Clinical implications of detection of Ureaplasma urealyticum in the amniotic cavity with the polymerase chain reaction

Clinical implications of detection of Ureaplasma urealyticum in the amniotic cavity with the polymerase chain reaction Bo Hyun Yoon, MD, PhD, Roberto Romero, MD, Miha Kim, MD, Eui-Chong Kim, MD, Teresa Kim, MS, Joong Shin Park, MD, and Jong Kwan Jun, MD Seoul, Korea OBJECTIVE: The objective of this study was to determine the frequency and clinical significance of the detection of Ureaplasma urealyticum by means of the polymerase chain reaction with specific primers in the amniotic fluid of patients with preterm premature rupture of membranes. STUDY DESIGN: Amniocentesis was performed in 154 patients with preterm premature rupture of membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas. Ureaplasma urealyticum was detected by means of the polymerase chain reaction with specific primers. Patients were divided into the following 3 groups according to the results of amniotic fluid culture and polymerase chain reaction for U urealyticum: those with a negative amniotic fluid culture and a negative polymerase chain reaction (n = 99), those with a negative amniotic fluid culture but a positive polymerase chain reaction (n = 18), and those with a positive amniotic fluid culture regardless of the results of the polymerase chain reaction (n = 37). Contingency table and survival techniques were used for analysis. RESULTS: (1) U urealyticum was detected by polymerase chain reaction in 28% (43/154) of patients and by culture in 16% (25/154). (2) Among the 43 patients with a positive polymerase chain reaction for U urealyticum, amniotic fluid culture was negative in 42% (18/43). (3) Patients with a negative amniotic fluid culture for U urealyticum but a positive polymerase chain reaction had a significantly shorter median interval from amniocentesis to delivery and a higher amniotic fluid interleukin 6 and white blood cell count than did those with a negative amniotic fluid culture and a negative polymerase chain reaction (interval to delivery; median, 53 hours; range, 0.3-335 hours; vs median, 141 hours; range, 0.1-3552 hours; P < .05; amniotic fluid white blood cell count: median, 513 cells/mm3; range, 1-2295 cells/mm3; vs median, 1 cell/mm3; range, 0-7956 cells/mm3; amniotic fluid interleukin 6: median, 16.6 ng/mL; range, 0.3-53.0 ng/mL; vs median 0.4 ng/mL; range, 0-69.8 ng/mL; P < .0001 for all). (4) Patients with a positive polymerase chain reaction for U urealyticum but a negative amniotic fluid culture had a higher rate of significant neonatal morbidity than did those with both a negative culture and a negative polymerase chain reaction (P < .05). (5) No significant differences in perinatal outcome were observed between patients with a negative culture but a positive polymerase chain reaction and those with a positive amniotic fluid culture. CONCLUSION: (1) Culture techniques for mycoplasmas missed 40% of cases of microbial invasion of the amniotic cavity with U urealyticum. (2) Patients with a positive polymerase chain reaction but a negative amniotic fluid culture are at risk for adverse outcomes. (3) The use of molecular microbiologic techniques is likely to increase the detection of infection among patients with obstetric complications. (Am J Obstet Gynecol 2000;183:1130-7.)

Key words: Amniotic fluid, chorioamnionitis, mycoplasma, polymerase chain reaction, premature rupture of membranes, prematurity, Ureaplasma urealyticum

From the Departments of Obstetrics and Gynecology and Clinical Pathology, Seoul National University College of Medicine, and the Laboratory of Fetal Medicine Research, Clinical Research Institute, Seoul National University Hospital. Supported by grant 2000-N-NL-01-C-078 from the Korea Institute of Science and Technology Evaluation and Planning (KISTEP), Republic of Korea, and by grant 04-99-034 from Seoul National University Hospital Research Fund. Presented at the Twentieth Annual Meeting of the Society for MaternalFetal Medicine, Miami Beach, Florida, January 31–February 5, 2000. Reprint requests: Miha Kim, MD, Department of Obstetrics and Gynecology, Seoul National University Hospital, Seoul, 110-744, Korea. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/6/109036 doi:10.1067/mob.2000.109036

