Probe Ca3 Research Articles

Molecular and Histological Association Between Candida albicans from Oral Soft Tissue and Carious Dentine of HIV-Positive Children.

Candida albicans and caries are frequently investigated among healthy and immunosuppressed individuals. The objective of this study was to demonstrate the presence of C. albicans on both oral soft and hard tissue and to investigate, at molecular level, the genetic subtype of the organism from the two oral sites. Tongue swabs and dentine scrapings from 362 HIV-positive children, referred for the extraction of carious primary teeth, were cultured on CHROMagar and identified to species level with ID32C. Histological staining of extracted carious teeth was also done. In patients with positive C. albicans cultures from both the tongue and carious dentine, DNA fingerprinting of such paired isolates was performed, using Southern blot hybridisation with the Ca3 probe. Yeasts were cultured from the tongue of 151 (41.7 %) individuals and 57 (37.7 %) simultaneously yielded positive C. albicans cultures from carious dentine. Nine different yeast spp. were identified from the tongue using the ID32C commercial system, but C. albicans was the only species recovered from carious dentine and histological investigation demonstrated fungal elements penetrated into the dentine and not limited to superficial debris on the floor of the cavity. Twelve of 13 paired isolates of C. albicans revealed identical fingerprinting patterns. The findings from this study demonstrated that in a particular individual, the same genetic subtype of C. albicans was capable of colonising both oral soft tissue and carious dentine. This renders carious teeth a constant source, or reservoir, of potentially infectious agents and, particularly among immunosuppressed individuals, should therefore not be left unattended.

Increased Mortality in Young Candidemia Patients Associated with Presence of a Candida albicans General-Purpose Genotype

The yeast Candida albicans causes life-threatening candidemia. A general-purpose genotype (GPG; corresponds to clade 1) causes more infections than other C. albicans genotypes. To investigate if GPG strains also cause higher mortality, we developed a duplex PCR assay which was 98% accurate in identifying GPG strains in an international collection of strains typed with probe Ca3. We applied the assay to 635 European C. albicans candidemia isolates. Of these, 18% conformed to the GPG genotype, 4% were of a borderline genotype, and 78% were of a non-GPG genotype, broadly consistent with genotype distributions in earlier studies. The prevalence of GPG strains was increased in females and in younger patients, exceeding 40% in infants aged ≤1 year. Logistic regression confirmed sex and age as significant determinants of GPG prevalence. Across the entire patient cohort, there was no difference in mortality for patients infected with GPG strains or other strains (36% versus 37%). However, mortality in patients aged ≤48 years was 33% for infection with GPG strains but only 15% for infection with other strains (z test; P < 0.01). Mortality rates associated with GPG and non-GPG strains were comparable in older patients (39% versus 46%). A logistic regression analysis confirmed the age-dependent impact of genotype on mortality. Thus, GPG strains may be more virulent than other strains in younger patients. Because candidemia is usually caused by endogenous strains, our PCR assay could potentially be used as a risk assessment tool for identifying younger patients most at risk of death from candidemia.

Comparison of multilocus sequence typing and Ca3 fingerprinting for molecular subtyping epidemiologically-related clinical isolates ofCandida albicans

Southern hybridization with the complex probe Ca3 is a well established tool for molecular subtyping of Candida albicans. Multilocus sequence typing (MLST) is a DNA sequence-based subtyping method recently applied to C. albicans and shown to have a high degree of intraspecies discriminatory power. However, its utility for studying the molecular epidemiology of sequential isolates from recurrent disease has not been established. We compared Ca3 Southern hybridization and MLST using seven housekeeping genes (CaAAT1a, CaACC1, CaADP1, CaPMI, CaSYA1, CaVPS13, CaZWF1b) for their ability to discriminate among 37 C. albicans isolates from recurrent cases of oropharyngeal candidiasis (OPC) in ten HIV-positive patients from India and the US. Among the 37 isolates, MLST identified 23 distinct genotypes (index of diversity = 97%); Ca3 Southern hybridization identified 21 distinct genotypes (index of diversity = 95%). Both methods clustered isolates into seven genetically-related groups and, with one exception, isolates that were indistinguishable by MLST were indistinguishable or highly related by Ca3 Southern hybridization. These results demonstrate that MLST performs equally well or better compared to Ca3 Southern hybridization for defining genetic-relatedness of sequential C. albicans isolates from recurrent cases of OPC in HIV-positive patients.

