S. Japonicum and Pregnancy Outcomes

Each participant was followed until delivery to record if the outcome of the pregnancy was a live birth. Live births were defined as the complete expulsion or extraction from its mother of a product of conception, irrespective of the duration of the pregnancy, which, after such separation, breathes or shows any other evidence of life such as heartbeat, umbilical cord pulsation, or definite movement of voluntary muscles, whether the umbilical cord had been cut or the placenta was attached.

Hemoglobin concentration in a venous blood sample collected at 14 and 32 weeks gestation was measured using a multi-analyte analyzer. Each participant's change in hemoglobin concentration between the two timepoints was determined, and the mean and standard deviation for each group calculated.

To assess total body iron, one needs to assess the storage compartment, which will contain sequestered iron, and the functional compartment, which represents bioavailable iron. Body iron status is defined by the two laboratory measurements that reflect these compartments, ferritin and serum transferrin receptor. The serum transferrin receptor:ferritin ratio has been shown in quantitative phlebotomy studies to provide an accurate assessment of total body iron over the entire range of status. At 14 and 32 weeks gestation, a blood sample was collected for assessment of total body iron by determination of the transferrin receptor:ferritin ratio. Each participant's change in ratio was calculated, and the median and interquartile range were determined for each group.

Anemia of inflammation was assessed via maternal urine hepcidin levels. In response to inflammation, elevated serum levels of hepcidin is synthesized. Hepcidin causes sequestration of iron from bio-available forms to storage forms such as ferritin and decreases intestinal absorption of iron. Hepcidin was measured in participants' urine at 32 weeks gestation.

Maternal weight gain was assessed by measuring participants' weight in kilograms. The weight increase from 14 to 32 weeks was determined for each participant, and the mean and standard deviation calculated.

Maternal fat stores were measured by thigh skinfold thickness obtained using a Holtain skinfold caliper. The thickness increase from 14 to 32 weeks was determined for each participant, and the mean and standard deviation calculated.

To assess total body iron, the serum transferrin receptor:ferritin ratio was assessed in the infant. At delivery, a heel stick blood sample and a cord blood sample were collected for assessment of total body iron by determination of the transferrin receptor:ferritin ratio.

Parasitologic response to treatment was evaluated by counting S. japonicum eggs per gram of stool at screening and again at 22 weeks gestation. Success of treatment was pre-specified as greater than 90 percent reduction in egg count from screening to 22 weeks gestation.

Participants were observed in hospital for 24 hours after dosing for serious adverse events. Serious adverse events included any untoward medical occurrence that resulted in death; was life threatening; was a persistent/significant disability/incapacity; required in-patient hospitalization or prolongation thereof (for reasons other than the 24-hour observation period); resulted in a congenital anomaly/birth defect; or may have jeopardized the participant, or required intervention to prevent one of these outcomes.

Abortion was defined by the protocol as bleeding followed by fetal loss as supported by ultrasound before 20 weeks gestation. Abortion was an important safety outcome measure due to the fact that abortion would occur closer to the time of dosing than miscarriage or stillbirth. Participants were observed in hospital for 24 hours after dosing and asked to return for any bleeding at any time.

Toxicity to maternal bone marrow, kidney, and liver was assessed by laboratory parameters collected just before and 24 hours after dosing. Specifically, blood was drawn just before the dose at 12-16 weeks gestation to determine baseline complete blood count, including white blood count (WBC), platelets, and hemoglobin. Blood was then drawn 24 hours after the second part of the split dose and before discharge from the hospital to assess any changes in these parameters. White blood count was abnormal at or above 10,800 or at or below 3500 cells/square millimeter (sq mm), platelets were abnormal at or below 140,000 cells/sq mm, and hemoglobin was abnormal at or below 10.9 grams/deciliter (g/dL).

Toxicity to maternal kidney and liver was assessed by laboratory parameters collected just before and 24 hours after dosing. Specifically, blood was drawn just before the dose at 12-16 weeks gestation to determine baseline blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin. Blood was then drawn 24 hours after the second part of the split dose and before discharge from the hospital to assess any changes in these parameters. Any values that were 1.1 times the upper limit of normal or greater for the parameter were considered abnormal.

The newborn was examined by the midwife at delivery and within 2-6 days of delivery to assess the presence of congenital anomalies and well-being. The newborn was also examined by study pediatrician at 28 days of life.

Participants were assessed for the presence of pre-eclampsia at both the 22 and 32 week visits. Pre-eclampsia was defined by the presence of proteinurea (2+ protein on urine dipstick) and a single diastolic blood pressure reading of 100 millimiters of mercury (mm Hg) or above OR more than one reading, four hours apart, of 90 mm Hg or above.

Extra-placental mechanisms mediating improved outcomes in the PZQ group were planned to be evaluated with maternal serum cytokine levels, particularly TNF-alpha, TNF-alpha receptors I and II, IL-1, and IL-6. These assays were intended to be performed only if the primary objective of the study was met; therefore, there will be no results reported for this outcome measure.

Cytokine assays were planned to be performed with culture supernatant harvested from placental explant cultures. The cytokines were to be measured with a multi-analyte analyzer. These assays were intended to be performed only if the primary objective of the study was met; therefore, there will be no results reported for this outcome measure.

A study hypothesis was that peripheral serum obtained from S. japonicum infected, treated mothers would induce a lower level of apoptosis (programmed cell death) in cultured trophoblasts as measured by cytokeratin 18 neo-epitope staining compared to peripheral serum obtained from S. japonicum infected, PZQ untreated mothers. This assay was planned to be completed only if the primary objective was met; therefore, there will be no data for this outcome measure.

Two plasma samples were collected during the overnight hospitalization from approximately 200 subjects that remained at the time of study modification to incorporate PK studies. Subjects had samples collected based on one of two sample collection strategies: 4.5 and 8 hr after the first praziquantel dose or 6 and 10 hr after the first praziquantel dose. Subjects randomized to an even study number were assigned to the 4.5 and 8 hour schedule. Subjects randomized to an odd study number were assigned to the 6 and 10 hour schedule. Samples only from subjects randomized to receive praziquantel were analyzed for praziquantel. Samples drawn from subjects randomized to the control group were not analyzed. Praziquantel concentrations (ng/ml) were assayed using high performance liquid chromatography-electrospray mass spectrometry in the University of California at San Diego Pediatric Clinical Pharmacology Laboratory.

Since pregnancy is associated with increased cytochrome P450 activity and physiologic changes in the gastrointestinal tract that tend to reduce drug absorption, praziquantel pharmacokinetics may be affected by pregnancy. Thus, the metabolite-to-parent drug ratio may serve as a differential marker to help determine if variability in drug exposure following oral administration during pregnancy is due to altered metabolism or drug absorption. Samples that were collected from subjects who were randomized to receive praziquantel were analyzed for praziquantel and 4-hydroxy praziquantel concentrations. Assays were performed using high performance liquid chromatography-electrospray mass spectrometry in the University of California at San Diego Pediatric Clinical Pharmacology Laboratory. Descriptive statistics were obtained for concentrations at each of the four sparse sampling timepoints.