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Mycology

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

P. A. Reichart, L. P. Samaranayake, Y. H. Samaranayake, M. Grote, E. Pow, B. Cheung
P. A. Reichart
1Department of Oral Surgery and Dental Radiology, Charité, Medical Faculty, Humboldt University, Berlin, Germany
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L. P. Samaranayake
2Divisions of Oral Biosciences
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  • For correspondence: lakshman@hkucc.hku.hk
Y. H. Samaranayake
2Divisions of Oral Biosciences
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M. Grote
1Department of Oral Surgery and Dental Radiology, Charité, Medical Faculty, Humboldt University, Berlin, Germany
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E. Pow
3Oral Rehabilitation, Faculty of Dentistry, The University of Hong Kong, Hong Kong
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B. Cheung
2Divisions of Oral Biosciences
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DOI: 10.1128/JCM.40.12.4479-4485.2002
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ABSTRACT

Although Candida albicans is the most common human yeast pathogen, other Candida species such as C. krusei are now recognized as emerging agents, especially in patients with human immunodeficiency virus (HIV) disease. C. krusei is inherently resistant to the widely used triazole antifungal fluconazole and poses therapeutic problems, especially in systemic candidiasis. In a surveillance study of leprosy patients (with arrested or burnt-out disease) in a leprosarium in northern Thailand, we found a rate of oral carriage of C. krusei (36%) significantly (P < 0.05) higher than that for a healthy control group (10%). Among the Candida-positive patients, 16 of 35 (46%) carried C. krusei, while C. albicans was the second most common isolate (12 of 35 patients; 34%). The corresponding figures for the control group were 2 of 13 (15%) and 6 of 13 (46%), respectively. Studies of the antifungal resistance of the C. krusei isolates from patients indicated that all except one of the isolates were resistant to fluconazole, two isolates were resistant to ketoconazole, and all isolates were sensitive to amphotericin B. Evaluation of their genetic profiles by randomly amplified polymorphic DNA analysis with three different primers and subsequent analysis of the gel profiles by computerized cluster-derived dendrograms revealed that the C. krusei isolates from patients belonged to 10 disparate clusters, despite the origin from a single locale. These nascent findings indicate an alarmingly high prevalence of a Candida species resistant to a widely used antifungal in a part of the world where HIV disease is endemic.

Leprosy is caused by Mycobacterium leprae and is a disease of global prevalence (18). Reports from 91 countries indicate that at the beginning of the new millennium 641,091 patients with leprosy were registered for treatment, although the real numbers may be much higher (27). There are regional variations in the prevalence of leprosy; its global prevalence rate is estimated to be 1.25 per 10,000 population. The Thailand Leprosy Registry contained 2,291 cases at the beginning of 2000, yielding a prevalence rate of 0.4 per 10,000 habitants (27). Since the introduction of cocktail, multidrug therapy (MDT), consisting of a combination of rifampin, clofazimine, and dapsone, the prognosis of leprosy has improved dramatically, with more than 10 million patients cured by the end of 1999.

Leprosy may manifest as indeterminate, tuberculoid, borderline, and lepromatous variants (9). While all of these may be associated with orofacial pathology (14), most oral lesions are recorded in lepromatous leprosy patients (11). Facial lesions include granulomatous skin lesions and other cutaneous adnexa, particularly of the facial and trigeminal nerves (15). Leproma formation, especially in the soft palate, may lead to the extensive loss of soft tissue and palatal perforations (12). In addition, the gingiva and periodontium as well as the facial skeleton including the alveolar process may be involved (1, 13).

Arrested or chronically treated (colloquially termed burnt-out) leprosy has been defined as a case in which mycobacteria are no longer produced either as a consequence of a lifelong course of leprosy or due to MDT. Patients with arrested leprosy are characterized by severe mutilations of limbs and orofacial alterations. Many have a saddle nose and severe ocular pathology including blindness (14). Due to these severe disabling conditions, many of these relatively elderly patients are hospitalized or live permanently in leprosy rehabilitation centers, especially in Thailand, where they form small communities of various sizes. Patients live close together, sharing bedrooms and taking their meals in a community setting.

Extensive perusal of the electronic and archival data sources in the English-language literature indicated that there is virtually no information on the oral mycotic flora of leprosy patients. Hence, we embarked on the cross-sectional study described here to investigate an institutionalized resident population of arrested leprosy patients in Thailand. The study cohort from the McKean Rehabilitation Center, Chiangmai, Thailand, was particularly interesting as it comprised an elderly population that had been confined to the center for a prolonged period and that had undergone extended periods of medication. First, a pilot, cross-sectional study was undertaken to evaluate the prevalence of Candida species in this cohort. As this study indicated that Candida krusei was present in an unusually large numbers of people in the cohort, we proceeded to analyze in detail the genotypes and the antifungal sensitivities of the isolates in order to further characterize this uncommon oral yeast now considered an emerging pathogen, especially among compromised patient groups (17, 22).

