOS was defined as onset at 72 hours of age.18 Administration of penicillin G and gentamicin is the standard empirical treatment for early onset neonatal infection in our unit. Data on birth weight (BW), GA, gender, race, date of positive blood culture, the microorganisms involved, and the survival status were obtained from our perinatal database. Individual medical records were also reviewed for CRP values, and other relevant data.
C-Reactive Protein Measurements Vitros CRP slides methodology (Vitros 250 Chemistry System, Ortho-Clinical Diagnostics, Inc., Johnson & Johnson Co. Raritan, NJ) was utilized.19 The lowest limit of quantification of CRP was at 0.2 mg/dL. A serum CRP level of > 1.5 mg/dL was considered the diagnostic cutoff which is comparable to CRP level of > 1.0 mg/dL when measured by Beckman Array system.20
Statistical Analysis SAS 9.3 (SAS Inst. Inc., Cary, NC) was used to perform the statistical analysis. Descriptive statistical data are presented as mean SD or median (interquartile range; Q1, Q3) as appropriate. Mixed linear models procedure was used to compare trends in CRP between groups. The chi-square test was used to evaluate the association between the two GA groups and the presence of skin flora such as coagulase negative staphylococci (CONS) and anaerobes. A p value less than 0.05 was considered statistically significant.
Results During a 10-year period, 41,113 neonates were born in our maternity center. Overall, 147 neonates had positive blood cultures at 72 hours of life; 133 (90%) of the cultures were obtained on the first day of life. Overall, 74 (50%) neonates were male and 119 (81%) were black. Group A included 25 neonates with a mean GA of 27.8 3.1 weeks and a mean BW of 1,029 517 g. Six American Journal of Perinatology
Shah et al.
(24%) infants were small for GA, and 14 (56%) were born with a BW < 1,000 g. Overall, 7 (28%) infants died within 5 days of positive blood culture. All neonates who died had abnormal CRP values except for two infants who died within the first 12 hours of life. Group B included 122 infants with a mean GA of 38.5 2.3 weeks and a mean BW of 3,146 664 g. Overall, 18 (15%) infants were small for GA. Overall, 108 (89%) infants were born with a BW > 2,500 g. All infants in group B survived. Group A had a lower incidence of cesarean delivery than group B, 15% and 72%, respectively; data were available for 84 infants. Group A also had a higher incidence of exposure to antenatal antibiotics, 66 and 46%, respectively; data were available for 129 infants. Microorganisms that were recovered from the blood cultures and the CRP responses to the microorganisms are shown in ►Table 1. A total of 677 CRP samples were obtained with medians of 4 (3, 6) and 5 (3, 6) in groups A and B, respectively. There was a high incidence of CONS positive blood cultures (33%) in the population: 40% in group A and 30% in group B. However, this incidence was not significantly different. Overall, 54 (37%) infants had a positive blood culture with a normal CRP value (group A, n ¼ 9 [36%]; group B, n ¼ 45 [37%]), (p ¼ 0.56). The most common microorganisms recovered when CRP remained within normal levels were, CONS (n ¼ 28), followed by GBS (n ¼ 8) and Escherichia coli (n ¼ 4). Two infants from group A died of GBS sepsis within the first 12 hours of life; therefore, no follow-up CRP was obtained. The effects of GA and postnatal age on CRP responses showed that the CRP trends in both the groups were similar (►Fig. 1). Both groups responded similarly to sepsis with an increase in CRP (p ¼ 0.59). Each group had a significant change in CRP levels over the six time periods (p < 0.0001), and in both groups the degree of this change was at the same rate over time (p ¼ 0.74). Excluding skin flora such as CONS and diphtheroids from the analysis did not affect the results. In both the groups, CRP trended again in a similar fashion (►Fig. 2). The effect of GA was also evaluated against the highest (peak) CRP value that an infant attained and the time to peak CRP. No correlation was found between the GA and the peak CRP values (r < 0.1; p ¼ ns) or the GA and the time needed for CRP to peak (r < 0.1; p ¼ ns) (►Fig. 3).
Discussion In this study, we have shown that in EOS, premature infants mount CRP that are similar to their more mature counterparts. CRP levels increased in a similar manner in both GA groups. In instances when CRP responses were absent, this also occurred in similar frequencies and in a similar manner in both GA groups. Few reports suggested that CRP responses in preterm neonates to EOS were not as prominent as in term infants. The methodologies used in these studies have been different and this made it hard to compare them for more consistent conclusions. Some of these differences included a small
Downloaded by: University of Pittsburgh. Copyrighted material.
Preterm Infants Can Mount Appropriate CRP Responses to EOS
Preterm Infants Can Mount Appropriate CRP Responses to EOS
Shah et al.