Clade-related amphotericin B resistance among South African Candida albicans isolates

Molecular epidemiology revealed 5 distinct clades among clinical isolates of Candida albicans, using DNA fingerprinting with the complex Ca3 probe. Certain clades were found to be highly enriched in particular geographical areas (e.g., clade E in Europe and clade SA in South Africa, whereas clade II is completely absent in the southwest United States). From fingerprinting data, it is concluded that little interclade recombination takes place, and therefore, it would not be unusual to expect clade-specific phenotypic characteristics. The first clade-related phenotypic difference was found with 5-flucytosine resistance being almost exclusively restricted to clade I. When in vitro antifungal susceptibility testing revealed 8.4% of South African oral yeast isolates to be naturally resistant to amphotericin B, it was decided to investigate a possible clade relationship for this relatively high resistance. Thirty-eight amphotericin B–resistant C. albicans isolates were fingerprinted, and a mixed dendrogram was constructed, including previously fingerprinted isolates of known clade affiliation. With the exception of clade III, resistant isolates occurred in all clades (clade I = 3; clade II = 3; clade NG = 3; and clade SA = 29), except clade III. However, the higher number of resistant isolates that clustered in clade SA was statistically significant ( P ≤ 0.02, χ 2 = 11.32).

Retrospective survey of candidaemia in hospitalized patients and molecular investigation of a suspected outbreak

Episodes of candida infection at a teaching hospital were investigated. During a 3-year period from 1998 to 2000, there were 53 cases of candidaemia. Candida albicans (64.2 %) was the most common causative species, followed by Candida glabrata (17.0 %) and Candida parapsilosis (15.1 %). Molecular analysis of a cluster of eight infections from a single unit was performed using Southern blotting with Ca3 probe hybridization. This showed that the patients were each infected by unrelated strains of C. albicans. On occasion, isolates were found to be closely related within individual patients. Following Southern blot analysis, it was concluded that the infections were not part of an outbreak caused by a single, epidemic strain.

Candida albicans clades

DNA fingerprinting with the complex probe Ca3 has revealed the following five Candida albicans clades: group I, group II, group III, group SA and group E. These groups exhibit geographical specificity. Group SA is relatively specific (i.e., highly enriched) to South Africa, group E is relatively specific to Europe, and group II is absent in the Southwest USA and South America. The maintenance of deep-rooted clades side by side in the same geographical locale and the apparent absence of subclade structure suggest little recombination between clades, but higher rates of recombination within clades. Exclusive 5-fluorocytosine resistance in the majority of group I isolates reinforces the above conclusions on recombination, and demonstrates that clades differ phenotypically. The ramifications of these findings with regard to pathogenesis are discussed. In particular, these findings lay to rest the idea that one strain represents all strains of C. albicans, support the need for a worldwide analysis of population structure and clade-specific phenotypic characteristics, and demonstrate that in the future, pathogenic characteristics must be analyzed in representatives from all five clades.

Oropharyngeal candidiasis caused by non- albicans yeast in patients receiving external beam radiotherapy for head-and-neck cancer

Purpose To characterize non- albicans Candida oral infections in patients with head-and-neck cancer receiving external beam radiotherapy (EBRT) with or without concurrent chemotherapy. Methods and materials Thirty-seven patients with head-and-neck cancer received EBRT in 2.0-Gy daily fractions to a median dose of 60.4 Gy (range 38–82.8, mean 64.6). They were followed for oropharyngeal candidiasis (OPC) confirmed by positive examination, positive KOH smear, and/or positive swab or swish culture. Samples were identified and plated on chromogenic media to identify non- albicans yeasts. Colonies were plated on Sabouraud dextrose slants for microdilution antifungal susceptibility testing to fluconazole. DNA typing, including karyotyping, restriction fragment length polymorphism analysis, and Southern blot hybridization with the moderately repetitive Ca3 probe, was performed on selected isolates to confirm individual species. Results Of the 37 patients, 10 (27%) developed OPC, and 26 (70.3%) displayed Candida carriage state. The median EBRT dose at time of positive culture was 22.5 Gy and at time of OPC was 28.6 Gy. Of the 6 patients receiving chemotherapy and EBRT, 4 (66%) developed OPC at median dose of 27.6 Gy. Three (8%) of 37 patients were infected with non- albicans Candida, and 3 (30%) of all 10 infections were caused by these organisms. Conclusion Non- albicans Candida is emerging as a relatively common cause of OPC in head-and-neck cancer patients. Chromogenic media are helpful to screen these infections. Our data also suggest a greater likelihood of developing OPC in patients receiving concomitant chemotherapy and EBRT.

