Abstract
Objective. Antibiotic administration has become part of the standard of care for patients with preterm premature rupture of membranes (PROM). Yet, the natural history of intrauterine infection/inflammation during antibiotic therapy remains largely unknown. This study was conducted to determine if antibiotic administration to the mother eradicates intra-amniotic infection and/or reduces the frequency of intra-amniotic inflammation, a risk factor for impending preterm labor/delivery and adverse neonatal outcome.
Methods. A subset of patients with preterm PROM admitted to our institution underwent amniocenteses before and after antibiotic administration in order to guide clinical management. Amniotic fluid analysis consisted of a Gram stain, culture for aerobic and anaerobic bacteria as well as genital mycoplasmas, and amniotic fluid white blood cell (WBC) count. Microbial invasion of the amniotic cavity (MIAC) was defined as a positive amniotic fluid culture. Intra-amniotic inflammation was defined as an amniotic fluid WBC count ≥100/mm3. Patients were given antibiotics and steroids after the 24th week of gestation. Antibiotic treatment consisted of ampicillin and erythromycin for 7 days for patients without evidence of intra-amniotic inflammation or MIAC, and ceftriaxone, clindamycin and erythromycin for 10–14 days for those with intra-amniotic inflammation or MIAC.
Results. Forty-six patients with preterm PROM whose first amniocentesis was performed between 18 and 32 weeks (median 27.4 weeks) were included in the study. The overall prevalence of intra-amniotic inflammation in the first amniocentesis was 39% (18/46). Seven had a positive amniotic fluid culture for microorganisms. At the time of the second amniocentesis, six of the seven patients with a positive amniotic fluid culture had microorganisms. Of 18 patients with intra-amniotic inflammation at admission, only three showed no evidence of inflammation after antibiotic treatment. Among patients with no evidence of intra-amniotic inflammation at admission, 32% (9/28) developed inflammation despite therapy. Five of these nine patients had positive amniotic fluid cultures.
Conclusions. (1) Antibiotic administration (ceftriaxone, clindamycin, and erythromycin) rarely eradicates intra-amniotic infection in patients with preterm PROM; (2) intra-amniotic inflammation developed in one-third of patients who did not have inflammation at admission, despite antibiotic administration; (3) a sub-group of patients with documented inflammation of the amniotic cavity demonstrated a decrease in the intensity of the inflammatory process after antibiotic administration.
Introduction
Preterm premature rupture of membranes (PROM) is a leading identifiable cause of preterm birth Citation[1],Citation[2], accounting for approximately 40% of all cases Citation[3-5]. Microbial invasion of the amniotic cavity (MIAC) at presentation occurs in one-third of patients presenting with preterm PROM Citation[6],Citation[7] and is a risk factor for maternal and neonatal complications, as well as long-term disabilities in children Citation[8-14].
Antibiotic administration is part of the current standard of care for patients with preterm PROM, mainly due to evidence demonstrating that antibiotics prolong pregnancy and reduce the rate of short-term neonatal morbidity (respiratory distress syndrome, infectious morbidity, and necrotizing enterocolitis) Citation[15-21]. It is not known whether this effect is attributable to the eradication of intrauterine infection, a decrease in the intensity of the inflammatory process present in patients with MIAC, or the prevention of ascending microbial invasion from the cervix and vagina.
This study was conducted to determine if antibiotic administration to the patient with preterm PROM eradicates intra-amniotic infection and/or reduces the frequency of intra-amniotic inflammation.
