Determination of zidovudine concentration in serum by enzyme-linked immunosorbent assay and by time-resolved fluoroimmunoassay.

Two solid phase, nonradioactive immunoassays were developed and evaluated for the determination of zidovudine (Retrovir, ZDV, AZT) concentrations in serum. The first, an enzyme-liked immunosorbent assay (ELISA), used an anti-AZT monoclonal antibody (MAb) and subsequent alkaline phosphatase second antibody system for the detection method. The second, a time-resolved fluoroimmunoassay (TR-FIA), used a polyclonal anti-AZT antibody followed by a europium-labeled goat anti-rabbit immunoglobulin (IgG) as the fluorescent probe. The ELISA had a detection range from 125 to 4,000 nM and a 50% inhibitory concentration (IC50) of about 500 nM. Development of the TR-FIA was based on a very sensitive detection system of metal chelate chemistry and time-resolved fluorometry. The standard curve in the TR-FIA was from 5 to 4,000 nM with an IC50 of 200 nM. Intra- and interassay precision of the ELISA was good, with coefficients of variation from 3.9 to 7.3% and 3 to 17%, respectively, while the same values for the TR-FIA were 6.2 to 10.9% and 5.4 to 19.9%, respectively. The results obtained from each method were compared individually with those of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). There was good agreement among the results obtained by each method. These two methods enable laboratories not licensed for radioisotopes to analyze potentially infectious samples without aerosol-forming centrifugation.

Effect of acyclovir on various murine in vivo and in vitro immunologic assay systems.

In two in vitro tests, lymphocyte-mediated cytotoxicity and neutrophil chemotaxis, acyclovir showed no inhibitory effects at concentrations as high as 600 microM. The compound inhibited rosette formation with nonimmune mouse lymphocytes in vitro by approximately 50 percent at 15.8 microM. The significance of this inhibition is unclear. In four in vivo tests in mice which measured humoral and cell-mediated immunity (complement-dependent cellular cytotoxicity, complement-independent cellular cytotoxicity, delayed hypersensitivity and graft versus host reaction) acyclovir showed no inhibitory effects at single doses up to 200 mg/kg given on day 2 after antigenic stimulation. Four daily doses of acyclovir at 50 mg/kg per day had no effect on the numbers of hemolytic IgM antibody-forming cells in the spleen when assayed on day 4. At the higher dosage of 100 mg/kg per day for four days, there was a slight reduction in the numbers of these cells. There was no significant decrease in hemagglutinin or hemolysin antibody titers after four daily doses of acyclovir up to 200 mg/kg.

Scintillation proximity radioimmunoassay for the measurement of acyclovir.

A homogeneous, single-tube scintillation proximity radioimmunoassay (SPRIA) to quantitate acyclovir (Zovirax), ACV, (9-[(2[hydroxyethoxy)]methylguanine)] in human plasma is described. The reagents for the SPRIA are an anti-ACV monoclonal antibody (WACO4 MAb), tritiated ACV, and scintillation proximity reagent (goat anti-mouse immunoglobulin G (IgG) coupled to fluoromicrospheres). The ACV standard curve range in the SPRIA is from 0.7 ng ml-1 (3.0 nmol l-1) to 90.0 ng ml-1 (0.4 mumol l-1) with a 50% inhibitory concentration of 5.0 ng ml-1 (22.2 nmol l-1). However, the lower limit of quantification is 7 ng ml-1 at 1:10 dilution of plasma. Analytical recovery of ACV in spiked human plasma controls ranges between 90-110%. Intra- and inter-assay relative standard deviations were < 8%. This high throughput homogeneous assay is a rapid, convenient and simple alternative to the current radioimmunoassay that uses ammonium sulfate precipitation as the separation method. This technique is particularly attractive because it requires neither separation of bound from free drug nor use of scintillation fluid. The procedure was applied to quantitate ACV in samples from pre-clinical and clinical studies after the administration of valaciclovir, a prodrug of ACV (256U87, Valtrex, L-valyl ester of ACV). Automation of this assay will further improve efficiency in processing a larger number of samples.

Functional MRI activity in the thalamus and occipital cortex of anesthetized dogs induced by monocular and binocular stimulation.

