Serotyping of Cryptococcus neoformans by using a monoclonal antibody specific for capsular polysaccharide

The importance of epidemiological studies of cryptococcosis has increased since the beginning of the AIDS epidemic. Cryptococcus neoformans exists in two varieties defining four serotypes, Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). The varieties are easy to distinguish by their differences of growth on diagnostic media. We propose here an easy serotyping method combining diagnostic media and a direct immunofluorescence assay with one monoclonal antibody (E1) specific for cryptococcal polysaccharide. The method was validated by the blinded testing of four to five reference strains of each serotype. Immunofluorescence patterns were characteristic of a given serotype provided that the variety of the strain had been defined before. For C. neoformans var. neoformans, a bright, homogeneous staining with several cell aggregates was characteristics of serotype A, whereas only a few serotype D cells were positive. For C. neoformans var. gattii, a completely negative isolate was serotype C, whereas the population of serotype B included a majority of negative cells but also included positive cells with a speckled pattern. The method was then used to serotype 156 clinical isolates from France and isolates from areas where C. neoformans var. gattii was endemic before the AIDS epidemic (13 strains from Rwanda and Zaire and 5 strains from Australia). The specificity of E1 was defined by its reactivity with various Cryptococcus spp. and analyzed according to the described cryptococcal antigenic factors. We conclude from this study that E1 provides a rapid and reliable means to serotype multiple isolates of C. neoformans.