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The traditional view is to consider the amniotic cavity sterile; therefore, isolation of any microorganisms from the amniotic fluid constitutes evidence of microbial invasion. Microorganisms may gain access to the amniotic cavity and fetus using any of the following pathways: (1) ascending from the vagina and the cervix, (2) hematogenous dissemination through the placenta (transplacental infection), (3) retrograde seeding from the peritoneal cavity through the fallopian tubes, and (4) accidental introduction at the time of invasive procedures, such as amniocente-sis, percutaneous fetal blood sampling, CVS, or shunting. The most common pathway of intrauterine infection is the ascending route.177,178, and 179
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We have proposed a 4-stage process leading to intrauterine infection (Figure 25-11 modified from Kim et al).180 The first stage consists of an overgrowth of facultative organisms or the presence of the pathologic organisms (ie, Neisseria gonorrhoeae) in the vagina, cervix, or both. Bacterial vaginosis may be an early manifestation of stage I. Then, microorganisms gain access to the intrauterine cavity, stage II. followed in the amniotic proliferation leading to microbial invasion of the amniotic cavity (stage III). Rupture of the membranes is not a prerequisite for this condition, as microorganisms are capable of crossing intact membranes. Once in the amniotic cavity, the bacteria may gain access to the fetus (stage IV) by different ports of entry. Aspiration of infected fluid by the fetus may lead to congenital pneumonia. Otitis, conjunctivitis, and omphalitis are localized infections that occur by direct spread of microorganisms from infected amniotic fluid. Seeding from any of these sites to the fetal circulation may lead to bacteremia and sepsis.
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The frequency of microbial invasion of the amniotic cavity based on conventional cultivation techniques, differs according to gestational age, presence of labor, cervical dilation, and state of the fetal membranes, and it ranges from 0.4% in patients in the mid-trimester of pregnancy to 51.5% in patients with cervical incompetence (Table 25-7).59,179,181,182,183,184,185,186,187,188,189, and 190
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Diagnosis of Microbial Invasion of the Amniotic Cavity Using Conventional Culture Techniques
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The most common microbial isolates from the amniotic cavity from women with preterm labor and intact membranes and women with preterm prelabor rupture of the membranes are U urealyticum, Fusobacterium species, and M hominis (Table 25-8).59,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196, and 197
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Microbial Invasion of the Amniotic Cavity in Preterm Labor with Intact Membranes
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The mean rate of positive amniotic fluid cultures for microorganisms in patients with preterm labor and intact membranes is approximately 12.8% (379 of 2963)198,199 based upon the review of 33 studies (Table 25-9).179,191,192,195,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227, and 228 Women with positive amniotic fluid cultures generally do not have clinical evidence of infection at presentation, but they are more likely to develop clinical chorioamnionitis (37.5% [60 of 160] vs 9% [27 of 301]), to be refractory to tocolysis (85.6% [101 fo 118] vs 16.3% [8 of 49]), and to spontaneously rupture their membranes (40% [6 of 15] vs 3.8% [2 of 52]) than women with negative amniotic fluid cultures. Several investigators have demonstrated that the rate of neonatal complications is higher in neonates born to women with microbial invasion of the amniotic cavity than in those without infection. Moreover, the earlier the gestational age at preterm birth, the more likely that microbial invasion of the amniotic cavity was pre-sent.214
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Microbial Invasion of the Amniotic Cavity in Patients with Preterm PROM
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The rate of positive amniotic fluid cultures for microorganisms is approximately 32.4% (473 of 1462)181,182,183,184,185, and 186,188,211,213,220,222,225,229,230,231,232, and 234 in patients with preterm PROM (Table 25-10).199 Clinical chorioamnionitis is present in only 29.7% (49 of 165) of the cases with proven microbial inva-sion.199,235 The rate of microbial invasion in preterm PROM reported by these studies is probably an underestimation of the true prevalence of intrauterine infection. Indeed, available evidence indicates that women with preterm PROM and severely reduced volume of amniotic fluid have a higher incidence of intra-amniotic infection than those without oligohydramnios.185,236 Since women with oligohydramnios are less likely to have an amniocentesis, the bias in these studies is to underestimate the prevalence of infection. A similar bias is that women with preterm PROM admitted in labor generally do not undergo amniocentesis. These patients have a higher rate of microbial invasion of the amniotic cavity than those admitted without labor (39% [24 of 61] vs 25% [41 of 160], P < .05). Furthermore, of patients who are not in labor on admission, 75% have a positive amniotic fluid culture at the time of the onset of labor.186 Therefore, studies restricted to women not in labor provide a lower rate of microbial invasion of the amniotic cavity than those including patients in labor.