Table 1 Microorganisms isolated from blood cultures during the first 72 hours of life and the ratio of abnormal to total CRP level for each microorganism Group A (N ¼ 25)
Gram-positive
Group B (N ¼ 122)
n ¼ 11/20 (55%)
n ¼ 60/94 (73%)
Group B streptococci
5/8
30/35
Staphylococcus aureus
0
0/2
Viridans streptococci
1/1
9/10
Enterococcus faecalis
0
1/2
Coagulase negative staphylococci
4/10
15/38
Micrococcus
0
0/1
Diphtheroid species
1/1
4/4
Streptococcus (unidentified)
0
0/1
Aerococcus
0
1/1
n ¼ 5/5 (100%)
n ¼ 15/20 (75%)
Escherichia coli
4/4
13/17
Klebsiella pneumoniae
0
1/1
Gram-negative
Acinetobacter spp
0
0/1
Haemophilus influenzae
0
1/1
Raoultella ornithinolytica
1/1
0
n¼0
n ¼ 1/5 (20%)
Enterococcus sp and CONS
0
1/1
Viridans streptococci and CONS
0
0/3
Viridans streptococci and Branhamella catarrhalis
0
0/1
n¼0
n ¼ 1/3 (33)
0
1/3
Polymicrobial
Anerobes Bacillus spp
Abbreviations: CONS, coagulase negative staphylococci; CRP, C-reactive protein; N, number of neonates with positive blood culture. Note Group A infants at 32 weeks and group B infants at > 32 weeks. CRP level > 1.5 mg/dL was considered abnormal value.20
Fig. 1 CRP levels of infants in group A (GA 32 weeks) and group B (GA > 32 weeks) at 0, 12, 24, 48, 72, and 96 hours of septic work-up. Values are least square means. Both groups showed significant changes over time (p < 0.0001). However, the manner of the response and the change in the response rate over time was similar in both the groups, p ¼ 0.59 and 0.74, respectively. CRP, C-reactive protein; GA, gestational age. American Journal of Perinatology
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Microorganisms
Shah et al.
Fig. 2 After excluding skin flora, CRP levels of infants in group A (GA 32 weeks; n ¼ 15) and group B (GA > 32 weeks; n ¼ 76) at 0, 12, 24, 48, 72, and 96 hours of septic work-up. Values are least square means. Both groups showed significant changes over time (p < 0.0001). However, the manner of the response and the change in response rate over time was similar in both the groups, p ¼ 0.87, and p ¼ 0.66, respectively. CRP, Creactive protein; GA, gestational age.
Fig. 3 (A) Scatter plot showing no correlation between the GA and the peak CRP values and, (B) between the GA and the time for CRP to peak. CRP, C-reactive protein; GA, gestational age.
number of neonates with positive blood culture,21,22 an unspecified trigger source (infection/tissue injury),23 an absence of longitudinal CRP trends,17,22 no listing of the microorganisms involved,17 a different cutoff threshold for,17 and/ or a different CRP methodology.24 Two studies used 35 weeks as a cutoff to compare premature groups.17,23 Both studies reported lower CRP responses in the premature infant group. In a post hoc analysis in our study, increasing the cutoff to 35 weeks did not affect the main results. Both groups had similar CRP responses, data not shown. Overall, 33% of our blood cultures were positive for CONS; the CRP value was abnormal in 50% (3/6) of group A compared with 40% (17/42) in group B. A low response to CONS has been reported before. Schmidt et al10 reported that 64% of infants with CONS bacteremia had abnormal CRP values. They suggested that “CONS isolated from blood in the NICU is clinically a relevant finding when neonates are symptomatic and the CRP values are abnormal.” In addition to contamination, a low American Journal of Perinatology
CRP level in CONS sepsis has also been attributed to its low virulence.4,9,25,26 As CONS is a common microorganism detected in blood cultures of neonates, Hofer et al17 evaluated CRP levels in a small cohort after excluding positive cultures with skin flora. They reported lower median CRP values in EOS in 19 infants at < 37 weeks’ GA when compared with 14 infants 37 weeks’ GA (0.9 and 1.8 mg/dL, respectively). Overall, 56% of all their positive blood cultures were GBS; other organisms were not identified. However, when we excluded the infants with positive blood cultures for organisms such as CONS and diphtheroids, the results of our study remained the same; CRP trends remained similar in both the groups with no statistical difference. On the contrary, Seibert et al27 studied 125 preterm infants with a GA of 23 to 31 weeks within 3 days of life; 45 were < 28 weeks GA. Only 8 of 125 infants had positive cultures (organisms were not classified). Overall, 28 infants had a probable infection and 89 were considered as not
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Preterm Infants Can Mount Appropriate CRP Responses to EOS
Preterm Infants Can Mount Appropriate CRP Responses to EOS
References