Materials and methods
Patient population
The study population consisted of a subset of patients with preterm PROM admitted to Sótero del Río Hospital, Puente Alto, Chile, between March 1998 and December 2002, who underwent amniocentesis for the assessment of the microbiologic status of the amniotic cavity before and after antibiotic administration. During this period, 541 patients were admitted with preterm PROM. There were 481 patients who delivered before 5 days (because they had a spontaneous or indicated delivery) and 60 patients who delivered after 5 days. Of these, 14 patients did not undergo a second amniocentesis or fluid was not obtained. Therefore, this report includes 46 patients who had the first amniocentesis on admission, remained undelivered for at least 5 days, and underwent a second amniocentesis. Premature rupture of the membranes was diagnosed by a combination of speculum examination confirming pooling of amniotic fluid in the vagina, a positive nitrazine test, and a positive ferning test Citation[22]. Digital examinations were not performed. Patients were given antibiotics and steroids after the 24th week of gestation, unless intrauterine infection/inflammation was diagnosed before 24 weeks, in which case antibiotic administration was started at the time of the diagnosis. Antibiotic treatment consisted of ampicillin and erythromycin for 7 days (IV for 48 h and oral thereafter) for patients without evidence of MIAC or intra-amniotic inflammation, and ceftriaxone, clindamycin, and erythromycin for 10–14 days (IV for 5 days and oral thereafter) for patients with MIAC or intra-amniotic inflammation. Tocolysis was not used in patient management. Patients gave written informed consent for the use of biological samples and clinical information. Amniocenteses were performed in our institution to monitor treatment, given the availability of previous data indicating that one-third of all patients with preterm PROM have MIAC and that the frequency increases over time during expectant management Citation[6]. This is particularly relevant at our institution because of the lethality rate of neonatal sepsis in preterm neonates, the concerns about the emergence of antibiotic resistance strains of microorganisms Citation[23], and the uncertainty of continuing antibiotic treatment in patients with preterm PROM who remain undelivered. The second amniocentesis was performed at the discretion of the specialist, generally after completion of antibiotic treatment or due to the emergence of clinical symptoms and signs suggestive of intrauterine infection.
Retrieval of amniotic fluid and microbiological studies
In all cases, amniotic fluid was retrieved by transabdominal amniocentesis under ultrasonographic guidance. Amniotic fluid was plated within 24 hours on blood agar, MacConkey's agar, Columbia colistin–nalidixic acid agar, and chocolate agar (36°C in 8% carbon dioxide) for aerobic culture and blood agar, BBE (Bacteroides bile esculin agar)/LKV (laked kanamycin–vancomycin agar), and Martin Lewis agar (all pre-reduced) for anaerobic culture. Anaerobic cultures were grown in an anaerobic chamber in a Forma Scientific anaerobic system (Model 1024, Division of Malinckrodt, Marietta, OH, USA). Genital mycoplasma species were cultured using a commercially available medium (Mycotrim GU, Berkeley, CA, USA). Gram stain examination was performed in all samples using commercially available reagents (crystal violet, safranin, and Gram's iodine; Difco Laboratories, Detroit, MI, USA) under standard conditions. Stained slides were examined by trained technologists, and the presence or absence of microorganisms was noted. The results of the amniotic fluid Gram stain examinations and white blood cell (WBC) counts were communicated to the clinicians.
Criteria for the diagnosis of MIAC, intra-amniotic inflammation, clinical chorioamnionitis, histologic chorioamnionitis and funisitis
Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined as an amniotic fluid WBC count ≥100/mm3. Clinical chorioamnionitis was defined following the criteria proposed by Gibbs et al. Citation[24]. The diagnosis required a temperature elevation ≥37.8°C and two or more of the following criteria: uterine tenderness, malodorous vaginal discharge, and leukocytosis. Leukocytosis was defined as a WBC count >15 000/mm3. The diagnosis of histologic chorioamnionitis was based on the demonstration of acute inflammatory cells on a chorioamniotic membrane roll and/or chorionic plate, and acute funisitis was diagnosed by the presence of neutrophils in the wall of the umbilical vessels and/or Wharton's jelly using criteria previously described Citation[25].
Statistical analysis
McNemar's test was used to compare the frequency of MIAC and intra-amniotic inflammation before and after antibiotic administration. Paired non-parametric statistics were used to compare median amniotic fluid WBC counts between the two amniocenteses. The Mann–Whitney U test was employed to compare the median of continuous variables between patients with and without intra-amniotic inflammation.
Results
Forty-six patients with singleton pregnancies and preterm PROM were included in the study. describes the characteristics of the study population. The median (range) gestational age at the first and second amniocenteses were 27 (18–32) and 29 (19–35) weeks, respectively. Patients with intra-amniotic inflammation had a lower median gestational age at admission and at delivery than those without intra-amniotic inflammation (p < 0.05 for each comparison).
Table I. Clinical characteristics of the study population, according to the inflammatory status of the amniotic cavity at admission.
