Fetal blood sampling in baboons (Papio spp.): important procedural aspects and literature review.

BACKGROUND: The baboons (Papio cynocephalus) have similarities with human placentation and fetal development. Fetal blood sampling allows investigators to assess fetal condition at a specific point in gestation as well as transplacental transfer of medications. Unfortunately, assessing fetal status during gestation has been difficult and fetal instrumentation associated with high rate of pregnancy loss. Our objectives are to describe the technique of ultrasound guided cordocentesis (UGC) in baboons, report post-procedural outcomes, and review existing publications. METHODS: This is a procedural paper describing the technique of UGC in baboons. After confirming pregnancy and gestational age via ultrasound, animals participating in approved research protocols that required fetal assessment underwent UGC. RESULTS: We successfully performed UGC in four animals (five samples) using this technique. Animals were sampled in the second and third trimesters with fetal blood sampling achieved by sampling a free cord loop, placental cord insertion site or the intrahepatic umbilical vein. All procedures were without complication and these animals delivered at term. CONCLUSIONS: Ultrasound guided fetal umbilical cord venipuncture is a useful and safe technique to sample the fetal circulation with minimal risk to the fetus or mother. We believe this technique could be used for repeated fetal venous blood sampling in the baboons.

ED(27) trophoblast-like cells isolated from first-trimester chorionic villi are genetically identical to HeLa cells yet exhibit a distinct phenotype.

ED(27) trophoblast-like cells were prepared from human chorionic villus samples obtained at 9 weeks gestation and have been grown continuously in vitro without phenotypic drift for nearly a decade. These cells express many trophoblast markers, including cytokeratin, placental alkaline phosphatase (PLAP), secretion of 17beta-estradiol, and a microvillous apical surface. The ED(27) cell line is a useful model system for studies of placental cell biology and has been distributed to laboratories world-wide. However, experiments to investigate their relationship to primary villous cytotrophoblast have shown that these cells do not secrete detectable amounts of human chorionic gonadotropin in culture and, when digested with trypsin, disperse into individual cells. Furthermore, immunocytochemical studies demonstrated that, unlike villous cytotrophoblasts, ED(27) cells were immunoreactive with monoclonal antibodies recognizing some HLA Class I antigens. This was not HLA-G, however, as would be expected if these cells originated from extravillous cytotrophoblasts, but rather classical HLA-A, B which is thought not to be expressed by any trophoblast subpopulations. These inconsistencies prompted us to question the authenticity of the continuous cell line as it now exists. Genetic haplotype analysis using the polymerase chain reaction (PCR) revealed that ED(27) was genetically identically to the HeLa cell line. Inasmuch as HeLa cells have never been grown in the laboratory (DAK), the only possible origin of HeLa cell contamination of ED(27) cells was the WISH cell line, and further PCR analysis revealed that this cell line was also genetically identical to HeLa. Like ED(27) cells, HeLa cells and WISH cells synthesized small amounts of estrogen and were found to express PLAP and antigens recognized by the monoclonal antibodies ED822, directed against the syncytiotrophoblast, and J1B5 directed against villous cytotrophoblast. These results point out the need for adherence to rigorous and consistent quality control measures to assure the authenticity of cell lines used as in vitro model systems.

Optimal dosing of penicillin G in the third trimester of pregnancy for prophylaxis against group B Streptococcus.

OBJECTIVE: We wanted to determine the optimal dose of intravenous penicillin (PCN) in the third trimester of pregnancy for the prophylaxis of group B Streptococcus. STUDY DESIGN: Healthy women in the third trimester with a singleton pregnancy were recruited. Eligibility included no previous penicillin or cephalosporin allergy and no history of renal disease. We obtained a baseline 24-hour urine collection for total protein concentration and creatinine clearance. Two intravenous catheters were placed, and 1 million units of penicillin G (PCN G) sodium was infused through one catheter. Serial blood samples were obtained through the second catheter at 1, 5, 15, 30, 60, 90, 120, 150, 180, 210, and 240 minutes. Serum was stored at -80 degrees C until assays were performed. Reverse-phase high-performance liquid chromatography was used to determine serum concentrations. RESULTS: Fifteen patients met the requirements for eligibility. The average 24-hour urine sample for total protein concentration was 187 mg/dL (range, 11-252), and creatinine clearance was 191 mL/min (range, 137-245). Average maximum serum concentration (C(max)) was 67 microg/mL (range, 34-132) and was reached within 5 minutes. Average serum PCN concentration was 12 microg/mL (range, 9-25) after 4 hours of urine collection. CONCLUSION: The C(max) was 67 microg/mL (670 x minimum inhibitory concentration). One million units of intravenous PCN G exceeds MIC in the treatment of GBS. The dosing interval should be 4 hours to ensure anti-GBS activity in all patients. More frequent dosing does not increase activity. Current recommendations for GBS prophylaxis which use PCN G should be modified pending future studies of neonatal PCN concentrations.

