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Maternal serum and vaginal fluid C-reactive protein levels do not predict early-onset neonatal infection in preterm premature rupture of membranes

Abstract

Objective:

The aim of this study was to evaluate the usefulness of maternal serum and vaginal fluid C-reactive protein (CRP) determinations in the prediction of neonatal congenital infection.

Study Design:

Fifty women between 24 and 36 weeks gestation, complicated by preterm premature rupture of membranes (pPROM), were divided into two groups according to the presence (n=14) or absence (n=36) of early-onset newborns' infection.

Result:

Maternal serum and vaginal fluid CRP concentrations were comparable between both groups. Serum CRP levels 10 and 15 mg l–1 predicted neonatal infection with a sensitivity of 47 and 47%, specificity of 63 and 76%, positive predictive value (PPV) of 38 and 47%, and negative predictive value (NPV) of 72 and 76%, respectively. The cutoff value of vaginal CRP 2.4 mg l–1 predicted infection with a sensitivity of 71%, specificity of 47%, PPV of 34%, and NPV of 81%. Receiver-operating characteristic curve analysis revealed that the predictive performance of CRP was poor.

Conclusion:

Maternal serum and vaginal fluid CRP determinations after pPROM are of poor predictive value in neonatal early-onset infection prediction.

Introduction

Preterm premature rupture of the membranes (pPROM) is one of the most common obstetrical complications, and accounts for nearly 30% of all preterm labors.1 Microbial invasion of the amniotic cavity is present in about 50% of pPROM cases, and neonates from pPROM-affected pregnancies are at higher risk of infection-related morbidity.2, 3

Prompt diagnosis of intraamniotic infection, which usually is subclinical, is necessary for the application of the proper pattern of clinical management. To determine whether the patient has intraamniotic inflammation, and thus should be delivered rather than managed expectantly or even treated with tocolytic agents, is often a critical decision with clinical implications for both mother and fetus.4 Unfortunately, currently there is a lack in noninvasive tests that would provide reliable early screening for fetal infection detection and thereby would help the obstetrician to recognize the need for antibiotic treatment, cessation of tocolysis, or early delivery. Antenatal detection of neonatal infection would also enable the physicians to more accurately select the population of newborn infants that require antibiotic treatment.2, 5, 6, 7, 8 Therefore, there is a need for the development of sensitive tests for the prediction of inborn infection that are of value in the management of the immediate neonatal period.4, 9

Maternal serum is an easily reachable biological material that is collected in almost noninvasive way. Some inflammatory mediators may also be detected in vaginal or cervical secretions, and it is probable that their concentrations in the vaginal compartment, especially after rupture of membranes, reflect their intraamniotic concentrations.10, 11

C-reactive protein (CRP) is an acute phase reactant produced by liver cells in response to interleukin-6 synthesized during infection, and it was proved that increased CRP values strongly indicate infectious complications.12 Although maternal serum level of CRP slightly increases within gestational age, still this marker is used as an indicator of intrauterine infection, especially in pPROM cases13, 14, 15, 16, 17, 18 nevertheless reports that its benefits are conflicting.19, 20 On the contrary, there are only a few published data on CRP in vaginal fluid during pregnancy or its complications.

The purpose of this study was to determine whether maternal serum and vaginal fluid concentrations of CRP are of value in the prediction of early-onset neonatal infection in patients with pPROM.

Methods

This case–control study covered pregnant women, who were admitted to the university Hospital, and met the following criteria: (1) singleton pregnancy; (2) gestational age between 24 and 36 weeks; (3) evident leakage of clear amniotic fluid, no longer than 4 h; (4) no uterine contractions; and (5) no tocolytics, antibiotics, or steroids within 14 days before study entry. Finally, according to these criteria, in the period between November 2005 and October 2007, 50 patients and their newborns were enrolled into the study. None of these women showed clinical signs of infection or any other maternal or fetal complications at admission. All subjects provided a written informed consent and the University Human Subject Review Committee approved the study protocol. The study group comprised of 14 patients whose newborns had clinical and laboratory indices of congenital infection, and the control group of 36 women whose newborns were not infected. Gestational age was estimated on the basis of the last menstrual period and confirmed by an ultrasonographic examination, which was performed in the beginning of the second trimester. From admission to delivery, all patients received prophylactic antibiotics and in women before 34 weeks gestation, steroid therapy was additionally given. Patients were restricted to bed, and fetal heart monitoring together with uterine activity assessment was performed twice daily.

Rupture of membranes was diagnosed by sterile speculum examination (confirming pooling of amniotic fluid from the vagina) and a positive result of Nitrazine test. Vaginal fluid was aspirated with a 5-ml plastic syringe from the cervical canal or from the posterior vaginal fornix within 4 h of pPROM detection, before vaginal examination and administration of any drugs. Simultaneously, maternal venous blood samples were taken for CRP levels and white blood cells count measurements. If the pPROM-to-delivery interval was longer than 48 h maternal CRP, white blood cells count levels were monitored by repeated measurements (performed every 48 h). These measurements were clinically useful in the management of pPROM patients, but consecutive CRP values were not taken into consideration for prediction of neonatal infection.

