S. R. Fan, Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital, Shenzhen 518036, China. Tel.: +86 755 8392 3333-5505. Fax: +86 755 8306 1340. E-mail: firstname.lastname@example.org
Studies of the genetic diversity of Candida albicans strains and the correlation between the antifungal susceptibility and gene diversity of C. albicans were carried out and the results were found to be inconsistent. To investigate antifungal susceptibility and genotypes of C. albicans strains from patients with vulvovaginal candidiasis (VVC), the genotypes of C. albicans in patients with VVC were studied using a recently developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of CAI microsatellite method and antifungal susceptibility was tested using E-test methods. Twenty-six genotypes were identified from 89 strains of C. albicans isolated from patients with VVC. Candida albicans isolates were susceptible to amphotericin B, flucytosine, ketoconazole and fluconazole. The dominant genotypes (A, B, C, D) account for 69.7% (62/89) of C. albicans. The resistant rate of C. albicans genotype B to itraconazole (ITR) and that of C. albicans non-genotype B strains were 66.7% (14/21) and 4.4% (3/68) respectively at P < 0.05. We concluded that C. albicans genotype B from patients with VVC was more resistant to ITR.
Vulvovaginal candidiasis (VVC) is a common disease, affecting up to 75% of women in their child-bearing age, at least once in their lifetime and is predominately caused by Candida albicans. Resistance of Candida species to azole antifungals is the most prevalent type of resistance to antifungals. Vaginal C. albicans isolates were found to be resistant to fluconazole (FLU) and exhibited considerably higher resistance to itraconazole (ITR). Some of the VVC therapy failures were because of the resistance of yeast pathogens to the drugs used.1,2 Resistance can be caused by an alteration of the target enzyme, the cytochrome P-450 lanosterol 14α-demethylase, which is mediated by ERG11 gene, or the failure of azole antifungal agents to accumulate inside the yeast cell as a consequence of enhanced drug efflux, which is mediated by MDR genes and CDR genes.3 The genetic diversity of C. albicans strains and the correlation between the antifungal susceptibility and gene diversity of C. albicans were studied and the results were found to be inconsistent.4–14 Only few have studied the genetic diversity of C. albicans strains recovered from VVC and the correlation between the antifungal susceptibility and gene diversity of C. albicans.15,16
In recent years, microsatellites have been increasingly used as molecular markers for population genetics and genotyping of different organisms. Several polymorphic microsatellite loci have been identified in the genome of C. albicans. Among them, the locus called CAI located in a non-coding region appeared to be the most polymorphic one exhibiting a discriminatory power of 0.97–0.99.17–19
The aim of this work was to use the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of CAI microsatellite for rapid strain typing of C. albicans in patients with VVC and correlate the relationship between antifungal susceptibility and gene diversity of the C. albicans strains.
Materials and methods
The patients, case definition, vaginal samples, and results of the study have been published elsewhere.20 Briefly, from September 2005 to December 2005, a total of 89 subjects were studied. A sample from the lateral wall of the vagina was obtained with a sterile cotton-tipped swab. The swab was placed in a tube filled with saline for direct microscopic examination. Culture was performed at the same time for all the positive wet vaginal smear cases. All specimens were plated on CHROM agar (Biocell Laboratory Ltd, Zhengzhou, China) and incubated for 24–48 h at 37 °C in ambient air atmosphere. Strains were identified in standardised system API Candida (bioMerieux, Marcy l'Etoile, France) and were confirmed by 26S rRNA gene D1/D2 domain sequence analysis. Candida albicans strain ATCC 90028 was used as a reference.
DNA extraction and PCR
Nuclear DNA was extracted by the method of Kaiser et al. . The microsatellite locus CAI was amplified by PCR using a pair of primers (forward, 5′-ATG CCA TTG AGT GGA ATT GG-3′; reverse, 5′-AGT GGC TTG TGT TGG GTT TT-3′) described previously.19 PCR amplification was performed in a thermocycler (ICycler; Bio-Rad, Hercules, CA, USA) with a program consisting of an initial denaturing step at 95 °C for 4 min; 33 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 1 min; and a final extension step of 7 min at 72 °C. The fragments of CAI alleles from all the C. albicans strains studied were successfully amplified by PCR.
