In vitro susceptibilities and biotypes of Candida albicans isolates from the oral cavities of patients infected with human immunodeficiency virus.

Candida albicans strains were isolated from the oral cavities of 62 human immunodeficiency virus (HIV)-infected patients at different stages of HIV infection. Only patients with persistent generalized lymphadenopathy-acquired immunodeficiency syndrome (AIDS)-related complex or full-blown AIDS showed typical clinical symptoms for oral candidiasis. In general, the microbiological recovery of Candida strains from the oral cavity increased with more advanced stages of HIV infection. The antifungal activity of ketoconazole, itraconazole, nystatin, amphotericin B, and flucytosine against all 62 strains was evaluated by means of a photometer-read broth microdilution method for determination of the 30% inhibitory concentrations of the drugs. The 95% ranges of 30% inhibitory concentrations were as follows: less than or equal to 0.063 to 32 micrograms/ml for ketoconazole, less than or equal to 0.063 to 8 micrograms/ml for itraconazole, 0.5 to 4 micrograms/ml for nystatin, less than or equal to 0.063 to 4 micrograms/ml for amphotericin B, and less than or equal to 0.063 to 8 micrograms/ml for flucytosine. Two strains were resistant to flucytosine, one was resistant to ketoconazole, and three were resistant to itraconazole. Isolates from patients with full-blown AIDS showed significantly less susceptibility to itraconazole, amphotericin B, and flucytosine. Strains were biotyped by using the API 20C carbohydrate assimilation system. The major biotype accounted for 63.9% of the isolates. At repeated evaluation, a change in biotype pattern was seen in 27.3%.     

Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests.

A collaborative comparison of macro- and microdilution antifungal susceptibility tests was performed in five laboratories. MICs of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined in all five centers against 95 coded isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. A standard protocol with the following National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Testing recommendations was used: an inoculum standardized by spectrophotometer, buffered (RPMI 1640) medium (pH 7.0), incubation at 35 degrees C, and an additive drug dilution procedure. Two inoculum sizes were tested (1 x 10(4) to 5 x 10(3) to 2.5 x 10(3) CFU/ml) and three scoring criteria were evaluated for MIC endpoint determinations, which were scored as 0 (optically clear), < or = 1 (slightly hazy turbidity), and < or = 2 (prominent decrease in turbidity compared with that of the growth control). Overall intra- and interlaboratory reproducibility was optimal with the low-density inoculum, the second-day readings, and MICs scored as either 1 or 2. The microdilution MICs demonstrated interlaboratory agreement with most of the four drugs higher than or similar to that of the macrodilution MICs. In general, there was good interlaboratory agreement with amphotericin B, fluconazole, and flucytosine; ketoconazole gave more variable results.     

Evaluation of serum gentamicin assay procedures for a clinical microbiology laboratory

Four methods for the measurement of serum gentamicin concentration were evaluated with respect to cost-effectiveness, accuracy, and precision. Gentamicin concentration was determined in 112 clinical samples by the Staphlococcus epidermidis agar diffusion bioassay procedure in routine service in our laboratory at the time this study was initiated. Appropriate portions of these clinical samples were frozen and later thawed for remeasurement of gentamicin by bioassay or for measurement of gentamicin in one of three other systems. These included the Enzymatic Radiochemical Assay, the Diagnostic Products Corporation Radioimmunoassay and the New England Nuclear Corporation Radioimmunoassay. In addition, gentamicin dissolved in horse serum at 2, 4, 6, 8, 10, and 12 micrograms/ml was aliquoted, frozen, and later thawed for assay in each of the above systems. The data were analyzed for evidence of constant and proportional bias as well as for accuracy and precision.     

Susceptibility testing of Cryptococcus neoformans: a microdilution technique.

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.     

Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy.

In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be > or = 6.25 micrograms/ml by the M27-P micro method and > or = 25 micrograms/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > or = itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole.     

Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp

The influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candida spp. clinical isolates (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida lusitaniae [10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (P < 0.01). A longer exponential phase and shorter lag phase were also observed. The A540 values at 24 h of incubation with medium containing glucose and an inoculum of 10(5) CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis (A540 = 0.26 +/- 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10(5) CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10(5) CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.     

Rapid susceptibility testing of fungi by flow cytometry using vital staining.

A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined.     

