Incidence of chorioamnionitis in patients with meconium-stained amniotic fluid

Objective: The goal of this study was to determine if meconium staining of the amniotic fluid (MSAF) is a marker for chorioamnionitis.Methods: In a retrospective, case-control investigation, we studied 100 patients with MSAF. Each patient was matched with a control who delivered during the same period but did not have MSAF. Subjects and controls were matched for age, parity, gestational age, mode of delivery, duration of rupture of membranes (ROM), length of internal monitoring, and number of examinations before and after ROM. The incidence of chorioamnionitis in controls and study patients was compared. The diagnosis of chorioamnionitis was based on clinical examination.Results: Thirteen of the 200 patients [6.5%, 95% confidence interval (CI), 2.5-10.5%] developed chorioamnionitis. Of the 100 women with MSAF, 10 (10%, 95% CI, 4-16) were infected compared with only 3 controls (3%, 95% CI, 0-6, P = 0.04). The odds ratio (OR) for this comparison was 3.3, and the 95% CI was 1.02-10.63.Conclusions: MSAF is associated with an increased frequency of chorioamnionitis. Several factors could explain this association. Infection may cause fetal stress, leading to the release of meconium. MSAF may enhance the growth of bacteria by providing a rich medium of essential nutrients or growth stimulants. MSAF also may impair the host immune system so that chemotaxis or phagocytosis is diminished, thus allowing accelerated growth of microorganisms.     

Evaluation of a universal real-time polymerase chain reaction for detection of amniotic fluid infection in premature rupture of membranes

Managing preterm rupture of membranes (PPROM) is a balance between benefits of prolonging gestation and the risks of perinatal infection. This study evaluates a real-time polymerase chain reaction (PCR) for the detection of amniotic fluid infection and neonatal complications by amniocentesis following PPROM. A total of 61 singleton pregnancies with PPROM were analyzed retrospectively, including histopathologic examination of the placenta and neonatal complications. The real-time PCR detects a highly conserved sequence of the bacterial 16S ribosomal DNA, and its efficacy was compared with standard tests including amniotic fluid glucose concentration, lactate dehydrogenase level, and maternal white cells count and C-reactive protein levels. Sensitivity and specificity were similar for PCR (64% and 85%) and standard tests (58% and 80%). However, the PCR technique has the additional advantage of possible identification of the bacteria through sequencing and has a much better positive predictive value in the occurrence of neonatal complications (60% for PCR versus 35% for standard tests). The latency period between premature rupture of the membranes and delivery was not related to the incidence of histopathologic signs of infection in the placenta or the umbilical cord and was inversely related to the incidence of neonatal complications.     

Prenatal diagnosis using polymerase chain reaction on amniotic fluid for congenital toxoplasmosis

OBJECTIVE: To evaluate sensitivity, specificity, and predictive values of a prenatal amniotic fluid (AF) polymerase chain reaction (PCR) test for diagnosis of congenital toxoplasmosis. METHODS: A multicenter prospective study was done on 271 women with proved primary Toxoplasma infection during pregnancy and who had amniocentesis for prenatal diagnosis by PCR. Live-born infants were eligible for analysis only if a serologic follow-up could assess a definitive infection status. RESULTS: Of the 270 evaluable cases, 75 were congenitally infected, 48 of whom had a positive PCR at prenatal diagnosis. Overall sensitivity of PCR on AF was estimated at 64% (95% confidence interval [CI] 53.1%, 74.9%), negative predictive value of 87.8% (95% CI 83.5%, 92.1%), whereas specificity and positive predictive value were 100% (95% CIs 98%, 100% and 92.3%, 100%, respectively). Among cases with congenital toxoplasmosis, there were no significant differences between those with positive or negative PCR with regard to median gestational age at maternal infection, interval between maternal infection and amniocentesis, or duration of treatment before amniocentesis. However, sensitivity of PCR was found to be significantly higher for maternal infections that occurred between 17 and 21 weeks' gestation (P <.02). CONCLUSION: A negative PCR of AF cannot rule out congenital infection. In this case, continuation of treatment with spiramycin combined with ultrasonographic follow-up and postnatal follow-up are warranted. Our results also suggest presumptive treatment combining pyrimethamine and sulfonamides in case of maternal infection occurring late in pregnancy.     

