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Amniotic Fluid Interleukin-6 and the Risk of Early-Onset Sepsis Among Preterm Infants
Archives of Medical Research 30 (1999) 198–202
ORIGINAL ARTICLE
Amniotic Fluid Interleukin-6 and the Risk of Early-Onset Sepsis Among Preterm Infants Ricardo Figueroa-Damián,* José L. Arredondo-García** and Javier Mancilla-Ramírez* *Departamento de Infectología e Inmunología, **Dirección de Investigación, Instituto Nacional de Perinatología, México, D.F., Mexico Received for publication November 25, 1998; accepted March 1, 1999 (98/149).
Background. High concentrations of interleukin-6 (IL-6) have been demonstrated in amniotic fluid (AF) from women with intra-amniotic infection. Recent studies have reported that IL-6 levels in AF were related to an increase in neonatal morbidity; moreover, higher IL-6 plasma levels have been observed in neonates with sepsis. Methods. A cohort study was carried out at the National Institute of Perinatology in Mexico City. Inclusion criteria were the following: 1) preterm singleton pregnancy; 2) intact membranes at time of enrollment, and 3) written informed consent. Women with other complications of pregnancy were excluded. Newborn sepsis during the first 72 h was defined as early-onset sepsis. Amniotic fluid was obtained at the moment of delivery. Amniotic fluid IL-6 (AF IL-6) was determined by enzyme-linked immunoassays. Results. Ninety-three women met the criteria for enrollment in the study and 31 (33%) of their newborns had early-onset neonatal sepsis. The mean AF IL-6 in mothers of septic newborns was 5779 6 2804 pg/ml compared to 729 6 382 pg/ml in mothers with noninfected neonates (p ,0.001). AF IL-6 concentrations higher than 1250 pg/ml were significantly associated with early-onset sepsis (OR 33.3; 95% CI 9.4–117.3) (p , 0.001). Gestational age under 32 weeks was also associated with neonatal sepsis (OR 2.56; 95% CI 1.2–9) (p 5 0.002). Women whose infants developed neonatal sepsis had a higher frequency of clinical chorioamnionitis (p 5 0.02). Conclusions. IL-6 determination in AF may be a useful indicator to identify neonates with higher risk of in utero bacterial infection. © 1999 IMSS. Published by Elsevier Science Inc. Key Words: Interleukin-6, Amniotic fluid, Preterm labor, Neonatal sepsis.
Introduction Intra-amniotic infection (IAI) is a complication of pregnancy that increases perinatal morbidity and may induce premature rupture of the membranes (PROM), preterm labor (PL), or puerperal infection (1). Early diagnosis of IAI allows the clinician to reduce other perinatal risks, such as fetal death, prematurity, or early-onset neonatal sepsis (2). However, early diagnosis is not easy because the clinical expression of IAI is frequently a late event or due to a subclinical course of IAI with premature uterine activity as the only manifestation (3). In order to establish an early identi-
Address reprint requests to: Dr. Ricardo Figueroa-Damián, Depto. de Infectología e Inmunología, 48 piso, Torre de Investigación, Instituto Nacional de Perinatología, Montes Urales 800, Col. Lomas Virreyes, 11000 México, D.F., México. Tel.: (1525) 520-9900, ext. 334; FAX: (1525) 520-0034.
fication of chorioamnionitis, several markers of infection have been tested in amniotic fluid (AF), such as gram stain, microbiologic cultures, glucose levels, leukocyte quantification and catalase activity, among others (4). Recently, interleukin-1 (IL-1), tumor necrosis factor-a (TNF), interleukin-6 (IL-6) and IL-8 have been proposed as markers for chorioamnionitis and could also be related to neonatal sepsis (5,6). Among these, IL-6 has shown high sensitivity and specificity in the diagnosis of IAI (7–9). IL-6 is a low molecular weight protein (26 kDa) that functions as an important mediator of immune and inflammatory responses (10). Interestingly, IL-6 may modulate the production of other pro-inflammatory cytokines, such as IL-1 and TNF at the level of transcription (11). The IL-6 present in AF seems to be produced by the decidua, with a possible fetal component (12). High concentrations of this cytokine have been demonstrated in AF from women with
0188-0128/99 $–see front matter. Copyright © 1999 IMSS. Published by Elsevier Science Inc. PII S0188-0128(99)00 0 1 5 - 9
Figueroa-Damián et al. / Archives of Medical Research 30 (1999) 198–202
IAI (13), suggesting that augmentation of IL-6 in AF may be of great value for diagnosis and prognosis of PL induced by infection (4,8,9). In a recent study by Romero et al. (14), IL-6 levels in AF were also related to a general increase in neonatal morbidity and mortality, whereas other authors and ourselves have reported the strong association of elevated IL-6 plasma levels in newborns with proven sepsis (15–17). Early-onset neonatal sepsis is one of the most important complications in infants born to mothers with IAI (18). If IL-6 is a good marker for IAI, its increase in AF could also be associated with the development of early neonatal sepsis a useful indicator in predicting sepsis in preterm newborns due to the fact that early sepsis is a more frequent as in low gestational age and low birth weight infants (19). The aim of this study was to determine the relationship between IL-6 concentrations in AF of mothers with preterm delivery without PROM and the development of early-onset neonatal sepsis in their infants.