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1 Fanaroff AA, Stoll BJ, Wright LL, et al; NICHD Neonatal Research
Network. Trends in neonatal morbidity and mortality for very low birthweight infants. Am J Obstet Gynecol 2007;196(2):147. e1–147.e8 2 Connell TG, Rele M, Cowley D, Buttery JP, Curtis N. How reliable is a negative blood culture result? Volume of blood submitted for culture in routine practice in a children’s hospital. Pediatrics 2007; 119(5):891–896 3 Philip AG, Mills PC. Use of C-reactive protein in minimizing antibiotic exposure: experience with infants initially admitted to a well-baby nursery. Pediatrics 2000;106(1):E4
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protein (CRP) response patterns in neonatal septicaemia. APMIS 1999;107(6):593–600 Chirico G, Loda C. Laboratory aid to the diagnosis and therapy of infection in the neonate. Pediatr Rep 2011;3(1):e1 Hofer N, Zacharias E, Müller W, Resch B. An update on the use of Creactive protein in early-onset neonatal sepsis: current insights and new tasks. Neonatology 2012;102(1):25–36 Benitz WE, Han MY, Madan A, Ramachandra P. Serial serum Creactive protein levels in the diagnosis of neonatal infection. Pediatrics 1998;102(4):E41 Forest JC, Larivière F, Dolcé P, Masson M, Nadeau L. C-reactive protein as biochemical indicator of bacterial infection in neonates. Clin Biochem 1986;19(3):192–194 Pourcyrous M, Bada HS, Korones SB, Baselski V, Wong SP. Significance of serial C-reactive protein responses in neonatal infection and other disorders. Pediatrics 1993;92(3):431–435 Schmidt BK, Kirpalani HM, Corey M, Low DE, Philip AG, Ford-Jones EL. Coagulase-negative staphylococci as true pathogens in newborn infants: a cohort study. Pediatr Infect Dis J 1987;6(11): 1026–1031 Chiesa C, Signore F, Assumma M, et al. Serial measurements of C-reactive protein and interleukin-6 in the immediate postnatal period: reference intervals and analysis of maternal and perinatal confounders. Clin Chem 2001;47(6):1016–1022 de Villiers WJ, Louw JP, Strachan AF, Etsebeth SM, Shephard EG, de Beer FC. C-reactive protein and serum amyloid A protein in pregnancy and labour. Br J Obstet Gynaecol 1990;97(8):725–730 Gitlin D, Biasucci A. Development of gamma G, gamma A, gamma M, beta IC-beta IA, C 1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, alpha 1-antitrypsin, orosomucoid, beta-lipoprotein, alpha 2-macroglobulin, and prealbumin in the human conceptus. J Clin Invest 1969; 48(8):1433–1446 Jaye DL, Waites KB. Clinical applications of C-reactive protein in pediatrics. Pediatr Infect Dis J 1997;16(8):735–746, quiz 746–747 Kääpä P, Koistinen E. Maternal and neonatal C-reactive protein after interventions during delivery. Acta Obstet Gynecol Scand 1993;72(7):543–546 Chiesa C, Natale F, Pascone R, et al. C reactive protein and procalcitonin: reference intervals for preterm and term newborns during the early neonatal period. Clin Chim Acta 2011;412(1112):1053–1059 Hofer N, Müller W, Resch B. Non-infectious conditions and gestational age influence C-reactive protein values in newborns during the first 3 days of life. Clin Chem Lab Med 2011;49(2): 297–302 Polin RA; Committee on Fetus and Newborn. Management of neonates with suspected or proven early-onset bacterial sepsis. Pediatrics 2012;129(5):1006–1015 Product manual VITROS Chemistry Products CRP Slides. Rochester, NY: Ortho-Clinical Diagnostics; 2010 Elabiad MT, Arheart KL, Korones SB, Pourcyrous M. Adjusting for bias in C-reactive protein levels when using a vitros slide method in infants. Am J Perinatol 2014;31(10):851–854 Kawamura M, Nishida H. The usefulness of serial C-reactive protein measurement in managing neonatal infection. Acta Paediatr 1995;84(1):10–13 Doellner H, Arntzen KJ, Haereid PE, Aag S, Austgulen R. Interleukin-6 concentrations in neonates evaluated for sepsis. J Pediatr 1998;132(2):295–299 Turner MA, Power S, Emmerson AJ. Gestational age and the C reactive protein response. Arch Dis Child Fetal Neonatal Ed 2004; 89(3):F272–F273 Philip AG. Response of C-reactive protein in neonatal Group B streptococcal infection. Pediatr Infect Dis 1985;4(2):145–148 Franz AR, Steinbach G, Kron M, Pohlandt F. Reduction of unnecessary antibiotic therapy in newborn infants using interleukin-8 and American Journal of Perinatology