Cidofovir in the treatment of cytomegaloviral disease.

OBJECTIVE: To review the clinical pharmacology and microbiology of cidofovir in the therapy of cytomegalovirus (CMV) disease. DATA SOURCES: Pertinent literature was identified via a MEDLINE search (October 1986-February 1997), and data from abstracts presented at recent scientific meetings were also included; unpublished information was provided by the manufacturer. STUDY SELECTION: Antiviral activity data were included if widely accepted methodology was used. All clinical data currently available from human studies were also included. DATA SYNTHESIS: Cidofovir is similar to ganciclovir in mechanism of action; however, cidofovir does not require viral enzymes for activation. Although the half-life of cidofovir in plasma is only 2.6 hours, the intracellular half-life may be much longer, allowing efficacy with biweekly maintenance dosing. In vitro, cidofovir appears to be equally or more effective than the other agents currently available for the treatment of CMV. In vivo, cidofovir appears to be effective in delaying the progression of CMV retinitis, although no clinical trials to date have directly compared cidofovir with either ganciclovir or foscarnet. Current intravenous dose recommendations are 5 mg/kg once weekly for two doses (induction), and then 5 mg/kg once every other week (maintenance). Since cidofovir is cleared almost entirely by the kidneys, dosage adjustments must be made in patients with impaired renal function. Disadvantages of cidofovir primarily include its risks of adverse drug reactions, such as nephrotoxicity, which is likely to occur in up to 50% of patients if appropriate preventative measures are not taken. Neutropenia and constitutional reactions to probenecid are also commonly encountered during the course of cidofovir therapy. CONCLUSIONS: Cidofovir is the first acyclic phosphonate nucleoside antiviral agent to be approved for general use in the US. In addition to delaying the progression of CMV retinitis, cidofovir may provide some protective benefits to patients at risk for developing the disease and may be active against certain strains of virus resistant to other currently available therapies. Another advantage of cidofovir is its infrequent dosage schedule, which may prove beneficial in patients who are not compliant with daily intravenous dosing regimens. When determining the appropriate treatment for a patient with CMV retinitis, the benefits of using cidofovir must be weighed carefully against the risk of potentially serious adverse effects.

Amphotericin B lipid complex compared with amphotericin B in the treatment of cryptococcal meningitis in patients with AIDS.

The study objective was to obtain preliminary information regarding the safety and efficacy of amphotericin B (AmB) lipid complex (ABLC) in the treatment of AIDS-associated cryptococcal meningitis. Of 55 patients randomly assigned to 6 weeks of therapy with ABLC (1.2-5.0 mg/[kg.d], with ascending doses for three sequential cohorts) or AmB (0.7-1.2 mg/[kg.d]), 46 received > or = 12 doses. Transfusion requirements, mean decreases in hemoglobin level, and mean increases in creatinine level were significantly greater with AmB than with ABLC. The total number of adverse events, infusion-related events, and occurrences of hypomagnesemia and hypokalemia associated with each form of therapy were similar. Among 21 recipients of ABLC at a dosage of 5 mg/kg (daily for 2 weeks and then thrice weekly for 4 weeks), symptoms and signs resolved for 18 (86%). Of those receiving > or = 12 doses of ABLC, cultures converted to negative for 8 (42%), were undeterminable for 3 (16%), and remained positive for 8 (42%) despite resolution of symptoms. Although preliminary, these data suggest ABLC has significant activity in patients with AIDS-associated cryptococcal meningitis. Because this formulation has less hematologic and renal toxicity than does AmB, further evaluation of ABLC is warranted.

Oral fluconazole versus amphotericin B bladder irrigation for treatment of candidal funguria.

