INTRODUCTION

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Opportunistic yeast pathogens are common residents of the mucosal surfaces of the human mouth, gastrointestinal tract, and genitourinary system (15). In recent years, the increases in the human population of infection with human immunodeficiency virus (HIV), organ transplantation, and chemotherapy have dramatically increased the incidence of candidiasis (4, 10, 16, 17, 20). The predominant causal agent of candidiasis is Candida albicans (4, 10, 16, 17, 20). It is often assumed that most cases of candidiasis originate from the commensal strain inhabiting the vaginal canal, oral cavity, or gastrointestinal tract prior to infection (7, 13, 18, 25, 31). However, only limited population genetic surveys support this concept (3, 6, 9, 17). In contrast, there is evidence of reduced genetic diversity among samples of C. albicans from the oral cavities of HIV-infected patients, suggesting the possibility that a commensal strain(s) is replaced by genetically more uniform strains before the inception of oral candidiasis in immunocompromised patients (1, 4, 22, 33, 34). Furthermore, an analysis of strains of C. albicans from various body sites of healthy women suggested that different body sites may select for certain genotypes (27). Other types of associations between genotypes and special host or ecological conditions have also been proposed (28).

Whether strains are replaced before the onset of candidiasis has significant implications for treatment and preventive strategies. For example, if candidiasis is caused by the host's original commensal strain(s), knowledge of the yeast microflora of high-risk patients prior to the manifestation of candidiasis (e.g., susceptibility to different antifungal drugs) could lead to improved strategies for prophylaxis and treatment. Conversely, if commensal strains are frequently replaced by certain genotypes, then identifying the routes of transmission of these potentially more virulent genotypes could lead to measures that limit their spread. These two hypotheses offer different predictions for populations of C. albicans. The "replacement hypothesis" predicts a significantly lower degree of genetic diversity and a higher degree of genetic similarity among strains associated with specific body sites and/or certain types of hosts. In contrast, the "persistent hypothesis" would predict that strains from these sources would display comparable levels of genetic diversity. Most previous studies either used limited types of samples (1, 12, 22, 23) or lacked appropriate controls for confounding factors such as sex, geographic origin, body site, or host condition (1, 3, 6, 9, 22, 23, 28, 33).

Advances in molecular biology in the last two decades have allowed the development of rapid molecular genotyping techniques for clinical and epidemiological analyses (3, 5, 6, 14, 21, 24, 28, 33). Among the current molecular techniques for the genotyping of yeast strains, PCR fingerprinting and random amplified polymorphic DNA (RAPD) analysis, are in wide use. Both genotyping methods have high discriminatory powers and reproducibilities; they require little starting material and are rapid and simple to perform (14).

The goal of this study was to use PCR fingerprinting and RAPD analysis to examine concurrently the roles of host condition and body site in the patterns of yeast genetic diversity among women from a single geographic area. Specifically, we were interested in the contributions of two commonly recognized factors associated with candidiasis in women: HIV infection status (4, 17) and pregnancy (15). We were particularly interested in determining whether isolates from a specific body site or host group might be genetically more similar to each other than to isolates from a different body site or host group.

	MATERIALS AND METHODS

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Strain collection. Female volunteers (ages 18 years and older) were recruited from among outpatients scheduled to undergo a pelvic examination as part of their routine care in the Obstetrics/Gynecology Clinic and the Adult Infectious Disease Clinic of the Duke University Medical Center. Three host groups were considered: women with AIDS and oral candidiasis (no one in this group was pregnant at the time of sampling; therefore, this group is called the HIV-positive and nonpregnant [HIV⁺, NP] group), women who were at least 3 months pregnant and who were not infected with HIV (HIV, P group), and healthy individuals who were neither pregnant nor infected with HIV (HIV, NP group). Swabs were taken from the vagina, rectum, and oral cavity of each volunteer and were cultured on Inhibitory Mold Agar (Difco, Detroit, Mich.) for yeast isolation. Morphologically distinct yeast colonies from each culture were transferred and stored on Sabouraud glucose agar slants for species identification and subsequent DNA fingerprinting. Yeast species were identified by the germ tube test and with API 20C identification kits (BioMerieux-Vitek). Growth at 45°C on Sabouraud glucose agar was used to distinguish between Candida albicans and Candida dubliniensis.

Strain typing. Genomic DNA was isolated from each isolate by a previously described protocol (21) and was stored at 20°C. For PCR fingerprinting, the M13 phage core sequence (5'-GAGGGTGGCGGTTCT-3') and the oligonucleotide 5'-AGTCAGCCAC-3' (primer PA03; Operon Technologies) were used as single primers. Amplifications were performed in volumes of 25 µl containing 10 ng of genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, dATP, dCTP, dTTP, and dGTP each at a concentration of 0.2 mM, 3 mM magnesium acetate, 10 ng of primer, and 1.5 U of AmpliTaq DNA polymerase. For primer M13, the PCR was performed in a Perkin-Elmer thermal cycler (model 480) with an initial denaturation of 97°C for 3 min, followed by 40 cycles of 20 s at 93°C, 60 s at 50°C, and 20 s at 72°C and a final cycle of 5 min at 72°C. For primer PA03, the PCR was performed with an initial cycle of 97°C for 3 min, followed by 45 cycles of 60 s at 93°C, 60 s at 36°C, and 120 s at 72°C and a final cycle of 5 min at 72°C.