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Microbial Invasion of the Amniotic Cavity in Patients with a Sonographic Short Cervix
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A sonographic short cervix is considered a powerful predictor of spontaneous pre-term delivery. Indeed, the shorter the sonographic cervical length in the mid-trimester of pregnancy, the higher the likelihood of spontaneous preterm delivery. Guzman et al237 studied 189 patients (125 singleton, 45 twin, and 19 triplet pregnancies) that underwent serial transvaginal cervical measurements with transfundal pressure between 15 and 24 weeks of gestation. The results showed that patients with a sonographic cervical length less than 20 mm had a significantly higher rate of acute inflammatory lesions in placental histologic examinations than those with a cervical length 20 mm or greater (48.6% vs 26.5%, P = .003). Hassan et al238 conducted a retrospective cohort study of 152 patients with a sonographic short cervix (<25 mm) at 14 to 24 weeks of gestation. Amniocenteses were performed in 57 patients and the prevalence of intra-amniotc infection was 9% (5 of 57); among these patients, the rate of preterm delivery at less than 32 weeks was 40% (2 of 5), suggesting that a short cervix may be the only manifestation of subclinical microbial invasion of the amniotic cavity.
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Microbial Invasion of the Amniotic Cavity in Patients Presenting with Acute Cervical Incompetence
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Women presenting with a dilated cervix, intact membranes, and few, if any, contractions before the 24th week are considered to have clinical cervical incompetence. In one study, 51.1% of these patients had a positive amniotic fluid culture for microorganisms.189 The outcome of patients with microbial invasion was uniformly poor because they developed subsequent complications (rupture of membranes, clinical chorioamnioni-tis, or pregnancy loss). Recently, Bujold et al239 conducted a study including 15 patients with suspected cervical insufficiency between 16 and 26 weeks of gestation. The diagnosis of intra-amniotic infection was confirmed in 47% (7 of 15), of whom 20% (3 of 15) were infected with more than one bacterial strain and 33% (5 of 15) with Ureaplasma species. This evidence suggests that infection is frequently associated with acute cervical incompetence. Whether intra-amniotic infection is the cause or consequence of cervical dilatation has not been determined. It is possible that clinical silent cervical dilatation with protrusion of the membranes into the vagina would lead to a secondary intrauterine infection.
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Microbial Invasion of the Amniotic Cavity in Patients with Twin Gestations and Preterm Labor
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The prevalence and outcome of microbial invasion of the amniotic cavity in twin gestation presenting with preterm labor and intact membranes was the subject of investigation almost 2 decades ago.187 Amniocenteses were performed on both sacs of 46 women with twin gestations, preterm labor, and intact membranes. Overall, 91.3% (42 of 46) of women delivered prematurely. A positive amniotic fluid culture of at least 1 sac was noted in 10.8% (5 of 46) of patients admitted in preterm labor and in 11.9% (5 of 42) of those who delivered preterm. Among those 5 patients with microbial invasion of the amniotic cavity, 3 had microorganisms present in the amniotic fluid isolated from both sacs. The presenting sac was involved in all cases, supporting an ascending route for microbial invasion of the amniotic cavity in twin gestation. Similar findings were observed by Mazor et al,240 who conducted a study including 74 consecutive twin pregnancies with spontaneous preterm labor with intact membranes that underwent transabdominal amniocentesis under sonographic guidance. The prevalence of preterm delivery was 91.9% (68 of 74). Amniotic fluid was retrieved from both sacs, and culture results were positive for microorganisms in 12.1% (9 of 74). Among patients with intra-amniotic infection, microorganisms were isolated from the presenting sac in 5 cases (55.6%), from both sacs in 3 patients (33.3%), and from the upper sac in the remaining case (11.1%). All patients with intra-amniotic infection delivered prematurely and had a more advanced cervical dilatation, a shorter interval from amniocentesis to de-livery, and a higher incidence of clinical chorioamnionitis than those with a negative amniotic fluid culture. These findings are in contrast to the 21.6% of abnormal amniotic fluid cultures observed in singleton gestations with pre-term labor and delivery,179 suggesting that intra-amniotic infection is a possible cause of preterm labor and delivery in twin gestation, but they do not support the hypothesis that intra-amniotic infection is responsible for the excessive rate of preterm delivery observed in these patients.
