High Oral Prevalence of Candida krusei in Leprosy Patients in Northern Thailand

Patients.The patients selected were all long-term residents at the McKean Rehabilitation Center, which especially caters to leprosy patients. The inclusion criteria for the patients were as follows: (i) the patients had to have arrested leprosy of long duration and a negative bacillary index, (ii) the patients could not have received any antileprosy therapy (MDT), and (iii) the patients had to be handicapped (e.g., mutilation of limbs) with or without the orofacial changes of the late stage of leprosy. None of the patients elicited a history of recent oral candidiasis and as such had not been exposed to antifungals. It is also noteworthy that neither the patients nor the controls included in the study were infected with human immunodeficiency virus (HIV), nor, to our knowledge, had they been in close contact with HIV-infected individuals. Furthermore, as the patients had arrested leprosy, they were not receiving any drugs (e.g., dapsone or thalidomide) that may have led to functional disturbances in systemic immunity.

All patients were examined by one examiner (P.A.R.) under an artificial light in a dental chair at the dental unit of the center. The following parameters were recorded: age, sex, type and duration of leprosy, period of hospitalization, type of previous antileprosy therapy, and period since a negative bacillary smear was recorded.

The oral cavities were examined for the presence of dentures and oral mucosal changes, in particular, oral candidiasis. If dentures were present, they were removed prior to sampling. The same collector obtained control samples from 20 adults residing in the same locale using sampling techniques identical to those used for the leprosy patients.

Sampling procedures and stock maintenance.For evaluation of candidal carriage, the tongue and palate of each patient were sampled by rigorously swabbing their surfaces with a sterile Fungi-Quick swab (Hain Diangostika, Berlin, Germany). Afterwards the swab was reinserted into the alginate transport medium within the tube and kept at room temperature for immediate transport to the Oral Biology Laboratory at the Prince Philip Dental Hospital, Hong Kong. The swabs were then cultured aerobically on Sabouraud dextrose agar at 37°C for 7 days. The cultures were inspected on a daily basis for yeast growth, and plates of pure isolates were obtained, if any were present. The pure cultures were stored in glycerol at −70°C until species identification and genetic fingerprinting.

Identification of Candida species.The Candida organisms were identified by the germ tube test, growth at 45°C, chlamydospore production, and API 20C AUX (Bio-Merieux, Marcy l'Etoile, France) assimilation tests; and the phenotype was further defined by using CHROMagar Candida plates (CHROMagar, Paris, France) (20). Their identities were reconfirmed with the new improved APILAB Plus system (Bio-Merieux) to exclude Candida dubliniensis. Carriage was defined as the presence of yeasts on inoculated plates; attempts were not made to quantify the oral yeast load per individual.

Antifungal sensitivity testing.Commercially available antifungal susceptibility gradient strips (E-test; AB BIODISK, Solna, Sweden) were used for direct quantification of MICs. The antifungal agents used were amphotericin B, ketoconazole, and fluconazole, and the range of concentrations of the antibiotics to which susceptibilities were tested was 0.002 to 256 μg/ml, respectively.

The procedures followed for antifungal susceptibility testing by the E-test were those recommended by the manufacturer (E-test technical guide number 4, Antifungal susceptibility of yeasts, AB BIODISK). The medium used to test the sensitivities was RPMI 1640 (American Bioorganics, Buffalo, N.Y.) with 2% glucose, as prescribed by the NCCLS; the depth of agar in each plate was 4.0 to 0.5 mm (8).

Well-isolated yeast colonies from a 24-h growth on Sabouraud agar were inoculated in 0.85% NaCl and adjusted spectrophotometrically at 530 nm to match the turbidity of a 0.5 McFarland standard. These suspensions were inoculated onto the agar with sterile cotton wool swabs by swabbing the entire surface evenly in three directions. After the surface was allowed to dry for 15 min, the E-test strips were applied. The plates were then incubated at 35°C for up to 48 h in a moist incubator until growth was seen. In the case of the azoles, the plates were first read at 24 h, and the reading was confirmed after 48 h. The lowest drug concentration on the E-test strip inhibiting 100% of the growth was defined as the MIC, according to the instructions provided by the manufacturer.

DNA isolation for randomly amplified polymorphic DNA (RAPD) analysis.For the isolation of DNA, the yeasts were grown on Sabouraud dextrose agar plates (Oxoid, Basingstoke, United Kingdom) for 24 h at 37°C. The yeast cells were scraped from the plate, washed in 1 M sorbitol, and incubated at 37°C for 1 h in 1 ml of SE buffer (1.2 M sorbitol, 0.1 M EDTA [pH 7.5]) containing 1 μl of β-mercaptoethanol (Sigma, St. Louis, Mo.) and 0.3 mg of yeast lyticase (Sigma). The resulting spheroplasts were harvested by centrifugation at 10,000 rpm for 1 min on a Beckman (Fullerton, Calif.) centrifuge (model G515R), washed once in SE buffer, and resuspended in 0.4 ml of 0.15 M NaCl-0.1 M EDTA (pH 7.5). They were lysed by the addition of proteinase K (final concentration, 400 μg/ml; Gibco BRL, Life Technologies, Inc., Gaithersburg, Md.) and sodium dodecyl sulfate (final concentration, 1% [wt/vol]) along with RNase A (final concentration, 400 μg/ml) (Sigma) at 55°C for 1 h. After centrifugation, the supernatants were extracted twice with phenol-chloroform-isoamyl alcohol and once with chloroform, and then the DNA was precipitated with 2-propanol. Once the DNA was extracted and purified, the DNA was dissolved in 50 μl of TE buffer (10 mM Tris, 0.1 mM EDTA [pH 8.0]) and stored at 4°C until further analysis (2).