Data analysis. The yeast recovery rate was calculated for each body site of the three host groups. Statistical significance between different patient types for the same body sites and between different body sites of the same patient type were calculated on the basis of a two-by-two chi-square table test (8, 26). When the expected count of the lowest cell in the chi-square test was less than five, Fisher's exact test was applied (26).

	RESULTS

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Rate of yeast recovery. Yeasts were recovered from at least one of the three body sites of all 49 women (Table 1). The rate of yeast recovery differed somewhat among host groups and body sites (Table 2). For example, yeast recovery rates were higher from oral and rectal swabs (63% from both body sites) than from vaginal swabs (42%) for patients with AIDS. The recovery rate from vaginal swabs (73%) was highest for the group that included HIV-negative pregnant women. However, none of these differences were statistically significant either between host groups or between body sites within a host group at a P value of <0.05. The biggest difference in yeast recovery rate was from vaginal swabs between HIV-infected (42%) and HIV-free but pregnant women (73%). The chi-square value between this pairwise comparison was 2.311 (degrees of freedom = 1; P > 0.1).

Species distribution and species diversity. Table 2 summarizes the number of isolates of each species isolated from each host group and body site. Species diversities for each group of women and body site are presented in Table 3. Overall, 73.8% of all isolates (n = 80) were C. albicans, 11.2% were Candida parapsilosis, 6.3% were Candida krusei, 3.7% were Candida glabrata, 2.5% were Saccharomyces cerevisiae, and 1.2% each were Candida tropicalis and Candida lusitaniae. At all three body sites, C. albicans was the most common yeast species (Table 2), and in all three host groups, oral swabs had lower species diversity than either vaginal or rectal swabs (Table 3). However, these differences were not statistically significant (by Fisher's exact test, P > 0.1).

Markers and species identification. For the set of 80 isolates, a total of 51 polymorphic PCR fragments of different sizes were detected and scored; 29 fragments were generated with the primer M13 core sequence and 22 were generated with primer PA03. A representative picture of the PCR products from each of the primers is presented in Fig. 1. The genetic similarity among all isolates is presented in Fig. 2. This UPGMA phenogram of PCR fingerprints shows the grouping of isolates from each species identified on the basis of morphological and biochemical markers. All 59 isolates with germ tubes and biochemical profiles typical of that for C. albicans were clustered together on the phenogram (Fig. 2). Similarly, multiple isolates of each of four other species, C. parapsilosis, C. glabrata, S. cerevisiae, and C. krusei, grouped together.

View larger version (110K): [in this window] [in a new window]   FIG. 1.   Examples of electrophoretic separation of PCR fingerprints obtained by amplifying genomic DNA from 34 strains of yeasts isolated in this study by using the M13 core sequence (5'-GAGGGTGGCCGGTTCT-3') (A) and PA03 (5'-AGTCAGCCAC-3') as single primers. Lanes: 1, 1-kb ladder from GIBCO-BRL; 2 to 35, strains in the order listed in Table 1 (from 01oy to 21rn).

View larger version (27K): [in this window] [in a new window]   FIG. 2.   UPGMA phenogram of yeast strains calculated from the DNA fingerprinting patterns obtained with the primers M13 core sequence and PA03. Strain designations are described in Table 1 (footnote d). Species identifications are based on those obtained with API 20C kits and are shown on respective basal branches. Underlined strains were associated with clinical symptoms of candidiasis. There is no significant genetic clustering of strains on the basis of host type, disease symptom, or body site.

Genotypic diversity. A total of 70 unique genotypes were found among the 80 isolates. One genotype was shared by three isolates, each from a different host (isolates 18on, 38vn, and 39o+n in Table 1 and Fig. 2). Eight other genotypes were shared by two isolates each; each pair came from different body sites of the same host (Fig. 2). The genotypic diversity at each body site and for each group of women is presented in Table 3. None of the pairwise comparisons of genotypic diversities between samples was significant at a P value of <0.05, regardless of body site or host group.

Similarity of strains within a host. Among the 49 volunteers, yeasts were isolated from all three body sites of 7 women (volunteers 04, 05, 15, 23, 29, 30, and 47), from two body sites of 12 women (volunteers 03, 08, 12, 14, 21, 24, 25, 28, 33, 39, 41, and 42), and from one body site of the remaining 30 women (Table 3).