A randomized trial was conducted to compare amphotericin B bladder irrigation (AmBBI) with oral fluconazole in terms of efficacy and safety in the treatment of candidal funguria. Fifty-three patients with two consecutive positive funal cultures of urine were randomized to undergo AmBBI (50 mg/L over 24 hours or 50 mg/L for 7 days) or to receive fluconazole (200 mg/d for 7 days). Urinary catheters were changed upon entry into the study and following therapy. Blood and urine specimens were obtained throughout the study. Candida albicans was the species isolated most frequently from urine cultures. Eradication rates for funguria at 24 hours and 5-9 days after therapy were 82.4% and 75%, respectively, with the 7-day AmBBI regimen; and 83.3% and 76.9%, respectively, with fluconazole. There were no differences in the posttherapy eradication rates between the regimens at 24 hours (P = .597) and at 5-9 days (P = .66). Candida glabrata was the predominant organism recovered from patients in the fluconazole group 5-9 days after the completion of therapy. Adverse events were limited to bladder fullness in a patient who underwent AmBBI and hypoglycemia in a patient who received concomitant therapy with fluconazole and glyburide. AmBBI (once or for 7 days) and fluconazole appear to be equally efficacious in the treatment of candidal funguria.

Enteric eradication of vancomycin-resistant Enterococcus faecium with oral bacitracin.

The emergence of vancomycin-resistant Enterococcus faecium (VREF) has produced a therapeutic dilemma. The colonization of the intestinal tract with VREF may predispose patients to infections by this organism and may contribute to its nosocomial spread. It is reasonable to attempt to eradicate VREF from colonized patients. The optimal regimen, however, is unknown and this study was designed to evaluate the efficacy of oral regimens of vancomycin and bacitracin for the elimination of VREF from the enteric tract. Enterococcal isolates were tested for susceptibilities to vancomycin, bacitracin, and ampicillin with median minimum inhibitory concentrations of > 512 micrograms/ml, 10 units/ml, and 128 micrograms/ml, respectively. All patients were given an initial trial of oral vancomycin 125 mg every 6 h for 10 days. Those who failed oral vancomycin were then given oral bacitracin 25,000 units every 6 h for 10 days due to its favorable in vitro activity. VREF was eradicated from the stools of 42% of patients (eight of 19) receiving oral vancomycin as compared with all eight patients receiving oral bacitracin (P < 0.01). The organism recurred in two bacitracin patients (25%) 8 and 20 days after completion of therapy. Whether prior vancomycin therapy predisposed patients to colonization by VREF was also examined. Ten (53%) of 19 patients had received prior vancomycin therapy before isolation of VREF from the stool. Our data suggest that oral bacitracin may be an effective alternative to commercially available oral vancomycin for the eradication of VREF from the enteric tract.

Effect of morphine or phenobarbital on teicoplanin elimination pharmacokinetics.

Elimination kinetics following a single dose of teicoplanin in rats pre-treated with morphine sulphate (MS), phenobarbital sodium (Pb), and normal saline (NS) were determined. A microbioassay was used to measure teicoplanin levels. A significant increase in the total clearance of teicoplanin was found in rats pre-treated with MS as compared to controls (P < 0.048). Wide variability was observed in the renal and non-renal clearances of teicoplanin. The mean renal clearance for rats pre-treated with MS, Pb and NS was 0.61 +/- 0.07.mL/min/kg, 0.60 +/- 0.13 mL/min/kg, and 0.46 +/- 0.02 mL/min/kg, respectively; the mean non-renal clearance was 0.33 +/- 0.18 mL/min/kg, 0.17 +/- 0.15 mL/min/kg, and 0.08 +/- 0.03 mL/min/kg, respectively. The differences among the groups for renal and non-renal clearance were not statistically significant. The mean apparent volume of distribution of teicoplanin at steady state was significantly lower in the Pb-pre-treated rats as compared to controls (P < 0.043). The mean half-life for MS-, Pb-, and NS pre-treated groups was 8.1 +/- 3.1 h, 5.9 +/- 3.3 h, and 34.6 +/- 20.7 h, respectively. The differences in mean half-life among the groups achieved statistical significance (P < 0.016). The increase in the total clearance of teicoplanin can best be explained by an increase in both renal elimination and hepatic metabolic pathways.

Effect of antibiotics on endotoxin release from gram-negative bacteria.

Antibiotics may inhibit bacterial growth or may kill bacteria by inhibiting cell wall synthesis or protein synthesis. The amount of endotoxin released during antibiotic action has been found to be clinically important. Nine antibiotics, representing seven classes, were studied for the amounts of endotoxin released during their action on susceptible strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. Staphylococcus aureus, which produces no endotoxin, was used as a control organism. Aztreonam induced the highest release of endotoxin, whereas other antibiotics such as imipenem and the quinolones induced the lowest release of endotoxin. Although the quantities of endotoxin released are not easily explained from the established mechanisms of antibiotic action, our findings may have implications for therapy of the acutely ill, septic patient in whom release of large quantities of endotoxin may be catastrophic.