The neuroanatomy of the mammalian visual system has received considerable attention through electrophysiological study of cats and non-human primates, and through neuroimaging of humans. Canine neuroanatomy, however, has received much less attention, limiting our understanding of canine vision and visual pathways. As an early step in applying blood oxygenation level dependant (BOLD) functional magnetic resonance imaging (fMRI) for veterinary use, we compared visual activity in the thalamus and occipital cortex of anesthetized dogs presented with binocular and monocular visual stimuli. Activity in the left and right thalamus and occipital cortex during monocular stimulation was also compared. Six beagles were presented with a vertical grating visual stimulus and scanned at 4 Tesla. Each dog was scanned twice under each of 3 anesthetic protocols (isoflurane, propofol, and fentanyl/midazolam). We found: 1) significant BOLD activation in the lateral geniculate nucleus (LGN) of the thalamus and the occipital cortex; 2) a significantly larger area of activation in the LGN during monocular stimulation than during binocular stimulation; and 3) that activity in the hemisphere contralateral to the stimulus was not significantly greater than that ipsilateral to it.

Unusual patterns of chronic photodamage through clothing.

Recent cancer statistics estimate that the yearly incidence of cutaneous malignancies in the United States is similar to the incidence of all other cancers combined. Ultraviolet radiation-induced photodamage of the skin plays a large role in the growth of these skin cancers. The case of a 47-year-old white man with peculiar patterns of actinic skin damage on the anterior chest and back is presented, and the relationship of these findings with his choice of clothing is reviewed. The transmission of ultraviolet radiation through clothing fabrics is addressed, with recommendations for the use of clothing as an effective form of photoprotection.

The effects of Dynavision rehabilitation on behind-the-wheel driving ability and selected psychomotor abilities of persons after stroke.

OBJECTIVE: Many conventional rehabilitation exercises, such as pencil-and-paper and computer tasks, may not train perceptual and motor skills as applied to a complex, multiskill activity such as driving. The present study examined the usefulness of the Dynavision apparatus for driving-related rehabilitation. The Dynavision was designed to train visual scanning, peripheral visual awareness, visual attention, and visual-motor reaction time across a broad, active visual field. METHOD: Ten persons with a cerebrovascular accident participated in the study. All had failed behind-the-wheel driving assessments. Subjects participated in a 6-week Dynavision training program using exercises designed to impose various motor, perceptual, and cognitive demands. RESULTS: Dynavision training resulted in significantly improved behind-the-wheel driving assessments as compared to expected outcomes. Comparisons between pretests, posttests, and follow-up tests on a number of Dynavision, response, and reaction time variables showed significant improvements and maintenance effects. Dynavision performance, and, to a lesser extent, choice visual reaction and response times, were found to differentiate between persons assessed as safe and unsafe to drive, and between older and younger drivers. Subject self-reports suggested that a variety of training-related improvements had occurred in everyday functioning. CONCLUSION: Dynavision training shows some rehabilitative promise for improving driving and basic psychomotor skills. Future research on the benefits and limitations of this apparatus should use finer laboratory skill measures and more comprehensive tests of driving and daily functioning to assess more thoroughly skill improvements in persons after stroke.

Radioimmunoassay for Retrovir, an anti-human immunodeficiency virus drug.

A direct radioimmunoassay (RIA) for the quantitation of Retrovir (zidovudine, azidothymidine, AZT) in biological fluids has been developed. The assay is sensitive with an I50 value of about 30 nM and with a lower limit of detection of about 3 nM. Intra-assay precision gave sample coefficients of variation that ranged from 1.77 to 8.65% for the standard curve with human plasma. Inter-assay precision and accuracy were within acceptable limits. The RIA was validated by comparing results obtained form the analysis of rat plasma samples by both this RIA and a high-performance liquid chromatography method. None of the crossreactivities recorded should interfere with the assay system. The affinity constant of the antibody chosen for use was 1.4 z 10(9) L/mol.

A competitive enzyme-linked immunosorbent assay to quantitate acyclovir and BW B759U in human plasma and urine.

A simple and sensitive enzyme-linked immunosorbent assay for the detection and quantitation of acyclovir in human plasma and urine was developed. Acyclovir immobilized on a solid phase and free acyclovir in the sample solution were allowed to compete for a limited amount of anti-acyclovir monoclonal antibody. The specific antibody bound to the immobilized acyclovir was detected by the use of alkaline phosphatase-conjugated anti-mouse immunoglobulin. The resulting enzyme activity was inversely related to acyclovir concentration in the sample. The Hill plot of standard acyclovir concentrations was linear over a 100-fold concentration range, with a lower detection limit of 0.2 nM and a concentration of soluble ligand displacing 50% of available antibody of approximately 1 nM. The metabolites of acyclovir cross-reacted minimally, and there was no detectable interference by various unrelated compounds tested in the assay. However, BW B759U [9-(2-hydroxy-1-hydroxymethylethoxy)methylguanine], a congener of acyclovir, cross-reacted significantly. As a consequence, the assay was found useful in measuring the concentrations of BW B759U in clinical samples devoid of acyclovir.

