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Candidiasis: the emergence of a novel species, Candida dubliniensis

Coleman, David C.1,2; Sullivan, Derek J.1; Bennett, Désirée E.1; Moran, Gary P.1; Barry, Hugh J.1; Shanley, Diarmuid B.1

Editorial Review

1The Department of Oral Medicine and Pathology, School of Dental Science and Dublin Dental Hospital, Dublin, Ireland.

2Requests for reprints to: Dr David Coleman, University of Dublin, School of Dental Science, Department of Oral Medicine and Pathology, Trinity College Dublin, Dublin 2, Republic of Ireland.

Sponsorship: Supported in part by the Irish Health Research Board grants 134/95 and 41/96 and by The Wellcome Trust grant 047204.

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The incidence of fungal infections in people has increased dramatically over the past 2 decades, as has the number and variety of infecting fungal agents [1–29]. These trends parallel a dramatic increase in the number of individuals with deficient cell-mediated immunity, particularly those infected with HIV and those receiving chemotherapeutic treatments which induce severe immunosuppression. These individuals include those having bone marrow and organ transplants and people receiving anti-cancer therapy. Furthermore, the use of broad-spectrum antibiotics, mucosal colonization by commensal yeasts, medical therapies which involve invasive surgical procedures and the use of indwelling central venous catheters render patients vulnerable to infection by a wide variety of opportunistic fungal pathogens, particularly Candida species [14,15,17,19,20,23,26,28,29].

Mycoses are among the most frequently encountered opportunistic infections in HIV-infected people and AIDS patients and are often among the earliest detectable clinical presentations of HIV-associated disease [1–10,12,13,15,18]. Oral candidiasis caused by yeast species from the genus Candida is by far the most common opportunistic infection observed in HIV-infected individuals, affecting the vast majority (90–95%) of subjects at some stage during the course of HIV disease progression [4,6,12,13,30–32]. In contrast, systemic or disseminated candidiasis is relatively rare in HIV-infected and AIDS patients [6,13]. Approximately one-third of AIDS patients develop oesophagitis at some stage during the course of their infection with HIV and retrospective and prospective endoscopic studies of symptomatic patients have found Candida to be the most common cause, occurring in 42–79% of cases [32]. Furthermore, although vaginal candidiasis is a common problem in immunocompetent women there is no definitive evidence to suggest that there is an increased incidence of this disease in HIV-positive women or in those suffering from AIDS [33,34].

Because of the growing number of immunocompromised patients it is perhaps inevitable that novel fungal species capable of causing opportunistic infections will be recognized. Many new fungal species are described each year and many of these have been reported to cause disease in humans [17,20,28]. The objectives of this review are to briefly summarize the clinical manifestations and epidemiology of oral candidiasis in HIV-infected and AIDS patients and to describe the discovery and characterization of Candida dubliniensis, a novel species of yeast associated with oral candidiasis in this patient group, in diverse geographical locations.

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Oral candidiasis in HIV-infected individuals

The clinical manifestations of oral candidiasis in HIV-infected individuals are essentially mucosal and infection often presents in multiple oral sites [6,13]. The frequency of clinical signs of oral candidiasis increases with advancing HIV disease progression, and infection is often recurrent and can sometimes be recalcitrant to therapy [2,6,13,30]. There are three main clinical presentations of oral candidiasis which manifest in HIV-infected subjects, including erythematous (atrophic) candidiasis, pseudomembranous candidiasis and angular cheilitis (Fig. 1.) [13]. The erythematous form is the most frequent and presents in approximately 50% of individuals with oral candidiasis [6,13]. Pseudomembranous candidiasis is the next most common and angular cheilitis is the least common of the three main presentations. Detailed descriptions of the clinical appearance of these lesions have been reported elsewhere [6,13]. However, in brief, erythematous candidiasis presents clinically as red lesions most commonly affecting the palate and the dorsum of the tongue, the latter of which often becomes depapillated. Erythematous candidiasis often precedes pseudomembranous candidiasis, which is observed predominantly in AIDS patients and presents as whitish yellow, semi-adherent patches or confluent membranes that can easily be removed from the mucosa leaving a red bleeding surface [6,13]. Pseudomembranous candidiasis most frequently affects the tongue, hard and soft palate and the buccal mucosa. Angular cheilitis presents at the angles of the mouth, unilaterally or bilaterally, as red fissured crusty lesions which may also be ulcerated [13].