Evaluation of a capacitance method for direct antifungal susceptibility testing of yeasts in positive blood cultures

The feasibility of using a capacitance method (CM) for direct antifungal susceptibility testing of yeasts in positive blood cultures was evaluated. The CM used the same test conditions as those recommended by the National Committee for Clinical Laboratory Standards. After direct inoculation of positive culture broths into module wells (Bactometer; bioMérieux, Inc., Hazelwood, Mo.), the end-point determination was made by monitoring the capacitance change in the culture broths with Bactometer. The MIC of amphotericin B was the lowest concentration at which yeast growth was completely inhibited, while the MICs of ketoconazole, flucytosine, and fluconazole were the concentrations at which a >/=80% reduction in capacitance change was observed. The MICs of the four drugs against each blood isolate obtained on subculture plates were also determined by the macrodilution method. For 51 positive blood cultures tested, the percent agreement (+/-2 log(2) dilutions) between the CM and the macrodilution method were as follows: amphotericin B (98%), ketoconazole (92%), flucytosine (84%), and fluconazole (96%). The CM was further used for breakpoint susceptibility testing of fluconazole (8 and 64 microg/ml) and flucytosine (4 and 32 microg/ml) against yeasts in positive blood cultures. After testing of 74 specimens by the CM, flucytosine and fluconazole produced one (1.4%) major error and two (2.8%) minor errors, respectively. All yeasts that displayed resistance to flucytosine or fluconazole were detected within 24 h after direct inoculation of the positive broths into Bactometer. The CM may be useful for the rapid detection of antifungal resistance in positive blood cultures containing yeasts.     

Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 microgram/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 micro g of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 microgram/ml, while the dynamic range was 0.06 to 1.7 microgram/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 microgram of anti-PA IgG per ml, the RDL was 0.016 microgram/ml, and the whole-serum equivalent MDC was 1.5 micrograms/ml. The dynamic range was 0.006 to 6.8 microgram/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.     

Nosocomial outbreak caused by Scedosporium prolificans (inflatum): four fatal cases in leukemic patients.

Four cases of fatal disseminated Scedosporium prolificans (inflatum) infection occurring in neutropenic patients are reported. Because of hospital renovation, the patients were cared for in a temporary hematologic facility. S. prolificans (inflatum) was isolated from blood cultures of these four patients, two of whom underwent full necropsy, and revealed abundant vegetative hyphae and ovoid conida with truncate bases in many organs. In vitro susceptibility testing of fungal strains showed all isolates to be resistant to amphotericin B, flucytosine, miconazole, ketoconazole, fluconazole, and itraconazole, with MICs greater than 16 micrograms/ml. The reported infections, two in each of two rooms, occurred over a period of 1 month, with very similar clinical outcomes. Circumstancial evidence suggested a nosocomial outbreak, but the environmental samples collected from the rooms, corridors, and adjacent areas did not yield S. prolificans (inflatum). Nevertheless, circumstantial evidence suggested a nosocomial outbreak of S. prolificans (inflatum) infection.     

Diffusion of fosfomycin into bone tissue in man

Fosfomycine diffusion into spongious and cortical bone was studied in 20 patients undergoing total replacement of the hip after an intravenous infusion of 4 g (1 g/h). The serum and bone samples were collected 1 h and 3 h after administration of the drug. Concentrations at 1 h were 105.0 +/- 12.4 micrograms/ml, 19.6 +/- 4.8 micrograms/g and 13.3 +/- 3.7 micrograms/g in serum, spongious bone and cortical bone respectively; at 3 h these concentrations had become respectively 67.8 +/- 15.9 micrograms/ml, 10.0 +/- 4.2 micrograms/g and 8.2 +/- 3.6 micrograms/g. Antibiotic levels in bone tissue decreased in direct ratio to corresponding serum values, the mean tissue/serum ratio remaining at 15 +/- 4%.     

Presence and measurement of sample histidine in the Ames test: quantification and possible elimination of a source of false-positive mutagenicity test results

Both histidine and dipeptides that can be converted to histidine can potentially interfere with the Ames test by increasing the number of spontaneous revertants. Such interference might be especially evident when urine and other biological samples are studied in this assay. We have developed a turbidimetric bioassay that utilizes a nonrevertible Salmonella typhimurium histidine auxotroph, NS1135. The assay is linear with histidine over at least a 300-fold range (0.015-5 micrograms/ml of L-histidine.HCl.H2O). Data indicate that several histidine-containing dipeptides can be utilized as efficiently as free histidine. Our data suggest that this assay may be used to measure biological samples accurately for their histidine content and thereby permit an adjustment for sample histidine during the setup of Ames assays, thus eliminating increased reversion caused by sample histidine.     