To obtain an equal amount of DNA loading for SSCP electrophoresis, the PCR product was first electrophoresed on agarose gel and the amount of DNA was estimated by its band intensity. Three to five microlitres of PCR products (approximately 100 ng DNA) were mixed with the same volume of denaturing loading buffer. Mixtures were heated at 95 °C for 10 min and then chilled on ice. Denatured PCR products were loaded on an 8% acrylamide : bis (29 : 1) non-denaturing gel, cast using the gel casting set provided in the DCode Universal Mutation Detection System (Bio-Rad). Electrophoresis was performed in the same system in prechilled 1× TBE buffer at 240 V for 10 h at 10 °C. After electrophoresis, silver staining of the gel was carried out using the procedure of Beidler et al. .
The E test was performed according to the manufacturer’s instructions (AB. Biodisk, Solna, Sweden). The interpretation of antifungal resistant-susceptible category among the yeast isolates was based on the NCCLS M27-A 2 criteria. Isolates with MICs between 16 and 32 μg ml−1 for FLU and 0.25–0.5 μg ml−1 for ITR or ketoconazole (KET) were considered susceptible-dose dependent. Isolates with MICs ≤ 8 μg ml−1 for FLU and 5FC, ≤0.125 μg ml−1 for ITR and KET, and ≤1 μg ml−1 for AmB were considered susceptible. Isolates with MICs ≥ 64 μg ml−1 for FLU, ≥32 μg ml−1 for 5FC, ≥1 μg ml−1 for ITR or KET, and ≥2 μg ml−1 for AmB were considered resistant.23
Statistical analysis was performed using the chi-squared test and t-test.
A total of 89 patients with VVC were enrolled in the study, which included gene typing using SSCP analysis and antifungal susceptibility using E-test methods. The mean age of patients with VVC was 30.33 (SD 5.15) years old. Twenty-six genotypes were identified from 69 strains of C. albicans isolated from patients with VVC. The dominant C. albicans genotypes (A, B, C, D) comprised 69.7% (62/89) (Fig. 1).
The antifungal susceptibility and genotypes of C. albicans strains from patients with VVC were shown in Table 1. Candida albicans was susceptible to amphotericin B, flucytosine, KET and FLU. The resistant rate of C. albicans to ITR was 19.1% (17/89). The resistant rate of C. albicans genotype B to ITR and that of C. albicans non-genotype B strains were 66.7% (14/21) and 4.4% (3/68) respectively at P < 0.05. All 17 cases of ITR resistant isolates were FLU susceptible.