The onset of clinical manifestations of oral candidiasis in HIV-infected people is usually preceded by oral colonization with Candida organisms. Many HIV-infected individuals at an early stage of the disease progression harbour substantially elevated levels of oral Candida organisms, relative to the levels present in normal healthy control individuals. This increase in the oral density of Candida occurs before the development of clinical signs of oral candidiasis indicating that defense mechanisms in the oral cavity are compromised in these people [35].

Because the precise events involved in the host immune reponse to Candida colonization and infection have yet to be established definitively for immunocompetent individuals, it is not surprising that the response to these organisms in HIV-infected individuals is poorly understood. The observation that tissue invasion and dissemination by Candida albicans is comparatively rare in AIDS patients suggests that the immune system is sufficiently effective at confining the infecting organisms to the mucosal layers of the oropharynx [36]. Chronic mucocutaneous candidiasis in non-HIV-infected subjects is usually associated with defects in cell-mediated immunity whereas hematogenous and disseminated infections mainly occur as a result of dysfunctional neutrophils and neutropenia [37].

Clinical and experimental data indicate that CD4+ cells are of critical importance in the host defence against candidal infection (a predominantly TH1-type pattern has been suggested) [37]. However, the nature of the specific antigen targets of this T-cell response and their protective nature in vivo are not known. Recent evidence, however, suggests that mannoproteins, which are major candidal cell wall constituents, may act as targets [38]. The finding that IL-2-activated CD8+ T lymphocytes are candidacidal and that they are particularly active against the hyphal form of C. albicans suggests that, in concert with neutrophils, these cells may help in the prevention of the dissemination of Candida infection [39]. Natural killer cells and lymphokine-activated killer cells have been demonstrated to bind directly to C. albicans. However, despite this extensive and intimate cell-cell contact and the concomitant production of γ-interferon, these cells apparently fail to induce the killing of Candida cells or the inhibition of their growth [40,41].

Because Candida infections in HIV-infected individuals are largely confined to the mucosal surface, the secretory immune response may be expected to play a role in the host response to colonization by Candida cells. Indeed, secretory IgA (sIgA) molecules have been implicated in the inhibition of the adherence of Candida cells to epithelia [42]. A number of studies have shown that, despite the fact that sIgA production is induced by T cells, AIDS patients infected with Candida have substantially higher levels of sIgA specific for Candida in whole and parotid saliva than those in control individuals [35,43]. These findings further indicate that defects in cell-mediated immunity rather than in the humoral immune response are responsible for the propensity of HIV-infected people and AIDS patients to develop oral candidiasis.

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Epidemiology of oral candidiasis in HIV-infected individuals

C. albicans is the most frequent cause of Candida infection in humans; it is the most pathogenic species of the genus Candida and often forms part of the oral and gastrointestinal commensal flora in healthy individuals [44,45]. Therefore, it is not surprising that most candidal infections in HIV-infected individuals are caused by this organism and most infections are likely to be endogenous in origin [30]. Most HIV-infected individuals appear to be colonized/infected with one or a small number of C. albicans strains, characteristic for each individual, which can persist through symptomatic and asymptomatic periods, even following antifungal drug therapy and the subsequent development of antifungal drug resistance [30,46–51]. A number of studies have also documented the transmission of oral C. albicans strains between HIV-infected partners [47,52,53]. However, detailed studies on populations of C. albicans oral isolates from HIV-infected and non-HIV-infected individuals indicate that particular types of C. albicans strains predominate in AIDS patients [30,48].