MATERIALS AND METHODS

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 MgCl2, 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 (SAB) 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 SAB 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 SAB 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 SAB. The program then searches for a strain(s) with the next highest SAB and groups them, with the branch point corresponding to the SAB. 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.

RESULTS

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).

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TABLE 1.

Identities of Candida species isolated from leprosy patients and control subjectsa

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).

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TABLE 2.

MICs of amphotericin B, fluconazole, and ketoconazole for 16 oral isolates of C. krusei derived from arrested leprosy patients

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.

FIG. 1.
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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 SABs (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 SAB 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.

FIG. 2.
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FIG. 2.

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

The dendrograms of these strains showed various relationships, with SAB 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 SAB 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.

DISCUSSION

There are no reports in the literature on the oral carriage of Candida species in cohorts of patients with either active or arrested leprosy. Hence, we embarked on this study to obtain baseline data on oral yeast carriage and for this purpose sought a group of reclusive individuals with minimal contact with the outside world. The residents of the McKean Rehabilitation Center were chosen, as the center is a long-established rural unit with a live-in population, with some of the patients having lived at the center for up to 70 years.

For practical reasons and because of the difficulty of airlifting (i.e., transport by air to another laboratory for analysis) of the samples, oral yeast carriage was evaluated by a swabbing technique, which is considered less reliable than the oral rinse technique (21). Nonetheless, the former technique is widely practiced in field studies and is well established (20). We were therefore surprised to note the very high oral yeast carriage rate (80%) in the patient cohort, despite the use of the less reliable technique. The oral yeast carriage rate in healthy populations ranges from 2.0 to 71% (20), and it may increase up to 95% in patients with particular disease states, such as in HIV infection (19).

Perhaps the most intriguing and interesting finding in our study was the significantly higher prevalence of C. krusei in the patient population than in the control population (36 versus 10%). C. albicans isolates, the most predominant species in humans, were present but were the second most common yeasts isolated. On comprehensive perusal of 44 publications in the literature for which statistics on rates of human carriage of C. krusei were available, the highest carriage rates were found to be 6.1% for the oral cavity, 10.3% for the gastrointestinal tract, and 12.5% for the vagina either in health or in disease (22). Although we are unable to forward a hypothesis for this exceptionally high prevalence of C. krusei in our cohort, it is tempting to speculate that the institutionalization and the shared dietary regimens possibly originating from a common outlet may have played a role in this phenomenon. Nonetheless, the genomic data indicated clearly that the yeasts were not derived from a single source (see below).

While diet has been proven to be one of the main etiological factors in dental caries, its effect on general oral health and especially the oral microflora is less clear. The flow rate and buffering capacity of whole saliva, but not the numbers of mutans group streptococci and lactobacilli, have been shown to increase following the transition from a mixed diet to a lactovegetarian diet in one Swedish study (5), whereas the stimulated salivary flow rate in lactovegetarians was lower than that in nonvegetarians in another Scandinavian study (7). A diminished salivary flow rate, generally seen in older individuals, could contribute to the relatively high rate of oral carriage of Candida in our study. Yet, why this would predispose the individuals to the growth of C. krusei in preference to other species remains to be examined. Compared to other medically important Candida species, such as C. albicans, C. krusei has been isolated from a large variety of natural habitats. These include the atmosphere, fruits, sewage, silage, soil, wine, and beer (4). It is also found in chickens and seagulls (10). In recent years C. krusei has been recognized as an important agent of nosocomial candidiasis due to its increasing incidence and inherent resistance to azoles, especially fluconazole (22). Hence, it is considered a facultative saprophyte which may cause opportunistic infections only when host defenses are impaired.

The most widely used antifungal agents are the polyenes and the azoles, which include the imidazoles and the newer triazoles. As stated previously (8), almost all C. krusei strains are inherently resistant to fluconazole, and accordingly, all such isolates from leprosy patients demonstrated this resistance pattern. All 16 isolates were also uniformly sensitive to amphotericin B (MIC range, 0.032 to 2.000 μg/ml; normal MIC range, 0.05 to >6.25 μg/ml). However, 2 of the 16 isolates (isolates L21 and L38) were resistant to ketoconazole, according to recent NCCLS criteria (16). As far as we are aware, the cohort has not recently received azole therapy, and the ketoconazole resistance exhibited by 2 of 16 C. krusei isolates therefore appears to be intrinsic in nature.

