Rapid presumptive identification of Cryptococcus neoformans

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Abstract
Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 C. neoformans isolates, 29 isolates of other Cryptococcus spp., 16 isolates of Candida spp., 2 Rhodotorula spp., 2 Torulopsis spp. and 1 Saccharomyces spp. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5 and 10 .mu.g/ml. Two-fold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained < 5 .mu.g of carbohydrate/ml, was suitable for a single rapid screening dilution and eliminated any cross-reaction from the C. laurentii isolates. This method provided a reliable differentiation of C. neoformans from other unknown yeast colonies in < 20 min exclusive of a Molisch determination.