Biomedical subjects
Publications and source records attributed to D W Warnock.
ELISA appears to be a less reliable method than counterimmunoelectrophoresis for the diagnosis of nasal aspergillosis in the dog. False-positive or false-negative results were recorded for anti-A. fumigatus IgG in nine animals with aspergillosis and in 27 disease-free dogs although this problem could be reduced with careful selection of antigen.
The response of precipitin-negative sera from non-selected asthmatic patients against a range of somatic and culture filtrate antigens of Aspergillus fumigatus in ELISA for anti-A. fumigatus IgG is described. Antibody to the various antigens was widely distributed but the precise distribution was dependent upon antigen type. To determine how the selection of negative reference sera from precipitin-negative sera can influence the discrimination between precipitin-positive and -negative sera in ELISA, panels of 10 sera were chosen from the extremes of the frequency distribution and by random selection. The closest agreement between precipitin testing and ELISA was seen when randomly-selected negative reference sera were incorporated in the assay, although the degree of correlation was dependent upon the detector antigen. These findings demonstrate the requirement for careful selection of sera included as negative reference sera in ELISA for anti-A. fumigatus IgG.
Three strains of Candida albicans from patients with chronic mucocutaneous candidosis (CMC) who relapsed during prolonged treatment with ketoconazole were compared with three other Can. albicans strains by means of an agar incorporation MIC method, a broth microdilution method for IC30 determination, and a hyphal elongation assessment method. The MICs of ketoconazole, tioconazole and miconazole were difficult to interpret, but were much higher for the three CMC strains if low inoculum concentrations and short incubation times were used. The IC30S of the three imidazoles were much higher for the CMC strains than for the others. The imidazoles all caused minimal inhibition of hyphal elongation by the three CMC strains at concentrations up to 50 mg/l. Concentrations of 0.5 mg/l caused marked inhibition of the other strains.
A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37 C and vigorous agitation during incubation enabled the total test time required for the procedure to be reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive-negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.
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Eight healthy adults were given single oral doses of ketoconazole (200, 400, 600, and 800 mg) in the fasting state and with a standard breakfast at weekly intervals according to a balanced block design. Concentrations in serum were measured up to 32 h after the dose. Food did not reduce ketoconazole absorption or significantly alter peak ketoconazole concentrations in serum, though there was a food-related delay in achieving peak concentrations. At the 400- and 600-mg doses, food appeared to enhance absorption, but this effect was not seen at the 800-mg dose. With an increase in dose, the half-life and area under the serum concentration-time curve increased disproportionately, suggesting that the pharmacokinetics of ketoconazole may be dose dependent. Up to the 800-mg dose, the elimination of ketoconazole did not appear to be saturable. Administration of the drug with food is unlikely to be a cause of therapeutic failure.
A range of 6 somatic and culture filtrate antigens of Aspergillus fumigatus were evaluated in a rapid ELISA procedure for anti-A. fumigatus IgG where the component incubation times had been reduced to 10 min. Sera from patients with allergic aspergillosis, patients with suspected allergic aspergillosis, and asthmatic patients with or without A. fumigatus precipitins were tested. For all antigens, levels of anti-A. fumigatus IgG were higher in patients with allergic aspergillosis than in the other 3 groups. Low levels of specific IgG were, however, detected in asthmatic patients who had no precipitins against A. fumigatus. None of the antigen preparations enabled all patients with proven or suspected allergic aspergillosis to be separated from the other 2 groups of asthmatic patients. Positive-negative discrimination in ELISA was achieved by the inclusion of 10 pools of precipitin test-negative sera from the 50 asthmatics without A. fumigatus precipitins. The number of sera that were classed as positive in ELISA ranged from 9 to 15 in the allergic aspergillosis group, depending on the antigen used; in the suspected aspergillosis group, the number of positive reactions ranged from 1 to 8, while in the asthmatics with precipitins, the number ranged from 0 to 2.
Eighteen resistogram strains of Candida albicans were found among isolates obtained from 10 oral sites sampled prior to treatment in 22 patients with denture stomatitis. Eight strains were isolated from single patients, but two were found in four patients and one was found in five patients. Fifteen of the 18 strains were isolated from sites including the fitting surface of the denture or the denture-bearing mucosa. Twelve patients, sampled on a second occasion after treatment, harboured at least one strain on both occasions.