Materials and Methods Study design. A cohort study was carried out to examine the relationship between AF concentrations of IL-6 and risk for early-onset neonatal sepsis. This study consisted of women who were admitted to the National Institute of Perinatology Hospital in Mexico City between March 1, 1995 and October 30, 1996, with PL without PROM, and who met the following criteria: (a) preterm singleton pregnancy (26–36 weeks); (b) intact membranes at time of enrollment; and (c) written informed consent for the use of AF for research purposes. Women with complications of pregnancy such as preeclampsia, immune diseases previously diagnosed non-bacterial infectious diseases, such as rubella, toxoplasmosis or HIV infection, and women who were having antimicrobial treatment at the time of admission were excluded. The study project was approved by the research review board of the Institute.
Study population. Participants were at gestational ages of 28–36 weeks, determined by menstrual dating or ultrasonographic examination. Preterm labor was defined as regular uterine contractions at a frequency of ,10 min and documented cervical dilatation. All women had intact membranes at the time of enrollment. Newborn anthropometric status was characterized by the neonatal staff based on standard charts of expected weight, length, and head circumference for Mexican preterm neonates, using a one-tenth percentile of birth weight for gestational age as a cut-off point (20). Newborn sepsis during the first 72 h was defined as early-onset sepsis. Diagnosis of sepsis was made by the attending neonatologist, who was unaware of the AF IL-6 values according to the criteria previously described (19).
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Amniotic fluid studies. The AF sample was immediately aliquoted for determination of IL-6 and microbiologic evaluation including cultures for aerobic and anaerobic bacteria and for genital mycoplasma, according to methods previously described (21). The AF samples were centrifuged at 10,000 3 g for 5 min and stored at 2708C in pyrogenic-free containers until assayed for IL-6, which was determined by commercial enzyme-linked immunoassays (Sanofi-Pasteur, Marnes, France) that had been validated for plasma, serum, and AF. Standard positive controls of IL-6 were run simultaneously with the study specimens. The lower limit of detection was 27 pg/mL. Amniotic fluid IL-6 levels above 1250 pg/mL were considered elevated; this value was the median for the entire distribution of IL-6 concentrations, and was chosen as the cut-off point. Similar values have been reported and used by others to determine associations between elevated cytokines in AF, infection and preterm delivery (7). Statistical analysis. The associations between potential risk factors and the occurrence of early-onset neonatal sepsis were examined first by unadjusted odds ratio (OR) and confidence intervals. Either Fisher’s two-tailed test or x2 test was used for categoric data, and Student’s t test was applied to continuous variables. In order to control potentially confounding variables, a stratified analysis was performed by means of the Mantel-Haenszel chi-square procedure. Multivariable logistic regression models were constructed to assess the association between AF IL-6 levels and neonatal sepsis, considering adjustment for birth weight, gestational age, clinical chorioamnionitis, pathogenic bacteria in the AF, prenatal care at the Institute and use of prophylactic antibiotics during delivery.
Results Clinical characteristics. Ninety-three women met the study criteria, and 31 (33%) of their newborns had early-onset neonatal sepsis. The study population was stratified according to their clinical characteristics and the presence or absence of neonatal sepsis (Table 1). No significant differences were observed between groups for maternal age, positive AF cultures, mode of delivery, prenatal care, gestational age, and birth weight. Women whose infants developed neonatal sepsis had a higher frequency of clinical chorioamnionitis (p 5 0.02) and also delivered their babies at earlier gestational ages (p ,0.01). Positive AF cultures were obtained from 18 women; Escherichia coli being the most common bacteria isolated from women whose infants had early sepsis, whereas Mycoplasma hominis and Ureaplasma urealyticum were commonly isolated from the women whose newborns did not have sepsis. Although clinical sepsis was proven in 31 neonates, only 10 had positive cultures. Escherichia coli and
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Table 1. Characteristics of the study population according to the presence or absence of early-onset sepsis
Factor
Early onset sepsis
Characteristics Maternal age (years, mean 6 standard deviation) Clinical chorioamnionitis Positive amniotic fluid culture Prenatal care Prophylactic antibiotics at delivery Cesarean delivery Gestational age at birth [weeks, median (range)] ,32 weeks of gestation Small for gestational age
Table 2. Univariate analysis of exposure and risk factors for early-onset neonatal sepsis
absent (n 5 62)
present (n 5 31)
26.7 6 7.1 3 (4.8%) 10 (16.1%) 36 (58.0%) 36 (58.0%) 34 (54.8%)
25.3 6 6.7 8 (25.8%) 8 (25.8%) 12 (41.9%) 23 (74.1%) 19 (61.2%)
NS 0.02 NS NS NS NS
33 (2,836) 21 (33.9%) 14 (22.5%)
32 (28–35) 21 (67.7%) 8 (25.8%)
NS ,0.01 NS
p Value
NS 5 not significant.