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Diagnosis of Microbial Invasion of the Amniotic Cavity Using Molecular Microbiology Techniques
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The prevalence of microbial invasion of the amniotic cavity previously described is based on the results of standard microbiologic methods. A positive culture in amniotic fluid can only be obtained if the culture conditions in the laboratory are able to support the growth of a particular microorganism. Since the growth requirements of all microorganisms are unknown, a negative culture should not be taken to definitively exclude the presence of microorganism. In other words, while a positive culture is indicative of microbial invasion of the amniotic cavity, a negative culture indicates that the laboratory was not able to grow bacteria from the specimen, either because bacteria was absent (a true negative result) or because the laboratory conditions did not support the growth of a specific microorganism (a false-negative result). Interestingly, only 1% of the whole microbial world can be detected by cultivation techniques ("the great plate count anomaly"). Thus, the frequency of microbial invasion of the amniotic cavity reported in the literature represents minimum estimates that are likely to change with the introduction of more sensitive methods for microbial recovery and identification. Indeed, several investigators have demonstrated that the prevalence of mi-crobial invasion of the amniotic cavity is higher when molecular microbiologic techniques are used to detect conserved sequences in prokaryotes (eg, bacterial 16S rDNA with PCR). Two strategies have been used to detect mi-croorganisms in the amniotic fluid using PCR. The first, also known as broad-range PCR, uses primer pairs designed to anneal with highly conserved DNA regions of all bacteria. A positive result indicates the presence of bacteria; however, identification of the specific microorganism requires subsequent sequencing of the PCR products. The second approach is to use specific primers for a particular microorganism.
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Blanchard et al241 were the first to report the recovery of U urealyticum using specific primers for the urease structural genes from 293 amniotic fluid samples collected by amniocentesis at cesarean section. The samples were cultured for bacteria, mycoplasmas, and chlamydiae, and had PCR performed for U urealyticum. Among the 10 PCR-positive amniotic fluid samples, only 4 were also culture positive. Three subsequent studies applied broad-spectrum bacterial 16S rDNA PCR for the detection of bacteria in the amniotic fluid. Jalava et al242 studied 20 amniotic fluid samples from patients with PROM and 16 control samples: PCR detected 5 microorganisms (U urealyticum [n = 2], Haemophilus influenzae [n = 1], Streptococcus oralis [n = 1], and Fusobacterium species [n=1]), whereas only 2 samples were positive in a traditional bacterial cul-ture. Two patients developed infectious complications, and both were correctly identified by PCR. Hitti et al243 studied 69 women in preterm labor with intact membranes. PCR was positive in 94% of the culture-positive amniotic fluid samples (15 of 16), and 36% (5 of 14) of patients with a negative culture had an elevated IL-6 concentration. Markenson et al218 examined amniotic fluid samples from 54 women in preterm labor without clinical evidence of intra-amniotic infection. Interestingly, 55.5% of amniotic fluid samples were PCR positive, whereas only 9.2% of the cultures recovered a microorganism (P < .05).
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Oyarzun et al224 described a PCR amplification technique aimed at detecting the 16 microor-ganisms most commonly cultured from the amniotic fluid of patients in preterm labor (U urealyticum, M hominis, Gardnerella vaginalis, E coli, Fusobacterium species, Peptostreptococcus anaerobius, Bacteroides fragilis, Chlamydia trachomatis, H influenzae, N gonorrhoeae, and Streptococcus species). Amniotic fluid samples were examined with bacterial culture and PCR in 50 patients with preterm labor and intact membranes and 23 patients not in labor. All control samples were both culture and PCR negative. A higher proportion of samples were positive by PCR compared to culture (46% [23 of 50] vs 12% [6 of 50], P < .05). However, there were patients with positive PCR who delivered at term without maternal or neonatal complications.
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The question of the clinical significance of microbial invasion of the amniotic cavity detected purely by molecular microbiology techniques, but which cannot be detected by cultivation techniques, was evaluated by Yoon et al in patients with preterm PROM234 or preterm labor and intact membranes.244 Patients with a positive PCR assay of amniotic fluid, but a negative culture, had a stronger amniotic fluid inflammatory reaction (higher amniotic fluid white blood cell count and IL-6 concentrations), a shorter interval to delivery, as well as a higher rate of histologic chorioamnionitis, funisitis, and neonatal morbid events than those with a negative amniotic fluid culture and negative amniotic fluid PCR assay for U urealyticum. These studies suggest that patients with preterm PROM or preterm labor with intact membranes and a negative amniotic fluid culture, but a positive PCR assay for U urealyticum, have worse pregnancy outcomes than those with a sterile amniotic fluid culture and negative PCR.
