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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 985-989
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 985-989
First Characterization
of Candida albicans by Random Amplified Polymorphic DNA Method in Nicaragua
and Comparison of the Diagnosis Methods for Vaginal Candidiasis in Nicaraguan
Women
Martha Darce Bello/*/+,
Alcides Gonzalez*, Christian Barnabé**, Georges Larrouy
Laboratoire de Parasitologie Médicale
et d'Ecologie Humaine, 37, Allées Jules-Guesde 31062 Toulouse Cedex,
France *Centro Nacional de Diagnostico y Referencia, Ministerio de Salud de
Nicaragua, Managua, Nicaragua **Unité de Recherche: "Génétique
des Maladies Infectieuses", Centre National de la Recherche Scientifique/Institut
de Recherche pour le Développement, Montepellier, France
+Corresponding author. Fax:
+33-5-61-14.5979. E-mail: madarcebello@wanadoo.fr
Received 7 November 2001
Accepted 29 July 2002
Code Number: oc02222
A total of 106 women with vaginitis
in Nicaragua were studied. The positive rate for the identification of
Candida species was 41% (44 positive cultures out of 106 women with vaginitis).
The sensitivity of microscopic examination of wet mount with the potassium hydroxide
(KOH) was 61% and 70% with Gram's stain when using the culture of vaginal fluid
as gold standard for diagnosis of candidiasis. Among the 44 positives cultures,
isolated species of yeast from vaginal swabs were C. albicans (59%),
C. tropicalis (23%), C. glabrata (14%) and C. krusei (4%).
This study reports the first characterization of 26 C. albicans stocks
from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis
in this small C. albicans population showed the existence of linkage
disequilibrium, which is consistent with the hypothesis that C. albicans
undergoes a clonal propagation.
Key words: vulvovaginal candidiasis
- diagnosis - Candida albicans - molecular typing - random amplified
polymorphic DNA - population structure - Nicaragua
The genital candidiasis is one of
the pathogenic demonstrations of yeast. Candida albicans is the most
frequent species; it is usually isolated in 85 to 90% from the vaginal mycoses
(Odds et al. 1988).
Vaginal candidiasis affects females
at least once during their lifetime, at an estimated rate of 70 to 75%, of whom
40 to 50% will experience a recurrence (Sobel 1999).
In Nicaragua, we know very little
about the prevalence and incidence of vaginal candidiasis and no study of the
biology of C. albicans has been carried out. In this country, diagnosis
of vaginal candidiasis is mainly based on the clinical presentation. Laboratories
of the hospitals and health centres (peripheral laboratories) carry out only
the microscopic diagnosis from the vaginal fluid. In the laboratory of the National
Centre of Diagnosis and Reference of Nicaragua (CNDR), the yeast identification
is based on the observation of the microscopic aspects, culture and biochemical
tests.
Most of the genetic studies revealed
that C. albicans is predominantly clonal (Pujol et al. 1993, Helstein
et al. 1993, Lockhart et al. 1995, Xu et al. 1999). Some authors have proposed
that clonal propagation with a remaining capacity of recombination, shape the
population structure of C. albicans (Caugant & Sandven 1993, Gräser
et al. 1996, Tibayrenc 1997).
Pujol et al. (1997) demonstrated
that the fingerprinting of C. albicans randon amplified polymorphic DNA
(RAPD), multilocus enzyme electrophoresis (MLEE) and Southern blot hybridization
with the moderately repetitive DNA Ca3 probe, not only clustered moderately
related isolates in a similar fashion but also afforded similar levels of resolution
of microevolution within a clonal population.
The goal of this study was to use
the RAPD method to examine the patterns of yeast genetic diversity among women
with vaginitis from a single geographic area. We were specifically interested
to know the frequency of yeast in vulvovaginal secretions. We also compared
the conventional methods of yeast diagnosis from vaginal samples used in Nicaragua
and yeast culture method.
MATERIALS AND METHODS
The vaginal swabs were taken from
106 women exhibiting symptoms of vulvovaginitis, who were attended in the outpatient
ward of the CNDR in Managua, Nicaragua, between June and August 1997. Swabs
were processed by the method routinely used for the detection of germinated
yeast pathogens: microscopic examination of wet mount, with a 10% potassium
hydroxide (KOH) preparation, and the Gram's stain. Samples were inoculated into
Sabouraud-glucose agar, supplemented with chloramphenicol, and were incubated
at 37°C for 48 h. For identification of C. albicans, isolates were
placedin foetal calf serum for 4 h to test for the production of germ tubes
and were incubated on Rice-Agar-Tween (RAT®) BioMérieux
Laboratories, France for 48 h to induce chlamydospores. All yeast isolates were
preliminary identified to the species level according to the CHROMagar Albicans®
Test (Mycoplasme International, Toulon, France). This medium contains a chromogene
substrate for immediate identification of C. albicans (green), C.
tropicalis (metallic blue), C. glabrata (pink) and C. krusei (pale
pink). Yeast species were confirmed with ID 32C identification kits (bioMérieux,
Marcy-l'Etoile, France). Growth at 45°C on Sabouraud glucose agar was used
to distinguish between C. albicans and C. dublinensis.
