JOURNAL OF CLINICAL MICROBIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
May 2000, p. 1974–1976Vol. 38, No. 5
Prevalence of Cryptococcus neoformans var. neoformans
(Serotype D) and Cryptococcus neoformans var. grubii
(Serotype A) Isolates in New York City
JUDITH N. STEENBERGEN1AND ARTURO CASADEVALL1,2*
Departments of Microbiology and Immunology1and Medicine,2
Albert Einstein College of Medicine, Bronx, New York 10461
Received 21 December 1999/Returned for modification 25 January 2000/Accepted 15 February 2000
Analysis of 40 New York City Cryptococcus neoformans isolates revealed that 39 were typeable, of which 85 and
12.5% were Cryptococcus neoformans var. grubii (serotype A) and Cryptococcus neoformans var. neoformans
(serotype D), respectively. The prevalence of serotype D isolates in New York City appears to be significantly
higher than indicated by previous studies of North American isolates.
Cryptococcus neoformans is an encapsulated yeast that can
cause life-threatening meningitis in immunocompromised pa-
tients (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 de-
tected 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 caus-
ative agent of cryptococcosis in patients afflicted with AIDS or
immunocompromised in some other way (3).
There is increasing evidence that there are clinical differ-
ences 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 sero-
type 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 require-
ments for the degree of doctor of philosophy in the Sue Gold-
ing Graduate Division of Medical Sciences, Albert Einstein
College of Medicine, Yeshiva University, Bronx, N.Y.)
The lab stock contained 40 clinical strains that were ob-
tained 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 con-
tinuous 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, 106formaldehyde-
killed cells were immobilized on polylysine-coated slides (Sig-
ma, St. Louis, Mo.) and incubated for 2 h at room temperature
with 10 ?g of either MAb 12A1 or 13F1 per ml. The cells were
washed with PBS and incubated with fluorescein isothiocya-
nate-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 iso-
lates (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 immunofluo-
* Corresponding author. Mailing address: Albert Einstein College
of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718)
430-4259. Fax: (718) 430-8701. E-mail: firstname.lastname@example.org.
rescence results, serotypes were confirmed using an agglutina-
tion 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 aggluti-
nated 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 immunofluores-
cence 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 immunofluores-
cence 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. neofor-
mans 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 geograph-
ical 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.
A.C. is supported by NIH awards AI33774, AI3342, and HL-59842
and is a recipient of a Burroughs Wellcome Fund Scholar Award in
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aThe sum of percentages for is ?100 because those studies also report sero-
types B and C.
TABLE 2. New York City isolate serotyping results
Pattern with MAb
VOL. 38, 2000 NOTES1975
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1976 NOTESJ. CLIN. MICROBIOL.