Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix.

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

Oyster-to-oyster variability in levels of Vibrio parahaemolyticus.

This study examined the variability in the levels of total and pathogenic Vibrio parahaemolyticus in individual oysters. Twenty oysters were collected on three occasions (in June, July, and September 2001) from a site near Mobile Bay, Ala. Ten of these oysters were tested immediately, and 10 were tested after 24 h of storage at 26 degrees C. Levels of total and pathogenic V. parahaemolyticus were determined by alkaline phosphatase-labeled DNA probe procedures targeting the thermolabile hemolysin and thermostable direct hemolysin genes, respectively. Similar V. parahaemolyticus levels (200 to 2,000 CFU/g) were found in nearly 90% of the oysters (for all sampling occasions) prior to storage. The log-transformed densities (means +/- standard deviations) of V. parahaemolyticus in oysters immediately after harvest were 2.90 +/- 0.91, 2.88 +/- 0.36, and 2.47 +/- 0.26 log10 CFU/g for June, July, and September, respectively. After storage for 24 h at 26 degrees C, the mean V. parahaemolyticus densities increased approximately 13- to 26-fold. Before storage, pathogenic V. parahaemolyticus was detected in 40% (10 to 20 CFU/g) of the oysters collected in June and July but was not detected in any oysters collected in September. After storage, pathogenic V. parahaemolyticus was detected in some oysters at levels of > 100 CFU/g. These data should aid in the development of sampling protocols for oyster monitoring programs and in the determination of exposure distributions associated with raw oyster consumption.

Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks.

AIMS: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north-western United States between 1988 and 1997. METHODS AND RESULTS: All of the clinical and environmental isolates tested in this study exhibited the presence of the thermolabile haemolysin gene, tl, confirming that all of the isolates were V. parahaemolyticus. Furthermore, the V. parahaemolyticus isolates that contained either the thermostable direct haemolysin gene, tdh, or the thermostable direct haemolysin-related gene, trh, or both, were also positive for urease. Isolates from infected human patients belong to serogroups O1 and O4, whereas, the isolates from oysters belong to serogroups O1, O4 and O5. These results suggest that the presence of a V. parahaemolyticus serogroup O1 and O4 could indicate the presence of a virulent strain of this pathogen. In this study, the presence of the haemolysin genes, serogroup profiles and urease production in V. parahaemolyticus isolated from human patients correlated with the oysters collected during the outbreaks. However, no significant correlation of the plasmid profiles was detected, based on their distribution and molecular weights, between V. parahaemolyticus isolated from infected human patients and from oysters collected during this outbreak. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: It is apparent from this study that the identification of the haemolysin genes by multiplex PCR amplification, in conjunction with serogroup analysis and urease production, can be used to monitor shellfish for the presence of potentially pathogenic strains of V. parahaemolyticus.

Changes in regional cerebral blood flow after electroconvulsive therapy for depression.

Fifteen patients with major depression and normal results of magnetic resonance imaging or computed tomographic studies were treated by electroconvulsive therapy (ECT). The regional cerebral blood flow (rCBF) of these patients was imaged using Tc-99m hexamethylpropylene amineoxime single-photon emission computed tomography before and after treatment, and their images were compared with a population of 11 healthy volunteers. Before ECT treatment, the patients had hypoperfusion of the frontal region compared with the controls, and they had multiple areas of altered perfusion throughout the brain. Five of the patients had an excellent clinical response to ECT; these patients also showed changes toward normal in rCBF. The remaining patients had minimal to moderate clinical response and showed no significant change in rCBF. These results indicate that improvement in clinical status as a result of ECT is correlated with a change toward normal in rCBF.

Neonatal morbidity and mortality associated with triplet pregnancy.

OBJECTIVE: To compare neonatal morbidity and mortality in a large cohort of triplet pregnancies with singleton and twin neonates managed at a single tertiary center over a short time. METHODS: Records from all triplet pregnancies managed and delivered from 1992 to 1996 were reviewed for neonatal outcome data. Pregnancies delivered before 20 weeks' gestation and neonates with lethal congenital anomalies were excluded. The comparison group comprised all singleton and twin neonates managed in the same neonatal intensive care unit (NICU) during the same period. RESULTS: During the 5-year period, 55 triplet pregnancies and their resulting 165 neonates were managed and delivered at this center. Their outcomes were compared with those of 959 singleton and 357 twin neonates born at similar gestational ages. The median gestational age at delivery for triplets was 32.1 weeks, and 149 of the 165 infants were admitted. Sixteen triplet neonates were not admitted to our neonatal intensive care unit, 12 because of previable gestational age, three because of stillbirth, and one because of a lethal congenital anomaly. The crude perinatal mortality rate in triplets was 121 per 1000 births, and there was no significant difference in outcome based on triplet birth order. There were no significant differences in survival rates between singleton, twin, and triplet neonates, with an overall neonatal survival of 95%, 95%, and 97%, respectively. The only significant differences in morbidity were an increased incidence of mild intraventricular hemorrhage (relative risk [RR] 6.20; 95% confidence interval [CI] 2.64, 14.61), mild retinopathy of prematurity (RR 20.05; 95% CI 3.59, 111.79), and severe retinopathy of prematurity (RR 46.69; 95% CI 6.25, 348.85) in triplets compared with singletons, and severe retinopathy of prematurity (RR 6.83; 95% CI 1.24, 37.56) in triplets compared with twins. CONCLUSION: When stratified by gestational age, triplet neonates delivered at 24-34 weeks' gestation have similar outcomes as singleton and twin neonates, with the only clinically significant difference being an increased incidence of retinopathy of prematurity in triplets.