The overall prevalence of intra-amniotic inflammation was 39% (18/46) before antibiotic treatment and 53% (24/45) after antibiotic administration (p > 0.05, McNemar's test). One patient did not have an amniotic fluid WBC count available at the second amniocentesis. Likewise, the prevalence of a positive amniotic fluid culture was 15% (7/46) in the first amniocentesis, a rate that increased to 28% (13/46) in the second amniocentesis, after completion of antibiotic treatment (p > 0.05, McNemar's test). describes the types of microorganisms isolated from amniotic fluid, WBC counts and glucose concentrations in amniotic fluid from both amniocenteses, as well as labor status and gestational age at delivery for patients who had a positive amniotic fluid culture for microorganisms from the first amniocentesis. The most common microorganism isolated from amniotic fluid was Ureaplasma urealyticum. All patients had spontaneous or induced deliveries before 30 weeks of gestation. The median amniotic fluid WBC count was higher after antibiotic treatment, but the difference did not reach statistical significance (Wilcoxon signed ranks test, see ).
Table II. Types of microorganisms isolated from amniotic fluid, white blood cell count and glucose concentration in amniotic fluid from both amniocenteses, as well as labor status and gestational age at delivery for patients who had positive amniotic fluid culture for microorganisms from the first amniocentesis.
Table III. Amniotic fluid parameters at the first and second amniocenteses.
Seven patients had positive amniotic fluid cultures for microorganisms. At the time of the second amniocentesis, six of these seven patients had a persistent positive amniotic fluid culture. Of 18 patients with intra-amniotic inflammation, three (17%) showed no evidence of inflammation after antibiotic treatment. Among the 28 patients without evidence of intra-amniotic inflammation at admission, 32% (n = 9) developed inflammation, despite therapy. Of these nine patients, five had a positive amniotic fluid culture. describes the changes of both the inflammatory (A) and microbiological (B) status of the amniotic cavity before and after antibiotic treatment.
Figure 1 (A) Flow chart showing amniotic fluid inflammatory findings (based on white blood cell count) before and after antibiotic treatment. One patient did not have an amniotic fluid white blood cell count available at the second amniocentesis. (B) Flow chart showing amniotic fluid microbiological findings (based on amniotic fluid culture) before and after antibiotic treatment.
In summary, intra-amniotic inflammation persisted in 83% (15/18) of patients with preterm PROM after antibiotic treatment. Moreover, inflammation developed in nine out of 28 cases (32%) where the amniotic cavity did not have evidence of inflammation at admission (). Similarly, MIAC remained in 86% (6/7) of patients with preterm PROM after antibiotic treatment, while a positive amniotic culture was found in 18% (7/39) of cases with a negative amniotic fluid culture at admission ().
Twenty-nine placentas were available for histological studies. Overall, the prevalence of histologic chorioamnionitis was 59% (17/29), while funisitis was present in 15 cases (52%). Patients with persistent intra-amniotic inflammation had histological chorioamnionitis and funisitis in 67% (8/12) and 58% (7/12) of cases, respectively. Cases without intra-amniotic inflammation or those who changed their inflammatory status in the amniotic fluid from positive to negative had a lower prevalence of histological chorioamnionitis 23% (3/13).
Discussion
The major finding of this study is that antibiotic treatment rarely eradicates MIAC in patients with preterm PROM. Indeed, 83% or more of patients with preterm PROM and either intra-amniotic inflammation or a positive amniotic fluid culture maintained the same microbiological/inflammatory status after antibiotic treatment.
The most common organism isolated from the amniotic cavity was Ureaplasma urealyticum, which has been demonstrated to be associated with a robust host response in amniotic fluid, maternal and fetal compartments in patients presenting with preterm labor or preterm PROM. The clinical significance of intra-amniotic infection with this organism has been emphasized in several studies Citation[26-29]. Compared to patients with sterile amniotic fluid, those who have a positive culture for Ureaplasma urealyticum in amniotic fluid have a higher amniotic fluid concentration of pro-inflammatory cytokines including tumor necrosis factor-alpha, interleukin (IL)-1β and IL-6, higher plasma concentrations of IL-6 in umbilical cord blood, higher prevalence of histologic chorioamnionitis, and higher risk of impending preterm delivery and adverse perinatal outcome Citation[26-29].
