Kristin Pullen

Bacterial flora-typing with targeted, chip-based Pyrosequencing

BackgroundThe metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the < 1,600 bp 16S rRNA gene will be affected by such limitations.ResultsWe describe a method for identifying the phylogenetic content of bacterial samples using high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques.Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region.ConclusionThis study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of high-throughput Pyrosequencing on human flora samples will accelerate the study of the relationship between the microbial world and ourselves.

Magnesium sulfate compared with nifedipine for acute tocolysis of preterm labor: a randomized controlled trial.

OBJECTIVE: To compare the efficacy and side effects of intravenous magnesium to oral nifedipine for acute tocolysis of preterm labor. METHODS: A multicenter randomized trial was performed. Patients in active preterm labor who were at 24 to 33 weeks and 6 days of gestation were randomly assigned to receive magnesium sulfate or nifedipine. The primary outcome was arrest of preterm labor, defined as prevention of delivery for 48 hours with uterine quiescence. RESULTS: One hundred ninety-two patients were enrolled. More patients assigned to magnesium sulfate achieved the primary outcome (87% compared with 72%, P=.01). There were no differences in delivery within 48 hours (7.6% magnesium sulfate compared with 8.0% nifedipine, P=.92), gestational age at delivery (35.8 compared with 36.0 weeks, P=.61), birth before 37 and 32 weeks (57% compared with 57%, P=.97, and 11% compared with 8%, P=.39), and episodes of recurrent preterm labor. Mild and severe maternal adverse effects were significantly more frequent with magnesium sulfate. Birth weight, birth weight less than 2,500 g, and neonatal morbidities were similar between groups, but newborns in the magnesium sulfate group spent longer in the neonatal intensive care unit (8.8±17.7 compared with 4.2±8.2 days, P=.007). CONCLUSION: Patients who received magnesium sulfate achieved the primary outcome more frequently. However, delay of delivery, gestational age at delivery, and neonatal outcomes were similar between groups. Nifedipine was associated with fewer maternal adverse effects. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00185900 LEVEL OF EVIDENCE: I

Maintenance nifedipine tocolysis compared with placebo: a randomized controlled trial.

OBJECTIVE: To estimate whether maintenance nifedipine tocolysis after arrested preterm labor prolongs pregnancy and improves neonatal outcomes. METHODS: A prospective, randomized double-blind, multicenter study was conducted. After successful tocolysis, patients were randomly assigned to receive 20 mg nifedipine or an identical-appearing placebo every 4–6 hours until 37 weeks of gestation. The primary outcome was attainment of 37 weeks of gestation. Patients were enrolled between 24 weeks and 34 weeks if they had six or fewer contractions per hour, intact membranes, and less than 4 cm cervical dilation. Exclusion criteria were placental abruption or previa, fetal anomaly incompatible with life, or maternal medical contraindication to tocolysis. Sixty-six patients were required for 80% power to detect a 50% reduction in birth before 37 weeks, with a two-tailed alpha of 0.05. Data were analyzed by intent to treat. RESULTS: Seventy-one patients were randomly assigned. Two patients were excluded after randomization and one was lost to follow-up. Thirty-five patients received placebo, and 33 received nifedipine. There were no maternal demographic differences between groups; the placebo group was significantly more dilated and effaced at study entry. There was no difference in attainment of 37 weeks (39% nifedipine compared with 37% placebo, P>.91), mean delay of delivery (33.5±19.9 days nifedipine compared with 32.6±21.4 days placebo, P=.81) or delay of delivery for greater than 48 hours or 1, 2, 3, or 4 weeks. Neonatal outcomes were similar between groups. CONCLUSION: When compared with placebo, maintenance nifedipine tocolysis did not confer a large reduction in preterm birth or improvement in neonatal outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00185952 LEVEL OF EVIDENCE: I

Maintenance nifedipine tocolysis compared with placebo: A randomized controlled trial