Levels of anti-Candida albicans immunoglobulin A (IgA) and IgG were measured by enzyme-linked immunosorbent assay in serum and cervicovaginal secretions from 64 non-pregnant women with vaginal candidosis and 158 uninfected non-pregnant women. Specific IgA and IgG were detected in the serum and secretions of all 222 women. There was no significant difference between the mean levels of specific IgA or IgG in secretions from women with candidosis and those of uninfected women. Neither was there a significant difference between mean levels of specific IgA or IgG when women using oral contraception were compared with others who were not. There was a significant correlation between the levels of IgA and IgG in serum and secretions from women with candidosis and from uninfected women. Blastospore and hyphal forms of C. albicans were seen in vaginal smears from 29 of the 64 women with culture-proven candidosis: in nine, both IgA- and IgG-coated C. albicans cells were recovered from the genital tract; in a tenth, IgG-coated cells were found.
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A rapid enzyme-linked immunosorbent assay (ELISA) was developed to detect IgG antibodies against a cytoplasmic antigen of Candida albicans. The optimum conditions and time required for each step were investigated. Pre-equilibration at 37 degrees C and constant agitation of immunoreactants resulted in a total test time of 1 h for examination of single serum dilutions. Initial binding of the antigen at 37 degrees C to the solid phase occurred within 4-6 min and was dependent on concentration. A serum dilution of 100-fold resulted in adequate discrimination between precipitin test-positive and -negative sera. Interaction of antibody and the bound antigen was maximal after 6 min incubation at 37 degrees C and was dependent upon the precipitin titre of the serum. Ten minutes was selected as the optimal incubation time for each of the stages, by which time maximal binding had occurred, irrespective of antibody affinity for antigen. The ELISA was completed by incubation at 37 degrees C for 10 min with alkaline phosphatase-conjugated anti-human IgG. Discrimination between positive and negative test sera was achieved by incorporating in each test run precipitin test-positive and -negative reference sera from groups of persons with or without C. albicans colonization or infection. Closest agreement between sera positive for C. albicans precipitins by counter-immunoelectrophoresis and the rapid ELISA was seen when reference negative sera were selected from non-colonized individuals.
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A computer-assisted method for the positive/negative discrimination of ELISA data is described. This method was applied to a rapid ELISA procedure for IgG class antibodies to Aspergillus fumigatus in which the performance time of the test was reduced to 1 h. The method gives results which compare well with those by agar gel double diffusion (AGDD). The computer-assisted reading, calculation and tabulation of ELISA results from one microtitre plate is performed in less than 2 min and permits analysis of large numbers of specimens.
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Ketoconazole 200 mg was given twice daily for five days to eight subjects. The serum pharmacokinetics were examined with the first dose on day 1 and with the ninth dose on day 5. The serum half life of ketoconazole was 74.8 +/- 13.9 min (mean +/- S.D.) on day 1, and 155.9 +/- 32.7 min on day 5. On day 1 the area under the serum concentration time curve was 626.9 +/- 230.1 mg/l.min, and 1441 +/- 897.3 mg/l.min on day 5. Antipyrine kinetics were determined before and after the course of ketoconazole. There was no significant change in the clearance and half life of antipyrine. Ketoconazole in the doses used in this study does not appear to alter hepatic microsomal enzyme activity in man. The increased ketoconazole half life and area under the serum concentration time curve on day 5 were in part caused by food. Other mechanisms for these changes such as saturation of a tissue compartment or a change in the elimination of ketoconazole during chronic dosing, may also be responsible.
Three imidazole antifungals (ketoconazole, tioconazole and miconazole) were examined for their effect on germ tube development by synchronous blastospore populations and on actively growing germ tubes of three strains of Candida albicans. After incubation in serum for 4 h the largest differences in germ tube elongation between strains were detected at concentrations (0.005 mg/l) where minimal inhibition was observed. Germ tube elongation in antifungal-containing serum diminished with increasing concentration. Maximum inhibition occurred at concentrations of 0.5 mg/l. All three antifungals inhibited the rate of hyphal elongation by the strains over a 6 h period at this concentration. Tests where germinated blastospores of strain 3281 were transferred to serum containing 0.5 mg/l of antifungal indicated that all three drugs inhibited further elongation. However, the rate of inhibition was dependent upon the length of time germination had proceeded in serum alone prior to transfer to antifungal-containing serum.
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