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infected. CRP responses in infants < 28 weeks’ GA were the same as the infants at 29 to 31 weeks regardless of being considered infected or not. Although this study did not include older GA infants, it at least supports the idea that infants 32 weeks’ GA have similar CRP responses and can be grouped together similarly to what our study did. This is also supported by the absence of an association between the GA and the time to peak CRP or GA and the highest CRP level reached. Although positive blood culture is considered as “gold standard” for diagnosis of sepsis, not all positive blood cultures in asymptomatic infants should be considered as true sepsis.9,10 Isolation of microorganisms in a blood culture in a clinically well infant may reflect asymptomatic bacteremia or contamination of blood specimen. As described by Benitz28, “bacteremia means detection of bacteria in the blood stream, but sepsis occurs when invasion of bacteria results in systemic inflammatory response.” In neonates, most sepsis work-ups are performed for maternal indications or the presence of nonspecific clinical signs in neonates. Antenatal antibiotics either for preterm labor, prolonged rupture of membranes, or maternal fever may result in partially treated infant and a blunted CRP response.3 Therefore, clinical signs and abnormal laboratory tests should be carefully evaluated before confirming the presence of true infection. Limitations of our study include its design as a retrospective cohort study in a predominately black population and less number of patients with positive blood cultures in the lower GA group. Although the study included a large number of infants with positive blood cultures, it was still underpowered to safely avoid a type II error. Another limitation was the availability of mode of delivery data on only 57% of the infants. Although most of group A infants were delivered by spontaneous vaginal delivery secondary to preterm labor, their trend in CRP responses did not differ from those infants in group B who were mostly delivered by cesarean delivery. These circumstances may have triggered an earlier CRP response in group A and confounded the data. On the contrary, their ability to mount such a response to arguably noninfectious causes does support the findings of this study regarding the maturity of their immune system. In conclusion, this study demonstrated that in EOS, premature infants have similar CRP responses when compared with more mature infants.
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Preterm Infants Can Mount Appropriate CRP Responses to EOS
27 Seibert K, Yu VY, Doery JC, Embury D. The value of C-reactive
protein measurement in the diagnosis of neonatal infection. J Paediatr Child Health 1990;26(5):267–270 28 Benitz WE. Adjunct laboratory tests in the diagnosis of early-onset neonatal sepsis. Clin Perinatol 2010;37(2):421–438
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C-reactive protein as markers of bacterial infections. Pediatrics 1999;104(3 Pt 1):447–453 26 Laborada G, Rego M, Jain A, et al. Diagnostic value of cytokines and C-reactive protein in the first 24 hours of neonatal sepsis. Am J Perinatol 2003;20(8):491–501
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American Journal of Perinatology
Preterm Infants Can Mount Appropriate C-Reactive Protein Responses to Early Onset Sepsis Sanket D. Shah, MD1 Ajay J. Talati, MD1,2 Mohamad T. Elabiad, MD, MS1 Ramasubbareddy Dhanireddy, MD1,2 Massroor Pourcyrous, MD1,2,3 1 Department of Pediatrics, University of Tennessee Health Science
Center, Memphis, Tennessee 2 Department of Obstetrics and Gynecology, University of Tennessee Health Science Center, Memphis, Tennessee 3 Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee
Address for correspondence Massroor Pourcyrous, MD, Division of Neonatology, Department of Pediatrics, College of Medicine, 853 Jefferson Avenue, 2nd floor, Memphis, TN 38163 (e-mail: [email protected]).
Abstract
Keywords
► ► ► ►
CRP early onset sepsis GBS CONS
Objective This study aims to evaluate whether infants born at 32 weeks’ gestational age (GA) can mount C-reactive protein (CRP) responses during early onset bacterial sepsis that are comparable to infants born at > 32 weeks’ GA. Methods Retrospectively (2003–2012) infants with a positive bacterial culture during the first 72 hours of life were identified and grouped into two categories based on their GA: 32 weeks (group A) and > 32 weeks (group B). Results Group A included 25 and group B included 122 infants. Both groups responded similarly to sepsis with an increase in CRP (p ¼ 0.59). Each group had a significant change in intragroup CRP levels over time (p < 0.0001). However, in both groups, the degree of this change was at the same rate over time (p ¼ 0.74). Conclusion CRP responses to bacterial sepsis during the first 72 hours of life in infants born at 32 weeks’ GA are comparable to infants born at > 32 weeks’ GA.