When the phenotypes of the C. krusei isolates were compared, they could be classified into four distinct biotypes according to the API 20C AUX system. All isolates were positive for glucose, and this was the only positive reaction seen with 12 isolates (75%; with a profile of 2000000). Of the remainder of the isolates, two isolates (isolates L21 and L39; 12.5%) were positive for glucose and N-acetyl-glucosamine (with a profile of 2000100); one isolate (isolate L38) was positive for glucose, glycerol, and N-acetyl-glucosamine (with a profile of 6000100); and another isolate (isolate L24) was positive for glucose and glycerol (with a profile of 6000000).

The biotyping system described above is relatively nondiscriminatory, as C. krusei species are notorious for their weak reactivities with the standard chemicals used in commercial systems such as the API 20C AUX system that we used. Furthermore, the method is inherently flawed, as it compares phenotypes, thus running the risk of separating strains which are genetically similar yet phenotypically dissimilar or vice versa (26). Recently, several methods for the genotyping of C. krusei and the generation of strain-specific profiles have been described (2a). These include (i) pulsed-field gel electrophoresis, a technique which improves chromosomal separation with excellent resolving power, (ii) the restriction fragment length polymorphism analysis method, in which the yeast DNA is cleaved by various restriction enzymes to generate strain-specific profiles, and (iii) the RAPD analysis method, in which specific short oligonucleotide fragments can be arbitrarily primed at multiple positions of the yeast genome for PCR assays. We used the last technique, as it has proved to be a useful tool for C. krusei strain delineation in our hands (3), especially in combination with the recently developed computer-assisted system with Dendron software for gel analysis (24). Furthermore, a number of workers have found that the RAPD technique is reliable and versatile for clinical epidemiological analysis of Candida populations, as little DNA sample is required and also because of the simplicity of the technique (for recent reviews, see references 2a and 26).

As in previous work (6), we used a total of three primers in order to generate various brand profiles for the 16 isolates (Fig. 1). When these results were analyzed by use of the Dendron program, which clusters organisms according to their SABs, the data obtained with primers OBU 1, OBU2, and RSD11 generated 14, 12, and 10 clusters, respectively, with an SAB of >0.8 (Fig. 2). Accordingly, primer OBU1 was the most discriminant, while primer RSD11 was the least. These results suggest that genetic shuffling does occur among the oral C. krusei isolates from the patients studied, despite the restricted, ring-fenced, tight geographic niche in which they live and despite the lack of extraneous contaminant sources by virtue of the stigmatized disease state of the patients.

On comparison of the phenotypes and the genotypes of the isolates, it was evident that strains L24 and L38, which had unique phenotypes, were also genetically similar, as they formed a closely related cluster in assays with both primer OBU2 and primer RSD11. Subspecies variations in C. krusei have previously been documented (18, 25). The current data add credence to these observations. Further research is warranted to reclassify these variants.

In conclusion, our data provide a tantalizing glimpse of the oral mycotic flora in an as yet undescribed patient population. Most importantly, the findings presented here reveal the high prevalence of C. krusei in the cohort studied and the intriguingly divergent genomic profiles of the strains isolated. In biological terms, the prolific diversity of such a relatively innocuous opportunistic pathogen is surprising and warrants further study.

In view of the predominance of a triazole-resistant C. krusei strain in the population studied and the high prevalence and incidence of HIV infection in northern Thailand, which in turn predisposes individuals to candidiasis, similar prevalence studies should be pursued with other regional population groups. If such data indicate a predominance of this yeast in the general populace, this may have implications for the protocols guiding the management of HIV-related candidiasis, in which fluconazole is the drug of choice.

FOOTNOTES

    • Received 29 January 2002.
    • Returned for modification 31 March 2002.
    • Accepted 5 September 2002.
  • Copyright © 2002 American Society for Microbiology

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High Oral Prevalence of Candida krusei in Leprosy Patients in Northern Thailand
P. A. Reichart, L. P. Samaranayake, Y. H. Samaranayake, M. Grote, E. Pow, B. Cheung
Journal of Clinical Microbiology Dec 2002, 40 (12) 4479-4485; DOI: 10.1128/JCM.40.12.4479-4485.2002

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High Oral Prevalence of Candida krusei in Leprosy Patients in Northern Thailand
P. A. Reichart, L. P. Samaranayake, Y. H. Samaranayake, M. Grote, E. Pow, B. Cheung
Journal of Clinical Microbiology Dec 2002, 40 (12) 4479-4485; DOI: 10.1128/JCM.40.12.4479-4485.2002
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KEYWORDS

Candida
Candidiasis, Oral
Carrier State
leprosy

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