Development of resistance in candida isolates from patients receiving prolonged antifungal therapy.

The impact of prolonged antifungal therapy on the development of resistance was examined in 61 patients with oropharyngeal thrush. Fifty-nine patients had symptomatic human immunodeficiency virus infection, one had lung cancer, and one had metastatic prostate cancer. Cultures of pharyngeal samples from all patients were positive for yeasts and included 57 (93.4%) Candida albicans, 3 (4.9%) Candida glabrata, and 1 (1.6%) Candida krusii. Of 61 patients, 32 (52.5%) were receiving or had recently received antifungal therapy. Clotrimazole was the most commonly prescribed azole, followed by ketoconazole and fluconazole. Two patients had received amphotericin B therapy and one had received flucytosine. The duration of therapy with clotrimazole, ketoconazole, and fluconazole ranged from 3 to 240, 14 to 44, and 7 to 138 days, respectively. There was no overall difference in the susceptibilities of the clinical isolates from treated and untreated patients to amphotericin B, nystatin, flucytosine, clotrimazole, ketoconazole, and fluconazole. A.C. albicans isolate from one patient who had clinically failed on ketoconazole, fluconazole, and amphotericin B was resistant to these drugs. The lack of difference in the susceptibility pattern indicates that clinically significant emergence of resistance does not occur in those patients who receive prolonged antifungal therapy.

Epidemiology of ciprofloxacin resistance among patients with methicillin-resistant Staphylococcus aureus.

During the first twelve months after ciprofloxacin was introduced for clinical use at our institution, 65 new patients were found to be either infected or colonized by methicillin-resistant Staphylococcus aureus (MRSA) which were also ciprofloxacin resistant (CR-MRSA). Only 18 of these patients (28%) had been previously exposed to this antibiotic. Nine (50%) of the 18 patients had received ciprofloxacin for treatment for a pathogen other than MRSA. Although the initial cases of colonization or infection with CR-MRSA can be directly related to ciprofloxacin use, many of the subsequent cases of colonization and infection were not the consequence of ciprofloxacin therapy but rather hospital transmission of existing CR-MRSA.

Imipenem resistance in a case of AIDS with relapsing Pseudomonas meningitis.

We describe an AIDS patient who had a recurrence of Pseudomonas meningitis to illustrate three points. First, the use of sulfamethoxazole-trimethoprim in AIDS patients for prophylaxis of Pneumocystis carinii pneumonia may cause the various body sites to be colonized with resistant species such as P aeruginosa. Second, Pseudomonas meningitis can recur in a patient with AIDS after a month of appropriate therapy. Finally, imipenem is a poor choice for Pseudomonas meningitis, even when alternative therapies appear much less attractive. High doses of imipenem should not be used for fear of seizures, and lower doses only produce resistant organisms in the CSF.

Pharmacokinetics, cerebrospinal fluid concentration, and safety of intravenous rifampin in pediatric patients undergoing shunt placements.

The objectives of this study were to characterize the pharmacokinetics and determine the cerebrospinal fluid concentrations and safety of intravenous rifampin in pediatric patients undergoing shunt placement. Nine patients (mean age 5.6 y) received a single dose of rifampin, 20 mg.kg-1, administered intravenously 1 h prior to surgery. The peak serum concentrations ranged from 13.5-26.7 micrograms.ml-1; cerebrospinal fluid concentrations ranged from 0.12-3.0 (mean: 1.4) micrograms.ml-1. The mean total clearance, apparent distribution volume, and elimination half-life were 0.29 l.kg-1.h-1, 1.11.kg-1, and 2.8 h. The concentrations of rifampin achieved in the cerebrospinal fluid exceeded the minimum inhibitory concentrations by 100- to 1000-fold against Staphylococcus epidermidis. However, 5 of 9 patients developed cutaneous reactions during intravenous rifampin prophylactic therapy. Because of the high frequency of adverse effects and more than adequate rifampin concentrations achieved in the cerebrospinal fluid, rifampin doses lower than that used in this study may be evaluated in future studies.

Pharmacokinetics and cerebrospinal fluid (CSF) concentrations of vancomycin in pediatric patients undergoing CSF shunt placement.