Table 1. Antifungal susceptibility and genotypes of Candida albicans strains from patients with vulvovaginal candidosis
The resistance of Candidas to azole antifungals continues to be a significant problem in fungal infections.1–3 Resistance to these drugs can contribute to treatment failures. The mechanism(s) of antifungal resistance are complicated and less understood.1–3
White et al. studied two of the most common point mutations ERG11, D116E and E266D. The two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. They emphasise the diversity of mechanisms that result in a phenotype of azole resistance.3 Miyazaki et al. found FLU treatment is effective against a C. albicans erg3/erg3 mutant in vivo despite in vitro resistance.4 Niimi found overexpression of C. albicans CDR1, CDR2 or MDR1 does not produce significant changes in Echinocandin susceptibility.5 Coste found that a mutation in Tac1p is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in C. albicans.6
Advances in molecular biology have allowed the development of rapid molecular genotyping techniques for clinical and epidemiological analyses. Jain et al.  demonstrated a homogeneous banding pattern for all sensitive strains that was distinct from that obtained in case of the resistant strains. Rogers et al.  found 14 genes were differentially expressed in association with the azole-resistant phenotype. Rustad et al. reported homozygosity at the C. albicans MTL locus associated with azole resistance.9 Pujol et al. demonstrated that isolates that are naturally 5FC resistant are restricted to group I and group I isolates are generally less susceptible to 5FC than non-group I isolates.10 Dodgson et al. reported that clade-specific flucytosine resistance is because of a single nucleotide change in the FUR1 gene of C. albicans.11 Mercure et al. found the correlation between the presence of a self-splicing intron in the 25S rDNA of C. albicans and isolate susceptibility to fluorocytosine.12 Pujol et al. further demonstrated the resistance is not directly affected by mating type locus zygosity in C. albicans.13 Balashov et al. found that the FKS1 gene mutations were associated with the resistance of C. albicans to caspofungin.14
Up to date, only a few have studied the genetic diversity of C. albicans strains recovered from VVC and the correlation between the antifungal susceptibility and gene diversity of C. albicans.15,16 Ribeiro et al. studied the expression of the MDR1, CDR1, CDR2 and ERG11 genes in all of the C. albicans isolates from VVC. They found that the strains with reduced susceptibility to FLU had increased expression of CDR1 when compared with the FLU-sensitive strains.15 Cernicka and Subik investigated the molecular mechanisms of resistance in 22 randomly selected FLU-resistant vaginal C. albicans isolates. Twelve isolates were found to be cross-resistant to ITR and 15 to voriconazole. Most of them also displayed decreased susceptibility to terbinafine. Northern blot analyses revealed overexpression of the MDR1 gene in all isolates, which in some isolates was accompanied by elevated levels of CDR1/CDR2 and ERG11 expression. They concluded that decreased susceptibilities of vaginal yeast isolates to clinically used azole derivatives are the result of a combination of several molecular mechanisms involving drug efflux and alterations in the structure or cellular amount of 14-alpha-lanosterol demethylase.16
In this study, the recently developed PCR-SSCP of CAI microsatellite with a high discriminatory power was used to detect the molecular variations in the genome of C. albicans isolates obtained from patients with VVC. Dominate C. albicans genotypes (A, B, C, D) accounting for 69.7% of C. albicans isolates were susceptible to amphotericin B, flucytosine, KET and FLU. Candida albicans genotype A was less resistant to azole antifungals, in contrast, C. albicans genotype B was more resistant to ITR. All ITR resistant isolates, however, were FLU susceptible. The presence of the dissociation in resistance between azoles (namely, FCZ and ITZ) and cross-resistance has also been described.24
The evolution of azole resistance can occur via different pathways, e.g., increased activity of transcription factors that regulate drug pumps or mutations in the ergosterol biosynthetic pathway and etc.3,9 Several mechanisms may facilitate the expression of azole resistance in Candida spp. As many determinants of resistance are often expressed in the same strain, it is difficult to assess the contribution of each mechanism on the overall level of resistance. Resistance may develop secondary to accumulation of many resistance factors over time. These may explain why azole resistance has been relatively slow to emerge among Candida spp. and why most instances in which resistance did occur were in patients receiving prolonged therapy. The potential ways of overcoming or preventing drug resistance may be by using combination therapy and by elucidating the mechanisms that contribute to the development of resistance.25,26
Polymerase chain reaction-single strand conformation polymorphism was used to identify specific base changes between isolates. By comparing the SSCP patterns of DNA fragments from susceptible and resistant isolates, it was possible to identify DNA fragments containing genomic alterations, which may contribute to the resistance to azoles.19,20
In conclusion, C. albicans from patients with VVC was susceptible to amphotericin B, flucytosine, KET and FLU. Candida albicans genotype B was more resistant to ITR. PCR-SSCP analysis may be a useful tool for studying the mechanisms of actions of Candida resistance to antifungal agents.
This research was supported by National Key Technologies R & D Programme Grant 2004BA720A05-01, and by Shenzhen Science and Technology Grant JA 200505270419B, 200702108, 200703031.