Staphylococcus coagulase negative were the most frequent isolations. The same microorganism was cultured from both neonatal blood and AF in seven cases: E. coli in four cases, Enterobacter cloacae, Streptococcus agalactie, and Citrobacter freundii in one case each, respectively. Amniotic fluid interleukin-6 and early-onset sepsis. Mothers of newborns who had early-onset sepsis showed significantly higher mean concentrations of IL-6 in their AF samples (Figure 1). The mean of AF IL-6 in the mothers of septic newborns was 5779 6 2804 pg/mL, compared to 729 6 382 pg/ mL in mothers of non-infected neonates (p ,0.001). Amniotic fluid IL-6 levels were higher than 1250 pg/mL in 26 of the 31 (83.8%) mothers whose newborns experienced early sepsis. Risk factors for early-onset sepsis. Table 2 summarizes the univariate analysis for various risk factors of neonatal sepsis. High concentrations of AF IL-6 were significantly asso-
IL-6 .1250 pg/ml Clinical chorioamnionitis Gestational age ,32 weeks Prophylactic antibiotics Positive amniotic fluid culture Small for gestational age Prenatal care
Odds ratio
95% CI
p Value
40.8 5.0 4.1 2.8 1.8 1.4 0.7
12 – 137.8 1.4 – 17.3 1.6 – 10.2 1.1 – 7.1 0.6 – 5.0 0.5 – 3.6 0.3 – 1.6
,0.001 0.008 0.002 0.020 NS NS NS
CI: confidence interval; NS: not significant.
ciated with early-onset sepsis. Clinical chorioamnionitis, gestational age under 32 weeks and the use of prophylactic antibiotics were also associated with early-onset sepsis. Positive AF cultures were weakly related to the development of neonatal sepsis. The stratified analysis showed that AF IL-6 concentrations higher than 1250 pg/mL remained significantly associated with early-onset sepsis after adjustment for diverse factors. This analysis also proved that clinical chorioamnionitis, gestational age less than 32 weeks, prenatal care and prophylactic antibiotics were significant confounding variables (Table 3). The relation of AF IL-6 levels with neonatal sepsis was also analyzed by a logistic regression model constructed with the confounding variables identified. Amniotic fluid IL-6 concentrations higher than 1250 pg/mL showed a significant relationship with early-onset newborn sepsis (OR 33.3; 95% confidence interval 9.4–117.3) (p ,0.001). Gestational age less than 32 weeks also was significantly associated with early neonatal sepsis (OR 2.56; 95% CI 1.2–9) (p ,0.01). Discussion Perinatal morbidity and mortality are complex public health problems due to the close relation between medical problems of pregnant women and the outcome of their infants. Infection is one of the common complications during pregnancy, leading to an adverse outcome in the neonate. Newborns from women with chorioamnionitis have a higher risk of becoming infected. Gilstrap et al. (22) studied 312 women with chorioamnionitis and 45% of their neonates were suspected for sepsis, which was confirmed in 5.4% of Table 3. Confounding variables identified by stratified analysis of the association of amniotic fluid IL-6 higher than 1250 pg/ml with early-onset sepsis Confounding variable
Figure 1. Interleukin-6 concentrations in amniotic fluid were significantly higher in mothers giving birth to septic neonates compared to mothers with non-infected newborns.
Prophylactic antibiotics Clinical chorioamnionitis Prenatal care Gestational age ,32 weeks
OR adjusted
(95% CI)
45.3 36.5 36.2 28.6
(12 – 170.5) (10.2 – 130.4) (10.4 – 126.2) (7.9 – 91.2)
OR: odds ratio; IL-6: interleukin-6; CI: confidence interval.