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DiGiulio et al used broad-range end-point and real-time PCR assays to amplify, identify, and quantify ribosomal DNA (rDNA) of bacteria, fungi, and archaea from amniotic fluid of 166 consecutive women with spontaneous preterm labor with intact membranes.246 The combined use of molecular and culture methods revealed a greater prevalence (15%) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6%; 11 taxa). A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100%. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval. This study shows that the amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery. The strength, temporality, and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.246 We also analyzed the amniotic fluid of 204 consecutive women with preterm PROM using both cultivation and molecular methods.247 The prevalence of microbial invasion of the amniotic cavity was 34% (70/204) by culture, 45% (92/204) by PCR, and 50% (101/204) by both methods combined. The number of bacterial species revealed by PCR (44 species-level phylotypes) was greater than that by culture (14 species) and included as-yet uncultivated taxa. Some taxa detected by PCR have been previously associated with the gastrointestinal tract (e.g., Coprobacillus sp.), the mouth (e.g., Rothia dentocariosa) or the vagina in the setting of bacterial vaginosis (e.g., Atopobium vaginae). Bacterial rDNA abundance exhibited a dose relationship with gestational age at delivery. A positive PCR was associated with lower mean birthweight, and with higher rates of respiratory distress syndrome and necrotizing enterocolitis. These results suggest that MIAC in preterm PROM is more common than previously recognized and is associated in some cases with uncultivated taxa, some of which are typically associated with the gastrointestinal tract.247
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Gerber et al248 identified U urealyticum using PCR in 11% (29 of 254) of amniotic fluid samples obtained between 15 and 17 weeks of gestation. Patients that were positive for U urealyti-cum had a significantly higher rate of subsequent preterm labor (58% [17 of 29] vs 4% [10 of 225]; P < .0001) and preterm birth (24% [7 of 29] vs 0.4% [1 of 225]; P < .0001) than those who had a negative result. In a smaller study249 of 78 patients undergoing genetic amniocentesis, bacterial 16S ribosomal DNA was detected in 18% (14 of 78) of the amniotic fluid samples. In this study, no samples tested positive for Mycoplasma species DNA, although it was specifically sought using a PCR/enzyme-linked immunosorbent assay. In contrast to previous and subsequent results, no association was found between the recovery of bacteria at the time of amniocentesis and either increased IL-6 concentration in the amniotic fluid or pre-term delivery. Nguyen et al250 identified M hominis in 6.4% (29 of 456) of amniotic fluid samples obtained between 15 and 17 weeks of gestation from 456 women. The rate of preterm labor in women with a positive PCR for M hominis was higher than in those with negative PCR (14% vs 3%; P = .01). Finally, Perni et al251 performed genetic amniocentesis in 179 asymptomatic women between 15 and 19 weeks of gestation. M hominis and U urealyticum were detected by PCR in 6.1% and 12.8% of cases, respectively. Preterm PROM occurred in 2.8% (5 of 179) of women, and the same proportion of patients had a spontaneous preterm birth with intact membranes. All women with preterm PROM were positive for either M hominis or U urealyticum, whereas none of those with spontaneous pre-term birth were. These observations suggest that microbial invasion of the amniotic cavity could be subclinical in the mid-trimester of pregnancy and chronic in nature, and that pregnancy loss or preterm delivery due to infection may take weeks to occur.
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Importance of the Diagnosis
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Accumulating evidence supports an association between microbial invasion of the amniotic cavity and preterm labor and delivery.190 Microbial invasion of the amniotic cavity has been reported in 21.6% of women with spontaneous preterm labor and intact membranes who subsequently delivered a preterm neo-nate.179 This condition is often subclinical in nature and requires microbiologic studies of amniotic fluid for diagnosis. The early identification of microbial invasion of the amniotic cavity is a desirable clinical goal because neonates born to mothers with intra-amniotic infection are at higher risk for both infectious and noninfectious complications (Table 25-11).179,181,182,183, and 184,195,201,210,215,229,230 More-over, patients with microbial invasion of the amniotic cavity are at risk for the development of clinical chorioamnio-nitis, failure to respond to tocolysis, subsequent rupture of membranes, and puerperal endometritis (see Table 25-11).190 Despite the importance of microbial invasion of the amniotic cavity, an early diagnosis is difficult to accomplish. Clinical signs of infection (fever, maternal and/or fetal tachycardia, uterine tenderness, foul-smelling vaginal discharge, and maternal leukocytosis) occur late and are found only in 12% of these pa-tients.179 However, amniotic fluid cultures, considered the gold standard for diagnosis, are not immediately available for clinical management, and their results may take several days. Consequently, there has been considerable interest in the development of rapid tests for the diagnosis of this condition by analysis of amniotic fluid.
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Amniotic Fluid Collection
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The method of amniotic fluid collection for microbiologic studies is critical. The 2 techniques that have been used are transabdominal amniocentesis and transcervical retrieval either by needle puncturing of the membranes or by aspiration through an intrauterine catheter. Transcervical amniotic fluid collection is associated with an unacceptable risk of contamination with vaginal flora and is contraindicated in both spontaneous preterm labor as well as nonlaboring patients with preterm PROM. Successful retrieval of amniotic fluid by ultrasonographically monitored am-niocentesis is possible in virtually all patients with preterm labor with intact membranes, and in more than 90% of patients with PROM, making it the method of choice to obtain amniotic fluid from these patients.
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Tests for Diagnosis of Microbial Invasion of the Amniotic Cavity
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In the following section, we review the clinical value of amniotic fluid Gram stain, acridine orange tests, limulus amebocyte lysate assays, white blood cell count, as well as glucose, IL-6, and matrix metalloproteinase-8 (MMP-8) concentrations in the identification of microbial invasion of the amniotic cavity.