CRP concentrations were measured quantitatively by immunoturbidometry with the Olympus AU 560 system (Olympus Diagnostica, Hamburg, Germany). The coefficients of variation for the assays were <10%. According to the studies by Gibbs et al.21 and Bañkowska et al.,22 two cutoff values for analysis of maternal serum CRP levels, that is 10 and 15 mg l–1, were chosen. White blood cells count was determined automatically with Celldyn 1700 and Celldyn 3500 Abbott instruments (Abbott Laboratories, IL, USA). An application of critical cutoff value of 15.0 G l–1 was based on data published by Baud et al.23

Vaginal fluid, as well as neonatal swabs from ear, nasopharynx, umbilicus, and anus were immediately placed into transport media and sent to the microbiological laboratory. Aerobic cultures were incubated for 48 h at 35 °C on standard sheep blood agar, McConkey's medium, Chocolate PVX medium, and Sabouraud's glucose agar. Anaerobic cultures were incubated in an anaerobic chamber at 35 °C for 72 h on Schaedler's agar. Neonatal blood samples were incubated in BacT/Alert system (bioMerieux) for 5 days. Isolated bacteria were identified by standard procedures.

Fetal outcome was evaluated by a neonatologist according to the clinical signs and microbial status of the neonate. Blood cultures were taken from all examined infants. Early-onset neonatal infection was assumed whether it occurred within 48 h after delivery based on maternal history and the presence of three or more of the following clinical signs:

  • skin color (pallor, jaundice, cyanosis),

  • respiratory (apnea, tachypnea > 60 per minute, grunting, nasal flaring, intercostal or sternal retractions, need for high ventilator settings or oxygen),

  • cardiovascular (brady/tachycardia, poor peripheral perfusion, hypotension),

  • neurologic (hypotonia, irritability, lethargy, seizures),

  • gastrointestinal (abdominal distention, green or bloody residuals, vomiting, temperature instability), and/or

  • positive blood culture.

Chest X-rays and laboratory tests routinely performed in the management of infection, including CRP levels, white blood cells count, platelet count, and the immature/total neutrophil ratio, contributed to the final diagnosis. Neonatal sepsis was recognized as proven when it was confirmed by positive blood culture. In other cases, it was defined as suspected (clinical) sepsis.6, 8, 9, 24, 25 All infected newborns received antibiotics after delivery.

For the diagnosis of histological chorioamnionitis, microscopic analysis of the placenta was examined at 400 magnification, and earlier described diagnostic criteria of chorioamnionitis (>10 polymorphonuclear leukocytes in 10, nonadjacent microscopic fields from the extraplacental membranes, chorionic plate, or umbilical cord blood vessels) were applied.26

Statistical analysis was performed using Statistica 5.5 and MedCalc 7.2 software. The Shapiro–Wilk test was used to check the distribution of analyzed parameters. Differences between the groups were analyzed by Mann–Whitney U-test and χ2 test. P-values <0.05 were considered significant. To establish the cutoff values that optimized the prediction of neonatal infection, receiver-operating characteristic (ROC) curve analysis was used. Sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs) for obtained cutoffs, as well as for generally accepted critical values of maternal serum CRP, were then calculated. Areas under the ROC curves were evaluated and compared. In addition, likelihood ratios were calculated, and their values exceeding 5.0 were considered as useful.

Results

The demographic and clinical characteristics of the groups are shown in Table 1. None of the women included into the study showed clinical signs of infection at admission. The pPROM-to-delivery interval was comparable between both groups. Rate of cesarean sections was significantly higher in the study group. The indications included intraamniotic infection (n=5) and breech presentation (n=2). In the control group, nine women delivered through cesarean section because of indication of breech presentation (n=5), fetal distress (n=3), or intraamniotic infection (n=1).

Table 1 Clinical characteristics of the study and control groups

Maternal serum levels, as well as vaginal fluid CRP concentrations, were comparable between the study group (N=14) and control group (N=36) (Table 2). Two cutoff values were chosen for maternal serum CRP concentrations. CRP levels 10 and 15 mg l–1 predicted neonatal infection with a sensitivity of 47 and 47%, a specificity of 63 and 76%, a PPV of 38 and 47%, a NPV of 72 and 76%, and likelihood ratio of 1.29 and 1.94, respectively. The cutoff value of vaginal fluid CRP2.4 mg l–1 predicted neonatal infection with a sensitivity of 71%, a specificity of 47%, a PPV of 34%, a NPV of 81%, and likelihood ratio of 1.35 (Table 3). ROC curve analysis revealed that the predictive performance of maternal serum and vaginal fluid CRP determinations were poor and comparable (Figures 1 and 2). The difference between areas under the ROC curves was 0.08; s.e., 0.11; 95% CI, −0.13 to 0.29 (z=0.77; P=0.44).