Figueroa-Damián et al. / Archives of Medical Research 30 (1999) 198–202
the infants. Other authors have described pneumonia and early sepsis in 24% of neonates from women with chorioamnionitis (23). Additionally, Hillier et al. (24) in a prospective study demonstrated a positive correlation between chorioamnionitis and PL. Similar data were proved as an independent association by Krohn et al. (25). IL-6 has been related to PL in the presence of positive bacterial cultures of AF (8,9,13,26) or chorioamnionitis confirmed by histologic studies (7,27,28). IL-6 is secreted by decidua chorion, trophoblastic and amniotic cells as an early response to bacterial infections (13) and is considered as a good marker of IAI (29). However, a direct correlation between the type of bacteria isolated and AF IL-6 levels has not been demonstrated (26). In other studies, similar bacteria have been isolated (30) and Mycoplasma has also been the predominant infectious agent. Other studies have reported anaerobes as the main causative agents (31). In our study, enterobacteria and Gram-positive cocci were more frequent in AF in women whose newborns developed earlyonset sepsis, while mycoplasma were isolated mainly from women with non-septic infants. IL-6 is valuable as an indicator of infection in wide ranges from 600 pg/mL to 375 ng/mL (7,9,13,19,26,28). This variability depends on the method used to measure this cytokine. Our data suggest a high correlation between elevated levels of IL-6 in the AF of women with PL without PROM and early-onset sepsis in their newborns. Maternal AF IL-6 might be useful in identifying pregnant women whose infants will be at high risk of acquiring a bacterial infection in utero. Romero et al. (14) reported an OR of 13.8 for IL-6 values above 30 ng/mL in AF and general neonatal morbidity. Yoon et al. (28) also observed higher levels of IL-6 in AF of women giving birth to infants with neonatal complications. In our study, the OR 33.3 for AF IL-6 and early-onset sepsis strongly supports the hypothesis of such an association. The findings of the current study allow us to support the hypothesis that the determination of AF IL-6 in women with preterm labor without PROM may identify newborns with high risk for developing early-neonatal sepsis and to begin in these neonates an early antibiotic treatment. Nevertheless, the high cost of the test carries the limitation for the clinical use of the determination of AF IL-6.
References 1. Newton E. Chorioamnionitis and intraamniotic infection. Clin Obstet Gynecol 1993;36:795. 2. Gilstrap L, Cox S. Acute chorioamnionitis. Obstet Gynecol Clin North Am 1989;16:373. 3. Yáñez-Velasco L, García-Marquina S, Salinas-Velázquez JC, CortésPérez J, Figueroa-Arredondo P, Calderón-Jaimes E. Infección durante el embarazo como factor causal de ruptura prematura de membranas y de parto pretérmino. Salud Públ Méx 1990;32:288. 4. Figueroa-Damián R, Garduño-Espinosa J. Pruebas diagnósticas de infección intraamniótica. Revisión de la literatura. Ginec Obstet Mex 1997;65:17.
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5. Foulon W, Van Liedekerke D, Demanet C, Decatte L, Dewaele M, Naessens A. Markers of infection and their relationship to preterm delivery. Am J Perinatol 1995;12:208. 6. Lencki S, Maciulla MB, Eglinton GS. Maternal and umbilical cord serum interleukin levels in preterm labor with clinical chorioamnionitis. Am J Obstet Gynecol 1994;170:1345. 7. Hillier S, Witkin S, Krohn M, Watts H, Kiviat N, Eschenbach D. The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis and chorioamnion infection. Obstet Gynecol 1993;81:941. 8. Greig P, Ernest JM, Teot L, Erikson M, Talley R. Amniotic fluid interleukin-6 level correlates with histologic chorioamnionitis and amniotic fluid cultures in patients in premature labor with intact membranes. Am J Obstet Gynecol 1993;169:1035. 9. Romero R, Yoon B, Kenney J, Gomez R, Allison A, Sehgal P. Amniotic fluid interleukin-6 determinations are of diagnostic and prognostic value in preterm labor. Am J Reprod Immunol 1993;30:167. 10. Kishimoto T. The biology of interleukin-6. Blood 1989;74:1. 11. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark S, Dinarello CA. Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 1990;75:40. 12. Silver R, Schwinzer B, McGregor J. Interleukin-6 levels in amniotic fluid in normal and abnormal pregnancies: preeclampsia, small-forgestational-age fetus and premature labor. Am J Obstet Gynecol 1993;169:1101. 13. Romero R, Avila C, Santhanam V, Sehgal PB. Amniotic fluid interleukin-6 in preterm labor. J Clin Invest 1990;85:1392. 14. Romero R, Yoon BH, Mazor M, Gomez R. The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and Gram stain in patients with preterm labor and intact membranes. Am J Obstet Gynecol 1993;169:805. 15. Lehrnbecher T, Schrod L, Kraus D, Roos T, Martius J, Von Stockhausen HB. Interleukin-6 and soluble interleukin-6 receptor in cord blood in the diagnosis of early onset sepsis in neonates. Acta Paediatr 1995;84:806. 16. Panero A, Pacifico L, Rossi N, Mancuso G, Stegagno M, Chiesa C. Interleukin-6 in neonates with early and late onset infection. Pediatr. Infect Dis J 1997;16:370. 