Several non-albicans Candida species, including Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei, have also been associated with oral candidiasis in HIV-infected individuals and AIDS patients, although to a much lesser extent than C. albicans, and particularly after prolonged antifungal drug therapy [54]. A number of studies indicate that the widespread therapeutic use of antifungal drugs to suppress oral candidiasis in HIV-infected individuals has been a significant factor in the selection of these organisms as opportunistic pathogens because they have an inherent reduced susceptibility relative to C. albicans, to azole antifungal drugs [30,55–57]. However, to date, insufficient studies have been performed to confirm this hypothesis unequivocally.

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Identification and taxonomy of Candida species

Classically, yeast species have been placed in the genus Candida on the basis of the absence of a detectable sexual mode of reproduction [58]. However, several species including C. krusei, Candida guillermondii, Candida kefyr, Candida lusitaniae and Candida norvegensis are now known to be the asexual forms of sexually reproductive yeasts, and yet they are currently retained within the genus [54,58].

For practical purposes, identification of Candida species has relied on the analysis of biochemical reactions and a limited number of morphological characteristics [58,59]. However, because phenotypic properties, such as substrate assimilation, colony morphology, cell wall composition and production of extracellular proteases, can vary considerably within a species such as C. albicans, defining a species is not a simple matter [54,60–64]. This has been exacerbated by a number of other anomalies in the taxonomy of Candida species (e.g. Candida clausenii, Candida langeronii and type I and type II Candida stellatoidea have been shown to be synonyms of C. albicans) [54,65–69]. Given these and numerous other irregularities in the taxonomy of Candida species it is not surprising that, as the numbers and types of Candida organisms being isolated from human infections has increased, further anomalies have become apparent [54].

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Identification of C. dubliniensis: species novus

Despite the prevalence of candidiasis caused by C. albicans over the past decade, there has been a substantial increase in the number of reports implicating non-albicans Candida species, including amongst others C. tropicalis, C. glabrata, C. parapsilosis and C. krusei, in human infection, particularly in individuals infected with HIV [4,12,17,20,23,27,28,30,54,70]. However, over the past 6 years a small number of reports have described the recovery from HIV-infected individuals of atypical oral Candida isolates (Table 1). These reports described isolates recovered from HIV-infected individuals in Ireland, Australia, Switzerland and England. On the basis of classical mycological tests, such as germ tube and chlamydospore production, these isolates were found to be very similar to C. albicans [30,48,71–76]. However, in our view they had some phenotypic and genetic properties which were not contingent with their definitive identification as C. albicans. In the light of these results, our studies [30,54,71] suggested that the unusual isolates corresponded either to an atypical subgroup of C. albicans, its closely related synonym C. stellatoidea, or to a hitherto undescribed species of Candida [71].

After the discovery of the atypical oral Candida isolates in the population of AIDS patients we studied in Dublin [30,71], we decided to perform detailed studies on these organisms to unequivocally determine their relationship to C. albicans and to the other Candida species frequently recovered from people. A total of 55 atypical oral Candida isolates recovered between August 1988 and September 1994 from separate Irish HIV-infected patients, of whom 37 had AIDS, were selected for study [69]. For comparison, six of the atypical oral C. albicans isolates recovered from five Australian AIDS patients by McCullough et al. [72,73] were also investigated, as were three atypical oral isolates from Irish HIV-negative individuals. All of the isolates were subjected to detailed phenotypic, molecular and phylogenetic studies and were compared with reference isolates of C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei. The results of the study demonstrated unequivocally that the Irish and Australian atypical isolates formed a clearly distinct group within the genus Candida for which we have proposed the name C. dubliniensis after the city of Dublin [69]. Subsequent analysis of several of the atypical C. albicans oral isolates from the Swiss HIV-infected intravenous drug users described by Boerlin et al. [74], and atypical oral Candida isolates recovered from HIV-infected patients in England and Argentina has revealed that they are identical to C. dubliniensis [77].