Measurement of plasma fibrin D-dimer levels with the use of a monoclonal antibody coupled to latex beads

Recently, monoclonal antibody (DD-3B6) to fibrin D-dimer was prepared and coupled to latex beads to provide a specific test (Dimertest) for fibrinolysis. The purpose of this study was to evaluate the Dimertest assay as a clinical laboratory test for the measurement of plasma fibrin D-dimer derivatives. The Dimer-test assay specifically detected 2 micrograms/mL of purified fibrin D-dimer or fibrin D-dimer/fragment E complex added to afibrinogenemic plasma but did not detect 500 micrograms/mL of either fibrinogen fragments X, D, E, or 160 micrograms/mL cross-linked fibrinogen. The fibrin(ogen) degradation product (FDP) assays of American Dade or Wellcome Diagnostics detected 5.0 micrograms/mL of fibrin D-dimer and from 1 to 10 micrograms/mL of the other FDPs. Twenty-eight percent of 150 random plasma samples assayed from hospitalized patients were positive for fibrin D-dimer derivatives. Plasma samples from 152 patients suspected of having disseminated intravascular coagulation (DIC) were assayed for serum FDP (Wellcome Diagnostics) and plasma fibrin D-dimer derivatives. Samples from 69% of patients with serum FDP levels less than 10 micrograms/mL, and more than 90% of those with serum FDP levels greater than 10 micrograms/mL, were positive for fibrin D-dimer derivatives. Dimertest results were not modified by heparin, streptokinase, freeze-thawing, or clotting plasma. Serum fibrinogen-related antigens were immunoadsorbed from Dimer-test positive sera by anti-fibrinogen antibody and formalin-fixed Cowan I strain Staphylococcus aureus. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein blotting with the use of monoclonal antibody DD-3B6 demonstrated a protein band with similar mobility to purified D-dimer. The measurement of plasma fibrin D-dimer derivatives by the Dimertest assay is a rapid, sensitive, and specific laboratory test for fibrinolysis. The Dimertest assay has proven to be a useful addition to the clinical laboratory and should be helpful in the diagnosis and management of patients with diseases associated with fibrinolysis.     

Comparative assessment of in vitro inactivation of gentamicin in the presence of carbenicillin by three different gentamicin assay methods

Inactivation of gentamicin (G) is known to occur secondarily to the formation of complexes with certain beta-lactam antibiotics. However, aminoglycosides in the presence of aminoglycoside-beta-lactam complexes may not be recognized uniformly by all assay methods. We tested this hypothesis by using mixtures of G plus carbenicillin (C), with and without the addition of penicillinase, in pooled sera under several in vitro conditions: at 25 and 35 degrees C and at low and high C concentrations. Samples were assayed for G with the EMIT and TDx systems, and a microbiological assay was performed with a strain of Klebsiella pneumoniae resistant to C. In the presence of C (500 micrograms/ml) at 35 degrees C, the initial G concentration of 5 micrograms/ml decreased markedly over 48 h as assessed by all three assay methods. However, significantly greater degradation was noted when samples were measured by microbiological assay and TDx than by EMIT. Differences between assays were less marked when mixtures were studied at a lower temperature and with a lower G to C ratio (5 micrograms of G plus 100 micrograms of C per ml). The addition of penicillinase to the antibiotic mixtures prevented the degradation of G over time when measured by all three assay systems. We concluded (i) that EMIT measures higher serum concentrations of G than do TDx or microbiological assays when complexes of G and C are present and (ii) that the addition of penicillinase to serum samples containing C and G would be effective in preventing G degradation during prolonged (greater than 24-h) periods between the time of sampling and assay.     

Cloxacillin concentration in suction fluid following flash antibiotic therapy during insertion of a hip prosthesis

Since 1982 we have administered cloxacillin intraoperatively during total hip replacement. 1 g cloxacillin is injected intravenously at induction of anesthesia, followed by 1g every hour until the end of the procedure or a total of 6 g. In our study, cloxacillin concentrations were determined in the fluid collected from the deep suction catheter inserted at the end of the surgical procedure. Fluid samples were collected 2 h, 4 h, 6 h, 8 h and 24 hours after the last injection of cloxacillin. Serum samples were taken 2 h, 6 h and 24 hours after the last injection. Cloxacillin was assayed using an agar-diffusion microbiologic method. 18 patients were studied. Each had received 3 to 5 g cloxacillin over 3 to 5 hours. Mean suction catheter fluid concentrations were to 69.7 micrograms/ml, 37.6 micrograms/ml, 24.2 micrograms/ml, 15.5 micrograms/ml, and 6.8 micrograms/ml respectively in the samples collected 2 h, 4 h, 6 h, 8 h and 24 hours after the last injection of cloxacillin. Mean serum concentrations were 34.1 micrograms/ml, 4.2 micrograms/ml, and 0 microgram/ml respectively 2 h, 6 h and 24 hours after the last injection. Our results indicate that cloxacillin concentrations within the hip joint are probably effective against staphylococci for 8 to 12 hours.     

Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole.