17. Mancilla-Ramirez J, Arredondo-García J, Vannier E, Dinarello CA. Interleucina-1, interleucina-6 y factor de necrosis tumoral en sepsis neonatal. Perinatol Reprod Hum (Mex) 1996;10:230. 18. Maberry M, Gilstrap L. Intrapartum antibiotic therapy for suspected intraamniotic infection: impact on the fetus and neonates. Clin Obstet Gynecol 1991;34:345. 19. Klein J, Marcy M. Bacterial sepsis and meningitis. In: Remington JS, Klein J, editors. Infectious Diseases of the Fetus and Newborn Infant. 4th ed. Philadelphia: WB Saunders;1995. p. 835. 20. Peñuelas-Olaya MA, Fernández-Carrocera LA, Velazco-Pasillas M, Baptista-González H, Udaeta-Mora E. Curvas de crecimiento del neonato pretérmino durante el primer año de vida. Bol Med Hosp Infant Méx 1991;48:643. 21. Balow A, Hausler W, Herrmann K, Iseberg H, Shadomy AJ, editors. Manual of clinical microbiology. 5th ed. Washington, D.C.: American Society for Microbiology;1991. 22. Gilstrap LC, Leveno KJ, Cox S, Burris J, Mashburn M, Rosenfeld CR. Intrapartum treatment of acute chorioamnionitis: impact on neonatal sepsis. Am J Obstet Gynecol 1988;159:579. 23. Hauth JC, Gilstrap L, Hankins G, Connor K. Term maternal and neonatal complications on acute chorioamnionitis. Obstet Gynecol 1985;66:59. 24. Hillier S, Martius J, Krohn M, et al. A case-control study of the chorioamnionic infection and histologic chorioamnionitis in pregnancy. N Engl J Med 1988;319:972. 25. Krohn M, Hillier S, Nugent RP, et al. The genital flora of women with intraamniotic infection. J Infect Dis 1995;171:1475. 26. Andrews W, Hauth J, Goldenberg R, Gómez R, Romero R, Cassell G.
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Amniotic fluid interleukin-6: correlation with upper genital tract microbial colonization and gestational age in women delivered after spontaneous labor versus indicated delivery. Am J Obstet Gynecol 1995;173:606. 27. Matsuzaki N, Taniguchi T, Shimoyo K. Placental interleukin-6 production is enhanced in intrauterine infection but not in labor. Am J Obstet Gynecol 1993;168:94. 28. Yoon B, Romero R, Kim C, Jun J, Gomez R, Choi J, et al. Amniotic fluid interleukin-6: a sensitive test for antenatal diagnosis of acute inflammatory lesions of preterm placenta and prediction of perinatal morbidity. Am J Obstet Gynecol 1995;172:960.
29. Dudley DJ, Trautman MS, Araneo BA, Edwin S, Mitchell MD. Decimal cell biosynthesis of interleukin-6: regulation by inflammatory cytokines. J Clin Endocrinol Metab 1992;74:884. 30. Gather D, Meyer W. Comparison of Gram stain, leukocyte esterase activity, and amniotic fluid glucose concentration in predicting amniotic fluid culture results in preterm premature rupture of membranes. Am J Obstet Gynecol 1992;167:1092. 31. Font G, Gauthier D, Meyer W, Myles T, Janda W, Bieniarz A. Catalase activity as a predictor of amniotic fluid culture results in preterm labor or premature rupture of membranes. Obstet Gynecol 1995;85:656.
ORIGINAL ARTICLE
Amniotic Fluid Interleukin-6 and the Risk of Early-Onset Sepsis Among Preterm Infants Ricardo Figueroa-Damián,* José L. Arredondo-García** and Javier Mancilla-Ramírez* *Departamento de Infectología e Inmunología, **Dirección de Investigación, Instituto Nacional de Perinatología, México, D.F., Mexico Received for publication November 25, 1998; accepted March 1, 1999 (98/149).
Background. High concentrations of interleukin-6 (IL-6) have been demonstrated in amniotic fluid (AF) from women with intra-amniotic infection. Recent studies have reported that IL-6 levels in AF were related to an increase in neonatal morbidity; moreover, higher IL-6 plasma levels have been observed in neonates with sepsis. Methods. A cohort study was carried out at the National Institute of Perinatology in Mexico City. Inclusion criteria were the following: 1) preterm singleton pregnancy; 2) intact membranes at time of enrollment, and 3) written informed consent. Women with other complications of pregnancy were excluded. Newborn sepsis during the first 72 h was defined as early-onset sepsis. Amniotic fluid was obtained at the moment of delivery. Amniotic fluid IL-6 (AF IL-6) was determined by enzyme-linked immunoassays. Results. Ninety-three women met the criteria for enrollment in the study and 31 (33%) of their newborns had early-onset neonatal sepsis. The mean AF IL-6 in mothers of septic newborns was 5779 6 2804 pg/ml compared to 729 6 382 pg/ml in mothers with noninfected neonates (p ,0.001). AF IL-6 concentrations higher than 1250 pg/ml were significantly associated with early-onset sepsis (OR 33.3; 95% CI 9.4–117.3) (p , 0.001). Gestational age under 32 weeks was also associated with neonatal sepsis (OR 2.56; 95% CI 1.2–9) (p 5 0.002). Women whose infants developed neonatal sepsis had a higher frequency of clinical chorioamnionitis (p 5 0.02). Conclusions. IL-6 determination in AF may be a useful indicator to identify neonates with higher risk of in utero bacterial infection. © 1999 IMSS. Published by Elsevier Science Inc. Key Words: Interleukin-6, Amniotic fluid, Preterm labor, Neonatal sepsis.