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Characteristics of C. dubliniensis

Phenotypic characteristics

Isolates of C. dubliniensis grow well at 30°C and 37°C on all mycological culture media suitable for the growth of C. albicans [69]. However, C. dubliniensis isolates grow poorly, or not at all, at 42°C [69], unlike isolates of C. albicans which grow well at this temperature [78]. The poor growth of C. dubliniensis isolates at 42°C is a feature shared with type I C. stellatoidea [69,78]. As mentioned previously, this is a very close relative of C. albicans and is now considered to be a synonym [58]. Like C. albicans, isolates of C. dubliniensis produce germ tubes and chlamydospores. These are the main phenotypic characteristics used for the definitive identification of C. albicans [59], features which are also exhibited by its synonym C. stellatoidea [79]. Germ tubes are cylindrical outgrowths from blastospore yeast cells and are the first step in hyphal formation. In the laboratory, germ tube production is assayed usually by incubating the yeast strain in the presence of serum for 2 h at 37°C. Chlamydospores are thick-walled, refrac-tile cells of unknown function. In vitro they are produced on nutrient-poor solid media (such as rice agar or cornmeal agar) and their production is enhanced in the presence of the detergent Tween 80 [69]. Staining with lactophenol cotton blue facilitates differentiation of chlamydospores from hyphal and pseudohyphal cells and the suspensor cells to which they are attached [69]. However, the production of chlamydospores by C. dubliniensis is radically different from that of C. albicans. In the latter species chlamydospores are generally produced singly at the ends of hyphae or pseudohyphae [59]. In contrast, in C. dubliniensis chlamydospores are produced much more abundantly and in unusual configurations, often in pairs (sometimes contiguously), triplets and occasionally in larger clusters of several chlamydospores attached to the same suspensor cell (Fig. 2) [69].

C. albicans can be divided into two serogroups (A and B) on the basis of agglutination reactions with antiserum raised against Candida antigenic factor no. 6 [54]. However, all C. dubliniensis isolates examined so far belong to C. albicans serotype A [54,69,73]. This finding allows C. dubliniensis isolates to be distinguished from type I C. stellatoidea, which belongs exclusively to C. albicans serotype B [66]. One of the most commonly used methods for identifying clinical isolates of Candida to the species level involves the use of commercially available yeast identification kits, such as the API ID 32C system (bioMérieux, Marcy l'Etoile, France), which record the ability of isolates to assimilate a variety of compounds that can be used as the sole source of carbon or nitrogen [30,44,59]. Using such a system, assimilation results for each isolate are transformed into a numerical code which can then be compared with a database to obtain a predictive value of an isolate's identity together with an estimate of how closely the profile corresponds to the most typical set of assimilation reactions in the database. Thus the database provides a list of species in order of their probability with a confidence estimate for each identification. However, there can be problems associated with the interpretation of these results because particular assimilation test results may not be consistent and can vary from one occasion to another [80,81]. C. dubliniensis isolates yield assimilation profiles with the API ID 32C yeast identification system which according to the API APILAB database correspond to poor identification of Candida sake, C. albicans 2 (i.e., C. stellatoidea) or C. albicans [69,71]. This finding has been demonstrated for atypical oral Candida isolates now known to be C. dubliniensis from HIV-infected patients in Ireland, England, Switzerland, Australia and Argentina [54,69,71,77,82].

C. dubliniensis isolates can be readily differentiated from C. albicans on the basis of colony colour after growth on the recently developed commercially available chromogenic CHROMagar Candida medium (CHROMagar, Paris, France) [81]. C. dubliniensis colonies present as dark green in colour after 48 h growth at 37°C in contrast to C. albicans colonies, which present as light blue-green in colour. Use of this medium is particularly advantageous for distinguishing between colonies of these two species in mixed culture and can be recommended as a very useful practical means for tentatively identifying a variety of Candida species in clinical specimens, including C. dubliniensis [82,83].

