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Journal of Clinical Microbiology, October 1999, p. 3204-3209, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Epidemiological Evidence for Dormant Cryptococcus
neoformans Infection
Dea
Garcia-Hermoso,
Guilhem
Janbon,* and
Françoise
Dromer
Unité de Mycologie, Institut Pasteur,
Paris, France
Received 13 May 1999/Returned for modification 28 June
1999/Accepted 22 July 1999
 |
ABSTRACT |
To date, the time of acquisition of a Cryptococcus
neoformans infectious strain has never been studied. We selected
a primer, (GACA)4, and a probe, CNRE-1, that by randomly
amplified polymorphic DNA (RAPD) analysis and restriction fragment
length polymorphism (RFLP), respectively, regrouped strains from
control samples of C. neoformans var. grubii
environmental isolates according to their geographical origins. The two
typing techniques were then used to analyze 103 isolates from 29 patients diagnosed with cryptococcosis in France. Nine of the 29 patients lived in Africa a median of 110 months prior to moving to
France; 17 of the patients originated from Europe. Results showed a
statistically significant clustering of isolate subtypes from patients
originating from Africa compared to those from Europe. We conclude that
the patients had acquired the C. neoformans infectious
strain long before their clinical diagnoses were made.
| |
INTRODUCTION |
Cryptococcus neoformans
is a ubiquitous and opportunistic yeast that causes life-threatening
meningoencephalitis in 3 to 30% of patients with AIDS (24).
This encapsulated basidiomycete exists in three varieties: C. neoformans var. grubii (serotype A) (14) and
C. neoformans var. neoformans (serotype D), both with worldwide distributions, as well as C. neoformans var.
gattii (serotypes B and C), which is limited to tropical and
subtropical regions (21). Cryptococcosis, like many other
fungal infections, is thought to begin with inhalation of airborne
fungi from an environmental source. Basidiospores, which are smaller,
more easily aerosolized, and much more resistant to desiccation than
yeast cells, are most likely to be the infectious particles (22,
35). It has been reported that this yeast's most important
natural source is weathered pigeon droppings or soil contaminated with avian guano (11, 12). Data reported in the literature
indicate that C. neoformans can be found as a transient
commensal organism on humans or as an incidental colonizer in the
respiratory tract or on the skin of healthy subjects or even patients
with bronchopulmonary disorders (19, 26).
Several observations converge toward the hypothesis that the infectious
particles can be acquired long before the infection develops and is
diagnosed. First, a high percentage of healthy subjects have
anticryptococcal antibodies, which suggests prior contact with the
fungus (7, 18). Second, patients coming from tropical areas
can be diagnosed with C. neoformans var. gattii cryptococcosis long after they have left their countries of origin (8). Finally, unlike French patients, African patients
living in France and diagnosed with cryptococcosis are rarely infected with C. neoformans var. neoformans strains
(10). To verify this hypothesis, a well-characterized group
of patients and a molecular method able to distinguish between isolates
of the same serotype but from different geographical regions should be
selected. The molecular typing methods currently available are reported
to be unable to regroup Cryptococcus neoformans var.
grubii isolates by their geographical origins (4, 5,
33). On the other hand, Kwon-Chung and Bennett, using standard
immunological methods of serotyping, have described a nonrandom
distribution of serotypes around the world (21). Although
serotyping is not sufficiently discriminative to determine the
geographical origin of a cryptococcal isolate, it provides good
evidence that a technique capable of clustering strains from the same
geographical region might exist.
In this study, we addressed the question of the time of acquisition of
the infecting organism, an issue that has never before been raised.
Using control samples of environmental isolates and two typing methods
capable of clustering strains based on their geographical origins, we
were able to demonstrate that patients diagnosed with cryptococcosis in
France but born in Africa had acquired their infectious strains a long
time ago, prior to emigrating from their countries of origin.
| |
MATERIALS AND METHODS |
Patients and strains.