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Amniotic Fluid Gram Stain
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The Gram stain is the most frequently used rapid diagnostic test for detecting intra-amniotic infection and has been used extensively for making clinical decisions in patients at risk for intra-amniotic infection. Some technical aspects should be taken into account to obtain optimal results using this test: (1) the slide should be prepared with fluid obtained directly from the syringe because swabs absorb both fluid and cells, decreasing the likelihood of observing organisms in a smear or culture; and (2) although centrifugation of amniotic fluid at low speed does not improve the detection of bacteria with the Gram stain,208 the use of a cytocentrifuge increases the concentration of bacteria in the sediment and probably allows an easier identification of microorganisms.
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Table 25-12 shows the diagnostic accuracy of the Gram stain as a diagnostic tool in the detection of microbial invasion of the amniotic cavity in different studies.179,181,182,183, and 184,186,188,192,195,200,201,203,206,210,211,213,215,229,230,252,253 The sensitivity increases significantly with a higher inoculum (>105 colony-forming units per milliliter).208 It is important to note that Mycoplasma species, which are frequently isolated in the amniotic fluid of patients in preterm labor with intact membranes or preterm PROM, are not visible on Gram stain examination.
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Acridine Orange Stain
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Acridine orange stain is a fluorochrome dye that, when buffered at low pH, binds to the nucleic acid of bacteria and stains them an orange color. The nucleus of human epithelial and inflammatory cells shows a green to yellow fluorescence, with no orange or green fluorescence in the cytoplasm. Therefore, bacteria should be readily identified as bright orange structures against a dark background. Several investigators have claimed that acridine orange stain is more sensitive than the Gram stain in the detection of bacteria in biologic fluids. Data generated by our group, how-ever, indicate that acridine orange stain cannot replace the Gram stain as a method to diagnose microbial invasion of the amniotic fluid, because the sensitivity, specificity, and predictive values of the test are not superior to those obtained when using Gram stain (Table 25-13).254 The potential use of acridine orange stain would be to identify mycoplasmas, but this potential advantage needs to be weighed against the risk of increasing the false-positive diagnosis.
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Limulus Amebocyte Lysate Assay
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Limulus amebocyte lysate assay is a rapid, inexpensive, and sensitive bioassay for the detection of bacterial lipopolysac-charide. It is based on the gelation of the lysate of blood cells (amebocyte) of the horseshoe crab, Limulus polyphemus, in the presence of endotoxin. Gram-negative organisms are frequently found in microbial invasion of the amniotic cavity. Their cell walls contain lipopolysaccharide, which can be detected either free after cell lysis or as part of the intact bacteria.
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Gram stain and limulus amebocyte lysate have comparable diagnostic performances for the diagnosis of microbial invasion of the amniotic cavity (sensitivity of 69%, specificity of 95%), but when used in combination, there is a remarkable improvement of the sensitivity (from 69% to 96%) with a small decrease in the specificity (from 95% to 86%).255 Although the limulus amebocyte lysate assay test would be expected to detect only gram-negative microorganisms, it also identifies 33% of pure gram-positive infections and cases of intra-amniotic infection produced by mycoplasmas or Candida. Two explanations have been proposed for these findings. The infection could have included gram-negative organisms that were not grown in culture because of their fastidious nature or because they were not visible. In the latter case, free lipopolysaccharide released at the time of cell death could lead to a positive limulus amebocyte lysate result, although the organism may not grow in culture. An alternative explanation is that cross-reacting microbial products could induce gelation of the limulus lysate.255 For example, peptidoglycans, a component of gram-positive bacteria, and mannan, a carbohydrate molecule isolated from Candida species, have been reported to produce a positive limulus amebocyte lysate test result.