RAPD analysis.RAPD analysis of C. krusei with primers OBU1 (5′CAC ATG CTT3′), OBU2 (5′CAC ATG CCT3′), and RSD11 (5′GCA TAT CAA TAA GCG GAG GAA AAG3′) was carried out in a 50-μl final volume containing 1 μM primer, 2 mM MgCl_2, 1× PCR buffer (200 mM Tris-HCl [pH 8.4], 500 mM KCl), 200 μM deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (Gibco BRL, Life Technologies, Inc.), and 200 ng of yeast DNA as the template (3). These primers were chosen after extensive pilot studies with a battery of six primers (unpublished data) due to their relatively high discriminatory powers, as described previously (2, 2a, 3). The first 5 cycles included 30 s of denaturation at 94°C, 2 min of annealing at 27°C (for primers OBU1 and OBU2) or 52°C (for primer RSD11), and 2 min of primer extension, followed by 45 cycles of 30 s of denaturation at 94°C, 2 min of annealing at 32°C (for primers OBU1 and OBU2) or 57°C (for primer RSD11), and 2 min of primer extension at 72°C. The reaction was held at 72°C for 10 min. Control tubes with no template DNA were included in each run, and the reproducibility of the reaction was checked. The amplification products were separated on 1.2% agarose gels in 0.5× TBE (Tris-borate-EDTA) buffer, stained with ethidium bromide, and visualized on a UV transilluminator.

Analysis of DNA fingerprints.The RAPD fingerprint patterns of each isolate were analyzed according to their band positions. The data for two banding patterns (lanes A and B) can be classified by the binary values 0 and 1, where 0 indicates no band at a position and 1 indicates a band at that position. The similarity coefficient (S_AB) for each pair of strains, A and B, was calculated by the following formula: $$mathtex$$\[S_{AB}{=}1{-}\frac{\sqrt{b{+}c}}{a{+}b{+}c}\]$$mathtex$$ where a is the number of bands common for both lanes A and B (coded 1,1), b is the number of bands in lane A with no counterpart in lane B (coded 1,0), and c is the number of bands in lane B with no counterpart in lane A (coded 0,1). An S_AB value of 0.80 was arbitrarily used as the threshold for clustering of similar strains, since it is roughly halfway between the average value for dissimilarity and identity.

A computerized dendrogram method (the Dendron software program) based on S_AB was used to cluster the isolates (23) on the basis of the pairwise similarity coefficient matrix. In the Dendron program, the two strains with the highest similarity coefficients are grouped, with the branch point corresponding to the S_AB. The program then searches for a strain(s) with the next highest S_AB and groups them, with the branch point corresponding to the S_AB. The process continues sequentially until all strains are included and then derives a tree of similarity. This method was used to generate the dendrogram profiles for all 16 isolates with each of the three primers used in the RAPD analysis.

Demographic data for patients and controls.A total of 44 patients (22 men, 22 women) with a mean age of 72.8 years (age range, 60 to 89 years) were examined. On average, the patients had spent 25.7 years as inpatients (range, 60 months to 60 years), with a mean period of leprosy of 42.9 years (median, 48 years; range, 7 to 70 years).

The majority of patients (n = 28; 63.6%) had lepromatous leprosy; 2 (4.5%) had a history of tuberculoid leprosy, and 14 (31.8%) had borderline (tuberculoid) leprosy. The average period since these patients had had a negative bacillary smear was 11.5 years (median, 17.5 years). The patients had been microbiologically negative for mycobacteria for between 2 and 45 years.

Dentures were present in 8 of 44 patients, and 1 patient had clinical manifestations of denture stomatitis. Other oral mucosal findings were median rhomboid glossitis (n = 2 patients), leukoedema (n = 12), geographic tongue (n = 1), oral lichen planus (n = 1), oral leukoplakia (n = 1), lichenoid reaction (n = 1), fibroma (n = 1), and betel chewer's mucosa (n = 2). Seven patients had a complete or partial loss of the uvula due to leproma at a previous stage of the disease, and three others had incompetent lips due to facial disfigurement.

The control group of nonleprosy patients, also from the McKean Rehabilitation Center, were long-term residents because of neurological diseases and debilitation. They lived in separate wards for men and women situated some distance away from the leprosy wards. None of them had acute oral infections or were receiving antifungal agents; none of the controls wore dentures.

