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Print version ISSN 0074-0276
Mem. Inst. Oswaldo Cruz vol.97 no.7 Rio de Janeiro Oct. 2002
Vol. 97(7): 985-989, October 2002
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
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.
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).
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).
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.
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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