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Glucose Concentration in Amniotic Fluid
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Glucose determination has been used extensively to diagnose infection in other body fluids (ie, cerebrospinal fluid, pleural fluid, and synovial fluid). It does not require sophisticated interpretation by trained personnel. Amniotic fluid glucose concentration is significantly lower in patients with microbial invasion of the amniotic cavity (identified by a positive amniotic fluid culture or clinical signs of infection) and in patients with preterm PROM who develop clinical infection.210,211,213,215,230,256 The mechanism responsible for the lower amniotic fluid glucose concentration in the setting of infection has not been established, but probably relates to glucose metabolism by both microorganisms and polymorphonuclear leukocytes.213
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Table 25-14 displays the diagnostic indices of amniotic fluid glucose concentration in the diagnosis of microbial invasion of the amniotic cavity according to various published reports. Collectively, these data indicate that the sensitivity and specificity of amniotic fluid glucose concentrations for the diagnosis of intra-amniotic infection range from 57% to 87% and from 51% to 100%, respectively, when using different cutoff values. As in the case of white blood cell count, the false-positive rates of amniotic fluid glucose concentration are high (8% to 48%) (see Table 25-14, and 25-15). The importance of a false-positive result is dependent on the action taken after an abnormal test result. If the course of action is to perform additional tests for the identification of intra-amniotic infection, then a false-positive result is not clinically problematic; however, if the course of action is to deliver a preterm neonate believed to be in-fected, then there is the potential for serious consequences.210
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White Blood Cell Count in Amniotic Fluid
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Neutrophils are infrequently found in the amniotic fluid of women who are not in labor. Their presence in the amniotic fluid indicates the existence of an inflammatory reaction, usually caused by microbial invasion of the amniotic cavity. Table 25-15 summarizes the results of 3 different studies examining the performance of amniotic fluid white blood cell count in the identification of microbial invasion of the amniotic cavity in patients with spontaneous preterm labor and intact membranes and in women with preterm PROM.212,215,230 In a study of 195 patients in preterm labor with intact membranes who underwent amniocentesis for the assessment of the microbiologic status of the amniotic cavity, a white blood cell count greater than or equal to 50 cells/mm3 had a sensitivity of 80% (20 of 25) and a specificity of 87.6% (149 of 170) in the detection of a positive amniotic fluid culture.212 Although the sensitivity was higher than the Gram stain (80% vs 48%, P < .05), the specificity was lower (87.6% vs 98.8%, P < .05); thus, the false-positive rate was high (12.4%). Interestingly, patients with an amniotic fluid white blood cell count greater than or equal to 50 cells/mm3 but a negative amniotic fluid culture are at risk for preterm delivery.212 Therefore, independently of the culture results, an elevated amniotic fluid white blood cell count identifies a subset of patients at risk for failure to respond to tocolysis and impending preterm delivery. These patients may have microbiologic invasion of the amniotic cavity that escapes detection with standard microbiologic techniques, or a noninfectious process may have driven neutrophil recruitment into the amniotic cavity. We have proposed a different cutoff value of white blood cell count for the diagnosis of microbial invasion of the amniotic cavity in patients with preterm PROM (30 cells/mm3) (Figure 25-12).230
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Amniotic Fluid Interleukin-6 Concentrations
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Microbial invasion of the amniotic cavity, elevated pro-inflammatory cytokines in the amniotic fluid and histologic chorioamnionitis are part of the "inflammatory cluster" related to patients with preterm parturition.254 Intra-amniotic infection and/or inflammation is present in approximately one-third of the patients with spontaneous preterm labor,199,258,259 is associated with development of fetal inflammatory response syn-drome,220,260 and is considered a risk factor for fetal injury.261,262,263,264,265,266,267,268,269,270,271, and 272
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Interleukin-6 has been implicated as a major mediator of the host response to infection and tissue damage. A growing body of evidence indicates that preterm parturition in the setting of infection is associated with dramatic alterations in the amniotic fluid concentration of several cytokines, including IL-6.209,274,275,276,277, and 278 This cytokine is a glycoprotein that is produced in a wide variety of cells, such as fibroblasts, monocyte/macrophages, endothelial cells, keratinocytes, and endometrial stromal cells. Interleukin-6 expression is induced by several in-flammation-associated cytokines, including IL-1, tumor necrosis factor and interferons, bacterial products, RNA- and DNA-containing viruses, and second messenger agonists (diacylglycerol, cAMP, and Ca2+) that activate any of the 3 major signal transduction pathways. Interleukin-6 elicits major changes in the biochemi-cal, physiologic, and immunologic status of the host, including the acute phase plasma protein response, activation of T and natural killer cells, and stimulation and proliferation of immunoglobulins production by B cells. Inter-leukin-6 also induces the production of C-reactive protein (CRP). This may be important in the context of intra-amniotic infection, because clinical studies have indicated that an increase in maternal serum CRP often pre-cedes the development of clinical chorioamnionitis and the onset of preterm labor in women with preterm PROM. In addition, it has been demonstrated that IL-6 stimulates prostaglandin production by human amnion and decidual cells in primary cultures.280
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Interleukin-6 has been studied as a rapid test for the detection of microbial invasion of the amniotic cav-ity.211,215,230 Patients with a positive amniotic fluid culture have significantly higher amniotic fluid IL-6 concentrations than patients with a negative culture. In patients with preterm labor and intact membranes, a cutoff level of 11.3 ng/mL has a sensitivity of 100% and a specificity of 82.6% in the detection of microbial invasion of the amniotic cavity (Table 25-16).279 Among patients with PROM, a cutoff level of 7.9 ng/mL results in a sensitivity of 80.9% and a specificity of 75% (Table 25-17).230 Moreover, an elevated amniotic fluid IL-6 is an independent predictor of the likelihood of preterm delivery and a risk factor for neonatal complications.215,230
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Another important clinical role for IL-6 determination has been proposed by our group in patients undergoing mid-trimester amniocentesis.281 Amniotic fluid IL-6 concentrations were higher in patients with a pregnancy loss than in patients with normal pregnancy outcome. An amniotic fluid IL-6 concentration greater than or equal to 2.8 ng/mL has been associated with an odds ratio of 8.1 (95% CI, 1.9 to 36.3) for pregnancy loss. We propose that a preexisting but subclinical inflammatory process is an important risk factor for pregnancy loss after mid-trimester amniocentesis.281 This finding may have relevant clinical and legal implications.