Ca3 fingerprinting of Candida albicans bloodstream isolates from the United States, Canada, South America, and Europe reveals a European clade.

It was previously demonstrated by a cluster analysis that 26 unrelated U.S. isolates of Candida albicans separated into three distinct groups (groups I, II, and III) while South African isolates separated into four distinct groups (groups I, II, III, and SA). To verify the absence or underrepresentation of SA isolates in North America, and to identify which groups are represented in Europe and South America, collections of bloodstream isolates from each geographical locale were analyzed by cluster analyses based on genetic fingerprinting with the Ca3 probe. The results verify that North America is almost devoid of SA isolates (2%). However, the results reveal a new clade, designated group E, relatively specific to Europe. While 26% of a European collection of 46 isolates was composed of group E isolates, only 2% of the 164 North American isolates, 5% of 22 South American isolates, and 1% of 361 South African isolates were composed of group E isolates. The North American collection proved to be the least-diverse collection in regard to group representation. In a comparison of collections from the Northeast, Midwest, and Southwest regions of the United States, Canada, and South America, it was demonstrated that both the U.S. Southwest and the South American collections were devoid of group II isolates. Together these results identify for the first time a European-specific clade and demonstrate clear distinctions in the representations of the five demonstrated clades (groups I, II, III, SA, and E) in different geographical locales.

Microevolutionary changes and chromosomal translocations are more frequent at RPS loci in Candida dubliniensis than in Candida albicans

The Cd1 fingerprinting probe of Candida dubliniensis, which is extremely effective in identifying microevolutionary changes in infecting populations, generates hybridization patterns that are similar to those generated by the Candida albicans fingerprinting probe Ca3. Since Ca3 recognizes microevolutionary changes through the repeat sequence RPS, it was suggested that Cd1 also contains a RPS-like element. To test this possibility, the C. albicans RPS unit was used as a probe, and an RPS-like element, RPSd1, was cloned from C. dubliniensis. The sequence of RPSd1 was 76% homologous to that of the C. albicans RPS unit RPS620 and the organization, including the non-RPS 3′ and 5′ bordering sequences, was highly similar. This analysis revealed additional copies of the repeat extraalt element and short additional repeat (SAR) sequences in both RPSd1 and RPS620 not previously identified in the latter. This analysis has allowed us to develop a model of RPSd1 organization and to revise the model for RPS620 organization. An estimate of the average frequency of reorganization (duplication and deletion) per RPS unit in C. dubliniensis was similar to that for C. albicans, but the estimate of frequency of reorganization per C. dubliniensis genome was higher, most probably as a result of the higher estimated average number of RPS clusters in C. dubliniensis. These results demonstrate that the microevolutionary changes identified by the Cd1 fingerprinting probe are based on the reorganization of RPS-like elements and are, therefore, similar to the microevolutionary changes identified by the Ca3 probe of C. albicans. Linkage analysis of pairs of markers situated on either side of an RPS cluster on chromosome 7 further revealed frequent recombination between non-homologous chromosomes at the RPS cluster in C. dubliniensis strains, but not in C. albicans strains, suggesting that RPS clusters may function as recombination hot spots in C. dubliniensis.

Ca3 Fingerprinting of Candida albicans Isolates from Human Immunodeficiency Virus-Positive and Healthy Individuals Reveals a New Clade in South Africa

To examine the question of strain specificity in oropharyngeal candidiasis associated with human immunodeficiency virus (HIV) infection, oral samples were collected from 1,196 HIV-positive black South Africans visiting three clinics and 249 Candida albicans isolates were selected for DNA fingerprinting with the complex DNA fingerprinting probe Ca3. A total of 66 C. albicans isolates from healthy black South Africans and 46 from healthy white South Africans were also DNA fingerprinted as controls. Using DENDRON software, a cluster analysis was performed and the identified groups were compared to a test set of isolates from the United States in which three genetic groups (I, II, and III) were previously identified by a variety of genetic fingerprinting methods. All of the characterized South African collections (three from HIV-positive black persons, two from healthy black persons, and one from healthy white persons) included group I, II, and III isolates. In addition, all South African collections included a fourth group (group SA) completely absent in the U.S. collection. The proportion of group SA isolates in HIV-positive and healthy black South Africans was 53% in both cases. The proportion in healthy white South Africans was 33%. In a comparison of HIV-positive patients with and without oropharyngeal symptoms of infection, the same proportions of group I, II, III, and SA isolates were obtained, indicating no shift to a particular group on infection. However, by virtue of its predominance as a commensal and in infections, group SA must be considered the most successful in South Africa. Why group SA isolates represent 53 and 33% of colonizing strains in black and white South Africans and are absent in the U.S. collection represents an interesting epidemiological question.