Table 2 Comparison of C-reactive (CRP) levels between the mothers who born healthy or infected newborns
Table 3 Prognostic value of C-reactive protein (CRP) determinations in pregnant women with preterm premature rupture of membranes for early-onset neonatal infection
Figure 1
figure1

ROC curve that describes the performance of vaginal fluid CRP concentration after pPROMs for prediction of early-onset neonatal infection (area under the curve, 0.59; s.e., 0.09; 95% CI, 0.44 to 0.72).

Figure 2
figure2

ROC curve that describes the performance of maternal serum CRP concentration after pPROMs for prediction of early-onset neonatal infection (area under the curve, 0.51; s.e., 0.09; 95% CI, 0.36 to 0.65).

Neonatal blood cultures were positive in only five (35.7%) of the infected neonates. (Escherichia coli: 2; Klebsiella pneumoniae: 1; Streptococcus agalactiae: 1; and Enterococcus foecalis: 1). In this subgroup, mean vaginal fluid and maternal serum CRP values were 3.8±1.3 mg l–1 (range, 2.0 to 6.0) and 27.9±10.2 mg l–1 (range, 16.3 to 43.4), respectively. The remaining nine (64.3%) neonates from the study group had positive results of ear, nasopharynx, or anal microbiological cultures' analysis (E. coli: 3; Staphylococcus aureus: 3; Candida albicans: 2; and Streptococcus mitis: 1). None of the neonates died as a result of sepsis.

Placentas were examined in 71.4% of study individuals (n=10), as well as in 69.4% (n=25) of control patients. Signs of inflammatory process were found in 6 and 15 of them, respectively.

Discussion

We have found only two publications addressing the association between vaginal fluid CRP concentrations and newborns status. In the study of Di Naro,10 a significant correlation was found between vaginal fluid CRP concentrations after pPROM and both amniotic fluid and umbilical cord CRP levels. The median vaginal fluid CRP concentration was higher in fetuses with funisitis, and the concentration >0.8 ng ml–1 remained a predictor of intraamniotic infection and funisitis. On the other hand, Shim et al.3 had not shown any relationship between vaginal fluid CRP levels after pPROM and congenital neonatal infection development.

The relationships between early-onset neonatal infection and maternal serum CRP levels were widely analyzed. Yoon et al.12 noticed that mothers with pPROM, delivering newborns with significant morbidity, had significantly higher median CRP concentration than mothers delivering newborns without significant complications (6.5 versus 2.0 mg l–1). Kayem et al.2 observed that elevated maternal serum CRP concentration in patients with pPROM between 24 and 34 weeks gestation was associated with neonatal infection. In their study, the relatively high cutoff value of >20 mg l–1 predicted congenital infection with a sensitivity of 21%, a specificity of 95%, a PPV of 50%, and an NPV of 84%. On the contrary, the study of Torbé27 showed that maternal serum CRP levels 10 mg l–1, assayed within 4 h after pPROM detection, predicted early-onset neonatal infection with a sensitivity of 52%, a specificity of 76%, a PPV of 52%, and an NPV of 76%.

In the nonhomogonous group of 47 patients who delivered prematurely (57% with pPROM) Škrablin et al.17 showed significantly higher serum CRP levels in women who delivered newborns with congenital infection (20.5 versus 6.6 mg l–1) and a value higher than 8.9 mg l–1 had 84% sensitivity and 69% specificity in prediction of neonatal sepsis. In addition, Martius et al.,8 in the diversified group of 343 infants who were born before 35 weeks gestation (33.8% mothers with pPROM), showed a low prognostic value of maternal serum CRP measurements (cutoff >15 mg l–1) for this purpose.

The limitation of our study is only a single estimation of CRP assayed in the vaginal fluid that was collected within 4 h after pPROM detection. There was a latency period of 4.4±6.6 days between pPROM and delivery in the study group, and it is possible that intraamniotic infection has occurred or progressed during this time. And, on the other hand, from admission to delivery, all patients received prophylactic antibiotics, which could influence the newborn status.

However, after a few days since rupture of membranes, the volume of amniotic fluid is reduced and its leakage is usually rather low. In these conditions, collection of satisfactory fluid sample is usually very difficult. In addition, the origin of vaginal fluid CRP after pPROM is unknown. It may reflect amniotic fluid, cervical mucus, or vaginal fluid CRP levels.3 As spontaneous rupture of membranes usually occurs close to the cervical os, we reasoned that immediately after pPROM, vaginal fluid CRP concentrations might reflect those of the amniotic fluid. In 26% of women, serum CRP levels were evaluated more than once. However, as vaginal fluid CRP estimation was performed only once, only the initial maternal serum CRP values were taken into consideration for prediction of neonatal infection.

In conclusion, our findings suggest that maternal serum and vaginal fluid CRP measurements after preterm premature rupture of the membranes are of poor diagnostic value for early-onset neonatal infection prediction.

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Torbé, A., Kowalski, K. Maternal serum and vaginal fluid C-reactive protein levels do not predict early-onset neonatal infection in preterm premature rupture of membranes. J Perinatol 30, 655–659 (2010). https://doi.org/10.1038/jp.2010.22

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Keywords

  • C-reactive protein
  • maternal serum
  • neonatal infection
  • preterm premature rupture of membranes
  • vaginal fluid

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