Improved Diagnosis of Acute Pulmonary Histoplasmosis by Combining Antigen and Antibody Detection.

BACKGROUND: Acute pulmonary histoplasmosis can be severe, especially following heavy inoculum exposure. Rapid diagnosis is critical and often possible by detection of antigen, but this test may be falsely negative in 17% of such cases. Antibody detection by enzyme immunoassay (EIA) may increase sensitivity and permit the measurement of immunoglobulin M (IgM) and immunoglobulin G (IgG) classes of antibodies separately. METHODS: Microplates coated with Histoplasma antigen were used for testing of serum from patients with acute pulmonary histoplasmosis and controls in the MVista Histoplasma antibody EIA. Results for IgG and IgM were reported independently. RESULTS: IgG antibodies were detected in 87.5%, IgM antibodies in 67.5%, and IgG and/or IgM antibodies in 88.8% of patients with acute pulmonary histoplasmosis in this assay, while immunodiffusion, complement fixation, and antigen testing showed sensitivities of 55.0%, 73.1%, and 67.5%, respectively (n = 80). Combining antigen and antibody detection increased the sensitivity to 96.3%. CONCLUSIONS: The MVista Histoplasma antibody EIA offers increased sensitivity over current antibody tests while also allowing separate detection of IgG and IgM antibodies and complementing antigen detection. Combining antigen and EIA antibody testing provides an optimal method for diagnosis of acute pulmonary histoplasmosis.

Seroprevalence of histoplasmosis in Kampala, Uganda.

Histoplasmosis is endemic to the Midwestern United States, but cases have been reported nearly worldwide. A 1970 study found 3.8% skin test sensitivity to Histoplasma capsulatum in Uganda but no systemic study of histoplasmosis exposure has occurred since the onset of the human immunodeficiency virus (HIV) pandemic. This study investigated the seroprevalence of H. capsulatum and sought previously undetected cases of histoplasmosis in Kampala, Uganda. Serum, cerebrospinal fluid (CSF) and/or urine specimens were obtained from HIV-infected persons with suspected meningitis. Specimens were tested for H. capsulatum IgG and IgM by enzyme immune assay and Histoplasma antigen. 147 of the 257 subjects who were enrolled had cryptococcal meningitis. Overall, 1.3% (2/151) of subjects were serum Histoplasma IgG positive, and zero of 151 were IgM positive. Antigen was not detected in any serum (n = 57), urine (n = 37, or CSF (n = 63) samples. Both subjects with serum Histoplasma IgG positivity had cryptococcal meningitis. Histoplasma capsulatum IgG was detected at low levels in persons with HIV/AIDS in Kampala, Uganda. Histoplasmosis is not widespread in Uganda but microfoci do exist. There appears to be no cross-reactivity between Cryptococcus neoformans and Histoplasma antigen testing, and cryptococcosis appears to be at most, a rare cause of positive Histoplasma IgG.

Glycosylation and immunoreactivity of the Histoplasma capsulatum Cfp4 yeast-phase exoantigen.

The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

NMR investigation of the binding between human profilin I and inositol 1,4,5-triphosphate, the soluble headgroup of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is involved in the regulation of the actin cytoskeleton through interactions with a number of actin-binding proteins. We present here NMR titration experiments that monitor the interaction between the cytoskeletal protein profilin and inositol 1,4,5-triphosphate (IP(3)), the headgroup of PI(4,5)P(2). These experiments probe the interaction directly, at equilibrium, and with profilin in its native state. We show the binding between profilin and IP(3) can readily be observed at high concentrations, even though profilin does not bind to IP(3) under physiological conditions. Moreover, the titration data using wild-type profilin and an R88L mutant support the existence of at least three headgroup binding sites on profilin, consistent with previous experimentation with intact PI(4,5)P(2). This work suggests that various soluble inositol ligands can serve as effective probes to facilitate in vitro studies of PI-binding proteins that require membrane surfaces for high-affinity binding.

Development of a highly sensitive and specific blastomycosis antibody enzyme immunoassay using Blastomyces dermatitidis surface protein BAD-1.

Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

High affinity binding to profilin by a covalently constrained, soluble mimic of phosphatidylinositol-4,5-bisphosphate micelles.

Phosphoinositide (PI) lipids are essential regulators of a wide variety of cellular functions. We present here the preparation of a multivalent analogue of a phosphatidylinositol-4,5-bisphosphate (PIP(2)) micelle containing only the polar headgroup portion of this lipid. We show that this dendrimer binds to the cytoskeletal protein profilin with an affinity indistinguishable from that of PIP(2), despite the fact that profilin discriminates between PIP(2) and its monomeric hydrolysis product inositol-1,4,5-triphosphate (IP(3)) under physiological conditions. These data demonstrate that the diacylglycerol (DAG) moiety of PIP(2) is not required for high-affinity binding and suggest that profilin uses multivalency as a key means to distinguish between the intact lipid and IP(3). The class of soluble membrane analogues described here is likely to have broad applicability in the study of protein.PI interactions.