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Intrauterine infection has been implicated in the etiology of preterm birth,1 in the genesis of the long-term sequelae of prematurity,2, 3 and recently in term birth.4 Ureaplasma urealyticum is the microorganism most frequently isolated from the amniotic fluid with standard microbiologic techniques in women with term5 and preterm6-8 labor and with premature rupture of membranes.8-10 The isolation of this microorganism, however, requires special culture techniques that are not widely available. Recently, application of the polymerase chain reaction (PCR) has emerged as a sensitive and rapid method for the detection of microorganisms in biologic fluids. This

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technique has been used for the detection of bacteria and viruses in amniotic fluid.11-16 Indeed, several investigators have reported a higher detection rate of microorganisms in amniotic fluid with PCR-based methods than with standard microbial cultures.11-15 However, the clinical significance of a positive PCR assay of amniotic fluid for microorganisms has not been determined. In some studies14, 15 patients with preterm labor and positive PCR assays for microorganisms subsequently delivered at term and without complications. These cases may represent either false-positive results or patients colonized with a small number of microorganisms who were therefore not at risk for adverse pregnancy outcomes. The objective of this study was to determine the frequency and clinical significance of a positive PCR for U urealyticum in the amniotic fluid of patients with preterm premature rupture of membranes. Material and methods Study design. The study population consisted of consecutive patients admitted to Seoul National University Hospital (Seoul, Korea) with the diagnosis of preterm premature rupture of membranes (≤35 weeks’ gestation) and singleton gestation who underwent amniocentesis for assessment of microbiologic status of the amniotic cavity between January 1994 and December 1998. At our institution amniocentesis is routinely offered to all patients admitted with the diagnosis of preterm premature rupture of membranes. Patients were divided into 3 groups according to the results of amniotic fluid culture and PCR assay for U urealyticum: group 1 (n = 99), those with a negative amniotic fluid culture and a negative PCR assay; group 2 (n = 18), those with a negative amniotic fluid culture but a positive PCR; and group 3 (n = 37), those with a positive amniotic fluid culture for microorganisms regardless of the results of PCR. Written informed consent was obtained from all subjects. The study was conducted under the approval of the institutional review board of our institution. Retrieval, culture, white blood cell count, and interleukin 6 (IL-6) determination in amniotic fluid. Amniotic fluid was collected by transabdominal amniocentesis and was cultured for aerobic and anaerobic bacteria and genital mycoplasmas according to methods described previously elsewhere.17 An aliquot of fluid was examined in a hemocytometer chamber, and the amniotic fluid white blood cell count was determined. Fluid not used for diagnostic studies was centrifuged and stored at –70°C. IL-6 concentrations were measured with a commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn). The sensitivity of the test was <1.0 pg/mL. Both interassay and intra-assay coefficients of variation were <10%. PCR assay for U urealyticum in amniotic fluid. PCR assay for U urealyticum was performed with stored amniotic

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fluid. Among the 158 patients who underwent amniocentesis during the study period, amniotic fluid was available for PCR in 154. After thawing, a 1-mL aliquot of amniotic fluid was centrifuged at 15,000g for 10 minutes at room temperature. The pellet was resuspended in 1 mL phosphate-buffered saline solution and centrifuged for 10 minutes, and the supernatant was discarded. A 20-µL volume of deionized water was then added, and the solution was boiled for 10 minutes and quickly cooled on ice. A 5-µL aliquot of the supernatant was taken for PCR assay, and known U urealyticum (ATCC 37819, serotype 7) obtained from the American Type Culture Collection (American Type Culture Collection, Manassas, Va) was used as a positive control preparation. Deoxyribonucleic acid (DNA) amplification was identified with β-globin primer. The primers chosen were in the urease gene: 5´CCAGGAAAACTAGTACCAGGAGC-3´ (14b) and 5´CTCCTAATCTAACGCTATCACC-3´ (c72b).18 PCR with the primers 14b and c72b amplified 460–base pair DNA fragments of all serotypes of U urealyticum. The DNA sequence analysis of 10 amplified PCR products by cycle sequencing with dye-labeled terminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kits; PE Biosystems, Foster City, Calif) revealed >97% homology with the urease gene from U urealyticum. PCR was performed in a total volume of 50 µL containing 0.3 pmol of each primer, 0.4-mmol/L deoxynucleotide triphosphate, 1.25 units Taq polymerase (Takara Bio Healthcare Co, Ltd, Shiga, Japan), 20-mmol/L tris(hydroxymethyl) aminomethane hydrochloride (pH 8.3), 100-mmol/L potassium chloride, 3-mmol/L magnesium chloride, and 5 µL extracted DNA template. Positive and negative control (sterile water) preparations were included in each PCR experiment. The thermal cycling (PTC-200; MJ Research, Inc, Waltham, Mass) profile consisted of 94°C for 4 minutes, 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1.5 minute, elongation at 72°C for 1 minute, and an extension step at 72°C for 7 minutes. The PCR products were separated by electrophoresis in a 1.5% agarose gel at 100 V for 30 minutes and visualized by ethidium bromide staining (Fig 1). Diagnoses of neonatal morbidity, funisitis, and chorioamnionitis. Congenital neonatal sepsis, suspected sepsis, respiratory distress syndrome, pneumonia, bronchopulmonary dysplasia, intraventricular hemorrhage, and necrotizing enterocolitis were diagnosed according to criteria previously described in detail elsewhere.2, 3, 17 Clinical chorioamnionitis was diagnosed when temperature was elevated to 37.8°C and two or more of the following criteria were present: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (>15,000 cells/ mm3), maternal tachycardia (>100 beats/min), and fetal tachycardia (>160 beats/min). Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton jelly, and histologic chorioam-