Radioimmunoassay for desciclovir, 2-[(2-amino-9H-purin-9-yl)methoxy]ethanol, a prodrug for the antiviral acyclovir.

A direct radioimmunoassay for the detection of desciclovir (DCV)(2) in biological fluids has been developed. The radioimmunoassay was validated by comparing results obtained from human plasma samples analyzed by both this RIA and a gas chromatographic method. None of the crossreactivities noted interfere with the assay system. Although the succinylated antigen has a slightly higher affinity constant, the non-succinylated tritiated antigen was chosen for routine use. The assay is sensitive with an I50 value of 20 nM and with a lower limit of detection of about 3 nM. Intra-assay precision gave sample coefficients of variation which ranged from 2.2 to 9.6% for the standard curve with human plasma and from 2.0 to 8.2% for the standard curve with human urine. Inter-assay precision and accuracy were within acceptable limits.

Rapid acyclovir radioimmunoassay, using charcoal adsorption.

Charcoal adsorption of unbound acyclovir rather than ammonium sulfate precipitation of bound acyclovir to facilitate the separation of bound antigen from free antigen gave rise to a radioimmunoassay which was quicker yet still as sensitive and accurate as that previously used.

Differential assay of zidovudine and its glucuronide metabolite in serum and urine with a radioimmunoassay kit.

We developed an ancillary procedure for the ZDV-Trac RIA (Incstar) to allow simultaneous determination of both zidovudine (3'-azido-3'-deoxythymidine, ZDV, AZT, Retrovir) and its metabolite, the glucuronide of ZDV (3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine, ZDVG, GAZT), in human serum and urine. Using the ZDV-Trac RIA, we measured ZDV concentrations before and after ZDVG in samples was hydrolyzed to ZDV by beta-glucuronidase (EC 3.2.1.31); ZDVG concentration was calculated as the difference between the two results. This method enables rapid evaluation of a large number of samples with a total turn-around time of 6 h. The lower detection limit of the RIA was 0.27 micrograms/L; the measurements varied linearly with ZDV concentrations from 0.27 to 217 micrograms/L, with the 50% inhibitory concentration being approximately 10 micrograms/L. Analytical recoveries of inhouse serum and urine controls for both ZDV and ZDVG exceeded 90%. Coefficients of variation (CVs) of serum controls were less than 6% for ZDV and less than 11% for ZDVG; for urine controls, CVs for both ZDV and ZDVG were less than 6%. Results for ZDVG concentrations obtained by HPLC and by the ZDV-Trac RIA system compared well: r = 0.978, slope 1.0, for serum samples, and r = 0.993, slope 1.09, for urine samples.

A single-tube radioimmunoassay for the antiviral agent 2,6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U).

A direct radioimmunoassay for the detection of A134U in biological fluids and extracts has been developed. The entire assay, including scintillation counting, is performed using 12 X 55-mm centrifuge tubes and results in a sensitive, reliable, and relatively inexpensive procedure. The log-logit transformation is linear over a range of 1 to 30 pmol per sample. Intra-assay precision was found to be excellent with a coefficient of variation ranging from 4.1 to 14.5% for the standard curve with plasma and a coefficient of variation ranging from 4.0 to 17.8% for the standard curve with urine. Interassay precision and accuracy were also found to be good. With the antisera chosen for use, no cross-reactivity was found with acyclovir or its two known metabolites, while some cross-reactivity was seen with the corresponding two derivatives of A134U. Only very minor cross-reactivities were seen with a small number of other compounds out of a large number tested.

Concurrent zidovudine levels in semen and serum determined by radioimmunoassay in patients with AIDS or AIDS-related complex.