Reduced nitric oxide in amniotic fluid of patients with chorioamnionitis

OBJECTIVE: Levels of nitric oxide (NO) and cytokines were assessed in amniotic fluid obtained from patients with severe chorioamnionitis (CAM) and appropriate controls. METHODS: Amniotic fluid was obtained from 12 patients with CAM (17-24 weeks of gestation) and 89 patients undergoing diagnostic amniocentesis (16-18 weeks of gestation). The concentrations of NO, interleukin-6 (IL-6), and leukocyte elastase (LE) in amniotic fluid were then measured and compared. RESULTS: The concentrations of NO, IL-6, and LE were all higher in CAM cases than in normal pregnant women. Furthermore, an inverse correlation between NO and LE was suggested in the CAM group. CONCLUSIONS: These results indicate that in severe CAM, the action of NO might be reduced, not only due to blockage of action but also by degradation, despite increased production.     

Prediction of congenital toxoplasmosis by polymerase chain reaction analysis of amniotic fluid

OBJECTIVE: To determine the accuracy of polymerase chain reaction (PCR) analysis of amniotic fluid for fetal toxoplasmosis according to clinical predictors of outcome and study centre. DESIGN: Prospective cohort study. SETTING: Nine European centres. POPULATION: Women with suspected toxoplasma infection identified by prenatal screening. METHODS: Logistic regression was used to examine the effects of gestational age at maternal seroconversion, treatment and timing of amniocentesis, on PCR accuracy, and to calculate the post-test probability of congenital toxoplasmosis. MAIN OUTCOME MEASURES: Infants had congenital toxoplasmosis if specific IgG persisted beyond 11.5 months. Uninfected infants had undetectable IgG in the absence of anti-toxoplasma treatment. RESULTS: Of 593 PCR results, 64 were positive (57 confirmed infected), and 529 were negative (23 confirmed infected). The likelihood ratio for a positive PCR result decreased significantly with trimester at seroconversion, but did not change significantly for a negative result. Weak associations were detected between sensitivity and, inversely, with specificity, and gestational age at maternal seroconversion. There was no significant association between sensitivity and centre, type or duration of treatment, or timing of amniocentesis. Specificity differed significantly between centres (P < 0.001). The change in pre- to post-test probability of infection was maximal for a positive PCR after first trimester seroconversion, affecting 1% of women tested, and a negative PCR after third trimester seroconversion, affecting half the women tested. CONCLUSIONS: Prediction of the risk of congenital toxoplasmosis should combine estimates of test accuracy and maternal-fetal transmission, which take account of the gestational age at which the mother seroconverted. Local laboratory standards will affect the generalisability of these results.     

Leukocyte esterase activity in human amniotic fluid for the rapid detection of chorioamnionitis

Chorioamnionitis plays an important role in perinatal morbidity and mortality. Fast and accurate diagnosis poses a major problem. A prospective study was performed to assess the value of positive leukocyte esterase test (Chemstrip 9) for the diagnosis of chorioamnionitis during labor. We evaluated 21 patients with chorioamnionitis in labor at term and used 21 matched control subjects. The sensitivity and specificity of leukocyte esterase activity were compared with those of amniotic fluid cultures, Gram stains, maternal pyrexia and leukocytosis, and fetal tachycardia. The sensitivity in diagnosing chorioamnionitis was 91% and the specificity was 95%. The use of this test strip could provide a rapid, inexpensive screening test for chorioamnionitis.     

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

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/mm(3); range, 1-2295 cells/mm(3); vs. median, 1 cell/mm(3); range, 0-7956 cells/mm(3); 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.     