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Table I. Comparison of amniotic fluid culture and PCR Amniotic fluid culture Amniotic fluid culture for U urealyticum including U urealyticum PCR for U urealyticum

Positive

Negative

Positive

Negative

Positive (n = 43) Negative (n = 111) TOTAL (N = 154)

23 2 25

20 109 129

25 12 37

18 99 117

nionitis was considered to be present in the face of acute inflammatory changes on examination of a membrane roll and chorionic plate of the placenta, according to criteria previously published elsewhere and used in our other studies.3, 17 Statistical analysis. Proportions were compared with the Fisher exact test. The Kruskal-Wallis analysis of variance test was used for comparison of continuous variables among the groups. Multiple comparisons among groups were performed with the Mann-Whitney U test. The generalized Wilcoxon test for survival analysis was used to compare the intervals from amniocentesis to delivery. Cases of patients delivered for maternal or fetal indications were treated as censored observations, with a censoring time equal to the interval from amniocentesis to delivery. Cox proportional hazards model analysis was used to examine the relationship between the interval from amniocentesis to delivery and the results of amniotic fluid culture and PCR assay after adjustment for gestational age. Results One hundred fifty-four patients were included in this study. The prevalence of positive amniotic fluid culture results was 24% (37/154). U urealyticum was detected by PCR assay in 28% (43/154; Fig 1) and by culture in 16% (25/154). Other microorganisms isolated by culture of the amniotic fluid included Candida species (n = 4), Escherichia coli (n = 3), and 1 isolate each of coagulase-negative staphylococci, Staphylococcus epidermidis, Staphylococcus hominis, Streptococcus mitis, Streptococcus intermedius, Streptococcus anginosus, Corynebacterium species, and Peptostreptococcus species. Table I compares the culture and PCR results. Among the 43 patients with a positive PCR for U urealyticum, amniotic fluid culture was negative in 42% (18/43) and culture for U urealyticum was negative in 47% (20/43). The sensitivity of PCR to detect a positive U urealyticum culture was 92% (23/25), and that of culture to detect a positive PCR assay for U urealyticum was 53% (23/43). Table II describes the clinical characteristics and outcomes of the study population according to the results of amniotic fluid culture and PCR for U urealyticum. The difference in the median gestational age at amniocentesis among the 3 groups of patients did not reach statisti-

Fig 1. Electrophoretic analysis of PCR products for U urealyticum from amniotic fluid. Lane M, 100–base pair (bp) ladder marker; lane 1, positive control (ATCC 37819; 460 base pairs); lane 2, negative control; lanes 3 through 5, positive PCR for U urealyticum; lanes 6 through 9, negative PCR for U urealyticum.