Introduction Intra-amniotic infection (IAI) is a complication of pregnancy that increases perinatal morbidity and may induce premature rupture of the membranes (PROM), preterm labor (PL), or puerperal infection (1). Early diagnosis of IAI allows the clinician to reduce other perinatal risks, such as fetal death, prematurity, or early-onset neonatal sepsis (2). However, early diagnosis is not easy because the clinical expression of IAI is frequently a late event or due to a subclinical course of IAI with premature uterine activity as the only manifestation (3). In order to establish an early identi-
Address reprint requests to: Dr. Ricardo Figueroa-Damián, Depto. de Infectología e Inmunología, 48 piso, Torre de Investigación, Instituto Nacional de Perinatología, Montes Urales 800, Col. Lomas Virreyes, 11000 México, D.F., México. Tel.: (1525) 520-9900, ext. 334; FAX: (1525) 520-0034.
fication of chorioamnionitis, several markers of infection have been tested in amniotic fluid (AF), such as gram stain, microbiologic cultures, glucose levels, leukocyte quantification and catalase activity, among others (4). Recently, interleukin-1 (IL-1), tumor necrosis factor-a (TNF), interleukin-6 (IL-6) and IL-8 have been proposed as markers for chorioamnionitis and could also be related to neonatal sepsis (5,6). Among these, IL-6 has shown high sensitivity and specificity in the diagnosis of IAI (7–9). IL-6 is a low molecular weight protein (26 kDa) that functions as an important mediator of immune and inflammatory responses (10). Interestingly, IL-6 may modulate the production of other pro-inflammatory cytokines, such as IL-1 and TNF at the level of transcription (11). The IL-6 present in AF seems to be produced by the decidua, with a possible fetal component (12). High concentrations of this cytokine have been demonstrated in AF from women with
0188-0128/99 $–see front matter. Copyright © 1999 IMSS. Published by Elsevier Science Inc. PII S0188-0128(99)00 0 1 5 - 9
Figueroa-Damián et al. / Archives of Medical Research 30 (1999) 198–202
IAI (13), suggesting that augmentation of IL-6 in AF may be of great value for diagnosis and prognosis of PL induced by infection (4,8,9). In a recent study by Romero et al. (14), IL-6 levels in AF were also related to a general increase in neonatal morbidity and mortality, whereas other authors and ourselves have reported the strong association of elevated IL-6 plasma levels in newborns with proven sepsis (15–17). Early-onset neonatal sepsis is one of the most important complications in infants born to mothers with IAI (18). If IL-6 is a good marker for IAI, its increase in AF could also be associated with the development of early neonatal sepsis a useful indicator in predicting sepsis in preterm newborns due to the fact that early sepsis is a more frequent as in low gestational age and low birth weight infants (19). The aim of this study was to determine the relationship between IL-6 concentrations in AF of mothers with preterm delivery without PROM and the development of early-onset neonatal sepsis in their infants.
Materials and Methods Study design. A cohort study was carried out to examine the relationship between AF concentrations of IL-6 and risk for early-onset neonatal sepsis. This study consisted of women who were admitted to the National Institute of Perinatology Hospital in Mexico City between March 1, 1995 and October 30, 1996, with PL without PROM, and who met the following criteria: (a) preterm singleton pregnancy (26–36 weeks); (b) intact membranes at time of enrollment; and (c) written informed consent for the use of AF for research purposes. Women with complications of pregnancy such as preeclampsia, immune diseases previously diagnosed non-bacterial infectious diseases, such as rubella, toxoplasmosis or HIV infection, and women who were having antimicrobial treatment at the time of admission were excluded. The study project was approved by the research review board of the Institute.
Study population. Participants were at gestational ages of 28–36 weeks, determined by menstrual dating or ultrasonographic examination. Preterm labor was defined as regular uterine contractions at a frequency of ,10 min and documented cervical dilatation. All women had intact membranes at the time of enrollment. Newborn anthropometric status was characterized by the neonatal staff based on standard charts of expected weight, length, and head circumference for Mexican preterm neonates, using a one-tenth percentile of birth weight for gestational age as a cut-off point (20). Newborn sepsis during the first 72 h was defined as early-onset sepsis. Diagnosis of sepsis was made by the attending neonatologist, who was unaware of the AF IL-6 values according to the criteria previously described (19).