Neonatal sepsis is a leading cause of mortality and morbidity in preterm infants.1 There still remains a difficulty in diagnosing neonatal sepsis because of the nonspecific signs of infection. Although blood culture is considered as the “gold standard” for the diagnosis of sepsis, it can be falsely positive because of the contamination of blood samples. Conversely, false-negative blood cultures are not uncommon and are usually because of the small volume of blood sample, an intermittent presence and low density of circulating pathogens, and antepartum antibiotic administration.2–4 Among the ancillary tests, C-reactive protein (CRP) is the most frequently studied and used laboratory test to screen for neonatal infection.5,6 Serial CRP measurements have high sensitivity and negative predictive value in neonatal sepsis.3,7–10 Fetuses are capable of producing CRP as early as 4 to 5 weeks of gestational age (GA), and CRP does not cross the placenta. Therefore, an abnormal level is an indication of
neonatal origin.11–15 Compared with other infection markers, measurement of CRP is simple, reliable, cost effective, and with a quick turn-around time.5 Recently, a few investigators had expressed concern for delayed or diminished CRP responses to early onset sepsis (EOS) in premature infants.16,17 We hypothesized that in EOS, premature infants can mount CRP responses that are similar to those of more mature infants. We thus conducted a retrospective chart review with an objective to compare CRP responses to EOS between infants born 32 weeks’ GA and those born > 32 weeks’ GA.
received January 7, 2015 accepted after revision May 13, 2015
Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
Methods and Design Study Population This retrospective chart review was performed at the neonatal intensive care unit (NICU) at the Regional One Health, in Memphis, TN, between January 2003 and December 2012.
DOI http://dx.doi.org/ 10.1055/s-0035-1555127. ISSN 0735-1631.
Downloaded by: University of Pittsburgh. Copyrighted material.
Am J Perinatol
This study was approved by the Institutional Review Board at the University of Tennessee Health Science Center in Memphis, TN. Infants with a positive bacterial culture during the first 72 hours of life were identified through our perinatal database and were grouped into two categories based on their birth GA at 32 weeks (group A) and > 32 weeks (group B). On the basis of our unit guidelines, when sepsis work-up was required, blood culture was obtained before starting antibiotic therapy. CRP levels were measured every 12 hours for three times and then every 24 hours if values remained elevated. Common indications for sepsis work-up included hyperthermia/hypothermia, meconium aspiration, respiratory distress, apnea/bradycardia, or abnormal glucose homeostasis. Sepsis work-up was also initiated because of the obstetrical risk factors, that is, intra- or postpartum fever, prolonged rupture of membranes, chorioamnionitis, premature onset of labor, urinary tract infection, or group B streptococcus (GBS) colonization without appropriate treatment. An “illness severity scoring” is not routinely calculated in our unit. EOS was defined as onset at 72 hours of age.18 Administration of penicillin G and gentamicin is the standard empirical treatment for early onset neonatal infection in our unit. Data on birth weight (BW), GA, gender, race, date of positive blood culture, the microorganisms involved, and the survival status were obtained from our perinatal database. Individual medical records were also reviewed for CRP values, and other relevant data.
C-Reactive Protein Measurements Vitros CRP slides methodology (Vitros 250 Chemistry System, Ortho-Clinical Diagnostics, Inc., Johnson & Johnson Co. Raritan, NJ) was utilized.19 The lowest limit of quantification of CRP was at 0.2 mg/dL. A serum CRP level of > 1.5 mg/dL was considered the diagnostic cutoff which is comparable to CRP level of > 1.0 mg/dL when measured by Beckman Array system.20
Statistical Analysis SAS 9.3 (SAS Inst. Inc., Cary, NC) was used to perform the statistical analysis. Descriptive statistical data are presented as mean SD or median (interquartile range; Q1, Q3) as appropriate. Mixed linear models procedure was used to compare trends in CRP between groups. The chi-square test was used to evaluate the association between the two GA groups and the presence of skin flora such as coagulase negative staphylococci (CONS) and anaerobes. A p value less than 0.05 was considered statistically significant.
Results During a 10-year period, 41,113 neonates were born in our maternity center. Overall, 147 neonates had positive blood cultures at 72 hours of life; 133 (90%) of the cultures were obtained on the first day of life. Overall, 74 (50%) neonates were male and 119 (81%) were black. Group A included 25 neonates with a mean GA of 27.8 3.1 weeks and a mean BW of 1,029 517 g. Six American Journal of Perinatology
Shah et al.