cal significance (P = .09, Kruskal-Wallis test). However, patients with a negative amniotic fluid culture but a positive PCR (group 2) had a significantly higher rate of adverse outcome, including lower gestational age at birth, lower birth weight, and higher significant neonatal morbidity, than those with a negative amniotic fluid culture and a negative PCR (group 1). In contrast, no differences were found between patients with a negative culture but a positive PCR (group 2) and those with a positive amniotic fluid culture regardless of the results of PCR (group 3; P > .1). Fig 2 illustrates the interval from amniocentesis to delivery according to the results of amniotic fluid culture and PCR for U urealyticum. In 53 cases delivered because of maternal or fetal indications (fetal lung maturity or evidence of infection according to amniotic fluid analysis, 26; abnormal results of either nonstress testing or biophysical profile, 10; clinical chorioamnionitis, 9; preeclampsia, 2; vaginal bleeding, 2; and other indications, 4), the procedure-to-delivery interval was censored. Patients with a negative amniotic fluid culture but a positive PCR (group 2) had a significantly shorter median interval from amniocentesis to delivery than those with a negative amniotic fluid culture and negative PCR (group 1; P < .05). However, there was no significant difference in the median interval from amniocentesis to delivery between patients with a negative amniotic fluid culture but a positive PCR (group 2) and those with a positive amniotic fluid culture (group 3; P > .1). Multivariate survival analysis demonstrated that the interval from amniocentesis to delivery among patients with a negative amniotic fluid culture but a positive PCR (group 2) was significantly shorter than that with a negative culture and a negative PCR (group 1) after adjustment for gestational age at amniocentesis (hazards ratio, 4.2; 95% confidence interval, 2.28.3; P < .001; Cox proportional hazards model analysis). Fig 3 compares the amniotic fluid white blood cell counts and IL-6 concentrations of the 3 study groups.

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Table II. Characteristics of study population according to amniotic fluid culture and PCR assay for U urealyticum Negative culture Negative PCR (n = 99)

Positive PCR (n = 18)

Maternal age (y, mean ± SD) 29.5 ± 4.6 29.1 ± 4.0 Nulliparity (No.) 58 (59%) 12 (67%) Gestational age at amniocentesis (wk, median 32.6 (20.4-35.0) 30.0 (21.7-35.0) and range) Clinical chorioamnionitis (No.) 9 (9%) 2 (11%) Histologic chorioamnionitis (No.) 35/74 (47%) 12/13 (92%) Funisitis (No.) 16/74 (22%) 8/13 (62%) Gestational age at birth (wk, median and range) 34.7 (22.3-42.4) 30.6 (23.0-35.1) Birth weight (g, mean ± SD) 2289 ± 609 1620 ± 549 1-min Apgar score <7 (No.) 40 (40%) 11 (61%) 5-min Apgar score <7 (No.) 20 (20%) 7 (39%) Cord arterial pH at birth (mean ± SD) 7.24 ± 0.10 7.26 ± 0.06 Significant neonatal morbidity‡§ (No.) 21/93 (23%) 8/16 (50%) Congenital neonatal sepsis (proven)§ (No.) 1/93 (1%) 0/16 (0%) Congenital sepsis (proven or suspected)§ (No.) 9/93 (10%) 2/16 (13%) Early pneumonia§ (No.) 1/93 (1%) 3/16 (19%) Congenital infectious morbidity (proven or 9/93 (10%) 5/16 (31%) suspected sepsis or early pneumonia)§ (No.) Respiratory distress syndrome§ (No.) 4/93 (4%) 2/16 (13%) Necrotizing enterocolitis§ (No.) 1/93 (1%) 0/16 (0%) Intraventricular hemorrhage (at least grade II)§ 9/93 (10%) 3/16 (19%) (No.) Bronchopulmonary dysplasia§ (No.) 2/93 (2%) 3/16 (19%)

Statistical significance* NS NS NS NS P < .010 P < .010 P < .001 P < .001 NS P = .090 NS P < .050 NS NS P < .010 P < .050

Positive culture (n = 37) 29.9 ± 4.2 14 (38%) 31.4 (22.4-35.0)

Statistical significance† NS P = .08 NS

7 (19%) 26/27 (96%) 19/27 (70%) 31.6 (23.1-35.0) 1592 ± 553 24 (65%) 12 (32%) 7.23 ± 0.13 23/32 (72%) 0/32 (0%) 10/32 (31%) 4/32 (13%) 12/32 (38%)

NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS

5/32 (16%) 2/32 (6%) 13/32 (41%)

NS NS NS

P < .010

7/32 (22%)

NS

NS, Not significant. *Comparison between a negative culture with a negative PCR and a negative culture with a positive PCR. †Comparison between a negative culture with a positive PCR and a positive culture. ‡Significant neonatal morbidity was considered to be the presence of respiratory distress syndrome, pneumonia, congenital sepsis (proven or suspected), intraventricular hemorrhage of at least grade II, bronchopulmonary dysplasia, or necrotizing enterocolitis. §Thirteen neonates who died in utero (n = 2) or immediately after birth because of extreme prematurity (n = 7), unknown cause (n = 3), or congenital anomaly (n = 1) and thus could not be evaluated with respect to the presence or absence of complications were excluded from the analysis.