Rapid determination of fetal sex using amniotic fluid cells and the polymerase chain reaction

Summary We determined the sex of 50 fetuses by an amplification of the Y-chromosome specific fragment using the polymerase chain reaction (PCR). Amniotic fluid cells were collected by amniocentesis from pregnant women at 14 to 17 weeks of gestation. Total DNA was purified from cells in 1 ml of amniotic fluid. When only the expected 130 base pair X-chromosome specific fragment was detected, we identified the fetus as female, while when both the expected 170 base pair Y-chromosome specific and X-chromosome specific fragments were detected, we identified it as male. In all cases, identification was confirmed either by chromosome analysis or post partum.     

The clinical significance of detecting Ureaplasma urealyticum by the polymerase chain reaction in the amniotic fluid of patients with preterm labor

OBJECTIVE: This study was undertaken to determine the clinical significance of a detection of Ureaplasma urealyticum by using the polymerase chain reaction (PCR) in the amniotic fluid of patients with preterm labor and intact membranes. STUDY DESIGN: Amniocentesis was performed in 257 patients with preterm labor and intact membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria as well as genital mycoplasmas. U urealyticum was detected by PCR using specific primers. Patients were divided into 3 groups according to the results of amniotic fluid culture and PCR for U urealyticum: those with a negative culture and negative PCR (n=228), those with a negative culture but positive PCR (n=6), and those with a positive culture regardless of the results of PCR (n=23). RESULTS: The prevalence of positive amniotic fluid culture was 9% (23 of 257). U urealyticum was detected by PCR in 6% (15 of 254) of cases. Of the 15 cases with positive PCR for U urealyticum, amniotic fluid culture was negative in 40% (6 of 15). Patients with a negative culture but positive PCR for U urealyticum had significantly shorter median amniocentesis-to-delivery interval and higher amniotic fluid interleukin-6 and white blood cell count than those with a negative amniotic fluid culture and negative PCR (P<.01 for each). Patients with a positive PCR for U urealyticum but a negative amniotic fluid culture had a higher rate of significant neonatal morbidity than those with a negative culture and negative PCR (P<.05). However, no significant differences in perinatal outcome were observed between patients with a negative culture but positive PCR and those with a positive amniotic fluid culture. CONCLUSION: Patients with preterm labor and a positive PCR for U urealyticum but negative amniotic fluid culture are at risk for impending preterm delivery and adverse perinatal outcome.     

Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii

OBJECTIVE: Our purpose was to evaluate Toxoplasma gondii concentration in amniotic fluid (AF) samples as a prognostic marker of congenital toxoplasmosis. STUDY DESIGN: A retrospective study was carried out in 88 consecutive AF samples from 86 pregnant women, which were found positive by prospective polymerase chain reaction (PCR) testing. Parasite AF concentrations were estimated by real-time quantitative PCR and analyzed in relation to the clinical outcome of infected fetuses during pregnancy and at birth, taking into account the gestational age at maternal infection. RESULTS: A significant negative linear regression was observed between gestational age at maternal infection and T gondii DNA loads in AF. After adjusting for time at maternal seroconversion by multivariate analysis, higher parasite concentrations were significantly associated with a severe outcome of congenital infection (odds ratio [OR]=15.38/log (parasites/mL AF) [95% CI=2.45-97.7]). CONCLUSION: PCR quantification of T gondii in AF can be highly contributive for early prognosis of congenital toxoplasmosis. Maternal infections acquired before 20 weeks with a parasite load greater than 100/mL of AF have the highest risk of severe fetal outcome.     

Prevalence of Mycoplasma bacteria in amniotic fluid at the time of genetic amniocentesis using the polymerase chain reaction

OBJECTIVE: To use the polymerase chain reaction (PCR) to detect the presence of bacteria and Mycoplasma in amniotic fluid at the time of genetic amniocentesis and to assess their relationship to amniotic fluid interleukin-6 (IL-6) concentration and preterm delivery. STUDY DESIGN: A prospective study was performed on 78 asymptomatic pregnancies presenting for genetic amniocentesis. Amniotic fluid samples were analyzed by PCR for the detection of bacterial 16S ribosomal RNA, by PCR/enzyme-linked immunosorbent assay (ELISA) for Mycoplasma and by ELISA analysis for IL-6 concentration. RESULTS: All 78 samples were analyzed, and bacterial RNA was detected in 18% of them. However, no sample tested positive for Mycoplasma. The mean IL-6 concentration was 435 pg/mL. There was no difference in IL-6 levels or gestational age between bacteria-positive and -negative groups. CONCLUSION: Amniotic fluid may not be sterile in the midtrimester, yet the presence of bacteria, as detected by PCR, does not always result in an inflammatory response or adverse perinatal outcome.     