(24%) infants were small for GA, and 14 (56%) were born with a BW < 1,000 g. Overall, 7 (28%) infants died within 5 days of positive blood culture. All neonates who died had abnormal CRP values except for two infants who died within the first 12 hours of life. Group B included 122 infants with a mean GA of 38.5 2.3 weeks and a mean BW of 3,146 664 g. Overall, 18 (15%) infants were small for GA. Overall, 108 (89%) infants were born with a BW > 2,500 g. All infants in group B survived. Group A had a lower incidence of cesarean delivery than group B, 15% and 72%, respectively; data were available for 84 infants. Group A also had a higher incidence of exposure to antenatal antibiotics, 66 and 46%, respectively; data were available for 129 infants. Microorganisms that were recovered from the blood cultures and the CRP responses to the microorganisms are shown in ►Table 1. A total of 677 CRP samples were obtained with medians of 4 (3, 6) and 5 (3, 6) in groups A and B, respectively. There was a high incidence of CONS positive blood cultures (33%) in the population: 40% in group A and 30% in group B. However, this incidence was not significantly different. Overall, 54 (37%) infants had a positive blood culture with a normal CRP value (group A, n ¼ 9 [36%]; group B, n ¼ 45 [37%]), (p ¼ 0.56). The most common microorganisms recovered when CRP remained within normal levels were, CONS (n ¼ 28), followed by GBS (n ¼ 8) and Escherichia coli (n ¼ 4). Two infants from group A died of GBS sepsis within the first 12 hours of life; therefore, no follow-up CRP was obtained. The effects of GA and postnatal age on CRP responses showed that the CRP trends in both the groups were similar (►Fig. 1). Both groups responded similarly to sepsis with an increase in CRP (p ¼ 0.59). Each group had a significant change in CRP levels over the six time periods (p < 0.0001), and in both groups the degree of this change was at the same rate over time (p ¼ 0.74). Excluding skin flora such as CONS and diphtheroids from the analysis did not affect the results. In both the groups, CRP trended again in a similar fashion (►Fig. 2). The effect of GA was also evaluated against the highest (peak) CRP value that an infant attained and the time to peak CRP. No correlation was found between the GA and the peak CRP values (r < 0.1; p ¼ ns) or the GA and the time needed for CRP to peak (r < 0.1; p ¼ ns) (►Fig. 3).
Discussion In this study, we have shown that in EOS, premature infants mount CRP that are similar to their more mature counterparts. CRP levels increased in a similar manner in both GA groups. In instances when CRP responses were absent, this also occurred in similar frequencies and in a similar manner in both GA groups. Few reports suggested that CRP responses in preterm neonates to EOS were not as prominent as in term infants. The methodologies used in these studies have been different and this made it hard to compare them for more consistent conclusions. Some of these differences included a small
Downloaded by: University of Pittsburgh. Copyrighted material.
Preterm Infants Can Mount Appropriate CRP Responses to EOS
Preterm Infants Can Mount Appropriate CRP Responses to EOS
Shah et al.
Table 1 Microorganisms isolated from blood cultures during the first 72 hours of life and the ratio of abnormal to total CRP level for each microorganism Group A (N ¼ 25)
Gram-positive
Group B (N ¼ 122)
n ¼ 11/20 (55%)
n ¼ 60/94 (73%)
Group B streptococci
5/8
30/35
Staphylococcus aureus
0
0/2
Viridans streptococci
1/1
9/10
Enterococcus faecalis
0
1/2
Coagulase negative staphylococci
4/10
15/38
Micrococcus
0
0/1
Diphtheroid species
1/1
4/4
Streptococcus (unidentified)
0
0/1
Aerococcus
0
1/1
n ¼ 5/5 (100%)
n ¼ 15/20 (75%)
Escherichia coli
4/4
13/17
Klebsiella pneumoniae
0
1/1
Gram-negative
Acinetobacter spp
0
0/1
Haemophilus influenzae
0
1/1
Raoultella ornithinolytica
1/1
0
n¼0
n ¼ 1/5 (20%)
Enterococcus sp and CONS
0
1/1
Viridans streptococci and CONS
0
0/3
Viridans streptococci and Branhamella catarrhalis
0
0/1
n¼0
n ¼ 1/3 (33)
0
1/3
Polymicrobial
Anerobes Bacillus spp
Abbreviations: CONS, coagulase negative staphylococci; CRP, C-reactive protein; N, number of neonates with positive blood culture. Note Group A infants at 32 weeks and group B infants at > 32 weeks. CRP level > 1.5 mg/dL was considered abnormal value.20
Fig. 1 CRP levels of infants in group A (GA 32 weeks) and group B (GA > 32 weeks) at 0, 12, 24, 48, 72, and 96 hours of septic work-up. Values are least square means. Both groups showed significant changes over time (p < 0.0001). However, the manner of the response and the change in the response rate over time was similar in both the groups, p ¼ 0.59 and 0.74, respectively. CRP, C-reactive protein; GA, gestational age. American Journal of Perinatology
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Microorganisms
Shah et al.
Fig. 2 After excluding skin flora, CRP levels of infants in group A (GA 32 weeks; n ¼ 15) and group B (GA > 32 weeks; n ¼ 76) at 0, 12, 24, 48, 72, and 96 hours of septic work-up. Values are least square means. Both groups showed significant changes over time (p < 0.0001). However, the manner of the response and the change in response rate over time was similar in both the groups, p ¼ 0.87, and p ¼ 0.66, respectively. CRP, Creactive protein; GA, gestational age.