Patients with a negative amniotic fluid culture but a positive PCR (group 2) had a significantly higher median amniotic fluid white blood cell count and IL-6 concentration than those with a negative amniotic fluid culture and a negative PCR (group 1; P < .0001 for each). However, there was no significant difference in the median amniotic fluid white blood cell count and IL-6 concentration between patients with a negative amniotic fluid culture but positive PCR (group 2) and those with a positive amniotic fluid culture (group 3; P > .1 for both; Fig 3). Table III displays the clinical characteristics, laboratory results, histopathologic characteristics of the placenta, and pregnancy and neonatal outcomes of 20 patients with a positive PCR but a negative amniotic fluid culture for U urealyticum. All patients delivered before 35 weeks of gestation and had histologic or biochemical evidence of intrauterine inflammation (histologic chorioamnionitis, high amniotic fluid white blood cell count, or high amniotic fluid IL-6 concentration). Comment Our results indicate that the use of a PCR assay for U urealyticum in amniotic fluid samples results in a higher rate of detection of this microorganism than

Fig 2. Survival analysis of interval from amniocentesis to delivery according to results of amniotic fluid culture and PCR for U urealyticum. Patients with a negative amniotic fluid culture for U urealyticum but a positive PCR had a significantly shorter median interval from amniocentesis to delivery than those with a negative amniotic fluid culture and a negative PCR (median, 53 hours; range, 0.3-335 hours; vs median, 141 hours; range, 0.1-3552 hours; P < .05). Circles, Negative culture and negative PCR; squares, negative culture and positive PCR; triangles, positive culture.

that observed with standard microbiologic cultures for genital mycoplasmas. Moreover, patients with a positive PCR assay of amniotic fluid but a negative culture had

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Fig 3. Amniotic fluid white blood cell counts and IL-6 concentrations according to results of amniotic fluid culture and PCR for U urealyticum. Patients with a negative culture but a positive PCR had a significantly higher median amniotic fluid white blood cell count and IL-6 concentration than those with a negative culture and a negative PCR (white blood cell count: median, 513 cells/mm3; range, 1-2295 cells/mm3; vs median, 1 cell/mm3; range, 0-7956 cells/mm3; IL-6: median, 16.6 ng/mL; range, 0.3-53.0 ng/mL; vs median, 0.4 ng/mL; range, 0-69.8 ng/mL; P < .0001 for each). No significant differences were found between patients with a negative culture but a positive PCR and those with a positive culture (white blood cell count: median, 270 cells/mm3; range, 0-13,248 cells/mm3; vs IL-6: median, 17.3 ng/mL; range. 0.06-166.7 ng/mL; P > .1 for each).

a stronger amniotic fluid inflammatory reaction (as gauged by amniotic fluid IL-6 concentration or white blood cell count), a shorter interval to delivery, and a higher rate of histologic chorioamnionitis, funisitis, and neonatal morbid events than those with a negative amniotic fluid culture and a negative PCR assay for U urealyticum. Collectively, these data suggest that patients with a positive PCR assay for U urealyticum have a worse pregnancy outcome than those with sterile amniotic fluid. Genital mycoplasmas are the organisms most frequently isolated from the amniotic fluid in patients with preterm and term gestations, and they have been implicated in the pathogenesis of clinical chorioamnionitis, puerperal endometritis, postoperative wound infections, neonatal sepsis, and meningitis and in the genesis of bronchopulmonary dysplasia.9, 19, 20 However, their isolation in clinical specimens in obstetrics remains a challenge. Microbial culture for genital mycoplasmas, the gold standard in clinical microbiology, requires special media and handling, it is expensive, and results may take 2 to 5 days. Indeed, culture results are generally not available in time for clinical management decisions involving the mother. Genital mycoplasmas could be detected with molecular hybridization with DNA probes, a technique that is specific but insensitive. Serologic studies provide another possibility but would be impractical and expensive. Therefore the detection of these microorganisms by PCR offers considerable promise in clinical obstetrics.

