The sensitivity for detection of antigen is lower in serum than

The sensitivity for detection of antigen is lower in serum than in urine. was 99.0% in controls including healthy subjects and individuals in whom histoplasmosis or blastomycosis were excluded. Precision and reproducibility were good and superb respectively. These findings demonstrate improvement in level of sensitivity without reduction in specificity precision or reproducibility after heat-EDTA treatment. The level of sensitivity for detection of antigenemia is lower than for antigenuria. For example in the quantitative MVista antigen enzyme immunoassay (EIA) among individuals with AIDS and progressive disseminated histoplasmosis (PDH) antigenuria was recognized in 95 to 100% compared to 92 to 95% for antigenemia (1 3 Previously we mentioned improvement in the level of sensitivity for detection of antigenuria after ultrafiltration (2). In the Platelia EIA pretreatment of serum at 104°C in the presence of EDTA is essential for detection of antigenemia. The presumed mechanisms for improvement in level of sensitivity include dissociation of antigen-antibody complexes and denaturation of the freed antibody. Screening for both antigenemia and antigenuria gives several advantages over screening for antigenuria only. First in some cases antigenuria may be undetectable but antigenemia may be present. Second urine may not be available in patients with renal failure. Third antigenuria levels early in the infection often are above the reportable range of the MVista antigen EIA (1 3 Clearance of antigenemia may provide a better marker for response to therapy in such cases. Fourth antigenuria is usually more likely to be affected by hydration status and consequently urine volume and concentration than is usually antigenemia making it a more accurate marker for fungal burden. The objective of this investigation was to evaluate the effect of preheating serum to 104°C in the presence of EDTA on detection of antigenemia. MATERIALS AND METHODS Clinical samples. Serum and urine specimens were obtained from AIDS patients with PDH treated with amphotericin B followed by itraconazole (4) or with itraconazole alone (5). The criteria for diagnosis included clinical findings of histoplasmosis supported by laboratory confirmation: positive culture 4-Hydroxyisoleucine histopathology or antigen. Positive cultures or histopathology was the basis for diagnosis in 89% and antigenuria in 11%. Serum and urine 4-Hydroxyisoleucine specimens had been frozen at ?20°C since 1996 to 1998 in a study conducted by the Mycoses Study Group (4) and since 1991 for an AIDS Clinical Trials Group study (5). For this analysis specimens obtained before or during antifungal therapy that were unfavorable or <0.6 ng/ml in the quantitative MVista antigen EIA were evaluated with or without pretreatment at 104°C 4-Hydroxyisoleucine in EDTA. Additional serum specimens from patients with probable histoplasmosis based upon detection of antigenuria in the MVista EIA or positive serologic findings were tested. Other clinical or laboratory information was not available from these patients. Clinical controls Gja5 included nine patients with probable blastomycosis based on repeatedly positive urine specimens in the antigen assay and patients in whom histoplasmosis was excluded based upon clinical and laboratory findings in a study approved by the institutional review board at Clarian Health Partners Indianapolis IN. Control specimens from healthy subjects were purchased (Houchin Blood Lender Bakersfield CA; SeraCare Milford 4-Hydroxyisoleucine MA). MVista antigen assay. The MVista EIA was performed as previously described (1). The results were quantitated in ng/ml by extrapolation from a human source material antigen calibration curve matched to primary reference galactomannan standards. Specimens with optical density values that exceeded the cutoff for the assay but that are less than the 0.6-ng/ml standard were reported as positive (<0.6 ng/ml) and those with results exceeding the 39-ng/ml standard are reported as positive (>39 ng/ml). Testing was performed at MiraVista Diagnostics Indianapolis IN. Pretreatment of serum at 104°C with EDTA. The procedure was modified after that used in the Platelia EIA (7). A total of 200 μl of EDTA was added to 600 μl of serum vortex mixed and placed in a heat block (Fisher Scientific) at 104°C for 6 min. The modifications included doubling the volume of EDTA and serum to provide sufficient supernatant for robotic pipetting and use of a heat block rather than a water bath. After that the specimen was centrifuged and the supernatant was removed for testing in 4-Hydroxyisoleucine the.