The Fungitell assay (Associates of Cape Cod, Inc.) is a commercial test that detects (1-3)-␤-D-glucan (BG) and is intended for diagnosis of invasive fungal infections. To evaluate the Fungitell assay, we tested serum and plasma samples from healthy blood donors and from patients with blood cultures positive for yeast or bacteria. All 36 blood donors were BG negative, and 13 of 15 candidemic patients were BG positive. Of 25 bacteremic patients, 14 (10 with gram-positive bacteremia) were BG positive. One of the latter patients with Staphylococcus aureus bacteremia also had invasive candidiasis, based on histological findings in a tissue biopsy; therefore, the BG result was a true positive. The sensitivity, specificity, and positive and negative predictive values of the Fungitell assay, by patient, for these three groups were 93.3%, 77.2%, 51.9%, and 97.8%, respectively. We also performed the Fungitell assay on sera that had been tested for Aspergillus galactomannan or Histoplasma antigen. All six Histoplasma antigen-positive patients and 31 of 32 Aspergillus galactomannan-positive patients were also BG positive. BG results for the 10 Histoplasma antigen-negative and the 32 Aspergillus galactomannan-negative patients varied, but we were unable to confirm many of the results. Between-run coefficients of variance (CVs) for the assay ranged from 3.2% to 16.8%; within-run CVs were <4.8%. The correlation coefficient for an interlaboratory reproducibility study was 0.9892. Concentrations of hemoglobulin, bilirubin, and triglycerides that caused 20% interference were 588, 72, and 466 mg/dl, respectively. Our results suggest that the Fungitel assay may be most useful for excluding invasive fungal infection.

We developed a multiplexed indirect immunofluorescence assay for antibodies to

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

We used a multiplexedfluorescent microsphere immunoassay to develop a sandwich capture assay to assess simultaneously the production of thymus helper (TH) 1- and TH2-type cytokines in tissue culture supernatant obtained from stimulated peripheral blood mononuclear cells. The assay then was used to assess the cytokine production of patients with hyperimmunoglobulinemia E syndrome and in cord blood from neonates. The multiplexed assay has a reportable range of less than 10 to 50,000 pg/mL. For linearity and recovery studies, R2 values for the 6 cytokines ranged from 0.988 to 0.999 for samples spiked with known concentrations of recombinant cytokine standards and for patient samples. The assay showed good specificity, with little cross-reactivity between cytokines. Results from supernatants of Staphylococcus aureus-stimulated peripheral blood mononuclear cells obtainedfrom 6 patients with hyperimmunoglobulinemia E syndrome showed significantly less interferon (IFN)-gamma production than cells from healthy control subjects. Cord blood cells from neonates produced significantly less interleukin 12 and IFN-gamma than cells from adults in group B streptococci-stimulated mononuclear cells. The fluorescent multiplexed microsphere immunoassay can be used to quantitate multiple cytokines from 1 sample and should be useful for further understanding of the cytokine role in disease.

dPneumococcal vaccination is frequently used to assess a patient's humoral immune function. The comparison of pre-and postvaccination levels of antipneumococcal antibodies is widely held to be the gold standard for documenting a response. However, many of the published criteria for defining an adequate response are based on assays that are no longer widely available. We compared the clinical classification of patient response by multiplex pneumococcal assays currently performed at three large reference laboratories using a variety of published criteria for defining responses in adults. The classification of responders agreed for 79% of the patients when using a threshold-based algorithm compared to 57 to 96% of the patients when using various fold-change-based algorithms. The highest rate of discordance was seen when the most stringent criteria for response were used (4-fold increase postvaccination in 70% of serotypes). The discordant samples tended to show similar patterns of response across all three assays, with small variations in the final number of serotypes converting postvaccination. We conclude that the use of published cut points for documenting response to pneumococcal vaccination can be affected by interlaboratory differences in pneumococcal assays, particularly for algorithms that require large fold changes for a response to be documented. However, the overall patterns of response were similar in virtually all samples, regardless of the assay used. P neumococcal vaccination is often used to evaluate a patient's response to polysaccharide antigens during a workup for deficiencies in antibody production. The adequacy of the specific vaccine response can be assessed by measuring postvaccination levels of antipneumococcal antibodies and by comparing these levels against predetermined cut points for either the absolute antibody level or the fold change relative to the baseline value. Measurements can be performed by a variety of analytical methods, including enzyme-linked immunosorbent assays (ELISAs) for total pneumococcal antibody level (1), ELISAs for antibodies against specific pneumococcal serotypes (2), and multiplex assays which measure the levels of a panel of serotype-specific antibodies (3-5). Because the vast majority of assays used for this purpose are lab-developed tests (LDTs) that were created and characterized by individual laboratories, the potential for interlaboratory variation in results exists. This may lead to difficulties when trying to interpret analytical results compared to the literature-based definitions for a therapeutic response to vaccination.The assessment of a patient's response when only a single (postvaccination) sample is available is usually done by comparing serotype-specific results against a "protective" threshold (6). However, the use of paired pre-and postvaccination samples is preferred because of the additional information provided for evaluating humoral immune function, which is the primary objective of these assays. Approaches for evaluating response from pa...