C. dubliniensis is phenotypically very similar to C. albicans. The ability of the former to produce germ tubes and chlamydospores, traits previously only associated with and diagnostic for C. albicans and its synonym C. stellatoidea, suggests that C. dubliniensis has probably been present in the community for a long time and has probably been misidentified as C. albicans. One confusing factor in our early studies was that a reference strain from the British National Collection of Pathogenic Fungi of C. stellatoidea (NCPF3108) possessed the characteristics of C. dubliniensis [71]. Before the designation of C. dubliniensis as a new species, we suggested that the atypical oral isolates from HIV-infected individuals in Dublin described by Sullivan et al. [71] may have been variants of type I C. stellatoidea. However, this possibility is extremely unlikely given that the latter are unable to assimilate sucrose and belong to C. albicans serotype B [66]. That this was indeed not the case was further borne out when in-depth molecular and phylogenetic comparisons between C. albicans, C. stellatoidea and C. dubliniensis were performed.

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Genotypic characteristics

Detailed molecular genetic analyses demonstrated the unique nature of C. dubliniensis compared with other Candida species, especially with regard to C. albicans and C. stellatoidea. The first evidence of this distinction was obtained from DNA fingerprinting analysis of C. dubliniensis genomic DNA with the cloned C. albicans- and C. stellatoidea-specific mid-repeat sequence probe 27A developed by Scherer and Stevens [84]. This probe hybridizes with repetitive sequences dispersed throughout the C. albicans and C. stellatoidea genomes [66,69,83], and usually hybridizes strongly to between 10 and 15 restriction endonuclease Eco R1-generated fragments of C. albicans genomic DNA ranging in size from 500 bp to 20 kb (Fig. 3) [30,69]. In the case of C. dubliniensis isolates, the 27A probe hybridizes poorly to between four and seven bands ranging in size from 5 to 20 kb (Fig. 3) [69]. These results indicate that the genomic organisation of C. dubliniensis is significantly different from that of C. albicans and C. stellatoidea. Further evidence in support of this conclusion is evident from examination of Hinf1-generated restriction fragment length polymorphism (RFLP) profiles of C. dubliniensis genomic DNA which are distinctly different from the corresponding profiles of C. albicans and C. stellatoidea [69]. Additional data confirming the unique genomic organisation of C. dubliniensis is evident from genomic DNA fingerprinting studies using short repeat sequence motif-containing oligonucleotide probes such as (GATA)4, (GACA)4 and (GTG)5 and by random amplified polymorphic DNA (RAPD) analysis using a range of oligonucleotide primers [69]. C. dubliniensis isolates also have distinctive karyotype patterns consisting of nine to 10 chromosome sized DNA bands, usually with one or more bands < 1 Mb in size [69]. This is also a characteristic of type I C. stellatoidea, but not of C. albicans isolates, which typically have eight pairs of chromosomes [65]. Preliminary data using cloned chromosome-specific genes from C. albicans as molecular probes in Southern hybridization experiments suggests that the large number of chromosome sized bands present in C. dubliniensis karyotype profiles is caused by fragmentation of the larger chromosomes (unpublished data).

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Molecular phylogenetic analysis of C. dubliniensis

Analysis of C. dubliniensis genomic DNA structure by DNA fingerprinting and karyotype analysis and studies of C. dubliniensis phenotypic characteristics demonstrated that C. dubliniensis isolates are clearly distinct from C. albicans and C. stellatoidea. However, these studies provided a purely qualitative estimate of difference between these organisms. One accurate method of determining a precise measure of the genetic relationships between individual species involves the comparison of the nucleotide sequence of the genes encoding ribosomal RNAs (rRNA) [85]. The rRNA genes are evolutionarily highly conserved and are present in eukaryotic genomes in multiple copies. Analysis of the V3 variable region of large ribosomal gene sequences has yielded very useful data on the phylogenetic relationships between a variety of marine yeasts [86,87]. Comparison of the sequence of the V3 variable region of the large ribosomal RNA subunit genes from eight isolates of C. dubliniensis recovered from HIV-infected individuals in Ireland and Australia and from reference strains of C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that the C. dubliniensis isolates comprised a homogeneous cluster phylogenetically distinct from the other Candida species examined (Fig. 4) [69]. These studies showed that C. albicans is the most closely related species to C. dubliniensis. This is not surprising given the close phenotypic similarities between the two taxons. Comparison of the nucleotide sequence of the entire small ribosomal RNA subunit gene (approximately 1.8 kb) from C. dubliniensis with the corresponding sequences from a variety of other Candida species confirmed these results (unpublished data).

