Previous Article | Next Article ![]()
Journal of Clinical Microbiology, May 2000, p. 1974-1976, Vol. 38, No. 5
Departments of Microbiology and
Immunology1 and
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 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.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Prevalence of Cryptococcus neoformans
var. neoformans (Serotype D) and Cryptococcus
neoformans var. grubii (Serotype A) Isolates in New
York City
![]()
ABSTRACT
Top
Abstract
Text
References
![]()
TEXT
Top
Abstract
Text
References
TABLE 1.
Prevalence of C. neoformans var.
grubii (serotype A) and C. neoformans var.
neoformans (serotype D) in various regions
(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.
| |
ACKNOWLEDGMENTS |
|---|
A.C. is supported by NIH awards AI33774, AI3342, and HL-59842 and is a recipient of a Burroughs Wellcome Fund Scholar Award in Experimental Therapeutics.
| |
FOOTNOTES |
|---|
* 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: casadeva{at}aecom.yu.edu.
| |
REFERENCES |
|---|
|
|
|---|
| 1. |
Bennett, J. E.,
K. J. Kwon-Chung, and D. H. Howard.
1977.
Epidemiologic differences among serotypes of Cryptococcus neoformans.
Am. J. Epidemiol.
105:582-586 |
| 2. |
Bottone, E. J.,
P. A. Kirschner, and I. F. Salkin.
1986.
Isolation of highly encapsulated Cryptococcus neoformans serotype B from a patient in New York City.
J. Clin. Microbiol.
23:186-188 |
| 3. | Bottone, E. J., I. F. Salkin, N. J. Hurd, and G. P. Wormser. 1987. Serogroup distribution of Cryptococcus neoformans in patients with AIDS. J. Infect. Dis. 156:242[Medline]. |
| 4. |
Cleare, W., and A. Casadevall.
1998.
The different binding patterns of two immunoglobulin M monoclonal antibodies to Cryptococcus neoformans serotype A and D strains correlate with serotype classification and differences in functional assays.
Clin. Diagn. Lab. Immunol.
5:125-129 |
| 5. | Criseo, G., and M. Gallo. 1997. Serotyping of Cryptococcus neoformans isolates from environmental and clinical sources in extreme southern Italy (Calabria and Sicily central Mediterranean area). Mycoses 40:95-100[Medline]. |
| 6. | Currie, B. P., and A. Casadevall. 1994. Estimation of the prevalence of cryptococcal infection among patients infected with the human immunodeficiency virus in New York City. Clin. Infect. Dis. 19:1029-1033[Medline]. |
| 7. | Dromer, F., S. Mathoulin, B. Dupont, and A. Laporte. 1996. Epidemiology of cryptococcosis in France: a 9-year survey (1985-1993). Clin. Infect. Dis. 23:82-90[Medline]. |
| 8. | Dromer, F., S. Mathoulin, B. Dupont, L. Letenneur, and O. Ronin. 1996. Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D. Clin. Infect. Dis. 23:91-96[Medline]. |
| 9. |
Ellis, D. H.
1987.
Cryptococcus neoformans var. gattii in Australia.
J. Clin. Microbiol.
25:430-431 |
| 10. |
Ellis, D. H., and T. J. Pfeiffer.
1990.
Natural habitat of Cryptococcus neoformans var. gattii.
J. Clin. Microbiol.
28:1642-1644 |
| 11. | Evans, E. D., and J. F. Kessel. 1951. The antigenic composition of Cryptococcus neoformans. J. Immunol. 67:109-114. |
| 12. | Evans, E. F. 1949. An immunologic comparison of twelve strains of Cryptococcus neoformans (Torula histolytica). Proc. Soc. Exp. Biol. Med. 71:644-646[CrossRef][Medline]. |
| 13. |
Franzot, S. P.,
I. F. Salkin, and A. Casadevall.
1999.
Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates.
J. Clin. Microbiol.
37:838-840 |
| 14. | Hsu, M. M., J. C. Chang, K. Yokoyama, K. Nishimura, and M. Miyaji. 1994. Serotypes and mating types of clinical strains of Cryptococcus neoformans isolated in Taiwan. Mycopathologia 125:77-81[CrossRef][Medline]. |
| 15. |
Ikeda, R.,
T. Shinoda,
Y. Fukazawa, and L. Kaufman.
1982.
Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates.
J. Clin. Microbiol.
16:22-29 |
| 16. | Kohno, S., A. Varma, K. J. Kwon-Chung, and K. Hara. 1994. Epidemiology studies of clinical isolates of Cryptococcus neoformans of Japan by restriction fragment length polymorphism. Kansenshogaku Zasshi 68:1512-1517[Medline]. |
| 17. | Kwon-Chung, K. J. 1975. A new genus, filobasidiella, the perfect state of Cryptococcus neoformans. Mycologia 67:1197-1200. |
| 18. | Kwon-Chung, K. J. 1976. A new species of Filobasidiella, the sexual state of Cryptococcus neoformans B and C serotypes. Mycologia 68:943-946. |
| 19. |
Kwon-Chung, K. J., and J. E. Bennett.
1984.
Epidemiologic differences between the two varieties of Cryptococcus neoformans.
Am. J. Epidemiol.
120:123-130 |
| 20. | Kwon-Chung, K. J., J. E. Bennett, and J. C. Rhodes. 1982. Taxonomic studies on Filobasidiella species and their anamorphs. Antonie Leeuwenhoek 48:25-38[CrossRef][Medline]. |
| 21. |
Kwon-Chung, K. J.,
I. Polacheck, and J. E. Bennett.
1982.
Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C).
J. Clin. Microbiol.
15:535-537 |
| 22. |
Mishra, S. K.,
F. Staib,
U. Folkens, and R. A. Fromtling.
1981.
Serotypes of Cryptococcus neoformans strains isolated in Germany.
J. Clin. Microbiol.
14:106-107 |
| 23. |
Mitchell, T. G., and J. R. Perfect.
1995.
Cryptococcosis in the era of AIDS 100 years after the discovery of Cryptococcus neoformans.
Clin. Microbiol. Rev.
8:515-548[Abstract].
|
| 24. | Padhye, A. A., A. Chakrabarti, J. Chander, and L. Kaufman. 1993. Cryptococcus neoformans var. gattii in India. J. Med. Vet. Mycol. 31:165-168[Medline]. |
| 25. | Poonwan, N., Y. Mikami, S. Poosuwan, J. Boon-Long, N. Mekha, M. Kusum, K. Yazawa, R. Tanaka, K. Nishimura, and K. Konyama. 1997. Serotyping of Cryptococcus neoformans strains isolated from clinical specimens in Thailand and their susceptibility to various antifungal agents. Eur. J. Epidemiol. 13:335-340[CrossRef][Medline]. |
| 26. | Rozenbaum, R., A. J. Goncalves, B. Wanke, M. J. Caiuby, H. Clemente, M. dos S. Lazera, P. C. Monteiro, and A. T. Londero. 1992. Cryptococcus neoformans varieties as agents of cryptococcosis in Brazil. Mycopathologia 119:133-136[CrossRef][Medline]. |
| 27. |
Salkin, I. F., and N. J. Hurd.
1982.
New medium for differentiation of Cryptococcus neoformans serotype pairs.
J. Clin. Microbiol.
15:169-171 |
| 28. | Sukroongreung, S., C. Nilakul, O. Ruangsomboon, W. Chuakul, and B. Eampokalap. 1996. Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand. Mycopathologia 135(2):75-78[CrossRef][Medline]. |
| 29. | Tortorano, A. M., M. A. Viviani, A. L. Rigoni, M. Cogliati, A. Roverselli, and A. Pagano. 1997. Prevalence of serotype D in Cryptococcus neoformans isolates from HIV positive and HIV negative patients in Italy. Mycoses 40:297-302[Medline]. |
| 30. | Villanueva, E., M. Mendoza, E. Torres, M. B. de Albornoz, M. E. Cavazza, and G. Urbina. 1989. Serotyping of 27 Cryptococcus neoformans strains isolated in Venezuela. Acta Cient. Venez. 40:151-154[Medline]. (In Spanish.) |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»