In our 14-valent Luminex assay for pneumococcal antibodies, we identified two groups of sera that caused false-positive results. The first group bound nonspecifically to the Luminex microspheres. The second group reacted specifically with bovine serum albumin (BSA). We describe here methods that eliminated the falsepositive reactivity of both groups.We previously described a multiplexed pneumococcal antibody assay based on the Luminex xMAP technology (11). Several other laboratories subsequently also described multiplexed Luminex assays for detecting these antibodies (1,6,15). In addition, the Luminex xMAP technology has been used for multiplexed assays to measure antibody responses to vaccines for other infectious diseases, including those caused by Neisseria meningitidis, Haemophilus influenza type b, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Bacillus anthracis, and papilloma virus (3,4,10,12,13,17). Waterboer et al. (18) documented an intrinsic problem with the use of the Luminex technology for serological assays. They found that some human sera bind directly to the carboxylated MicroPlex (formally MultiAnalyte) microspheres, causing a very high level of nonspecific background. These workers also found that SeroMAP microspheres, introduced by Luminex Corporation specifically for use in serological assays, reduced but did not eliminate the nonspecific-binding problem.Using our original protocol (11) for the 14-valent pneumococcal antibody assay with lot B MicroPlex microspheres, we encountered serum samples with very high-level false-positive results that were near or above the analytical measurement range (AMR) of the assay (Table 1). Approximately 15 of every 1,000 sera tested for pneumococcal antibodies exhibited this behavior. We termed these samples "polyspecific," although they did not react specifically to pneumococcal polysaccharides (PnPs). We tested a panel of 33 of these polyspecific sera and 1 control serum sample not showing polyspecific reactivity against an unconjugated MicroPlex microsphere and an unconjugated SeroMAP microsphere. The serum samples used in this study were submitted to ARUP Laboratories for pneumococcal antibody testing. All samples were deidentified according to protocols approved by the University of Utah Institutional Review Board (no. 7275). Serum samples were diluted 1:25 in phosphate-buffered saline (PBS), pH 7.2, with 5 g/ml pneumococcal C-polysaccharide (C-Ps) (Staten Serum Institut, Copenhagen, Denmark), 5 g/ml pneumococcal polysaccharide 22F (American Type Culture Collection, Manassas, VA), and 0.0015% bromcresol purple (BCP) (Sigma-Aldrich, St. Louis, MO). A MicroPlex (region 7) (Luminex Corporation, Austin, TX) microsphere and a SeroMAP (region 8) (Luminex Corporation, Austin, TX) microsphere were pelleted by centrifugation and resuspended in blocking/storage (B/S) buffer consisting of PBS with 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) or in BSA-free StabliGuard immunoassay stabilizer (SG01) (SurModics, Inc., Eden P...

A new commercial glycoprotein G-based enzyme immunoassay (gG-EIA) was compared with Western blotting (WB) for detection of herpes simplex virus type 1 (HSV-1) or HSV-2 type-specific antibodies in 193 serum samples. Sensitivity for HSV-1 was 95%; specificity was 96%. Sensitivity for HSV-2 was 98%; specificity was 97%. Twelve of 13 serum samples with equivocal gG-EIA results were serotyped by WB.

Both the in-house Luminex method and the eSensor trade mark DNA detection system reproducibly and unambiguously genotyped SNPs of CYP2C9 and CYP2C19 in the samples tested.