Two basic strategies have been used to detect microorganisms with PCR. The first uses primer pairs designed to anneal with highly conserved DNA regions of all bacteria, such as the 16S ribosomal DNA.12-14 A positive result indicates the presence of bacteria, but identification of the specific organism requires sequencing of the PCR products. The second approach consists of using specific primers for a particular microorganism. Both techniques have been used in amniotic fluid to assess the frequency of microbial invasion. Three studies12-14 have used primers to the conserved sequence, and two11, 15 have used specific primers. In all studies the rate of positive PCR assays is higher than that of microbial cultures. This has been interpreted as evidence that microbial invasion is more commonly detected with PCR than with culture techniques. However, issues of potential false-positive and false-negative results have emerged. For example, several patients with a positive PCR assay with the 16S ribosomal DNA method or with the specific primers technique had negative culture results and normal maternal and neonatal outcomes, thus calling into question the clinical significance of a positive PCR assay for microorganisms.12, 14, 15 In addition, Hitti et al13 reported a case of a positive amniotic fluid culture for Stomatococcus species, an organism occasionally found in the mouth flora, but with a negative PCR, which indicates that falsenegative PCR results may also occur. Our study is the first to demonstrate that patients with preterm premature rupture of membranes and a positive PCR assay for U urealyticum but a negative culture had

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Table III. Patients with a positive PCR for U urealyticum but a negative culture for U urealyticum Gestational Amniotic fluid age at Gestational Interval from Birth white blood Amniotic amniocentesis age at birth amniocentesis weight Histologic cell count fluid IL-6 Case (wk) (wk) to delivery (h) (g) chorioamnionitis Funisitis (cells/mm3) (ng/mL) 1 2

21.7 23.1

23.0 23.3

204 24

600 679

Present Present

Absent Present

87 83

NA 75.8

3

24.1

24.3

35

538

NA

NA

2295

20.6

4

25.6

27.6

335

990

Present

Present

893

20.8

5 6

27.1 28.4

28.7 29.0

264 87

1170 1060

Present NA

Present NA

>1000 432

17.2 1.8

7 8 9 10

28.6 28.7 28.7 28.9

30.6 30.4 29.7 29.0

334 278 168 24

1560 1560 1640 1350

Present Present Present NA

Present Present Present NA

737 10 >1000 >1000

1.4 26.4 NA 2.6

11 12

29.7 30.3

30.6 30.3

128 5

1590 1686

Present NA

Present NA

30 >1000

7.1 9.7

13 14 15

31.0 32.7 33.4

31.4 33.7 33.9

39 132 53

1610 1890 1990

Present NA Present

Present NA Present

50 42 3

1.1 4.2 53.0

16

33.6

33.9

53

1880

Present

Absent

288

45.9

17 18 19 20

33.9 34.0 34.7 35.0

33.9 34.1 35.0 35.1

0.3 23 49 23

2330 2030 2630 2130

NA Absent Present Present

NA Absent Absent Absent

>1000 1 900 12

15.4 16.0 20.7 0.3

Remarks Neonatal death Positive amniotic fluid culture for Candida tropicalis; clinical chorioamnionitis; neonatal death Respiratory distress syndrome, bronchopulmonary dysplasia, intraventricular hemorrhage (grade II), and nosocomial sepsis; neonatal death Respiratory distress syndrome; neonatal death Positive amniotic fluid culture for S hominis; neonatal death Pneumonia Congenital suspected sepsis Pneumonia, bronchopulmonary dysplasia Clinical chorioamnionitis Pneumonia, bronchopulmonary dysplasia Clinical chorioamnionitis, suspected congenital sepsis, intraventricular hemorrhage (grade III) Intraventricular hemorrhage (grade III) Neonatal death

NA, Not available.

a worse pregnancy outcome and a higher frequency of histologic chorioamnionitis and funisitis than patients with a sterile culture and a negative PCR. Moreover, there was no demonstrable difference in pregnancy outcome between patients with a positive amniotic fluid culture for microorganisms regardless of the results of PCR and those with a positive PCR for U urealyticum but a negative culture. Therefore the use of a PCR assay for U urealyticum improved the identification of patients at risk for adverse pregnancy outcomes. It is noteworthy that all 20 patients with a positive PCR assay but a negative culture for U urealyticum had laboratory or histologic evidence of inflammation (see Table III). Indeed, all these patients had elevated amniotic fluid IL-6, elevated amniotic fluid white blood cell count, or histologic chorioamnionitis. Only one patient had no histologic evidence of chorioamnionitis or funisitis. This patient had a dramatically elevated amniotic fluid concentration of IL-6 (16 ng/mL), and labor was induced.