Twenty environmental isolates of
C. neoformans var. grubii from different
geographical regions were used in this study: Japanese isolates J1, J2,
J3, J4, and J5 were kindly provided by S. Kohno (Nagasaki University
School of Medicine, Nagasaki, Japan) as M12, SH1311, MT11, SUMO1, and
SASO1, respectively (36); African isolates AF1 (Morocco),
AF2 (Togo), AF3 (Ivory Coast), AF4 (Burundi), and AF5 (Zimbabwe) were
provided by D. Swinne (Institute of Tropical Medicine, Antwerp,
Belgium) as RV45718, RV45880, RV46288, RV67312, and RV70273,
respectively; American isolates US1, US2, and US3 (Kentucky) as well as
US4 (New York) were provided by J. M. Clauson (Western Kentucky
University, Bowling Green) and A. Casadevall (Albert Einstein College
of Medicine, Bronx, N.Y.) as FE-1, PE-1, SSE-1, and B5 respectively
(6); and French isolates F1 through F6 were provided by S. Mathoulin and B. Couprie (Centre Hospitalo-Universitaire, Bordeaux,
France) as 115A, 57B, 109B, 13A, 110B, and 122A, respectively (16).
A total of 103 clinical C. neoformans var. grubii
isolates were recovered from 29 patients who had been diagnosed with
cryptococcosis in France and whose infections had been reported to the
National Reference Center for Mycoses during the first year (1997) of a multicentric clinical study, étude Crypto A/D (Direction
Générale de la Santé no. 970089). Detailed
information on clinical and epidemiological issues (particularly the
patients' trips and stays since childhood) and on all of the isolates
recovered at the time of diagnosis and during the course of the
infection were collected. Among the 29 patients, 17 had been born in
Europe and 9 had been born in Africa (see Table 1). The African
patients had been living in France for a median of 110 months before
cryptococcosis was diagnosed. The last trip back to Africa had occurred
as long as 13 years ago (patient P17).
The identification of all cultured organisms as
C. neoformans was confirmed by standard biochemical methods. Isolates
were
identified as
C. neoformans var.
grubii by
the use of canavanine-glycine-bromothymol
medium,
D-proline
assimilation, and a direct immunofluorescence
assay using a monoclonal
antibody (
9). All strains were stored
frozen in 40%
glycerol at
80°C and were grown overnight in YPD
medium (5 g of
yeast extract, 10 g of Bacto Peptone, and 10 g
of glucose per
liter) at 30°C.
Randomly amplified polymorphic DNA (RAPD) analysis.
C.
neoformans DNA was extracted as previously described
(31). The following primers were chosen from the literature
and tested for their discriminatory power on 20 environmental isolates under various annealing temperatures: (CA)8 RY
(25), (GTG)5, (GACA)4
(23), the phage M13 core sequence (27), and two
enterobacterial repetitive intergenic consensus sequences, ERIC1 and
ERIC2 (2). PCR was carried out in a thermal cycler
(Omnigene; Hybaid, Teddington, United Kingdom) in 100-µl reaction
volumes, each containing 50 ng of genomic DNA, 50 pmol of primer, 200 µM deoxynucleoside triphosphates, and 2 U of recombinant
Taq polymerase (Pharmacia Biotech, Uppsala, Sweden), with
the manufacturer's recommended buffers.
Two primers, ERIC1 and (GACA)
4, were then selected and used
to study clinical isolates under the following optimized conditions:
reactions were cycled 35 times, with a 4-min denaturation at 94°C,
1 min of annealing each at 28 and 48°C, and a 2-min primer extension
at
74°C. Amplification products were analyzed by electrophoresis
through
a 2% agarose gel and visualized under UV light after being
stained
with ethidium bromide. The reproducibility of this method
was confirmed
by reanalyzing another DNA preparation from five
clinical isolates. In
every case, identical profiles were obtained
(data not
shown).
RFLP analysis.
Restriction fragment length polymorphisms
(RFLPs) were detected by Southern blot hybridization after restriction
enzyme SstI digestion of total-DNA samples. The restriction
fragments obtained were then separated by electrophoresis through a
0.8% agarose gel and transferred onto positively charged nylon
membranes (Boehringer Mannheim, Mannheim, Germany). The DNA probe
(C. neoformans repetitive element CNRE-1), generously
provided by E. Spitzer and S. Spitzer (28), was labeled with
DIG digoxigenin-11-dUTP by using a DIG-High Prime Kit (Boehringer
Mannheim). After an overnight hybridization at 68°C and stringent
washes, bands were detected and exposed according to the
manufacturer's instructions. Reproducibility was confirmed by
reanalyzing another DNA preparation from five clinical isolates (data
not shown).
Data analysis.
