CrAg lateral flow assay for cryptococcosis.

UNLABELLED: Importance of field: Cryptococcal meningitis is a leading cause of death globally among people with AIDS. In sub-Saharan Africa, cryptococcosis is estimated to kill more people than tuberculosis. Cryptococcosis is also an important infectious disease among immunosuppressed patients in countries with advanced medical care. Early diagnosis is the key to effective treatment, particularly in patients in resource-limited settings. A new lateral flow immunoassay (LFA) for cryptococcal antigen (CrAg) allows for rapid and inexpensive diagnosis of cryptococcosis at or near the point of patient contact. AREAS COVERED: This article reviews the need for improved diagnostics for cryptococcal meningitis and describes the features of an ideal diagnostic. The design of a new LFA for CrAg is described as well as the results of initial clinical evaluation of the CrAg LFA. EXPERT OPINION: The CrAg LFA is recommended for use with serum, plasma or CSF for diagnosis of cryptococcal meningitis or non-meningeal cryptococcal disease in symptomatic patients. There is a need for further evaluation of LFA for screening of asymptomatic patients. However, the LFA is emerging as a valuable tool for screening of serum or plasma in ART-naive adults with CD4 counts less than 100 cells/mm(3) in geographic regions with a high prevalence of cryptococcal antigenemia. CrAg screening has the potential to identify patients with asymptomatic cryptococcal infection who should receive preemptive antifungal therapy.

Performance characteristics of a polyclonal enzyme immunoassay for the quantitation of Histoplasma antigen in human urine samples.

An antigen detection assay was prepared with rabbit anti- Histoplasma antibodies to detect and quantitate Histoplasma capsulatum antigen in urine samples. By using a 4-parameter curve fit, the assay calibration ranges from 2 to 1,000 enzyme immunoassay (EIA) units. We compared results for 99 urine samples with those of a reference laboratory, half of which tested positive or equivocal by that reference laboratory. Performance characteristics were further defined by studying the assay linearity, precision, percentage of positive agreement, and percentage of negative agreement. An acceptable correlation with the reference laboratory (R2=0.7174) was obtained with results ranging from less than 2 (negative samples) to 132 EIA units by the new method. Compared with the reference laboratory, the percentage of agreement for positive samples, excluding equivocal samples, was 92% and for negative samples was 98%. Crossreactivity occurred with culture filtrates of Paracoccidioides brasiliensis, Coccidioides immitis, and Blastomyces dermatiditis. No cross-reactivity was observed with Candida albicans or Aspergillus fumigatus culture filtrates. The current EIA for the detection and quantitation of H capsulatum antigen in urine specimens is a valid assay that agrees well with results from an established reference laboratory.

Effects of tumor necrosis factor alpha on dendritic cell accumulation in lymph nodes draining the immunization site and the impact on the anticryptococcal cell-mediated immune response.

Cell-mediated immune (CMI) responses and tumor necrosis factor alpha (TNF-alpha) have been shown to be essential in acquired protection against Cryptococcus neoformans. Induction of a protective anticryptococcal CMI response includes increases in dendritic cells (DC) and activated CD4(+) T cells in draining lymph nodes (DLN). During the expression phase, activated CD4(+) T cells accumulate at a peripheral site where cryptococcal antigen is injected, resulting in a classical delayed-type hypersensitivity (DTH) reaction. Induction of a nonprotective anticryptococcal CMI response results in no significant increases in the numbers of DC or activated CD4(+) T cells in DLN. This study focuses on examining the role of TNF-alpha in induction of protective and nonprotective anticryptococcal CMI responses. We found that neutralization of TNF-alpha at the time of immunization with the protective immunogen (i) reduces the numbers of Langerhans cells, myeloid and lymphoid DC, and activated CD4(+) T cells in DLN and (ii) diminishes the total numbers of cells, the numbers of activated CD4(+) T cells, and amount of gamma interferon at the DTH reaction site. Although TNF-alpha neutralization during induction of the nonprotective CMI response had little effect on cellular and cytokine parameters measured, it did cause a reduction in footpad swelling when mice received challenge in the footpad. Our findings show that TNF-alpha functions during induction of the protective CMI response by influencing the accumulation of all three DC subsets into DLN. Without antigen stimulated DC in DLN, activated CD4(+) T cells are not induced and thus not available for the expression phase of the CMI response.

Differences in components at delayed-type hypersensitivity reaction sites in mice immunized with either a protective or a nonprotective immunogen of Cryptococcus neoformans.

Cell-mediated immunity is the major protective mechanism against Cryptococcus neoformans. Delayed swelling reactions, i.e., delayed-type hypersensitivity (DTH), in response to an intradermal injection of specific antigen are used as a means of detecting a cell-mediated immune (CMI) response to the antigen. We have found previously that the presence of an anticryptococcal DTH response in mice is not always indicative of protection against a cryptococcal infection. Using one immunogen that induces a protective anticryptococcal CMI response and one that induces a nonprotective response, we have shown that mice immunized with the protective immunogen undergo a classical DTH response characterized by mononuclear cell and neutrophil infiltrates and the presence of gamma interferon and NO. In contrast, immunization with the nonprotective immunogen results in an influx of primarily neutrophils and production of tumor necrosis factor alpha (TNF-alpha) at the DTH reaction site. Even when the anticryptococcal DTH response was augmented by blocking the down-regulator, CTLA-4 (CD152), on T cells in the mice given the nonprotective immunogen, the main leukocyte population infiltrating the DTH reaction site is the neutrophil. Although TNF-alpha is increased at the DTH reaction site in mice immunized with the nonprotective immunogen, it is unlikely that TNF-alpha activates the neutrophils, because the density of TNF receptors on the neutrophils is reduced below control levels. Uncoupling of DTH reactivity and protection has been demonstrated in other infectious-disease models; however, the mechanisms differ from our model. These findings stress the importance of defining the cascade of events occurring in response to various immunogens and establishing the relationships between protection and DTH reactions.