Boerlin et al. [74] recently reported the recovery of germ tube- and chlamydospore-positive atypical oral isolates from HIV-infected intravenous drug users in Lausanne, Switzerland. These isolates were very similar to the Irish and Australian atypical isolates described by Sullivan et al. [71] and McCullough et al. [73], and which are now known to be C. dubliniensis [69]. Several of the atypical isolates described by Boerlin et al. [74] have been studied in our laboratory and have been shown to be identical to the Irish and Australian C. dubliniensis isolates referred to above [77]. In addition, Boerlin et al. [74] using the technique of multilocus enzyme electrophoresis (MLEE) showed that the allelic makeup of their C. dubliniensis isolates was clearly distinct from that of C. albicans and C. stellatoidea, thus confirming the unique taxonomic position of C. dubliniensis determined from the phylogenetic studies described above.

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Occurrence of C. dubliniensis

Because C. dubliniensis was first described as a new species in July 1995, there is only a limited amount of data on the incidence of C. dubliniensis in HIV-infected individuals [69]. However, some data can be gleaned from published studies describing the recovery of atypical oral Candida isolates. Table 1 shows data extracted from the five primary published studies describing the recovery of atypical oral Candida isolates phenotypically similar to C. albicans from HIV-infected subjects [48,71,73–75]. The isolates from three of these studies have subsequently been shown to be C. dubliniensis [69,77]. However, because of insufficient information it was only possible to calculate the incidence of recovery of C. dubliniensis from two of these studies; in the case of Sullivan et al. [71] eight out of 33 (24.2%) of the HIV-infected patients included in the study yielded C. dubliniensis, whereas in the case of McCullough et al. [72,73] nine out of 60 (15%) of the AIDS patients included in the study yielded C. dubliniensis (Table 1).

The most extensive data currently available on the incidence of C. dubliniensis in HIV-infected and non-HIV-infected subjects comes from studies of Irish subject cohorts (Table 2) [82] (unpublished data). Oral C. dubliniensis was recovered from 32% of AIDS patients presenting with clinical symptoms of oral candidosis and from 25% of asymptomatic AIDS patients. Similarly, a high incidence of recovery of C. dubliniensis was recorded for HIV-positive subjects, both with (27%) and without (19%) clinical symptoms of oral candidiasis. In contrast, C. dubliniensis was recovered from only 3% of HIV-negative normal healthy individuals without oral candidiasis and from 14.6% HIV-negative individuals with denture-associated oral candidiasis. These results suggest that C. dubliniensis is present at a low incidence (3%) as part of the normal oral flora in normal healthy individuals. However, the prevalence of C. dubliniensis in asymptomatic HIV-positive individuals is significantly higher (19%) and higher again in asymptomatic AIDS patients (25%). Furthermore, the incidence of C. dubliniensis in HIV-positive (27%) and AIDS (32%) patients with oral candidiasis is almost double, in each case, the incidence recorded in HIV-negative individuals with oral candidiasis (Table 2).

The vast majority (76%) of the HIV-positive and AIDS patients and the HIV-negative individuals (83%) who were found to harbour oral C. dubliniensis also harboured other Candida species in the oral cavity (Table 2). The most common of these was C. albicans, followed by C. glabrata, C. tropicalis and C. krusei. In many instances two or more of these Candida species were co-isolated with C. dubliniensis.

Because C. dubliniensis and C. albicans are phenotypically very similar, it is highly likely that isolates of C. dubliniensis have been misidentified as C. albicans or C. stellatoidea in the past. Evidence in support of this was obtained by performing detailed phenotypic and molecular investigations on two stored collections of Candida oral isolates which had originally been identified as C. albicans on the basis of their ability to produce germ tubes and chlamydospores. The results of the study revealed that two out of 110 (1.82%) of the presumptive C. albicans oral isolates recovered from asymptomatic normal healthy individuals and 13 out of 79 (16.46%) of the presumptive C. albicans oral isolates recovered from HIV-infected individuals were in fact C. dubliniensis (unpublished data). Furthermore, previous studies from this laboratory demonstrated that Candida strain NCPF3108, which was listed as C. stellatoidea in the British National Collection of Pathogenic Fungi, was indistinguishable from C. dubliniensis [69,71].