199
Amniotic fluid studies. The AF sample was immediately aliquoted for determination of IL-6 and microbiologic evaluation including cultures for aerobic and anaerobic bacteria and for genital mycoplasma, according to methods previously described (21). The AF samples were centrifuged at 10,000 3 g for 5 min and stored at 2708C in pyrogenic-free containers until assayed for IL-6, which was determined by commercial enzyme-linked immunoassays (Sanofi-Pasteur, Marnes, France) that had been validated for plasma, serum, and AF. Standard positive controls of IL-6 were run simultaneously with the study specimens. The lower limit of detection was 27 pg/mL. Amniotic fluid IL-6 levels above 1250 pg/mL were considered elevated; this value was the median for the entire distribution of IL-6 concentrations, and was chosen as the cut-off point. Similar values have been reported and used by others to determine associations between elevated cytokines in AF, infection and preterm delivery (7). Statistical analysis. The associations between potential risk factors and the occurrence of early-onset neonatal sepsis were examined first by unadjusted odds ratio (OR) and confidence intervals. Either Fisher’s two-tailed test or x2 test was used for categoric data, and Student’s t test was applied to continuous variables. In order to control potentially confounding variables, a stratified analysis was performed by means of the Mantel-Haenszel chi-square procedure. Multivariable logistic regression models were constructed to assess the association between AF IL-6 levels and neonatal sepsis, considering adjustment for birth weight, gestational age, clinical chorioamnionitis, pathogenic bacteria in the AF, prenatal care at the Institute and use of prophylactic antibiotics during delivery.
Results Clinical characteristics. Ninety-three women met the study criteria, and 31 (33%) of their newborns had early-onset neonatal sepsis. The study population was stratified according to their clinical characteristics and the presence or absence of neonatal sepsis (Table 1). No significant differences were observed between groups for maternal age, positive AF cultures, mode of delivery, prenatal care, gestational age, and birth weight. Women whose infants developed neonatal sepsis had a higher frequency of clinical chorioamnionitis (p 5 0.02) and also delivered their babies at earlier gestational ages (p ,0.01). Positive AF cultures were obtained from 18 women; Escherichia coli being the most common bacteria isolated from women whose infants had early sepsis, whereas Mycoplasma hominis and Ureaplasma urealyticum were commonly isolated from the women whose newborns did not have sepsis. Although clinical sepsis was proven in 31 neonates, only 10 had positive cultures. Escherichia coli and
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Figueroa-Damián et al./ Archives of Medical Research 30 (1999) 198–202
Table 1. Characteristics of the study population according to the presence or absence of early-onset sepsis
Factor
Early onset sepsis
Characteristics Maternal age (years, mean 6 standard deviation) Clinical chorioamnionitis Positive amniotic fluid culture Prenatal care Prophylactic antibiotics at delivery Cesarean delivery Gestational age at birth [weeks, median (range)] ,32 weeks of gestation Small for gestational age
Table 2. Univariate analysis of exposure and risk factors for early-onset neonatal sepsis
absent (n 5 62)
present (n 5 31)
26.7 6 7.1 3 (4.8%) 10 (16.1%) 36 (58.0%) 36 (58.0%) 34 (54.8%)
25.3 6 6.7 8 (25.8%) 8 (25.8%) 12 (41.9%) 23 (74.1%) 19 (61.2%)
NS 0.02 NS NS NS NS
33 (2,836) 21 (33.9%) 14 (22.5%)
32 (28–35) 21 (67.7%) 8 (25.8%)
NS ,0.01 NS
p Value
NS 5 not significant.
Staphylococcus coagulase negative were the most frequent isolations. The same microorganism was cultured from both neonatal blood and AF in seven cases: E. coli in four cases, Enterobacter cloacae, Streptococcus agalactie, and Citrobacter freundii in one case each, respectively. Amniotic fluid interleukin-6 and early-onset sepsis. Mothers of newborns who had early-onset sepsis showed significantly higher mean concentrations of IL-6 in their AF samples (Figure 1). The mean of AF IL-6 in the mothers of septic newborns was 5779 6 2804 pg/mL, compared to 729 6 382 pg/ mL in mothers of non-infected neonates (p ,0.001). Amniotic fluid IL-6 levels were higher than 1250 pg/mL in 26 of the 31 (83.8%) mothers whose newborns experienced early sepsis. Risk factors for early-onset sepsis. Table 2 summarizes the univariate analysis for various risk factors of neonatal sepsis. High concentrations of AF IL-6 were significantly asso-
IL-6 .1250 pg/ml Clinical chorioamnionitis Gestational age ,32 weeks Prophylactic antibiotics Positive amniotic fluid culture Small for gestational age Prenatal care
Odds ratio
95% CI
p Value
40.8 5.0 4.1 2.8 1.8 1.4 0.7
12 – 137.8 1.4 – 17.3 1.6 – 10.2 1.1 – 7.1 0.6 – 5.0 0.5 – 3.6 0.3 – 1.6
,0.001 0.008 0.002 0.020 NS NS NS
CI: confidence interval; NS: not significant.