DNA fingerprint patterns were analyzed by
using the software Taxotron, developed by P. D. Grimont (Institut
Pasteur, Paris, France) (17), which automatically identified
band positions and compared two profiles by calculating the Dice
coefficient complement (number of different bands per total number of
fragments in the two profiles). Dendograms were then generated by using the unweighted pair group method of average linkage (17).
Statistical analysis.
The distributions of the patients'
isolates by subtype according to the European or African origin were
analyzed by using Fisher's exact test.
| |
RESULTS |
Selection of a typing method for environmental isolates.
We
tested different typing methods to evaluate their abilities to cluster
environmental C. neoformans var. grubii strains according to their geographical origins. Because RFLP is known to be
reproducible and easy to perform, we first tested the ability of CNRE-1
to geographically classify the isolates. Figure
1 shows the Taxotron-derived schematic
representation of the RFLP profiles obtained after hybridization of
CNRE-1 to SstI-digested genomic DNA. Fifteen different
profiles were obtained for the 17 strains tested. Two Japanese (J5 and
J4) and two African (AF2 and AF1) isolates yielded identical
hybridization profiles with this probe. Partial clustering of the
Japanese (four of five) and African (three of five) isolates was
obtained, but no specific profile could be associated with a given
region. Therefore, we tested another molecular technique to determine
it could further differentiate isolates based on their geographical
origins.
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FIG. 1.
Schematic representation of and dendogram generated from
the Dice coefficient complement computed from the CNRE-1 patterns of
environmental isolates from Japan (J), France (F), the United States
(US), and Africa (AF).
|
|
Previous studies using RAPD techniques demonstrated geographical
clustering among isolates of
C. neoformans var.
gattii (
2,
27). RAPD analysis has been applied in
several epidemiological
studies of
C. neoformans; however,
some questions have been raised
concerning the interpretation of the
profiles generated. The advantages
RAPD offers over other methods,
particularly speed and ease of
execution, made it suitable for
investigation of the ability to
discriminate between
C. neoformans var.
grubii isolates according
to their
geographical
origins.
Six different primers previously used in some epidemiological studies
of
C. neoformans were chosen. Using different amplification
temperatures, they were tested on the control group of 17 environmental
isolates to which 3 more environmental isolates from France were
added.
Only primers ERIC1 and (GACA)
4 revealed roughly the same
patterns for strains coming from the same geographical region
and
different patterns for strains coming from different continents.
Representative RAPD profiles obtained with the primer
(GACA)
4 are shown in Fig.
2.
Using this primer, all five Japanese isolates
had profile II, five of
the six French isolates exhibited profile
I, and four of the five
African isolates gave profile V or VI.
Of the four North American
isolates, two exhibited profile I and
two showed profile II.
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FIG. 2.
Representative RAPD profiles of C. neoformans
isolates generated with the (GACA)4 primer. Profile
identifications are indicated as roman numerals (I through VI).
Profiles V and VI were obtained only with environmental isolates.
Molecular size standards were obtained with the 1-kb ladder (Gibco BRL,
Gaithersburg, Md.).
|
|
Evaluation of clinical isolates with the selected techniques.
One hundred and three C. neoformans var. grubii
clinical isolates were then analyzed with the two primers described
above. Four different profiles were obtained with the primer ERIC1.
However, no association between a given profile and the corresponding
patient's continent of origin could be established (data not shown).
Typing of the clinical isolates with primer (GACA)4
revealed four different RAPD profiles, whose distributions are reported
in Table 1. All of the strains exhibiting
profile III were isolated from European patients. Of the 15 patients
with profile I strains, 11 were born in Europe. None of the strains
generating profile II or IV were isolated from patients born in Europe.
It is important to note that all of the isolates recovered from a
particular patient gave the same profile with both primers. Thus, the
analysis of clinical isolates showed that the distribution of profiles
according to the geographical origin of the patients was not random.
This bias of distribution was statistically significant (P < 0.0005) when the European and African patients were being
compared (Table 2). None of the clinical
isolates tested yielded profile V or VI, both of which were
characteristic of environmental African isolates. On the other hand,
profiles III and IV were specific to clinical isolates in this study.
Finally, profile II, which was characteristic of the Japanese
environmental isolates, was generated mostly by strains isolated from
African patients. These results might be explained by the facts that
environmental and clinical African strains did not come from the same
country and that no Japanese patients were included in our study.