Ketoconazole and itraconazole were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Two isolates that had been previously identified as QC isolates for amphotericin B, fluconazole, and flucytosine (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were tested in accordance with the National Committee for Clinical Laboratory Standards M27-P guidelines. Each isolate was tested 20 times with the two antifungal agents in the five laboratories by using a lot of RPMI 1640 unique to each laboratory as well as a lot common to all five laboratories, thus generating 200 MICs per drug per organism. Overall, 96 to 99% of the MICs for each drug fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). By using these data, 3-log2 dilution QC ranges encompassing 98% of the observed MICs for three of the organism-drug combinations and 94% of the observed MICs for the fourth combination were established. These QC ranges are 0.064 to 0.25 micrograms/ml for both ketoconazole and itraconazole against C. parapsilosis ATCC 22019 and 0.125 to 0.5 micrograms/ml for both ketoconazole and itraconazole against C. krusei ATCC 6258.     

Initial use of a broth microdilution method suitable for in vitro testing of fungal isolates in a clinical microbiology laboratory.

Antifungal susceptibility testing methods currently lack a standardized procedure. Many factors, such as inoculum preparation, inoculum density, medium selection, pH, incubation time and temperature, and endpoint determination, affect results. We developed a workable procedure for fungal susceptibility testing, with a microtiter method based upon modifications of the proposed guidelines from the National Committee for Clinical Laboratory Standards, using two different growth media. For this procedure, the microtiter tray is prepared as a panel of 6 drugs (amphotericin B, flucytosine, fluconazole, ketoconazole, miconazole, and itraconazole) alone and in combination with amphotericin B. Eagle's minimal essential medium and RPMI 1640 are the two growth media. Two separate susceptibility trays are inoculated for each sensitivity test, with one tray incubated at 30 degrees C and the other incubated at 35 degrees C. After 48 h of growth, results for both temperatures and both media are recorded and interpreted. The four test environments (two media each at two temperatures) provided growth for 100 of the first 104 organisms that were submitted for testing. This approach provides a workable methodology for routine antifungal susceptibility testing in a clinical microbiology laboratory setting.     

Purification of a plasma protein that inhibits complement-mediated prevention of immune precipitation.

A pre-albumin glycoprotein that inhibits complement-mediated prevention of immune precipitation (PIP) has been purified from normal human serum by sequential affinity chromatography on IgG-Sepharose, protein A-Sepharose and Con A-Sepharose. A total of 4.7 mg of this protein were obtained from 50 ml of serum, representing a yield of 49% and a 253-fold degree of purification. We have named this glycoprotein gp60 as it has an apparent molecular weight of 60,000 on SDS-PAGE. The addition of gp60 to normal serum produced dose-dependent inhibition of both PIP and solubilization of immune precipitates. Maximum inhibition of PIP was achieved by a concentration of 600 micrograms/ml. A monospecific antiserum was produced by the immunization of rabbits, which enabled us to develop an enzyme-linked immunosorbent assay to measure serum concentrations of gp60. In 12 normal sera the mean concentration was 205 micrograms/ml (range 132-258 micrograms/ml), while that in 15 rheumatoid arthritis sera was 515 micrograms/ml (range 430-708 micrograms/ml). The serum concentration of this protein correlated with the level of inhibition of PIP (r = 0.91, P less than 0.01).     

Detection of immune complexes by a new assay, the polyethylene glycol precipitation-complement consumption test (PEG-CC).

A new assay for the detection of circulating immune complexes, the polyethylene glycol precipitation-complement consumption assay (PEG-CC), is described. The test is both simple and sensitive, and exhibits a high degree of specificity. Immune complexes are first isolated from serum by precipitation in 2.5% polyethylene glycol (PEG) and concentrated. They are then assayed functionally by measuring their ability to fix complement using a sensitive kinetic assay for total haemolytic complement. The test can detect aggregated IgG in serum at concentrations around 6.0 micrograms/ml (about 2.0 micrograms absolute. Using DNA-anti-DNA and ovalbumin-anti-ovalbumin immune complexes prepared in vitro, antigen concentrations less than 0.5 micrograms/ml can be detected. Interference by endogenous complement, polyanions and other factors in test sera has been virtually eliminted by the design of the assay. The increased specificity of the PEG-CC test for immune complexes, should prove useful in the diagnosis and monitoring of immune complex-mediated diseases.     

Hemagglutination assay for human serum fibronectin

A hemagglutination assay for human serum fibronectin (Fn) is described. The assay is based on the capacity of Fn to bind to gelatin. Purified and human serum Fn agglutinate chromic chloride treated sheep red cells coated with gelatin in a quantitative and reproducible manner. The lowest Fn concentration giving positive hemagglutination was found to be 7 micrograms/ml, a sensitivity making it suitable for measurement of Fn in both normal and pathologic sera. The assay is simple and rapid and does not require specialized equipment.