ciated with early-onset sepsis. Clinical chorioamnionitis, gestational age under 32 weeks and the use of prophylactic antibiotics were also associated with early-onset sepsis. Positive AF cultures were weakly related to the development of neonatal sepsis. The stratified analysis showed that AF IL-6 concentrations higher than 1250 pg/mL remained significantly associated with early-onset sepsis after adjustment for diverse factors. This analysis also proved that clinical chorioamnionitis, gestational age less than 32 weeks, prenatal care and prophylactic antibiotics were significant confounding variables (Table 3). The relation of AF IL-6 levels with neonatal sepsis was also analyzed by a logistic regression model constructed with the confounding variables identified. Amniotic fluid IL-6 concentrations higher than 1250 pg/mL showed a significant relationship with early-onset newborn sepsis (OR 33.3; 95% confidence interval 9.4–117.3) (p ,0.001). Gestational age less than 32 weeks also was significantly associated with early neonatal sepsis (OR 2.56; 95% CI 1.2–9) (p ,0.01). Discussion Perinatal morbidity and mortality are complex public health problems due to the close relation between medical problems of pregnant women and the outcome of their infants. Infection is one of the common complications during pregnancy, leading to an adverse outcome in the neonate. Newborns from women with chorioamnionitis have a higher risk of becoming infected. Gilstrap et al. (22) studied 312 women with chorioamnionitis and 45% of their neonates were suspected for sepsis, which was confirmed in 5.4% of Table 3. Confounding variables identified by stratified analysis of the association of amniotic fluid IL-6 higher than 1250 pg/ml with early-onset sepsis Confounding variable
Figure 1. Interleukin-6 concentrations in amniotic fluid were significantly higher in mothers giving birth to septic neonates compared to mothers with non-infected newborns.
Prophylactic antibiotics Clinical chorioamnionitis Prenatal care Gestational age ,32 weeks
OR adjusted
(95% CI)
45.3 36.5 36.2 28.6
(12 – 170.5) (10.2 – 130.4) (10.4 – 126.2) (7.9 – 91.2)
OR: odds ratio; IL-6: interleukin-6; CI: confidence interval.
Figueroa-Damián et al. / Archives of Medical Research 30 (1999) 198–202
the infants. Other authors have described pneumonia and early sepsis in 24% of neonates from women with chorioamnionitis (23). Additionally, Hillier et al. (24) in a prospective study demonstrated a positive correlation between chorioamnionitis and PL. Similar data were proved as an independent association by Krohn et al. (25). IL-6 has been related to PL in the presence of positive bacterial cultures of AF (8,9,13,26) or chorioamnionitis confirmed by histologic studies (7,27,28). IL-6 is secreted by decidua chorion, trophoblastic and amniotic cells as an early response to bacterial infections (13) and is considered as a good marker of IAI (29). However, a direct correlation between the type of bacteria isolated and AF IL-6 levels has not been demonstrated (26). In other studies, similar bacteria have been isolated (30) and Mycoplasma has also been the predominant infectious agent. Other studies have reported anaerobes as the main causative agents (31). In our study, enterobacteria and Gram-positive cocci were more frequent in AF in women whose newborns developed earlyonset sepsis, while mycoplasma were isolated mainly from women with non-septic infants. IL-6 is valuable as an indicator of infection in wide ranges from 600 pg/mL to 375 ng/mL (7,9,13,19,26,28). This variability depends on the method used to measure this cytokine. Our data suggest a high correlation between elevated levels of IL-6 in the AF of women with PL without PROM and early-onset sepsis in their newborns. Maternal AF IL-6 might be useful in identifying pregnant women whose infants will be at high risk of acquiring a bacterial infection in utero. Romero et al. (14) reported an OR of 13.8 for IL-6 values above 30 ng/mL in AF and general neonatal morbidity. Yoon et al. (28) also observed higher levels of IL-6 in AF of women giving birth to infants with neonatal complications. In our study, the OR 33.3 for AF IL-6 and early-onset sepsis strongly supports the hypothesis of such an association. The findings of the current study allow us to support the hypothesis that the determination of AF IL-6 in women with preterm labor without PROM may identify newborns with high risk for developing early-neonatal sepsis and to begin in these neonates an early antibiotic treatment. Nevertheless, the high cost of the test carries the limitation for the clinical use of the determination of AF IL-6.
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