Staphylococcus epidermidis has been established as the common pathogen causing cerebrospinal fluid shunt infections. In addition, clinical isolates of S. epidermidis from infected shunts are typically resistant to methicillin. Vancomycin is often used for neurosurgical prophylaxis due to its excellent in vitro activity against methicillin-resistant staphylococci. Limited data are available about the pharmacokinetics and cerebrospinal fluid concentrations of vancomycin in pediatric patients intraoperatively. The objectives of this study were to characterize the pharmacokinetics and determine the cerebrospinal fluid concentrations of vancomycin. Eight patients (mean age 8.3 +/- 7.0 years) received three doses of intravenous vancomycin, 15 mg/kg every 6 h. The first dose was administered 1 h prior to surgery. Blood samples were collected at 0, 0.5, 1, 2, 4, and 5 h after the end of the infusion. A cerebrospinal fluid sample was collected at the time of shunt insertion. Urine samples were collected over a 24-hour period. Vancomycin was measured with a fluorescence polarization immunoassay. The peak serum concentrations ranged from 15.6 to 33.7 micrograms/ml; cerebrospinal fluid concentrations ranged from less than 0.6 to 0.8 microgram/ml. The mean total clearance, renal clearance, apparent volume of distribution, and elimination half-life were 0.11 +/- 0.05 l/h/kg, 0.07 +/- 0.02 l/h/kg, 0.54 +/- 0.15 l/kg, and 4.8 +/- 4.0 h, respectively. Approximately 70% of total vancomycin dose was excreted in the urine. A 2- to 5-fold variation in total clearance and a 2.5-fold variability in renal clearance were observed. Low cerebrospinal fluid concentrations of vancomycin were present at the time of shunt insertion in these pediatric patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and cerebrospinal fluid concentration of nafcillin in pediatric patients undergoing cerebrospinal fluid shunt placement.

Postoperative infection is among the most common complications in patients with cerebrospinal fluid shunt placement. Nafcillin is often used for prophylaxis but not pharmacokinetic data are available perioperatively in pediatric patients. The objectives of this study were to characterize the pharmacokinetics and determine the cerebrospinal concentrations of nafcillin. Ten patients (mean age 8.0 +/- 5.6 years) received three doses of intravenous nafcillin, 50 mg/kg every 6 h; the first dose was administered 1 h prior to surgery. Multiple blood samples were collected during and after surgery and the cerebrospinal fluid sample was obtained at the time of shunt insertion. Urine samples were collected for 24 h after initiation of nafcillin. Nafcillin was analyzed with an HLPC method. The peak serum concentrations ranged from 22 to 107 micrograms/ml; cerebrospinal fluid concentrations ranged from 0.02 to 0.30 (mean 0.16 +/- 0.11) micrograms/ml. The mean total clearance, renal clearance, apparent volume of distribution, and elimination half-life were 0.90 +/- 0.55 l/kg/h, 0.12 +/- 0.04 l/kg/h, 0.70 +/- 0.52 l/kg, and 0.5 +/- 0.1 h, respectively. 16% of total nafcillin dose was excreted in the urine. A 4-fold variability in total clearance and a 10-fold variation in cerebrospinal fluid concentrations of nafcillin was observed in these patients. Further, the concentrations of nafcillin attained in the cerebrospinal do not appear to be adequate, based on its minimum inhibitory concentration of 0.5 micrograms/ml against very susceptible staphylococci. These data, in addition to the fact that an increasing number of staphylococci are becoming resistant to nafcillin, question the usefulness of prophylactic nafcillin in pediatric patients undergoing shunt procedures.

A rapid analysis of nafcillin using high-performance liquid chromatography.

A simple, rapid, and reproducible high-performance liquid chromatography method is described for the measurement of nafcillin using an internal standard (IS), 5-(p-hydroxyphenyl)-5-phenylhydantoin. A one-step protein precipitation was used for preparation of nafcillin and IS. The mobile phase included 0.02 M ammonium acetate buffered solution and acetonitrile (75:25 vol/vol). An Ultrasphere ODS reversed-phase column was used and the detector was set at 224 nm. The retention time of IS and nafcillin was 4.8 and 6.6 min, respectively. The absolute recovery for nafcillin ranged from 95-98%. The intraday coefficient of variation for the assay was 3.5% at nafcillin concentrations of 1.0 and 60 micrograms/ml. The interday coefficient of variation was 7.1 and 3.1% at the nafcillin concentrations of 1.0 and 60.0 micrograms/ml, respectively. The major advantages of this method include the single-step extraction and the use of a small volume (50 microliter) of serum samples for nafcillin measurement. The serum concentrations of nafcillin in a pediatric patient were determined to demonstrate the application of this method.