The neonate had no demonstrable complications. In view of the fact that all 20 of these patients had some laboratory or histologic evidence of inflammation in the amniotic or maternal compartment, we propose that they represent cases in which microbial invasion was correctly detected by the PCR assay. Several studies6, 17 have consistently demonstrated that among patients with preterm labor and preterm premature rupture of membranes there is a group with high concentrations of proinflammatory cytokines and negative amniotic fluid cultures who deliver preterm and often have histologic evidence of chorioamnionitis. These data have been interpreted as indicating the presence either of an inflammation of infectious etiology that escaped detection by standard microbiologic techniques or of an inflammatory process of noninfectious etiology. The observations in our study and those of Hitti et al13 and Markenson et al14 support the view that patients with elevated amniotic fluid cytokines have evidence of micro-

1136 Yoon et al

bial invasion that may be detected with molecular microbiologic techniques. Previous observations by Andrews et al21 suggest that this infection may be sufficiently established in the chorioamniotic space to be detected with standard microbiologic techniques if cultures are taken in this area but not by culturing amniotic fluid, although concentrations of cytokines in this compartment are increased. Two patients in our study had a positive culture for U urealyticum but a negative PCR assay result. Similar observations have been reported by other investigators13, 22 and have been attributed to degradation of bacterial DNA or inhibitor(s) of the PCR reaction in clinical samples. For example, blood is a poor biologic specimen for PCR-based methods, because hemoglobin can inhibit the PCR reaction. Blood contamination of amniotic fluid specimens is therefore a potential source of false-negative results. In 1 of our 2 cases of false-negative results, blood contamination was present. It is noteworthy that despite these potential problems the overall sensitivity of PCR for U urealyticum in the detection of microbiologically proven infection of the amniotic cavity by U urealyticum was 92% (23/25). The role of modern molecular microbiologic techniques in clinical obstetrics requires further study. PCRbased methods are at first difficult to establish and require scrupulous handling of specimens, not only in the laboratory but also in the clinical area, to avoid contamination and false-positive results. However, the technique is more sensitive, more rapid, and less expensive than standard microbial cultures. Molecular microbiology techniques may be particularly useful in the diagnosis of infections caused by fastidious microorganisms or agents that have not been discovered. For example, a PCR-based method allowed the discovery of unknown pathogens such as Tropheryma whippelii, the etiologic agent of Whipple disease, and Rochalimae henselae, the microorganism responsible for bacillary angiomatosis.23, 24 We would be surprised if all microorganisms responsible for perinatal disease have already been discovered, because conventional microbiologic techniques used thus far rely on the ability to support the growth, isolation, and specific identification of all pathogens. Recent surveys of terrestrial and aquatic ecosystems indicate that >99% of microorganisms cannot be cultured in the laboratory and can only be identified with molecular microbiologic techniques.25 It would be presumptuous to believe that we understand the growth requirements of all potential human pathogens. Therefore methods that are based on the identification of a microbial genotype are likely to yield important information about the role of microorganisms in the etiology of perinatal disease. This information may be more difficult to obtain if we rely on the incomplete knowledge of microbial phenotype that has been the basis of traditional microbiology.