We compared the microscopy and KOH
test with the culture (chosen as gold standard), which is at present considered
as the most sensitive method (Sobel 1999).
Sensitivity and specificity of each
technique were estimated with the culture results with the following formulas:
Sensitivity = True positives/True
positives + False negatives
Specificity = True negatives/True negatives + False positives
The identified C. albicans stocks
were used to study the genetic structure of this population. The stocks were
stored in 1.5 ml of a stationary phase culture (until reaching a density of
2 x 108 cells:ml), mixed with 0.1 ml of dimethyl sulfoxide and frozen
at -70°C until use. Four reference C. albicans strains of the American
Type Culture Collection (ATCC) were used as control strains: 90028, 64548, 64550
and 64551. Six stocks belonging to other species from the strains collection
of the Parasitology Laboratory of the Purpan Hospital of Toulouse, France, were
used as outgroups for phylogenetic analysis (C. krusei, C. tropicalis,
C. glabrata, C. parapsilopsis, Saccharomyces cerevisiae
and Geotrichum candidum).
DNA extraction- A modification
of procedure previously applied to S. cerevisiae was used (Philippsen
et al. 1991). Briefly, the yeast colonies were taken from each agar plate and
were inoculated in 15 ml of YPD (2% yeast extract, 1% peptone, 2% dextrose).
Yeast were grown overnight at 37°C to a cell density of approximately 2x108
cells/ml (early stationary phase). The yeast were collected by centrifugation
at 3200 g for 5 min, washed in 15 ml of water at 1700 g for 5 min and suspended
once again in 1.5 ml of a solution (pH 7.5) of 0.9 M sorbitol, 0.1 M EDTA, 50
mM dithiothreitol plus 0.5 mg of Zymoliase 20T (ICN, biomedicals inc, UK). After
1 h of incubation at 37°C, the cells were centrifuged at 1700 g for 5 min
and suspended in 0.5 ml of lysis buffer: 50 mM Tris HCl pH 8.0, 10 mM EDTA,
1% SDS (W/V), plus 5 µl of proteinase K 20 µg/ml. The mixture was
incubated at 56°C for 2 h. Eight µl of RNAse A (100 µg/ml) were
then added, followed by mixing and incubation at 37°C for 30 min. DNA purification
was carried out by classical phenol-chloroform extraction (Sambrook et al. 1989).
The DNA was precipitated with cold ethanol at -70°C for 1 h and centrifuged
at 5000 g for 30 min. The pellet was washed twice with 70% ethanol. The precipitate
was dried and suspended in 100 µl of purified water.
RAPD - Conditions for the
RAPD have been previously described (Williams et al. 1990). Briefly, a polymerase
chain reaction was performed in 0.5 ml microcentrifuge tubes in a final reaction
mixture (60 µl) containing 20 ng of C. albicans DNA, 6 µl of
10X buffer for Taq DNA polymerase, 0.9 U of Taq DNA polymerase (Boehringer Mannheim,
Meylan, France), 100 µM each dATP, dCTP, dGTP and dTTP (Boehringer Mannheim),
and 200 nM of the primers (Operon Technologies, Alameda, USA). The decameric
primers used were A2 (5' CAGGCCCTTC 3'); A3 (5' AGTCAGCCAC 3'); A5 (5' AGGGGTCTTG
3'); A9 (5' GGGTAACGCC 3'); A12 (5' TCGGCGATAG 3'); A15 (5' TTCCGAACCC 3');
A18 (5' AGGTGACCGT 3') and A20 (5' GTTGCGATCC 3'). Amplifications were performed
in a thermal cycler PTC-100 (MJ Research Inc., USA) programmed for 45 cycles
of 1 min at 94°C (denaturation), 1 min at 36°C (annealing) and extension
at 72°C for 2 min, with a final 10 min extension at 72°C for the last
cycle. Amplified DNA fragments were analyzed by electrophoresis on 1.6 % agarose
gels stained with ethidium bromide.