Moreover, the discriminatory power of this RAPD method was low.
We then tested the 103 strains with the CNRE-1 probe. As previously
described in other reports (
29,
32), we found that
isolates
from the same patient showed identical hybridization
patterns, although
there were a few examples of microevolution
(
15,
30). Thus,
for the remainder of the analysis, one representative
isolate from each
patient was chosen, and their hybridization
patterns are presented in
Fig.
3; 28 different profiles were
obtained
from 29 isolates studied.
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|
FIG. 3.
Schematic representation of and dendogram generated from
the Dice coefficient complement computed from the CNRE-1 patterns of 29 clinical isolates. African strains (in boldface) are regrouped in
clusters A and B (boxed in this figure). DRC, Democratic Republic of
Congo.
|
|
The Taxotron software analysis generated a dendogram in which two boxed
clusters, named A and B, can be seen (Fig.
3). Cluster
A contained the
seven strains with the profile II subtype as determined
by the
(GACA)
4 RAPD technique. Five of these strains were isolated
from patients born in Africa, one was from a patient born in Colombia,
and one was from a patient born in Cambodia. None of the strains
from
this cluster was isolated from a European patient. Cluster
B contained
four strains which were all the profile I subtype,
as determined by the
(GACA)
4 RAPD technique. Three of them were
isolated from
patients born in Africa, and one was from a patient
born in Europe. The
strain from the African patient P29 seemed
to be completely different
from the others. It was also the only
strain for which the profile IV
subtype was generated by the RAPD
technique.
| |
DISCUSSION |
After analyzing the epidemiology of cryptococcosis in France
(10), we were especially interested in learning more about the pathophysiology of this infection. An important issue was the time
of acquisition of the infecting isolate compared to the time of
diagnosis: were the infectious particles inhaled daily and killed as
long as host defense mechanisms were efficient, or, on the contrary,
would the immune system normally achieve local control without
eradication? Since this latter mechanism has already been evoked or
demonstrated for other microorganisms, such as Leishmania
spp. (1) and Histoplasma capsulatum
(34), and since several lines of evidence suggest that it
could also occur with C. neoformans, we looked for a way to
verify this hypothesis. To do so required control samples composed of
environmental isolates from remote areas (to ascertain their
geographical origin), clinical isolates recovered from patients whose
travels and clinical histories were known, and the assessment of
various typing methods since none has yet been able to correlate
geographical origin with a specific pattern.
Indeed, several groups have studied the molecular epidemiology of
C. neoformans infections and attempted to regroup isolates based on their geographical origins. However, the genetic
differentiation generated by CNRE-1 RFLP analysis showed no
geographical correlation among strains isolated from Brazil and the
United States (13). Using UT-4p, Varma and colleagues
reported no obvious clustering of the C. neoformans var.
grubii isolates according to geographical origin
(33), although Garcia-Hermoso and coworkers evoked the possibility of geographical clustering (16) when they
compared the patterns obtained for French isolates to those observed by Varma et al. (33) and Kohno (20). Finally, using
the multilocus enzyme electrophoresis technique, Brandt and colleagues
found that some subtypes were more common in some areas of the United States than in others; however, that finding was not confirmed when
another typing method (RAPD) was used (3). In light of our
results, we think that the lack of clear-cut regional differences in
the previously published studies may be due to the population sample
from which the clinical isolates were recovered (with its lack of
patients coming from remote places and with known travel histories),
the lack of environmental isolates from remote areas, and/or the
technique selected. Indeed, our data show that depending on the sample
chosen (environmental or clinical isolates) and the technique tested
(six primers selected for RAPD or CNRE-1 RFLP), we could have concluded
that either there was or was not a geographical clustering of isolates
and that the isolates tested exhibited or did not exhibit genetic
variability. These discrepancies clearly demonstrate the importance of
the sample choice and the technique selected to answer a specific
epidemiological question. To address the question of geographical
clustering, we needed an epidemiological tool with adequate
discriminatory power: not too high (to prevent further strain
delineation among isolates from the same geographical origin) and not
too low (to enable the differentiation of the isolates based on their
geographical origin). The RAPD method using the primer
(GACA)4 fulfilled this requirement.