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1. Gibbs RS, Romero R, Hillier SL, Eschenbach DA, Sweet RL. A review of premature birth and subclinical infection. Am J Obstet Gynecol 1992;166:1515-28. 2. Yoon BH, Romero R, Kim KS, Park JS, Ki SH, Kim BI, et al. A systemic fetal inflammatory response and the development of bronchopulmonary dysplasia. Am J Obstet Gynecol 1999;181: 773-9. 3. Yoon BH, Romero R, Park JS, Kim CJ, Kim SH, Choi JH, et al. Fetal exposure to an intra-amniotic inflammation and the development of cerebral palsy at the age of three years. Am J Obstet Gynecol 2000;182:675-81. 4. Grether JK, Nelson KB. Maternal infection and cerebral palsy in infants of normal birth weight. JAMA 1997;278:207-11. 5. Romero R, Nores J, Mazor M, Sepulveda W, Oyarzun E, Parra M, et al. Microbial invasion of the amniotic cavity during term labor: prevalence and clinical significance. J Reprod Med 1993;38:543-8. 6. Romero R, Yoon BH, Mazor M, Gomez R, Diamond MP, Kenney JS, et al. 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;169:805-16. 7. Yoon BH, Chang JW, Romero R. Isolation of Ureaplasma urealyticum from the amniotic cavity and adverse outcome in preterm labor. Obstet Gynecol 1998;92:77-82. 8. Gauthier DW, Meyer WJ, Bieniarz A. Correlation of amniotic fluid glucose concentration and intraamniotic infection in patients with preterm labor or premature rupture of membranes. Am J Obstet Gynecol 1991;165:1105-10. 9. Yoon BH, Romero R, Park JS, Chang JW, Kim YA, Kim JC, et al. Microbial invasion of the amniotic cavity with Ureaplasma urealyticum is associated with a robust host response in fetal, amniotic, and maternal compartments. Am J Obstet Gynecol 1998;179:1254-60. 10. Romero R, Mazor M, Morrotti R, Avila C, Oyarzun E, Insunza A, et al. Infection and labor. VII. Microbial invasion of the amniotic cavity in spontaneous rupture of membranes at term. Am J Obstet Gynecol 1992;166:129-33. 11. Blanchard A, Hentschel J, Duffy L, Baldus K, Cassell GH. Detection of Ureaplasma urealyticum by polymerase chain reaction in the urogenital tract of adults, in amniotic fluid, and in the respiratory tract of newborns. Clin Infect Dis 1993;17 Suppl 1: S148-53. 12. Jalava J, Mäntymaa ML, Ekblad U, Toivanen P, Skurnik M, Lassila O, et al. Bacterial 16S rDNA polymerase chain reaction in the detection of intra-amniotic infection. Br J Obstet Gynaecol 1996;103:664-9. 13. Hitti J, Riley DE, Krohn MA, Hillier SL, Agnew KJ, Krieger JN, et al. Broad-spectrum bacterial rDNA polymerase chain reaction assay for detecting amniotic fluid infection among women in premature labor. Clin Infect Dis 1997;24:1228-32. 14. Markenson GR, Martin RK, Tillotson-Criss M, Foley KS, Stewart RS Jr, Yancey M. The use of the polymerase chain reaction to detect bacteria in amniotic fluid in pregnancies complicated by preterm labor. Am J Obstet Gynecol 1997;177:1471-7. 15. Oyarzún E, Yamamoto M, Kato S, Gómez R, Lizama L, Moenne A. Specific detection of 16 micro-organisms in amniotic fluid by polymerase chain reaction and its correlation with preterm delivery occurrence. Am J Obstet Gynecol 1998;179:1115-9. 16. Wenstrom KD, Andrews WW, Bowles NE, Towbin JA, Hauth JC, Goldenberg RL. Intrauterine viral infection at the time of second trimester genetic amniocentesis. Obstet Gynecol 1998;92: 420-4. 17. Yoon BH, Romero R, Kim CJ, Jun JK, Gomez R, Choi JH, 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: 960-70. 18. Willoughby JJ, Russell WC, Thirkell D, Burdon MG. Isolation and detection of urease genes in Ureaplasma urealyticum. Infect Immun 1991;59:2463-9.

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19. Roberts S, Maccato M, Faro S, Pinell P. The microbiology of postcesarean wound morbidity. Obstet Gynecol 1993;81:383-6. 20. Cassell GH, Waites KB, Watson HL, Crouse DT, Harasawa R. Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns. Clin Microbiol Rev 1993;6:69-87. 21. Andrews WW, Hauth JC, Goldenberg RL, Gomez R, Romero R, Cassell GH. 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;173:606-12. 22. Abele-Horn M, Wolff C, Dressel P, Zimmermann A, Vahlensieck W, Pfaff F, et al. Polymerase chain reaction versus culture for de-

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tection of Ureaplasma urealyticum and Mycoplasma hominis in the urogenital tract of adults and the respiratory tract of newborns. Eur J Clin Microbiol Infect Dis 1996;15:595-8. 23. Relman DA, Schmidt TM, MacDermott RP, Falkow S. Identification of the uncultured bacillus of Whipple’s disease. N Engl J Med 1992;327:293-301. 24. Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis: an approach to the identification of uncultured pathogens. N Engl J Med 1990;323: 1573-80. 25. Relman DA. The search for unrecognized pathogens. Science 1999;284:1308-10.

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