Carriage frequency, intensity of carriage, and strains of oral yeast species vary in the progression to oral candidiasis in human immunodeficiency virus-positive individuals.

Candida samples were taken over a period of 2 years from 54 human immunodeficiency virus (HIV)-positive asymptomatic subjects to evaluate changes in yeast carriage, intensity of carriage, and genotype over time. Overall, we found that HIV-positive patients with CD4(+)-cell counts of between 200 and 400/microl had significantly more yeast colonization than healthy control subjects. Of the 54 patients, 11 developed thrush. We found that intensity of carriage in these 11 patients increased significantly in the progression from asymptomatic yeast carrier to an episode of oral thrush. Also, the most common yeast species isolated was Candida albicans; however, we did see a number of patients harboring multiple species at the same time. Using the C. albicans-specific probe Ca3, we found that 54% (n = 6) of the 11 patients who developed thrush maintained genetically similar strains throughout the study period, with minor genetic variations in all patients except one. Forty-six percent of these patients had either multiple strains throughout the study period (n = 2), strain replacement (n = 1), or species replacement (n = 2). Of the patients who had multiple strains, one (I4) was infected by two different strains of Candida dubliniensis distinguished by a recently developed species-specific probe. These results suggest that commensal strains colonizing HIV-positive individuals can undergo alterations prior to producing an episode of thrush.

A DNA polymorphism specific to Candida albicans strains exceptionally successful as human pathogens

A large proportion of infection-causing isolates of the yeast Candida albicans belong to a general-purpose genotype, identifiable by fingerprinting with the moderately repetitive sequence Ca3. The high prevalence of this group – up to 70% in some patient categories – suggests that its members possess genetic determinants, which enhance their success as pathogens compared to other strains. To find such determinants we are comparing the genomes of representatives of the general-purpose genotype cluster with the genomes of other strains. In this paper we describe the identification of a 985 bp HpaII fragment (MU13-4) specific to general-purpose genotype strains. The fragment was present in 90% of these strains, but only in 10% of other strains. The fragment did not hybridize with probe Ca3, used to define the general-purpose cluster. It contains elevated levels of repetitive DNA. Sequences homologous to MU13-4 are dispersed throughout the chromosomes of general-purpose strains but are rarer or absent in other strains, as judged by Southern hybridization. Using the Stanford C. albicans genome database, we have placed the MU13-4 fragment next to a CARE-1 element. We also found 79 significant homologies between parts of MU13-4 and 19 other contigs. Attempts to amplify the region surrounding the polymorphic fragment in non-general-purpose genotype strains suggest, as do the hybridization data, that the polymorphism is created by a deletion in non-cluster strains. These results show that it is possible to identify polymorphisms specific to general-purpose genotype strains. Primers against the fragment will allow PCR-based discrimination between general-purpose genotype strains and other strains, facilitating investigations aimed at determining morbidity and mortality caused by general-purpose genotype strains compared to other strains.

An investigation into the pathogenesis of vulvo-vaginal candidosis

Objective: To monitor yeasts isolated from women during and between episodes of recurrent vulvo-vaginal candidosis (VVC) to determine whether vaginal relapse or re-infection occurred. Methods: Women presenting at the genitourinary...

Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites.