Genetic analysis - Genetic
relationships among the stocks were estimated by the Jaccard's genetic distance
(Jaccard 1908). Each RAPD band was coded with a number, starting with 1 for
the slowest band. The distance was estimated based on the following formula:
D = 1 - [a/(a +b +c)]
a= number of bands that are common
to the two compared genotypes
b= number of bands present in the 1st genotype and absent in the 2nd
c= number of bands absent in the 1st genotype and present in the 2nd
The UPGMA method [Unweighted Pair-Group
Method with Arithmetic Averages, Sneath and Sokal (1973)] was used to cluster
the genotypes together according to their Jaccard's distances.
Population genetic analysis was based
on linkage disequilibrium statistics, with random mating as null hypothesis.
The following four probability values proposed by Tibayrenc et al. (1990) were
used: d1: the combinatorial probability of sampling the most frequent
genotype as often as or more often than actually observed if there were random
recombination; d2: the probability of observing any genotype as often
as or more often than the most common genotype actually observed if there were
random recombination; e: the probability of observing as few or fewer
genotypes than actually observed if there were random recombination; and f:
it gives the probability of observing linkage disequilibrium as high, or higher
than actually observed in the sample if there were random recombination. If
a probability is non significant (p > 5 X 10-2), random recombination
can not be rejected, but it is significant (p < 5 X 10-2), it
supports the nonrandom association of loci. The f test is based on Montecarlo
simulations with 104 iterations. A level of significance < 10-4
means that no case was observed out of 104 iterations (Tibayrenc
et al. 1991).
RESULTS
Frequency of candidiasis -
Yeast were isolated in 44 out of 106 women (42%), while no trace of yeast was
found in 62 women (58%). C. albicans was the most frequently isolated
species accounting for 26 (59%), followed by the next most frequent yeast species,
C. tropicalis, which was isolated from 10 (23%) of the women. Other species
of Candida were also cultured: C. glabrata from 6 (14%) and C.
krusei from 2 (4%).
Laboratory methods - The microscopic
examination gave 31 positive results and 13 negative (Table
I). Among the 44 positive in culture, the sensitivity of the microscopic
examination by the Gram's stain was 70%. The KOH test was positive in 27 cases
(61%). The specificity of both techniques was 100% (all negative results in
microscopic examination and KOH test were culture negative). The filamentation
test was positive for all the C. albicans stocksand negative for the
other species. The RAT confirmed the presence of chlamydospore of C. albicans,
whereas for the other species, we observed only the presence of yeast.
RAPD -The 26 C. albicans
stocks collected in this study were analyzed with 8 individual primers.
These primers were selected from 20 primers tested for their capacities to discriminate
variability and reproducibility. Eight primers for the C. albicans stocks
generated 37 bands. From the total samples (C. albicans and the others
species) the number of bands was 78. RAPD profiles were close to the four reference
stocks. The RAPD profiles obtained with the A3 and A20 primers were better for
differentiating the yeast stocks (Fig.
1).
Genetic diversity - All primers
tested showed the polymorphic bands. The level of RAPD resolution was high:
among 26 stocks, 23 different rapdemes were observed (genotype diversity = 0.88).
All the primers exhibit variability.
Phylogenetic clustering- The
UPGMA tree derived from RAPD data showed that all C. albicans fell into
a single cluster, in which Jaccard's genetic distances were fairly low, while
the other non-C. albicans stocks fell apart in a sharply distinct cluster
(Fig. 2). The most important genetic
distance among the stocks of our sample was 0.49, with an average of 0.26 ±
0.1, theoretical maximum is 1.0. The level of polymorphism was 0.75 and the
mean genetic diversity was 0.45.
Genetic population tests -
The linkage disequilibrium tests (d1, e, and f) were significant
within the C. albicans group with all stocks, but when repeated genotypes
were removed, the f test only showed a borderline p value (0.06).
DISCUSSION
Laboratory diagnosis- When
we used the culture as gold standard for detecting Candida sp. the sensitivity
of microscopic examination by Gram's stain was more effective than the KOH test.
These results are similar to a study made by Geiger et al. (1995). A positive
culture does not necessarily indicate that the yeast is responsible for the
vaginal symptoms. The diagnosis of Candida vaginitis requires a correlation
between clinical conditions, and laboratory results.