Zidovudine was present in the semen and serum of six patients with acquired immunodeficiency syndrome or the related complex who were receiving 200 mg of the drug orally every four to six hours. Mean semen zidovudine levels (as measured by a new radioimmunoassay) in samples collected 0.75 to 1.25 hours after oral dosing were 3.63 to 7.19 mumol/L. Levels in semen samples collected 3.0 to 4.5 hours after oral dosing were 1.68 to 6.43 mumol/L. These values are above the in vitro minimum inhibitory concentration for the human immunodeficiency virus type 1 (HIV-1). Mean serum concentrations at the early and late times after oral dosing were 0.22 to 3.07 mumol/L and 0.10 to 1.42 mumol/L, respectively. Ratios of semen/serum zidovudine levels ranged from 1.3 to 20.4. It is possible that a pH-dependent trapping mechanism, which has been described in the prostate for other antibiotics, was responsible for the relatively high semen levels observed.

Functional MRI as a tool to assess vision in dogs: the optimal anesthetic.

Functional magnetic resonance imaging (fMRI) is a recent advance in neuroimaging that provides a picture of brain activity with excellent spatial resolution. Current methods used to evaluate canine vision are poorly standardized and vulnerable to bias. Functional MRI may represent a valuable method of testing vision in dogs if the impacts of anesthesia on fMRI are understood. Six dogs were scanned during visual stimulation, each under three different anesthetic protocols (isoflurane, propofol, fentanyl/midazolam) to address the questions: (1) Can visually evoked fMR signals be reliably recorded in anesthetized dogs? and (2) Which anesthetic agent permits the least suppression of visually induced fMR signal in dogs? This study confirms that visual stimuli reliably elicit neural activity and fMR signal change in anesthetized dogs. No significant differences in images acquired under the three anesthetics were found, and there was no significant relationship between anesthetic dose and brain activity, within the range of doses used in this study. Images obtained during isoflurane anesthesia were more consistent between dogs than those obtained with the other two agents. This reduced variation may reflect the fact that inhalant anesthesia is more easily controlled than intravenous anesthesia under conditions associated with high field fMRI.

Single-dose pharmacokinetics of acyclovir.

The pharmacokinetics of intravenously administered acyclovir were studied in 10 patients with advanced malignancies. After doses of 0.5 and 1.0 mg/kg, the slow disposition half-life values (t1/2beta) ranged from 2.2 to 3.1 h for the 1-h infusions and from 1.8 to 3.7 h for the 6-h infusions. Plasma levels, measured by radioimmunoassay, reached a maximum at the end of the 1-h infusions and approached steady state at 3 to 4 h into the 6-h infusions. Mean peak plasma concentrations obtained at 0.5 and 1.0 mg/kg administered over 1 h were 3.03 and 5.99 microM, respectively. Mean peak levels for the 6-h infusions were 1.07 microM at 0.5 mg/kg and 2.58 microM at 1.0 mg/kg. The mean urinary elimination of acyclovir was 44.7% of the administered doses. No clinical or laboratory abnormalities were noted in the 10 patients studied.

Mycosis fungoides d'embleé: CD30-negative cutaneous large T-cell lymphoma.

The d'embleé variant of mycosis fungoides initially described patients with a rapid onset of tumors without progression through patch- and plaque-stage disease. We report a case of a patient with the clinical presentation of mycosis fungoides d'embleé and correlate the histologic and immunophenotypic data with those of a more updated classification scheme.

Colletotrichum species as emerging opportunistic fungal pathogens: a report of 3 cases of phaeohyphomycosis and review.

BACKGROUND: Numerous etiologic agents of subcutaneous phaeohyphomycosis have been reported. Colletotrichum spp, common plant pathogens, have been reported as a cause of ocular keratomycosis, but only one previous case of cutaneous disease (hyalohyphomycosis) has been attributed to this genus. OBJECTIVE: Our purpose was to describe 3 cases of subcutaneous phaeohyphomycosis due to Colletotrichum spp occurring in patients undergoing chemotherapy for hematologic malignancies. METHODS: Three cases of Colletotrichum-induced phaeohyphomycosis are reviewed. The clinical and histologic features of this infection are presented, the antifungal susceptibilities are reported, and treatment options are discussed. RESULTS: We describe the first report in which C coccodes and C gloeosporioides are implicated as etiologic agents of subcutaneous phaeohyphomycosis. Despite treatment, one patient died after the onset of visceral fungal disease. CONCLUSION: Colletotrichum spp may cause life-threatening phaeohyphomycosis in immunosuppressed patients. Prompt recognition and intervention with surgical and antifungal treatment may result in decreased morbidity and mortality associated with these infections.