Based on the RAPD profiles obtained, we showed that the distribution of
clinical isolates from nine African patients diagnosed with
cryptococcosis in France was significantly different from that of
clinical isolates recovered from the 17 European patients (P < 0.0005). Furthermore, a second, independent typing method (CNRE-1 RFLP) confirmed the results, showing two clusters that contained the isolates from eight of the nine African patients. This
finding suggests that the infecting organism can be acquired long
before the infection develops, since these patients had been living in
France a median of 110 months and had not been in contact with an
African environment for as long as 13 years. That African patients were
infected with African isolates strongly suggests that these isolates
had been sequestered and contained somewhere in the body, most likely
the alveolar macrophages. Then, as soon as some kind of immune system
defect occurred, which in most of the patients was AIDS, the fungus
could multiply, disseminate, and cause infection. The clinical
histories of these patients and the demonstration of a geographical
clustering of isolates based on the generated profiles are consistent
with a dormant phase of C. neoformans within all
individuals. Why infection would be caused by a dormant strain of
C. neoformans rather than a newly acquired one, what form
the dormant form of C. neoformans would assume, and why some
isolates would be more virulent than others remain to be determined.
The observation that the infecting organism had been (or at least could
have been) acquired long before the infection was diagnosed should be
taken into account in the prospective development of prophylactic
programs, such as vaccination or antifungal therapy, for populations at
particularly high risk of developing cryptococcosis, such as AIDS
patients in central or southern Africa, South Africa, or Southeast Asia
(24).
| |
ACKNOWLEDGMENTS |
We thank A. Casadevall (Albert Einstein College of Medicine,
Bronx, N.Y.), J. M. Clauson (Western Kentucky University, Bowling Green, Ky.), S. Kohno (Nagasaki University School of Medicine, Nagasaki, Japan), and D. Swinne (Institute of Tropical Medicine, Antwerp, Belgium) for supplying the environmental isolates used in this
study. We are grateful to Eric Spitzer and Silvia Spitzer for the
generous gift of the CNRE-1 probe. We are grateful for the
collaboration of the French Cryptococcosis Study Group, which includes
clinicians and microbiologists from various hospitals in France. Those
who participated in collection of the data used for this specific study
are listed here (in alphabetical order by city): M. E. Bougnoux,
S. Morelon, and E. Rouveix (Boulogne); C. Passa Gaudouen, B. Michel, G. Otterbein, and J. Roucoules (Bry-sur-Marne); J. M. Korach
(Châlon en Champagne); B. Salles and C. Sire
(Chalon/Saône); M. Gauthier and O. Salmon (Evry); F. Botterel and
J. F. Delfraissy (Le Kremlin Bicêtre); M. A. Desailly
and H. Maisonneuve (La Roche/Yon); D. Bouhour, E. Dannaoui, D. Peyramond, and M. A. Piens (Lyon); O. Morin and P. Poirier
(Nantes); M. Gari Toussaint and P. Dellamonica (Nice); and in Paris C. Chochillon, X. Duval, and J. L. Vildé (Hôpital
Bichat); M. Kazatchkine, V. Lavarde, and C. Piketty (Hôpital
Broussais); B. Dupont (Institut Pasteur); L. Baril, F. Bricaire,
J. Carrière, A. Datry, S. Herson, and C. Trivalle (Hôpital Pitié Salpétrière); G. Delzant and G. Kac (Hôpital Tenon); J. Gilquin (Hôpital St. Joseph); D. Toubas (Reims); B. Hery and J. Y. Leberre (St. Nazaire); M. F. Biava and C. Rabaud (Vandoeuvre les Nancy); and C. Fontier and E. Mazards (Valenciennes). We also thank Olivier Ronin for serotyping the
cryptococcal isolates, K. Sitbon and A. de Gouvello for help in
collecting the clinical data, and J. Jacobson for editorial assistance.
Financial support for this work was provided by SIDACTION (a
postdoctoral fellowship for G.J. and a grant for F.D.), Pfizer Laboratory (a scholarship for D.G.-H.), and the Pasteur Institute (Contrat de Recherche Clinique for F.D.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Mycologie, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris
Cedex 15, France. Phone: (33) 1 45688356. Fax: (33) 1 45688420. E-mail: janbon{at}pasteur.fr.
| |
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Journal of Clinical Microbiology, October 1999, p. 3204-3209, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