Fig. 3 (A) Scatter plot showing no correlation between the GA and the peak CRP values and, (B) between the GA and the time for CRP to peak. CRP, C-reactive protein; GA, gestational age.
number of neonates with positive blood culture,21,22 an unspecified trigger source (infection/tissue injury),23 an absence of longitudinal CRP trends,17,22 no listing of the microorganisms involved,17 a different cutoff threshold for,17 and/ or a different CRP methodology.24 Two studies used 35 weeks as a cutoff to compare premature groups.17,23 Both studies reported lower CRP responses in the premature infant group. In a post hoc analysis in our study, increasing the cutoff to 35 weeks did not affect the main results. Both groups had similar CRP responses, data not shown. Overall, 33% of our blood cultures were positive for CONS; the CRP value was abnormal in 50% (3/6) of group A compared with 40% (17/42) in group B. A low response to CONS has been reported before. Schmidt et al10 reported that 64% of infants with CONS bacteremia had abnormal CRP values. They suggested that “CONS isolated from blood in the NICU is clinically a relevant finding when neonates are symptomatic and the CRP values are abnormal.” In addition to contamination, a low American Journal of Perinatology
CRP level in CONS sepsis has also been attributed to its low virulence.4,9,25,26 As CONS is a common microorganism detected in blood cultures of neonates, Hofer et al17 evaluated CRP levels in a small cohort after excluding positive cultures with skin flora. They reported lower median CRP values in EOS in 19 infants at < 37 weeks’ GA when compared with 14 infants 37 weeks’ GA (0.9 and 1.8 mg/dL, respectively). Overall, 56% of all their positive blood cultures were GBS; other organisms were not identified. However, when we excluded the infants with positive blood cultures for organisms such as CONS and diphtheroids, the results of our study remained the same; CRP trends remained similar in both the groups with no statistical difference. On the contrary, Seibert et al27 studied 125 preterm infants with a GA of 23 to 31 weeks within 3 days of life; 45 were < 28 weeks GA. Only 8 of 125 infants had positive cultures (organisms were not classified). Overall, 28 infants had a probable infection and 89 were considered as not
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Preterm Infants Can Mount Appropriate CRP Responses to EOS
Preterm Infants Can Mount Appropriate CRP Responses to EOS
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Network. Trends in neonatal morbidity and mortality for very low birthweight infants. Am J Obstet Gynecol 2007;196(2):147. e1–147.e8 2 Connell TG, Rele M, Cowley D, Buttery JP, Curtis N. How reliable is a negative blood culture result? Volume of blood submitted for culture in routine practice in a children’s hospital. Pediatrics 2007; 119(5):891–896 3 Philip AG, Mills PC. Use of C-reactive protein in minimizing antibiotic exposure: experience with infants initially admitted to a well-baby nursery. Pediatrics 2000;106(1):E4
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protein (CRP) response patterns in neonatal septicaemia. APMIS 1999;107(6):593–600 Chirico G, Loda C. Laboratory aid to the diagnosis and therapy of infection in the neonate. Pediatr Rep 2011;3(1):e1 Hofer N, Zacharias E, Müller W, Resch B. An update on the use of Creactive protein in early-onset neonatal sepsis: current insights and new tasks. Neonatology 2012;102(1):25–36 Benitz WE, Han MY, Madan A, Ramachandra P. Serial serum Creactive protein levels in the diagnosis of neonatal infection. Pediatrics 1998;102(4):E41 Forest JC, Larivière F, Dolcé P, Masson M, Nadeau L. C-reactive protein as biochemical indicator of bacterial infection in neonates. Clin Biochem 1986;19(3):192–194 Pourcyrous M, Bada HS, Korones SB, Baselski V, Wong SP. Significance of serial C-reactive protein responses in neonatal infection and other disorders. Pediatrics 1993;92(3):431–435 Schmidt BK, Kirpalani HM, Corey M, Low DE, Philip AG, Ford-Jones EL. Coagulase-negative staphylococci as true pathogens in newborn infants: a cohort study. Pediatr Infect Dis J 1987;6(11): 1026–1031 Chiesa C, Signore F, Assumma M, et al. Serial measurements of C-reactive protein and interleukin-6 in the immediate postnatal period: reference intervals and analysis of maternal and perinatal confounders. Clin Chem 2001;47(6):1016–1022 de Villiers WJ, Louw JP, Strachan AF, Etsebeth SM, Shephard EG, de Beer FC. C-reactive protein and serum amyloid A protein in pregnancy and labour. Br J Obstet Gynaecol 1990;97(8):725–730 Gitlin D, Biasucci A. Development of gamma G, gamma A, gamma M, beta IC-beta IA, C 1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, alpha 1-antitrypsin, orosomucoid, beta-lipoprotein, alpha 2-macroglobulin, and prealbumin in the human conceptus. J Clin Invest 1969; 48(8):1433–1446 Jaye DL, Waites KB. Clinical applications of C-reactive protein in pediatrics. Pediatr Infect Dis J 1997;16(8):735–746, quiz 746–747 