Studies indicate that Candida
sp. may be isolated from lower genital tract of approximately 20% (occasional
studies set the upper limit at 55%) of asymptomatic healthy women (Drake &
Maibach 1973). Our results in symptomatic women showed that yeast were present
in 41% of the vaginal specimens. This study was based on the isolation of agent
from vaginal fluid in culture, which does not allow to differentiate pathogenic
from saprophytic Candida sp. As these microorganisms are common colonisers
of the female genital tract, it would be useful to have a gold standard for
identification of Candida sp. able to distinguish pathogenic forms from
saprophytic ones.
As previously observed in others
studies (Tietz et al.1995, Mendoza et al. 1999), C. albicans was
the most frequent specie of yeast isolated from these vaginal samples from Nicaragua,
with a total of 59%.
When we compared ours results with
the data from others tropical countries (Venezuela, Angola, and Madagascar),
no statistical difference in the C. albicans/non- C. albicans
distribution was observed (P = 0.75). However, compared to results from Dublin
(Al-Rawi et al. 1999), a statistical difference was observed (P < 0.002),
with a higher frequency of C. albicans in Dublin (Table
II). Recruitment strategy of each study could have modified these results,
but they suggest the possibility of differences linked to climate or to socio-economic
development, further studies on this thematic are necessary.
Genetic and phylogenetic diversity
of C. albicans - The present study confirmed early results and demonstrated
that stocks attributed to C. albicans by classical morphological and
biological criteria display a genetic and phylogenetic diversity. The results
recorded here confirm a tendency, already noted by Schmid et al. (1993), in
a population of C. albicans strains from vaginal samples using the DNA
fingerprints with the moderately repetitive sequence Ca3, observing that the
majority of the stocks of C. albicans are in a relatively homogeneous
group.A high genetic similarity was also reported, using the same Ca3 probe,
in strains isolated from women with vaginal candidiasis, from a same geographical
region (Schmid et al. 1999).
Population genetic analysis
-Like many other pathogens, the question of the population structure of C.
albicans has been the subject of intense debates (Tibayrenc 1997, Vilgalys
et al. 1997). Early studies addressing this question (Tibayrenc et al. 1991,
Caugant & Sandven 1993) have recorded low levels of linkage disequilibrium
(nonrandom association of genotypes occuring at different loci), by comparison
with other pathogens such as Trypanosoma or Leishmania (Tibayrenc
et al. 1990). By constrast, other authors (Hellstein et al. 1993, Pujol et al.
1993, Xu et al. 1999, Arnavielhe et al. 2000) have found considerable levels
of linkage in differents C. albicans populations and have concluded
that these populations propagate clonally. Tibayrenc (1997) has proposed that
the relevant boundary is not between clonal and sexual species, but rather between
species that are structured into stable evolutionary units (DTUs), and species
in which genetic exchange is frequent enough to render impossible that maintaining
of such stable subdivisions.
The significant tests of linkage
disequilibrium observed here in the population of 26 C. albicans allow
us to reject the hypothesis of a panmictic structure within these C. albicans
stocks from Nicaragua. However, when the repeated genotypes were removed, only
a limited, non-significant, linkage disequilibrium was found. A statistical
type II error (lack of power of the test used in our small sample) could explain
this fact. This population genetic analysis indicates that the two fundamental
consequences of sexual reproduction (segregation and recombination) are apparently
absent or rare in this C. albicans population. Now, it is impossible
to identify clear-cut subdivisions within the C. albicans cluster (Fig.
2). It is worth noting that for another pathogen, T. cruzi, the agent
of Chagas disease, clear subdivisions could be individualized by both isoenzyme
electrophoresis and RAPD typing with an even lower set of primers (Tibayrenc
et al. 1993). This result has been fully confirmed by a an other study involving
a broader range of primers (Brisse et al. 1998). The results obtained here:
significant linkage disequilibrium with apparent lack of stable and clear-cut
subdivisions, are consistent with the proposition that the propagation of C.
albicans is mainly clonal.
Nevertheless, complementary studies
with other molecular markers and with other C. albicans populations from
Nicaragua are necessary to ascertain the C. albicans population structure
in this country. In any case, confrontation of clinical and therapeutic data
with set of molecular data, such as RAPD, would be useful for a better understanding
of the epidemiological aspects of the candidiasis infections.
ACKNOWLEDGEMENTS
To Michel Tibayrenc for his support
in setting up our collaboration. To Rafaela Ruiz, Julissa Avila, Justo Reyes,
Sergio Lopez, and Brigitte Gras for providing laboratory assistance, Joaquin
V Martinez-Suarez for supplying the reference stocks, Pierre-Yves Bello for
critically reading the manuscript, Nikki Wilkinson Rodriguez and Ana Cristi
Martinez for the English-language revision.
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Cruz - Fiocruz
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