Genetic similarity between isolates from the same or different anatomical locations of different host groups. Table 6 summarizes the genetic similarity between isolates from the same or different anatomical locations of each of the three host groups. Two sample types were analyzed here; one included all the isolates and the other included only isolates of C. albicans. For all three groups of women, there is no evidence of greater similarity between strains from the same or different body sites at a P value of <0.05 (Table 6). These results do not support the hypothesis that women with HIV infection or pregnancy harbor isolates with higher genetic similarity. Isolates associated with clinical candidiasis were also dispersed throughout the phenogram (Fig. 2). However, there are two noteworthy features in Table 6. First, when all isolates were included in the analysis, oral isolates from all three host groups had a higher degree of genetic similarity to each other than strains from the other two body sites, but similar genetic similarities were observed among isolates from samples from all sites when only strains of C. albicans were analyzed. This was because a slightly higher percentage of oral strains were C. albicans. Second, the standard deviations for each estimate of genetic similarity were high, indicating a high degree of genetic heterogeneity of strains at each body site, as well as between body sites.

	DISCUSSION

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This study compared opportunistic pathogenic yeasts recovered from three body sites for each of three groups of women. Similar yeast recovery rates were found for all three body locations of all three groups (Table 2). The yeast recovery rates were within the range of those of previous studies based on similar groups of hosts and body sites (15, 17). Also consistent with previous studies, the most common species was C. albicans, regardless of body site and host status (4, 15, 17, 20).

PCR fingerprinting with the M13 core sequence and PA03 provided a very effective method for assessing the genetic similarities of all isolates from all the yeast species recovered in this study. In a previous study, standard reference strains from different species of yeasts pathogenic for humans were found to be different in their PCR fingerprinting profiles when the M13 core sequence was used as a single primer (14). Here, multiple clinical and natural isolates from each of five species were clustered together. Therefore, unlike individual species-specific DNA probes for Southern analysis of strains of a single species (5, 21, 24), PCR fingerprinting and RAPD analysis allow the concurrent identification of species and analysis of similarity among strains both within and between species.

In an earlier study of genetic similarity between isolates of C. albicans from 17 anatomical locations of 52 healthy women, Soll et al. (27) found that certain clusters of isolates were vagina or oral cavity specific. Brawner and Cutler (1) also found that oral strains of C. albicans from immunocompromised individuals were twice as likely to be serotype B than oral strains from immunocompetent individuals. Furthermore, Schmid et al. (22) suggested that in the early manifestation of AIDS in 11 AIDS patients from Leicester, England, indigenous commensal yeasts were replaced by a genetically more homogeneous group of C. albicans strains. However, in the present study, there was no convincing evidence for associations between genotypes of pathogenic yeasts and body site, HIV status, or pregnancy. Why is there a difference in these findings?

One possibility is that all the samples in the other studies were obtained from different geographic areas and different hosts (1, 3, 6, 9, 22, 27, 28). The strains in geographically different samples may have different patterns of relatedness. Isolates in the study by Soll et al. (27) were from Iowa City, Iowa. Strains in the study by Brawner et al. (1) were from different hospitals across the United States. However, in a comparison of two samples of C. albicans strains from patients infected with HIV from two geographically distant areas (one from Durham, N.C., and the other from Vitória, Brazil), little evidence of genetic differentiation was found between these two samples (34).

Methods of analysis and the controls for confounding factors may also contribute to the disparity in these studies. We have presented an overall assessment for correlation between genotypic similarities of isolates and body sites of isolation or host conditions for women in a single geographic area. In the study by Soll et al. (27), small clusters of isolates were first identified on the basis of DNA fingerprinting by Southern hybridization of the moderately repetitive genomic sequence Ca3, and then these small clusters were compared with each other. While this method of analysis might uncover small clusters of potentially ecologically specific genotypes, such a generalization can be risky. Even when we applied a method of analysis similar to that of Soll et al. (27) by breaking the UPGMA phenogram into smaller clusters, we failed to find any statistically significant cluster that would suggest a potential ecological specialization for certain genotypic groups. All major clusters in Fig. 2 had multiple isolates from each body site or host group.

Host histories may also affect colonization and infection. For example, different hosts may have different patterns of behavior that could influence the patterns of transmission and dynamics of yeast microflora. Since C. albicans and other yeasts can be sexually transmitted (15, 23), sexual behavior and the yeast microflora of sexual partners could affect the genetic diversity of yeast isolates.

We must emphasize that the failure to find any significant association between genotypes and body sites, HIV status, or pregnancy does not exclude the possibility that some other samples or genetic markers might detect a significant association (as shown previously by others). Appropriate sampling strategies and analytical methods would be required to control for the effects of geographic heterogeneity, socioeconomic status, and other risk factors.

We successfully applied PCR fingerprinting to investigate the patterns of genetic similarity among strains of opportunistic pathogenic yeasts from three different body sites of each of three groups of women. All body sites and groups of women harbored similar yeast species and genotypic diversities. We found no significant evidence of an association between genotype and body site, HIV status, or pregnancy. These results are consistent with the persistence hypothesis and inconsistent with the replacement hypothesis. However, the persistence hypothesis does not imply a view of a static yeast microflora. On the contrary, the yeast microflora can be very dynamic. A single host could harbor multiple yeast species or multiple genotypes of the same species at the same or different body sites. Furthermore, microevolution of colonizing strains may occur in hosts with recurrent Candida vaginitis (11, 12). It is clear that there is much to learn about mechanisms that influence the basic processes of colonization and the maintenance of yeast microflora in humans.