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An interesting observation is that the outcome of patients with spontaneous preterm labor with intra-amniotic inflammation and a negative amniotic fluid culture is similar to that of patients with microbiologically proven intra-amniotic infection. Yoon et al259 performed amniocentesis in 206 patients with spontaneous pre-term labor and intact membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria and mycoplas-mas, and the diagnosis of intra-amniotic inflammation was made on the basis of amniotic fluid concentrations of IL-6 greater than or equal to 2.6 ng/mL, a cutoff derived from a receiver operating characteristic (ROC) curve. Statistical analysis was conducted with contingency tables and survival techniques. Intra-amniotic inflammation (negative amniotic fluid culture but elevated amniotic IL-6) was more common than intra-amniotic infection (posi-tive amniotic fluid culture regardless of amniotic fluid IL-6 concentration; 21% [44 of 206 women] vs 10% [21 of 206 women]; P < .001]. Spontaneous preterm delivery at less than 37 weeks was more frequent in patients with intra-amniotic inflammation than in those with a negative culture and without inflammation (98% vs 35%; P < .001). Moreover, patients with intra-amniotic inflammation had a significantly higher rate of adverse outcome than patients with a negative culture and without intra-amniotic inflammation, including clinical and histologic chorioamnionitis, funisitis, early preterm birth, and significant neonatal morbidity. There were no significant differences in the rate of adverse outcomes between patients with a negative culture but with intra-amniotic inflammation and patients with intra-amniotic infection. The authors concluded that the outcome of patients with microbiologically proven intra-amniotic infection is similar to that of patients with intra-amniotic inflammation and a negative amniotic fluid culture. Similar observations have been reported in patients with preterm PROM.282
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Matrix Metalloproteinase-8 in Amniotic Fluid
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Matrix metalloproteinase-8, or human neutrophil collagenase, is a glycoprotein that is synthesized as a latent pro-enzyme during the myelocyte stage of neutrophil development and is released during inflammatory conditions. Matrix metalloproteinase-8 is contained in the secondary or specific granules of polymorphonuclear leukocytes and released after stimulation of polymorphonuclear leukocytes by proinflammatory stimuli. The active form of this enzyme has been implicated in the cleavage of collagen types I, II, and III, as well as in proteolytic damage to connective tissues in inflammatory conditions. Matrix metalloproteinase-8 plays an important role in microbial invasion of the amniotic cavity, preterm PROM, and term and preterm labor. It has been reported that spontaneous labor, both term and preterm, is associated with a significant increase in amniotic fluid concentrations of MMP-8.283 Similarly, microbial invasion of the amniotic cavity is associated with a significant increase in amniotic fluid MMP-8 concentration in patients with preterm labor and intact membranes,281 as well as in patients with preterm PROM.279,282 Indeed, among patients with spontaneous preterm labor, the median amniotic fluid concentration of MMP-8 is more than 50-fold higher in patients with intra-amniotic infection than in patients without, and is significantly higher in patients with preterm labor who delivered preterm than in those who delivered at term, even after the exclusion of patients with intra-amniotic infection.284
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Comparison between Amniotic Fluid Tests
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The diagnostic performance of amniotic fluid IL-6 determination, glucose concentration, white blood cell count, and Gram stain in patients with preterm labor and intact membranes215 and patients with PROM230 has been studied. Figure 25-13 shows the ROC curves for the performance of amniotic fluid IL-6, white blood cell count, and glucose in the detection of a positive amniotic fluid culture in patients with preterm labor and intact membranes.215 Diagnostic indices and prognostic values for each test and their potential combinations are displayed in Table 25-16. Interleukin-6 was the most sensitive test for the detection of microbial invasion of the amniotic cavity (100%), followed by glucose concentration (81.8%), white blood cell count (63.6%), and Gram stain (63.6%). The most specific test was Gram stain (99.1%), followed by white blood cell count (94.5%), IL-6 (82.6%), and glucose (81.6%).
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Figure 25-12 shows the ROC curves for amniotic fluid IL-6, white blood cell count, and glucose concentration in the detection of a positive amniotic fluid culture in patients with preterm PROM.230 Interleukin-6 was the most sensitive individual test in the detection of microbial invasion of the amniotic cavity (80.9%), followed by MMP-8 (76.1%), glucose concentration (71.4%), and Gram stain (34.8%). The most specific test for the detection of microbial invasion of the amniotic cavity was the Gram stain (96.4%), followed by white blood cell count (83.8%), IL-6 (75%), and glucose concentration (73.5%) (see Table 25-17).230 Interleukin-6 was the only proven test to have an independent relationship with the amniocentesis to delivery interval and likelihood of neonatal complications.