Kääpä P, Koistinen E. Maternal and neonatal C-reactive protein after interventions during delivery. Acta Obstet Gynecol Scand 1993;72(7):543–546 Chiesa C, Natale F, Pascone R, et al. C reactive protein and procalcitonin: reference intervals for preterm and term newborns during the early neonatal period. Clin Chim Acta 2011;412(1112):1053–1059 Hofer N, Müller W, Resch B. Non-infectious conditions and gestational age influence C-reactive protein values in newborns during the first 3 days of life. Clin Chem Lab Med 2011;49(2): 297–302 Polin RA; Committee on Fetus and Newborn. Management of neonates with suspected or proven early-onset bacterial sepsis. Pediatrics 2012;129(5):1006–1015 Product manual VITROS Chemistry Products CRP Slides. Rochester, NY: Ortho-Clinical Diagnostics; 2010 Elabiad MT, Arheart KL, Korones SB, Pourcyrous M. Adjusting for bias in C-reactive protein levels when using a vitros slide method in infants. Am J Perinatol 2014;31(10):851–854 Kawamura M, Nishida H. The usefulness of serial C-reactive protein measurement in managing neonatal infection. Acta Paediatr 1995;84(1):10–13 Doellner H, Arntzen KJ, Haereid PE, Aag S, Austgulen R. Interleukin-6 concentrations in neonates evaluated for sepsis. J Pediatr 1998;132(2):295–299 Turner MA, Power S, Emmerson AJ. Gestational age and the C reactive protein response. Arch Dis Child Fetal Neonatal Ed 2004; 89(3):F272–F273 Philip AG. Response of C-reactive protein in neonatal Group B streptococcal infection. Pediatr Infect Dis 1985;4(2):145–148 Franz AR, Steinbach G, Kron M, Pohlandt F. Reduction of unnecessary antibiotic therapy in newborn infants using interleukin-8 and American Journal of Perinatology
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infected. CRP responses in infants < 28 weeks’ GA were the same as the infants at 29 to 31 weeks regardless of being considered infected or not. Although this study did not include older GA infants, it at least supports the idea that infants 32 weeks’ GA have similar CRP responses and can be grouped together similarly to what our study did. This is also supported by the absence of an association between the GA and the time to peak CRP or GA and the highest CRP level reached. Although positive blood culture is considered as “gold standard” for diagnosis of sepsis, not all positive blood cultures in asymptomatic infants should be considered as true sepsis.9,10 Isolation of microorganisms in a blood culture in a clinically well infant may reflect asymptomatic bacteremia or contamination of blood specimen. As described by Benitz28, “bacteremia means detection of bacteria in the blood stream, but sepsis occurs when invasion of bacteria results in systemic inflammatory response.” In neonates, most sepsis work-ups are performed for maternal indications or the presence of nonspecific clinical signs in neonates. Antenatal antibiotics either for preterm labor, prolonged rupture of membranes, or maternal fever may result in partially treated infant and a blunted CRP response.3 Therefore, clinical signs and abnormal laboratory tests should be carefully evaluated before confirming the presence of true infection. Limitations of our study include its design as a retrospective cohort study in a predominately black population and less number of patients with positive blood cultures in the lower GA group. Although the study included a large number of infants with positive blood cultures, it was still underpowered to safely avoid a type II error. Another limitation was the availability of mode of delivery data on only 57% of the infants. Although most of group A infants were delivered by spontaneous vaginal delivery secondary to preterm labor, their trend in CRP responses did not differ from those infants in group B who were mostly delivered by cesarean delivery. These circumstances may have triggered an earlier CRP response in group A and confounded the data. On the contrary, their ability to mount such a response to arguably noninfectious causes does support the findings of this study regarding the maturity of their immune system. In conclusion, this study demonstrated that in EOS, premature infants have similar CRP responses when compared with more mature infants.
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Preterm Infants Can Mount Appropriate CRP Responses to EOS
27 Seibert K, Yu VY, Doery JC, Embury D. The value of C-reactive
protein measurement in the diagnosis of neonatal infection. J Paediatr Child Health 1990;26(5):267–270 28 Benitz WE. Adjunct laboratory tests in the diagnosis of early-onset neonatal sepsis. Clin Perinatol 2010;37(2):421–438
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C-reactive protein as markers of bacterial infections. Pediatrics 1999;104(3 Pt 1):447–453 26 Laborada G, Rego M, Jain A, et al. Diagnostic value of cytokines and C-reactive protein in the first 24 hours of neonatal sepsis. Am J Perinatol 2003;20(8):491–501
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American Journal of Perinatology