Oral yeast prevalence rates in leprosy patients.The rates of oral yeast carriage were relatively high in both the leprosy patients and the controls. Among the individuals in the patient group, 35 of 44 (80%) carried yeasts, whereas 13 of 20 (65%) of the control adults sampled carried yeasts (P > 0.05 by Fisher's exact test).

When the species of the organisms from the leprosy patients were identified, the majority of the yeasts were found to be C. krusei (16 of 35; 46%), while C. albicans was the second most common yeast (12 of 35; 34%), with the remainder belonging to the species Candida tropicalis, Candida guilliermondii, Candida rugosa, Candida famata, Candida parapsilosis, and Pichia ohmeri (Table 1). Two of the 13 Candida-positive patients in the control group carried C. krusei, while the others harbored C. albicans, C. tropicalis, C. parapsilosis, Saccharomyces cerevisiae, and Hansenula polymorpha. Considering the overall rate of oral candidal carriage in the two populations studied, the patients had a significantly higher oral prevalence of C. krusei (16 of 44; 36%) than the healthy controls (2 of 20; 10%) (P < 0.05).

Phenotypes of C. krusei.According to the API 20C AUX assimilation profiles, the 16 C. krusei isolates from the control group could be categorized into four different phenotypes. Thus, a total of 12 isolates belonged to classic phenotype 2000000, isolates L21 and L38 belonged to phenotype 2000100, isolate L24 belonged to phenotype 6000000, and isolate L38 belonged to phenotype 6000100.

Antifungal sensitivities of C. krusei isolates.All except one of the C. krusei isolates (isolate L24, for which the fluconazole MIC was 192 μg/ml) were resistant to fluconazole (MIC, >64 μg/ml), according to the breakpoint definitions proposed by Rex et al. (16) (Table 2).

All C. krusei isolates were sensitive to amphotericin B, with an MIC range of 0.032 to 2.00 μg/ml (normal range, 0.050 to >6.25 μg/ml) (8).

Due to the lack of consensus definitions of breakpoints for ketoconazole MICs, arbitrary values were established, according to those suggested in previous publications: susceptible for isolates for which MICs were <0.125 μg/ml, susceptible dose dependent for isolates for which MICs were between 0.25 and 0.5 μg/ml, and resistant for isolates for which MICs were >1 μg/ml. According to these criteria, 12% of our isolates were resistant to ketoconazole and another 12% were deemed susceptible dose dependent, while the remainder were susceptible (16).

Interestingly, the two isolates which exhibited resistance to ketoconazole (isolates L21 and L38) were the most resistant to amphotericin B.

RAPD genotypes and dendrogram profiles of C. krusei.We wished to determine whether the 16 strains of C. krusei isolated from leprosy patients belong to either a distinct genetic group or disparate genetic groups. For this purpose the C. krusei isolates were genotyped by the RAPD technique with three different primers, primers OBU1, OBU2, and RSD11. The RAPD profiles thus obtained are shown in Fig. 1. By examining the band profiles one can determine, even without the aid of computer assistance with the Dendron program, that the patterns of commensal C. krusei strains differed from each other by the positions of one or more bands. However, some patterns were similar. For example, with primers OBU1 and RSD11, the patterns of isolates L27 and L28 differed by only one intense band. Again, with primer OBU2, the patterns of isolates L40, L29, and L41 were almost identical.

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FIG. 1.

RAPD fingerprinting patterns of 16 C. krusei isolates (derived from the oral cavities of arrested leprosy patients) with primers OBU1, OBU2, and RSD11 after separation by electrophoresis on a 1.2% agarose gel. Lanes M, molecular marker (bacteriophage λ DNA cleaved with HindIII).

The relationships between strains and patterns are best visualized in dendrograms based on computed S_ABs (Fig. 2). Hence, to determine whether the 16 C. krusei strains from the patients represent a distinct genetic group, we further analyzed the dendrogram profiles of the gels obtained with the three different primers. An S_AB value of 0.80 was arbitrarily used as the threshold for clustering of similar strains. The three different primers (primers OBU1, OBU2, and RSD11) categorized the 16 isolates into 14, 12, and 10 clusters, respectively.

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FIG. 2.

Dendrograms generated from S_ABs computed for pairwise comparisons of 16 isolates of C. krusei and fingerprinted with three different primers (primers OBU1, OBU2, and RSD11). S_ABs were calculated by using band positions alone (see Materials and Methods).

The dendrograms of these strains showed various relationships, with S_AB values ranging from 0.99 for the relationship between isolates L27 and L28 obtained with primer OBU1 to 0.55 for the relationship between isolate L39 and the group containing all the other strains obtained with primer OBU2. A genetically identical cluster with an S_AB of 1 was noted with isolates L27 and L28 with OBU1 and isolates L40, L29, and L41 with OBU2. The dendrograms also revealed the various profiles that can be generated when different primers are used to characterize the genotypic profiles. In summary, it was clear from the dendrograms that there was considerable genetic diversity and the existence of genetic shuffling among the isolates within this particular locale, irrespective of the primer used.