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Cryptococcus neoformans is an encapsulated yeast that can cause life-threatening meningitis in immunocompromised patients (23). Based on biochemical, morphological, and genetic characteristics, C. neoformans was originally divided into two varieties: Cryptococcus neoformans var. gattii (serotypes B and C) and Cryptococcus neoformans var. neoformans (serotypes A and D) (17, 18, 20). These two varieties can be distinguished by biochemical tests (21, 27). Recently a proposal was made to further subdivide the C. neoformans var. neoformans strains into two varieties: C. neoformans var. neoformans (serotype D) and Cryptococcus neoformans var. grubii (serotype A) (13). The serotype classification is based on antigenic differences detected with rabbit adsorbed sera (11, 12, 15). C. neoformans var. gattii usually infects patients with normal immune status living in tropical and subtropical areas (9, 10). In contrast, C. neoformans var. neoformans and C. neoformans var. grubii are distributed throughout the world (1) and are usually the causative agent of cryptococcosis in patients afflicted with AIDS or immunocompromised in some other way (3).

There is increasing evidence that there are clinical differences between serotype A and serotype D infections (7, 8). For example, serotype D infections are more likely to result in skin involvement and afflict older patients (8). The prevalence of serotype D among clinical isolates has ranged from 0 to 100% depending on the region of the world in which samples were taken (Table 1). The majority of North American isolates pooled from various geographic locations have been reported to be serotype A isolates (Table 1). New York City has been a major site of cryptococcal infections in the United States, and in the early 1990s the prevalence of cryptococcal infection in that city alone was more than 1,000 cases per year (6). The majority of clinical isolates in New York City belong to serotype A or serotype D. Serotype B strains have occasionally been isolated in New York City (2). A previous study of a small number of isolates revealed no serotype D isolates in New York State (1). To our knowledge the prevalence of serotype A and serotype D in New York City has not been determined. In this study we characterized various New York City clinical isolates of C. neoformans by two methods and compared the results to those in literature studies.

(The data in this paper are from a thesis to be submitted by Judith N. Steenbergen in partial fulfillment of the requirements for the degree of doctor of philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.)

The lab stock contained 40 clinical strains that were obtained from cryptococcal meningitis patients between 1991 and 1999 in two New York City hospitals, Jacobi Medical Center (J strains) and Montefiore Medical Center (M strains) (Table 2). Strain 24067 was obtained from the American Type Culture Collection (Manassas, Va.), and MY2061 was obtained from Merck & Co., Inc. (Whitehouse Station, N.J.). These two strains were used as controls for serotype A and serotype D, respectively. Serotyping was done by factor sera agglutination with the Crypto-Check kit (Iatron Inc., Tokyo, Japan) and indirect immunofluorescence with monoclonal antibody (MAb) 13F1 (4; W. Cleare, M. E. Brandt, and A. Casadevall, Letter, J. Clin. Microbiol. 37:3080, 1999). All samples were prepared simultaneously to avoid variation in growth conditions. The isolates were grown on Sabouraud dextrose (SAB) broth (Difco Laboratories, Detroit, Mich.) agar for 48 h at 30°C. A single colony from each isolate was used to inoculate 10 ml of SAB broth. The SAB broth cultures were incubated with continuous shaking for 72 h to reach stationary phase. Stationary-phase cultures were washed in phosphate-buffered saline (PBS) (0.137 M NaCl, 0.003 M sodium phosphate [pH 7.4]) three times and fixed in 2.5% formaldehyde overnight. For indirect-immunofluorescence serotyping, 10⁶ formaldehyde-killed cells were immobilized on polylysine-coated slides (Sigma, St. Louis, Mo.) and incubated for 2 h at room temperature with 10 µg of either MAb 12A1 or 13F1 per ml. The cells were washed with PBS and incubated with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin M (Southern Biotechnology, Birmingham, Ala.) for 1 h at room temperature in the dark. Cells were washed in PBS and mounted on the polylysine slides using a solution of 50% glycerol-0.1 M n-propyl gallate (Sigma) in PBS. The slides were viewed with a fluorescein isothiocyanate filter-equipped Zeiss (Thornwood, N.Y.) Axiophot microscope, and serotypes were determined based on binding patterns as described previously (4; Cleare et al., letter). MAb 13F1 produces a punctate pattern on serotype D strains and an annular pattern on serotype A and AD isolates (4; Cleare et al., letter). MAb 12A1 produces an annular pattern on serotype A, D, and AD strains and was used as a control for distinguishing punctate and annular patterns (4; Cleare et al., letter). Without knowledge of the immunofluorescence results, serotypes were confirmed using an agglutination assay kit (Iatron Inc.). The agglutination patterns were analyzed as follows: serotype A strains agglutinated with both factors 1 and 7, and serotype D strains agglutinated with both factors 1 and 8, as per the manufacturer's instructions.

Table 2 shows the distribution of serotype A and serotype D in the 40 clinical New York City isolates. All isolates agglutinated rapidly with the Iatron Crypto-Check sera. Both control strains yielded the expected results: MY2061 was classified as C. neoformans var. grubii (serotype A) and strain 24067 was classified as C. neoformans var. neoformans (serotype D). Of the 40 samples, 33 were serotype A and 5 were serotype D. One strain, J50, did not agglutinate with either serum 7 or 8 and was not typeable using this method. Strain J3 agglutinated with both Iatron sera 7 and 8 and is therefore serotype AD. To confirm the agglutination results, indirect immunofluorescence analysis was performed using both the 12A1 and the 13F1 MAbs (Table 2). It is important to use both 12A1 and 13F1 antibodies on each strain because capsular differences between strains cause slight variations in binding. All strains grouped as serotype A by the Crypto-Check method produced annular fluorescence when stained with MAb 13F1. Strains grouped as serotype D produced punctate fluorescence when stained with MAb 13F1. The J3 strain produced annular immunofluorescence with MAb 13F1, consistent with the prior observation that MAb 13F1 produces annular binding on AD strains (4; Cleare et al., letter). Strain J50 could not be serotyped by the Crypto-Check method and produced annular immunofluorescence with MAb 13F1, suggesting that it may be an atypical serotype A isolate or an AD isolate.

In summary, of the 39 typeable strains, 85% were C. neoformans var. grubii (serotype A), 12.5% were C. neoformans var. neoformans (serotype D), and 2.5% were serotype AD. The percentage of serotype D strains in New York City was twice that reported in prior studies of North American isolates (1, 19). The occurrence of regional variability is illustrated by reports from northern (29) and southern Italy (5) in 1997 which revealed that the prevalence of serotype D isolates was 71 and 0%, respectively. The factors responsible for geographical variation in the prevalence of serotype A and serotype D are not understood. The relatively high prevalence of serotype D strains in New York City, combined with the variability in prior studies (Table 1), suggests a need for detailed regional surveys to ascertain the distribution of serotypes in various parts of the world.