Rapid detection of group B streptococcus and Escherichia coli in amniotic fluid using real-time fluorescent PCR

OBJECTIVE: To establish reliability and validity of real-time fluorescent PCR for early detection of bacterial invasion of the amniotic cavity. METHODS: Amniotic fluid samples from 40 patients undergoing mid-trimester genetic amniocentesis were incubated for 6 h at 37 degrees C and were cultured on media specific for group B streptococcus (GBS) and E. coli. Concurrently, samples were analyzed with real-time fluorescent PCR (Roche LightCycler) using DNA primers and probes designed to detect the CAMP factor encoding cfb gene and uidA gene of GBS and E. coli, respectively. For positive control and to simulate amniotic fluid colonization, 104 cfu/ml of GBS and E. coli were inoculated on sterile amniotic fluid and incubated for 6 h. Bacterial genomic DNA for the two organisms was extracted and purified via the two-step precipitation method using a commercial kit. The real-time PCR assays were also tested against 25 non-GBS and non-E. coli bacterial species. The lower limit of detection for each pathogen was established using serial dilution of bacterial genomic DNA. RESULTS: All patient samples were negative for evidence of GBS and E. coli with both culture and real-time PCR methods. Amniotic fluid samples inoculated with GBS and E. coli were positive with real-time PCR whereas the 25 bacterial species other than GBS or E. coli tested negative with the assay. Average total sample processing time including the pre-enrichment step was 7 h 40 min. The average cost for DNA extraction and PCR testing was 8.50 dollars per test. CONCLUSION: Real-time fluorescent PCR is a valid and reliable method for detection of specific pathogens in amniotic fluid. This technique is sensitive for low inoculation levels. Real-time fluorescent PCR has potential to impact clinical management as a rapid, reliable detection method for GBS and E. coli in chorioamnionitis.     

The use of the polymerase chain reaction to detect bacteria in amniotic fluid in pregnancies complicated by preterm labor

OBJECTIVES: The purpose of this investigation was to determine the feasibility of using the polymerase chain reaction to detect bacteria in amniotic fluid and to compare pregnancy outcomes in subsets of women categorized by amniotic fluid culture, polymerase chain reaction, and interleukin-6 findings. STUDY DESIGN: Amniotic fluid from 54 pregnancies with preterm labor and no clinical evidence of intraamniotic infection was evaluated with use of the polymerase chain reaction, interleukin-6, and bacterial culture. Gestational age, newborn weight, and time between amniocentesis and delivery were compared between subsets of women categorized by these tests. RESULTS: With use of the polymerase chain reaction <100 bacteria per milliliter could be detected in amniotic fluid. A total of 55.5% of the amniotic fluid samples were polymerase chain reaction positive, whereas 9.2% of culture results were positive. Birth weights and gestational age at delivery were less and time from amniocentesis to delivery was shorter in the polymerase chain reaction–positive group (p < 0.05). Nine samples (15%) had elevated interleukin-6 concentrations; of these, six were polymerase chain reaction positive. CONCLUSIONS: The polymerase chain reaction is a sensitive means of detecting bacteria in amniotic fluid. These results provide further evidence of an association between preterm delivery and intraamniotic infection. Not all amniotic fluid samples with elevated interleukin-6 levels have bacteria detectable by the polymerase chain reaction. We anticipate that the polymerase chain reaction will provide another avenue for the detection of bacteria in amniotic fluid.(Am J Obstet Gynecol 1997;177:1471-7)     

Differential amniotic fluid cytokine profile in women with chorioamnionitis with and without funisitis

Objectives: To evaluate whether the amniotic fluid (AF) cytokine profile in women with chorioamnionitis may differentiate between those with and without funisitis.