Maternal morbidity associated with triplet pregnancy.

The objective of this article is to describe maternal morbidity in a large cohort of triplet pregnancies managed by a single Maternal-Fetal Medicine group over a short period of time. Records from all triplet pregnancies managed and delivered from 1992 to 1996 were reviewed for maternal outcome data. Pregnancies delivered prior to 20 weeks were excluded. During the 4-year study period, 55 triplet pregnancies were managed and delivered at this center. The most common maternal complication was preterm labor, which occurred in 42 cases (76%). Preterm premature rupture of membranes occurred in 11 cases (20%). Pregnancy-induced hypertensive complications occurred in 15 cases (27%), which included severe preeclampsia 13 (24%), hemolysis, elevated liver function tests, and low platelets (HELLP) syndrome 5 (9%), and eclampsia 1 (2%). Other maternal antenatal complications included anemia 15 (27%), acute fatty liver of pregnancy 4 (7%), gestational diabetes 4 (7%), supraventricular tachyarrhythmias 2 (4%), dermatoses 2 (4%), urinary tract infection 2 (4%), and acute disc prolapse requiring surgery in 1 case (2%). Postnatal complications occurred in 18 cases (33%), including endometritis 13 (24%), postpartum hemorrhage 5 (9%), pneumonia 2 (4%), urinary tract infection 2 (4%), and diastasis of rectus muscles requiring surgery in 1 (2%). There were no maternal deaths. Antenatal and postnatal maternal complications occur in almost all triplet gestations, suggesting that such pregnancies be managed at centers that have appropriate multidisciplinary expertise available.

Decompression of fetal axillary lymphangioma to prevent dystocia.

BACKGROUND: Lymphangiomas are commonly diagnosed by prenatal ultrasound. These anomalies are most often cervical, but occasionally present in the axilla, thorax and abdomen. We present a case in which intrauterine drainage was followed by an uneventful vaginal delivery. CASE: A 30-year-old G4P3 female was referred to our ultrasound unit at 36 weeks of gestation for evaluation of a large cystic chest wall mass. Ultrasound demonstrated a large septated mass along the left chest wall up to the apex of the axilla of the fetus. The left arm was held in 90 degrees abduction due to the mass. Because of concern about shoulder dystocia at delivery the cystic mass was aspirated under ultrasound guidance and the patient underwent an uneventful induction of labor. CONCLUSIONS: Intrauterine decompression of large axillary lymphangiomas may obviate concern about a shoulder dystocia and avoid the need for cesarean sections.

Rhesus sensitization and alloimmune thrombocytopenia.

Significant progress has unquestionably occurred in the diagnosis and treatment of fetal and neonatal alloimmune thrombocytopenia. However, fundamental aspects of this disease are still not fully understood, especially those factors that account for the diverse clinical spectrum of this disease. This impacts our ability to counsel patients regarding the ultimate outcome of fetuses affected with AIT. IVIG appears to be a promising treatment for fetuses affected with this disease. In all likelihood, fetuses affected with AIT are at higher risk for serious bleeding complications related to fetal blood sampling procedures, but this risk remains to be further defined.

Dosimetric determination of I-131 activity in the treatment of recurrent, nontoxic, multinodular goiter.

Although surgical treatment of nontoxic multinodular goiter remains the most effective therapy, I-131 is a reasonable alternative in cases where thyroidectomy is not appropriate. Selection of I-131 activity in the management of nontoxic multinodular goiter has largely been empirical. The use of dosimetric measurements in guiding I-131 therapy in the treatment of a patient with a recurrent, nontoxic, multinodular goiter is described.

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice.

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.

Mutagenicity of ultrasound in cultured mammalian cells.

Chinese hamster V79 cells were exposed in suspension to 1 MHz continuous wave ultrasound at spatial peak intensities of 7 and 35 W/cm2. Exposure durations were from 10 to 180 sec, so as to encompass the first decade of decreased survival at each intensity. Exposure at 35 W/cm2 resulted in the induction of 6-thioguanine-resistant mutants. The mutation frequency was approximately one-tenth that induced by x rays at similar survival levels. No mutagenesis was observed at 7 W/cm2. Results are consistent with free radical formation as the mechanism responsible for ultrasound-induced mutagenesis.

Skinfold measurements in children. A comparison of Lange and McGaw calipers.

Two types of calipers to measure skinfolds have been compared in adults, but the same type of comparison has not been done in a pediatric population who generally would have smaller skinfolds. Biceps and triceps skinfold measurements were made in 240 pediatric patients ranging in age from 2 weeks to 18 years. Skinfold measurements obtained with a McGaw caliper were on the average 12.6 percent lower than those obtained with a Lange caliper on the same patient. Differences between measurements by two investigators using the same caliper on the same patient averaged 20.4 percent for the Lange caliper and 17.6 percent for the McGaw caliper (root mean square differences). We conclude that McGaw and Ross plastic calipers would be an adequate substitute where cost prohibits the use of Lange calipers.

Lack of effect of electric field exposure on rats: a data re-evaluation.

Three recent investigations of the possible effects of to Hz electric fields on rats have yielded apparently contradictory results. While one group of investigators reported that chronic exposure to a 15 kV/m field caused several biological effects, the other two groups did not find these effects at 25 kV/m and 100 kV/m, respectively. A re-examination of the data from the 15 kV/m experiments indicates that the reported effects were probably artifacts due to improper statistical analysis and to a cage design which resulted in transient electric shocks (spark discharges) to the exposed rats. Hence, the results of all three investigations are consistent with a lack of biological effects from chronic exposure to 15--100 kV/m electric fields.