Two large randomized clinical trials have demonstrated that administration of erythromycin and/or amoxicillin in patients with preterm PROM is associated with a reduction in maternal infectious-related morbidity and several neonatal complications Citation[16],Citation[17]. Similar results have been reported in a recent meta-analysis Citation[30]. Our observations show that eradication of MIAC may not be the primary mechanism by which antibiotics exert their beneficial effect on pregnancy prolongation and neonatal outcome. The possibility that antimicrobial agents may prevent the amniotic cavity colonization with ascending microorganisms should be considered in light of the fact that most patients with preterm PROM have a negative amniotic fluid culture when the antibiotic therapy is instituted Citation[6]. Furthermore, antibiotic therapy could potentially prevent the development of a systemic inflammatory response in the fetus from patients with an ongoing intra-amniotic infection. The fetal inflammatory response syndrome (FIRS) is associated with impending delivery Citation[31] and neonatal complications Citation[32-34]. This interpretation is supported by the observation that a reduction of the amniotic fluid WBC count was seen in a subset of patients with intra-amniotic inflammation, suggesting that the fetal inflammatory response may be reduced in these patients after antibiotic treatment. However, the majority of patients with intra-amniotic inflammation at the first amniocentesis showed evidence of a persistent inflammatory process after antibiotic treatment. Bendon et al. reported that there were no differences in the rate of histologic chorioamnionitis in patients with preterm PROM who received antibiotics and those who did not, suggesting that antibiotic therapy had no effect in the frequency of inflammatory changes in the extra-placental membranes Citation[35]. Our results are also in agreement with experimental data reported by Gibbs et al. Citation[36]. In a pregnant rabbit model using intracervical inoculation of Escherichia coli with delayed antibiotic therapy, Gibbs and colleagues showed that the administration of therapeutic doses of antibiotics did not consistently eradicate bacteria from the fetus and the amniotic fluid Citation[36].
Several case reports of eradication of intrauterine infection have been published in the literature Citation[37-39]. However, our study shows that this phenomenon occurs in very few cases and that eradication of MIAC is rarely achieved with the combination of ceftriaxone, clindamycin, and erythromycin. Factors that may explain the inability of this antimicrobial therapy to eradicate microorganisms include: (1) the timing of the antimicrobial therapy – experimental evidence suggests that antibiotic administration within 12 hours of inoculation, but not after 18 hours, reduced the rate of preterm delivery and increased neonatal survival in a rabbit model of ascending intrauterine infection Citation[40]; (2) low bioavailability of the antimicrobial agent in the amniotic fluid – the transplacental transfer of erythromycin is only 3%, allowing a fetal serum concentration of 0.06 µg/mL that is below the minimal inhibitory concentration (MIC) estimated for Ureaplasma (0.5–4 µg/mL) and for Mycoplasma (>128 µg/mL) Citation[41],Citation[42]; and (3) the antibiotic combination may not be optimal, and other antimicrobials should be considered. For example, clarithromycin has a placental transfer of 6%, allowing for a fetal serum concentration above the MIC for Ureaplasma Citation[42].
Intrauterine infection may become a chronic process in a significant proportion of patients with preterm PROM who prolong their pregnancies, in spite of aggressive antimicrobial treatment. It is also possible that the design of our study was unable to demonstrate a higher rate of microbial eradication from the amniotic fluid. This may be due, in part, to the following limitations of our study design: (1) only a fraction of all patients with preterm PROM are represented in our study (those with a latency period of at least 5 days) and, therefore, the microbiological state of the amniotic fluid may be different than that of the general population of patients with preterm PROM; (2) the high frequency of Mycoplasma species found in our study may be a characteristic of patients who remain undelivered after several days of rupture of membranes or failure to retrieve other species. However, eradication of Ureaplasma urealyticum or a decrease in the accompanying inflammatory process was exceptional in spite of the fact that all patients received erythromycin, the antibiotic with the best performance in the ORACLE trial; (3) we studied microorganisms in the amniotic fluid but not in the choriodecidual interface, a region where they are isolated more frequently Citation[43]. It is possible that higher concentrations of antibiotics in the chorion-decidua may be associated with a rate of microbial eradication higher than that observed in the amniotic fluid, a process that could explain the beneficial effect of antimicrobial agents in patients with preterm PROM.
There are implications derived from the findings reported herein: (1) more than 50% of fetuses of patients with preterm PROM who remain in utero are exposed to a persistent and/or developing inflammatory process in the amniotic cavity; and (2) these fetuses may develop FIRS, with short and long-term adverse outcomes Citation[32-34]. Therefore, we hypothesize that although the overall effect of antibiotic administration in patients with preterm PROM is beneficial in the short term, there is a subset of fetuses who will be exposed to a chronic intrauterine inflammatory process. It is unknown whether the adverse consequences derived from the potential development of FIRS in these cases may exceed the benefits of pregnancy prolongation. Long-term studies of neonates enrolled in studies about the effect of antibiotics in preterm PROM are highly desirable.
Acknowledgements
This research was supported (in part) by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services.
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