Subjects and methods: Forty women at high risk of chorioamnionitis were studied. Gestational age at study was 26.94. Amniocentesis, universal and specific polymerase chain reaction, and microbiological cultures were performed. AF IL-1β, IL-2, IL-4, IL-6, IL 8, IL-10, IL-12, TNF-alpha, IFN-gamma, and MMP-8 were measured by multiplex assay. After delivery, the placenta and umbilical cord were studied histologically. Comparisons were made between three groups: controls, and chorioamnionitis with and without funisitis.

Results: In 25 cases, the histological findings were normal (61.5%). The remaining 15 composed of 9 cases of chorioamnionitis alone (9/40; 23.1%) and 6 cases of chorioamnionitis plus funisitis (6/40; 15.4%). All AF cytokine levels were significantly higher in the cases with chorioamnionitis in comparison to controls, except for IFN-gamma. The comparisons between the three groups showed significant differences between chorioamnionitis alone and chorioamnionitis plus funisitis in IL-1β, IL-6, IL-10, IL-12, IL-8, and TNF-alpha, with the levels being higher when funisitis was present. Logistic regression found a powerful predictive model for funisitis including the following cytokinesIL-4, IL-10, IL-12, and IL-8.

Conclusions: Measurements of AF interleukins 4, 10, 12, and 8 allow to identify cases with funisitisin women at high risk of chorioamnionitis.     

Human papillomavirus detection for cervical cancer prevention with polymerase chain reaction in self-collected samples

OBJECTIVE: We studied the usefulness of self-sampling in cervical cancer prevention. STUDY DESIGN: A cross-sectional study was undertaken at screening services in Recife (Brazil); 253 women aged 16 to 88 years were included. Participants were randomly selected from a high-risk population for cervical neoplasia. All participants collected a self-sample with a cotton-tipped swab by rotating it against the vaginal epithelium and, possibly, the cervix. Physician-collected samples from the ectocervix and endocervix, respectively, with an Ayre's spatula and a Cytobrush endocervical brush (Medscand) were followed by thorough colposcopy. Human papillomaviruses were detected by consensus polymerase chain reaction and typed by restriction fragment length polymorphism. RESULTS: The difference among human papillomavirus results in samples that were self-collected versus physician collected was significant (P <.03). The agreements were poor among patients with cervical intraepithelial neoplasia (CIN) grade 3 (kappa <0.29) and cervical cancer (kappa < 0.10). Self-sampling missed 50% more cancers than did physician sampling (P =.04). CONCLUSION: Self-sampling with a cotton-tipped swab for human papillomavirus detection is not a safe method for the collection of samples that are aimed at primary cervical cancer screening.     

Human papillomavirus DNA detection in sperm using polymerase chain reaction

OBJECTIVE: To detect human papillomavirus (HPV) in semen and find if sperm washing removes HPV DNA. METHODS: Amplification by nested polymerase chain reaction (PCR) was used to detect viral DNA sequences in semen samples from 85 volunteers. Forty-five men had historical or clinical evidence of genital HPV infection (study group) and 40 were healthy, clinically HPV-negative semen donors. RESULTS: We detected HPV DNA in the sperm cells of 24 of 45 subjects (53%) with past or current HPV infections in contrast to three of 40 healthy subjects (8%) (P <.001). Overall, PCR detected HPV in 21 of 32 subjects (66%) with identifiable lesions and six of 53 (11%) without them (P <.001). Swim-up washings of all 27 prewash sperm cells with HPV reduced cellular HPV DNA below detectable levels in only two cases. CONCLUSION: HPV is present in sperm cells from infected and apparently healthy subjects, and sperm washing does not eliminate the risk of HPV transmission to recipients. We suggest that HPV DNA testing should be done on the semen of prospective donors, and those with positive tests should be excluded from donation.     

Isolation of Kingella denitrificans from amniotic fluid in a woman with chorioamnionitis. A case report.

Kingella denitrificans and Streptococcus agalactiae were isolated from the amniotic fluid of a woman with chorioamnionitis. She was treated with intravenous ampicillin/sulbactam, with a good response.