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The significance of oral C. dubliniensis in HIV-infected individuals

In recent years the importance of non-albicans Candida species in human infection, particularly in patients infected with HIV, has increased significantly [4,11,14,20,26,29–31,54,55,57,70,71,88–91]. Undoubtedly, the underlying severe immunosuppression present in these individuals renders them susceptible to a growing number of opportunistic yeast pathogens and reports from several studies suggest that the widespread therapeutic and prophylactic use of antifungal drugs, particularly in cases of recurrent disease, may also contribute significantly [18,55,57,71]. Many non-albicans Candida species such as C. glabrata, C. krusei and C. tropicalis are inherently less susceptible to commonly used antifungal drugs than C. albicans, and these could be selected in HIV-positive and AIDS patients after antifungal drug therapy [11,16,18,55,71, 92–95].

The significance of C. dubliniensis in HIV-infected patients is presently unclear. Whether all the Candida species present in the oral cavity of individuals with oral candidiasis contribute significantly to the disease process is nearly impossible to judge. However, it is easier to make such a judgment when only a single oral species is recovered, particularly when resolution of clinical symptoms can be correlated with the eradication of the organism after antifungal drug therapy. In the HIV-infected patient population we studied 27.6% of those with oral candidiasis yielded C. dubliniensis as the only Candida species recoverable after laboratory culture (Table 2). These results suggest indirectly that C. dubliniensis was responsible for oral candidiasis in these individuals. Data in support of this suggestion were available for eight of these individuals, whose clinical symptoms resolved after fluconazole therapy with subsequent failure to isolate the organism by culture from the oral cavity after the cessation of therapy.

Whether antifungal drug therapy has an influence on the prevalence of oral C. dubliniensis is presently unknown, but many of the HIV-infected patients from whom these isolates have been recovered were previously treated (some for prolonged periods) with fluconazole for oral candidiasis [71–74]. In one part of a study from our laboratory 20 clinical isolates of C. dubliniensis from 10 HIV-positive and five HIV-negative individuals were assessed for their susceptibility to fluconazole by broth microdilution [96]. Sixteen of the isolates were found to be fluconazole-susceptible (MIC range 0.125–1.0 µg/ml) and four (recovered from two separate AIDS patients) were fluconazole-resistant (MIC range 8–32 µg/ml). The three AIDS patients from whom the five fluconazole-resistant isolates were recovered had each been treated with fluconazole for oral candidiasis preceding the recovery of the resistant isolates. However, it should be noted that two of the other HIV-infected individuals who had received flu-conazole therapy previously yielded fluconazole-sus-ceptible C. dubliniensis isolates. These results demonstrate that fluconazole resistance does occur in clinical populations of C. dubliniensis and that it is very likely that this property provides these organisms with a survival advantage in vivo. Encouragingly these studies also show that existing methods for assessing antifungal drug susceptibility can be used with C. dubliniensis.

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C. dubliniensis is a recently identified species of Candida phenotypically similar to but genetically very distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of a substantial number of HIV-infected and AIDS patients from a variety of countries throughout the world. Evidence indicates that C. dubliniensis is present as a commensal organism in many of these individuals but has been indirectly implicated as the causative agent of oral candidiasis in some. The organism has also been identified as an oral commensal in HIV-negative individuals although at a significantly reduced incidence. The precise contribution of C. dubliniensis to oral disease in HIV-infected individuals has yet to be established. However, expression of fluconazole resistance by some oral isolates from these patients has important therapeutic and epidemiological consequences. Ongoing studies with C. dubliniensis are directed towards the analysis of pathogenicity, antifungal drug resistance and epidemiology.

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Oral candidiasis; Candida dubliniensis; opportunistic pathogen; HIV-infected; novel yeast; fluconazole resistance

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