The 11 kb complex DNA fingerprinting probe Ca3 is effective both in cluster analyses of Candida albicans isolates and in identifying microevolutionary changes in the size of hypervariable genomic fragments. A 2.6 kb EcoRI fragment of Ca3, the C fragment, retains the capacity to identify these microevolutionary changes, and when the C fragment is cleaved with SacI, the capacity is retained exclusively by a 1 kb subfragment, C1, which contains a partial RPS repeat element. The microevolutionary changes identified by Ca3, therefore, may involve reorganization of RPS elements dispersed throughout the genome. To test this possibility, hypervariable fragments from several strains of C. albicans were sequenced and compared. The results demonstrate that the microevolutionary changes identified by Ca3 are due to the insertion and deletion of full-length tandem RPS elements at specific genomic sites dispersed throughout the C. albicans genome. The RPS elements at these dispersed sites are bordered by the same upstream and downstream sequences. The frequency of recombination was estimated to be one recombination per 1000 cell divisions by following RPS reorganization in vitro. The results are inconsistent with unequal recombination between homologous or heterologous chromosomes, but consistent with intrachromosomal recombination. Two alternative models of intrachromosomal recombination are proposed: unequal sister-chromatid exchange and slipped misalignment at the replication fork.

Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection.

Epidemiological studies, using the probe Ca3, have shown that in a given patient population a single cluster of genetically related Candida albicans isolates usually predominates. The authors have investigated whether these local clusters are part of a single group, geographically widespread and highly prevalent as an aetiological agent of various types of candidiasis. An unrooted neighbour-joining tree of 266 infection-causing C. albicans isolates (each from a different individual) from 12 geographical regions in 6 countries was created, based on genetic distances generated by Ca3 fingerprinting. Thirty-seven per cent of all isolates formed a single genetically homogeneous cluster (cluster A). The remainder of isolates were genetically diverse. Using the maximum branch length within cluster A as a cut-off, they could be divided into 37 groups, whose prevalence ranged between 0.3% and 9%. Strains from cluster A were highly prevalent in all but one geographical region, with a mean prevalence across all regions of 41%. When isolates were separated into groups based on patient characteristics or type of infection, strains from cluster A had a prevalence exceeding 27% in each group, and their mean prevalence was 43% across all patient characteristics. These data provide evidence that cluster A constitutes a general-purpose genotype, which is geographically widespread and acts as a predominant aetiological agent of all forms of candidiasis in all categories of patients surveyed.

Elucidating the origins of nosocomial infections with Candida albicans by DNA fingerprinting with the complex probe Ca3.

Computer-assisted DNA fingerprinting with the complex probe Ca3 has been used to analyze the relatedness of isolates collected from individuals with nosocomial bloodstream infections (BSIs) and hospital care workers (HCWs) in the surgical and neonatal intensive care units (ICUs) of four hospitals. The results demonstrate that for the majority of patients (90%), isolates collected from commensal sites before and after collection of a BSI isolate were highly similar or identical to the BSI isolate. In addition, the average similarity coefficient for BSI isolates was similar to that for unrelated control isolates. However, the cluster characteristics of BSI isolates in dendrograms generated for each hospital compared to those of unrelated control isolates in a dendrogram demonstrated a higher degree of clustering of the former. In addition, a higher degree of clustering was observed in mixed dendrograms for HCV isolates and BSI isolates for each of the four test hospitals. In most cases, HCW isolates from an ICU were collected after the related BSI isolate, but in a few cases, the reverse was true. Although the results demonstrate that single, dominant endemic strains are not responsible for nosocomial BSIs in neonatal ICUs and surgical ICUs, they suggest that multiple endemic strains may be responsible for a significant number of cases. The results also suggest that cross-contamination occurs between patients and HCWs and between HCWs in the same ICU and in different ICUs. The temporal sequence of isolation also suggests that in the majority of cases HCWs are contaminated by isolates from colonized patients, but in a significant minority, the reverse is true. The results of this study provide the framework for a strategy for more definitive testing of the origins of Candida albicans strains responsible for nosocomial infections.

Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in North America by primary CHROMagar candida screening and susceptibility testing of isolates.