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Bedside Rapid Test for Diagnosis of Microbial Invasion of the Amniotic Cavity
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Significant progress has been made in the development of rapid tests for the diagnosis of microbial invasion of the amniotic cavity. Although Gram stain, white blood cell count, and glucose determination remain valuable tools for clinical decision making, and the determination of IL-6 in amniotic fluid seems to be better than any other current method for the detection of microbial invasion of the amniotic cavity, MMP-8 has demonstrated to be an excellent marker of intra-amniotic infection. A comparison between the ROC curves demonstrated that the diagnostic performance of amniotic fluid MMP-8 concentration is superior to that of IL-6 and amniotic fluid white blood cell count for the prediction of a positive amniotic fluid culture (P < .001).284
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Since the outcome of patients with microbiologically proven intra-amniotic infection is similar to that of patients with intra-amniotic inflammation and a negative amniotic fluid culture, our group has proposed that the management of patients with spontaneous preterm labor259 or preterm PROM279 should be based on the operational diagnosis of intra-amniotic inflammation rather than the diagnosis of intra-amniotic infection, because the results of the amniotic fluid culture will be available not earlier than 48 hours. Recently, a bedside test was developed to detect intra-amniotic inflammation that is based on the detection of an elevated concentration of MMP-8 in amniotic fluid.286 The configuration of the MMP-8 PTD (preterm deliv-ery) Check test (SK Pharma Co, Ltd, Kyunggi-do, Korea) is similar to a rapid pregnancy test, and some of its advantages are that it requires only 20 μL of amniotic fluid and no laboratory equipment, and that the results are available within 15 minutes. Our group conducted a prospective cohort study287 including 331 patients admitted with increased uterine contractility and intact membranes between 22 and 35 weeks of gesta-tion. The prevalence of intra-amniotic infection, intra-amniotic inflammation, and preterm delivery at less than 37 weeks of gestation were 7.3% (24 of 331), 11.5% (38 of 331), and 41.1% (136 of 331), respectively. Table 25-18 displays the diagnostic indices, predictive values, and likelihood ratios of the MMP-8 rapid test for the detection of intra-amniotic infection and inflammation. The efficiency of a positive MMP-8 rapid test result in the identification of intra-amniotic infection and intra-amniotic inflammation was 94% (311 of 331) and 97% (321 of 331), re-spectively. Table 25-19 shows the diagnostic indices, predictive values, and likelihood ratios of the MMP-8 rapid test for the identification of patients with spontaneous preterm delivery within 48 hours, 7 days, 14 days, and at less than 32 and less than 34 weeks of gestation. Moreover, patients with a positive MMP-8 rapid test result had a significantly shorter amniocentesis-to-delivery interval than patients with a negative MMP-8 rapid test result (mean, 3 days [95% CI, 1 to 4 days] vs 41 days [95% CI, 38 to 44 days]; log rank, P < .001). These results demonstrated that the MMP-8 PTD Check is a sensitive and specific test for the identification of both intra-amniotic infection and inflammation among patients with preterm labor and intact membranes. Inter-estingly, a patient with a positive MMP-8 rapid test result is at a substantial risk for spontaneous preterm delivery within 48 hours, 7 days, and 14 days, with likelihood ratios of a positive test ranging from 17 to 61.287 Further clinical trials are needed to determine whether, based on the MMP-8 rapid test results, treatment with antibiotics and/or anti-inflammatory agents may improve pregnancy outcome.
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Acknowledgments
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This work was conducted by members of the Perinatology Research Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH), and funded by the Intramural Program of NICHD/NIH.
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This chapter was published in previous editions of this book. The current version has been substantially changed. The authors wish to acknowledge the contribution of authors of previous chapters. In particular, the current authors and the editors gratefully acknowledge the intellectual contributions of Dr Eli Maymon.
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KEY POINTS
The most common indications for amniocentesis are genetic diagnosis, assessment of fetal lung maturity, and evaluation of microbial status of the amniotic cavity.
Amniocentesis performed after 15 weeks of gestation is a safe procedure, with a procedure-related pregnancy loss rate of approximately 1 in 300 to 500.
Early amniocentesis (procedure before 15 weeks of gestation) is associated with higher rates of pregnancy loss and complications than that of mid-trimester amniocentesis, which precludes its clinical use.
The use of the sonographically monitored technique reduced the rate of bloody taps, multiple needle insertions, and fetal injuries.
The frequency of microbial invasion of the amniotic cavity differs according to gestational age, presence of labor, cervical dilation, and state of the fetal membranes, and it ranges from 0.4% in patients in the mid-trimester of pregnancy to 51% in patients with cervical incompetence.