After tocolysis to arrest preterm labor, the patient remains at risk for preterm delivery and maintenance tocolysis is common. Few data are available on the use of nifedipine for maintenance tocolysis and the 2 randomized studies comparing nifedipine to no treatment were unblinded and had conflicting results. This prospective, randomized double-blind trial investigated the effectiveness of maintenance nifedipine tocolysis following arrested preterm labor to prolong pregnancy and improve neonatal outcomes. Between 2001 and 2007, 68 patients in active preterm labor were enrolled who were 24 to 34 weeks of gestational age, and had been treated with intravenous magnesium sulfate or oral nifedipine to arrest preterm labor. Inclusion criteria included 6 or fewer contractions per hour, intact membranes, and cervical dilation less than 4 cm. Patients were excluded who had signs of placenta previa, placental abruption, a fetal anomaly incompatible with life, intrauterine infection, or a maternal medical contraindication to ongoing tocolysis. Of the 68 patients, 33 received 20 mg nifedipine orally every 4 to 6 hours and 35 a placebo. Treatment was continued until 37 weeks of gestation. At baseline, the 2 groups were similar with regard to prior preterm birth, gestational age, and other maternal demographic characteristics. There was, however, significantly greater cervical dilation and shorter cervical length in the placebo group. The data showed that maintenance nifedipine did not prolong gestation. No difference was found among the nifedipine and placebo groups in the percentage of patients who reached 37 weeks of gestation (39% vs. 37%, respectively, P > 0.91). Among the 2 groups, there were no differences in mean delay of delivery (nifedipine vs. placebo: 33.5 ± 19.9 days vs. 32.6 ± 21.4 days, P > 0.81), in delay of delivery for more than 48 hours or 1,2,3, or 4 weeks, in the mean gestational age at delivery, or in episodes of recurrent preterm labor. No significant differences in neonatal outcomes were found. These findings are consistent with the conclusion of the American College of Obstetricians and Gynecologists that prolonged oral or parenteral tocolytic treatment is not effective in reducing preterm birth or improving neonatal outcomes.

Daily compared with 8-hour gentamicin for the treatment of intrapartum chorioamnionitis: A randomized controlled trial

OBJECTIVE: To assess whether daily gentamicin is as effective as 8-hour gentamicin for the treatment of intrapartum chorioamnionitis. METHODS: Women with a clinical diagnosis of chorioamnionitis between 32 and 42 weeks of gestation were randomly assigned in labor to receive either daily gentamicin (5 mg/kg intravenously (IV), then 2 placebo doses IV after 8 and 16 hours) or 8-hour gentamicin (2 mg/kg IV, then 1.5 mg/kg IV after 8 and 16 hours). Both groups received ampicillin (2 grams IV every 6 hours for a total of four doses). Patients who underwent cesarean delivery also received clindamycin (900 mg IV every 8 hours, for a total of three doses). The primary outcome was treatment success, defined by resolution of chorioamnionitis after 16 hours of treatment without development of endometritis. One hundred twenty-six patients were required to have 95% confidence that daily gentamicin is at worst 15% inferior to 8-hour dosing with an alpha of .05 and a beta of 0.2. RESULTS: One hundred twenty-six women were enrolled, of whom 63 received daily gentamicin and 63 received 8-hour gentamicin. One patient was excluded from data analysis. Baseline maternal and obstetric characteristics were similar between groups except for longer mean duration of ruptured membranes in the 8-hour group (679±514 compared with 469±319 minutes; P =.03). Treatment success was equal between groups (94% daily gentamicin compared with 89% 8-hour gentamicin, P =.53). There were no differences in maternal or neonatal morbidities, including neonatal sepsis and newborn hearing screen. CONCLUSION: Daily and 8-hour gentamicin appear equally effective for the treatment of intrapartum chorioamnionitis. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00185991. LEVEL OF EVIDENCE: I