Candida dubliniensis has been associated with oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV). C. dubliniensis isolates may have been improperly characterized as atypical Candida albicans due to the phenotypic similarity between the two species. Prospective screening of oral rinses from 63 HIV-infected patients detected atypical dark green isolates on CHROMagar Candida compared to typical C. albicans isolates, which are light green. Forty-eight atypical isolates and three control strains were characterized by germ tube formation, differential growth at 37, 42, and 45 degreesC, identification by API 20C, fluorescence, chlamydoconidium production, and fingerprinting by Ca3 probe DNA hybridization patterns. All isolates were germ tube positive. Very poor or no growth occurred at 42 degreesC with 22 of 51 isolates. All 22 poorly growing isolates at 42 degreesC and one isolate with growth at 42 degreesC showed weak hybridization of the Ca3 probe with genomic DNA, consistent with C. dubliniensis identification. No C. dubliniensis isolate but only 18 of 28 C. albicans isolates grew at 45 degreesC. Other phenotypic or morphologic tests were less reliable in differentiating C. dubliniensis from C. albicans. Antifungal susceptibility testing showed fluconazole MICs ranging from </=0.125 to 64 microgram/ml. Two isolates were resistant to fluconazole (MIC, 64 microgram/ml) and one strain was dose dependent susceptible (MIC, 16 microgram/ml). MICs of other azoles, including voriconazole, itraconazole, and SCH 56592, for these isolates were lower. C. dubliniensis was identified in 11 of 63 (17%) serially evaluated patients. Variability in phenotypic characteristics dictates the use of molecular and biochemical techniques to identify C. dubliniensis. This study identifies C. dubliniensis in HIV-infected patients from San Antonio, Tex., and shows that C. dubliniensis is frequently detected in those patients by using a primary CHROMagar screen.

Hospital specificity, region specificity, and fluconazole resistance of Candida albicans bloodstream isolates.

In a survey of bloodstream infection (BSI) isolates across the continental United States, 162 Candida albicans isolates were fingerprinted with the species-specific probe Ca3 and the patterns were analyzed for relatedness with a computer-assisted system. The results demonstrate that particular BSI strains are more highly concentrated in particular geographic locales and that established BSI strains are endemic in some, but not all, hospitals in the study and undergo microevolution in hospital settings. The results, however, indicate no close genetic relationship among fluconazole-resistant BSI isolates in the collection, either from the same geographic locale or the same hospital. This study represents the first of three fingerprinting studies designed to analyze the origin, genetic relatedness, and drug resistance of Candida isolates responsible for BSI.

Isolation of Candida species on media with and without added fluconazole reveals high variability in relative growth susceptibility phenotypes.

Mouthwashes from human immunodeficiency virus-positive individuals were sampled for yeasts by direct plating on a differential agar medium with and without added fluconazole and via enrichment broths with and without added fluconazole. The colonies of the yeasts isolated were tested for relative growth in the presence of single concentrations of itraconazole and fluconazole. Among 258 culture plates containing yeasts obtained via different isolation routes from 86 yeast-positive samples, 33 (12.7%) of the plates showed unexpectedly high colony-to-colony variation in relative growth. Intercolony variation was seen in 41 (47.7%) of the 86 isolates when relative growth data were analyzed for all colonies of an isolate tested, regardless of the medium used for isolation. The prevalence of relative growth variability with the azoles was highest for Candida glabrata (100% of 13 isolates), followed by Candida krusei (60% of 5 isolates) and Candida albicans (40% of 53 isolates), and the visual patterns of variability seen in scatter plots of the data showed species specificity. Relative growth phenotypes generally tended to be stable for each yeast colony in subcultures, whether or not the medium used for subculture contained antifungal agents. DNA fingerprinting of stable and variable C. albicans isolates showed changes in band patterns detected with the probe Ca3, suggesting that the variability may have resulted from selection of different subtypes of the yeasts during the isolation procedure. These findings suggest that the yeasts isolated from single clinical samples were often not clonal in nature. The relative growth test revealed colony variability more readily than conventional susceptibility testing.

Frequency, intensity, species, and strains of oral Candida vary as a function of host age.

While the age of the host has been suggested as a determining factor in yeast carriage, no studies in which the genetic relatedness of isolates has been assessed in combination with the frequency and intensity of carriage as a function of host age have been performed in a single geographical locale and over a short time window. Therefore, by using a simple plating protocol to determine the frequency and intensity of carriage, sugar assimilation patterns to determine species, and Southern blot hybridization with the DNA fingerprinting probe Ca3 combined with computer-assisted analysis to determine the genetic relatedness of strains of Candida albicans, yeast carriage was analyzed as a function of age. All test individuals lived in the Iowa City, Iowa, locale and, except for some of the 0.5- to 1.5-year-olds, were dentate. The results demonstrate that for this test population, the frequency, average intensity, predominant species, and genetic relatedness of C. albicans strains varied as a function of host age. In addition, comparison with oral commensal organisms from the Ann Arbor, Mich., locale confirms the geographical specificity of C. albicans strains and the existence of an Iowa City-enriched strain which is most prevalent in elderly individuals.