Unfortunately, there have been relatively few investigations of C. glabrata compared to other Candida species. Although this infection is second or third in frequency after C. albicans, difficult to treat, and associated with a high mortality rate, publications to date on C. glabrata account for only a small percentage of published studies on medically important fungal infections. Very little is known about the virulence of C. glabrata, and virtually nothing is known about the host defenses directed against the organism. There are only two established animal models of experimental C. glabrata infections (systemic and vaginal) (24, 41). Therefore, studies to understand the pathogenesis of C. glabrata infections are sorely needed. This review discusses what is currently known about C. glabrata infections and includes specific comparisons to C. albicans wherever possible. Specific topics discussed include its biology, epidemiology, pathogenesis, clinical perspectives, treatment, and antifungal resistance.

C. glabrata, together with other Candida species, belongs to the class Fungi Imperfecti, the order Moniliales, and the family Cryptococcaceae (91, 148). C. glabrata is a nondimorphic yeast that exists as small blastoconidia under all environmental conditions as a pathogen. In fact, C. glabrata is the only Candida species that does not form pseudohyphae at temperatures above 37°C. Figure 1 shows wet-mount preparations of C. glabrata and C. albicans at similar magnifications. It is clear that C. glabrata blastoconidia (1 to 4 μm) are considerably smaller than C. albicans blastoconidia (4 to 6 μm). On Sabouraud dextrose agar, C. glabrata forms glistening, smooth, cream-colored colonies which are relatively indistinguishable from those of other Candida species except for their relative size, which is quite small. On Chromagar, a relatively new agar that distinguishes different Candida species by color as a result of biochemical reactions, C. glabrata colonies appear pink to purple, in contrast to C. albicans colonies, which appear green to blue-green. A critical distinguishing characteristic of C. glabrata is its haploid genome, in contrast to the diploid genome of C. albicans and several other non-albicans Candida species (176). Finally, C. glabrata is distinguishable from C. albicans by its small-subunit rRNA (4).

FIG. 1 Size differential of C. glabrata and C. albicans. Shown are wet-mount slide preparations of C. glabrata (A) and C. albicans (B) on a hemocytometer. Magnification, ×400.

Most medically important Candida species can be easily differentiated from one another by either established commercially available biochemical tests or molecular biology techniques. With the advent of molecular genetics, newer identification methods have emerged. These methods use comparative analysis of chromosomal DNA to identify Candida species from each other and also to delineate different strains within a species. These newer methods include restriction fragment length polymorphisms, pulsed-field gel electrophoresis, randomly amplified polymorphic DNA, and DNA probes (77, 79, 95, 170). By using contour-clamped homogeneous electric field gel electrophoresis (CHEF), a form of pulsed-field gel electrophoresis, chromosomal DNA from C. glabrata can be separated based on the different chromosomal molecular weights and thus can be subjected to electrophoretic karyotyping (EK). The EK pattern of C. glabrata generally produces 10 to 13 bands (79, 170). Depending on the EK patterns, C. glabrata can be classified into several different strain types. To date, 28 strain types have been formally described (170), although more than 70 different strains have been identified (168). In contrast, CHEF usually separates C. albicans chromosomal DNA into eight chromosomal bands, with more than 90 different strain types identified to date (168). Figure 2 shows the CHEF-derived DNA-banding patterns characteristic of C. glabrata and C. albicans.

FIG. 2 CHEF of genomic DNA from representative isolates of C. albicans and C. glabrata. Lanes 1 to 3 and 5 are similar strains of C. albicans; lanes 4 and 6 are strains of C. glabrata.

The biochemical reactions of C. glabrata are also quite distinct. In contrast to C. albicans, which ferments and/or assimilates a number of sugars, C. glabrata ferments and assimilates only glucose and trehalose (91). In fact, this repertoire of sugar utilization is unique compared to the majority of Candida species and is used by several commercially available kits (API 20C, Uni-Yeast-Tek, and YeastIdent) to identify yeast to the level of genus and species.

Historically, C. glabrata was classified in the genus Torulopsis (91). The genus Torulopsis was described in 1894, while the genus Candida was not named until 1913. C. glabrata was originally placed in the genus Torulopsis due to its lack of pseudohypha production. However, in 1978, it was determined that the ability to produce pseudohyphae was not a reliable distinguishing factor for members of the genus Candida and it was proposed that T. glabrata be classified in the genus Candida (91). The incorporation of T. glabrata into the genus Candida required that the description relative to pseudohyphae for the genus Candida be changed from “pseudomycelial” to “pseudohyphae: absent, rudimentary, or well developed” (91). This change in nomenclature has taken considerable time to gain acceptance by the medical mycology community, and several publications still refer to C. glabrata as T. glabrata. Wherever possible, efforts should be made to use the contemporary nomenclature.

Candida species are ubiquitous organisms (115). An increasing incidence of fungal infections with Candida species has been noted in immunocompromised patients such as intensive-care, postsurgical, and neutropenic patients (7, 11, 14, 67, 90, 175). Candida species are most frequently isolated from the oral cavity and are detected in approximately 31 to 55% of healthy individuals (115). Colonization rates increase with severity of illness and duration of hospitalization (115, 170, 175). Historically, C. albicans accounted for 70 to 80% of the isolates recovered from infected patients. C. glabrata and C. tropicalis each accounted for approximately 5 to 8% of isolates, while other non-albicans Candida species occur only rarely (3, 7). However, more recent epidemiological data reveal a mycological shift from C. albicans to the non-albicans Candida species such as C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei (7, 90, 107, 180, 183, 184).

The changing patterns and the increasing incidence of disseminated Candida infection are also evident in a large autopsy series (11). The high mortality rate associated with bacterial infections has declined with the early administration of empirical antibiotics, while systemic fungal infections have become increasingly important in causing morbidity and mortality in immunocompromised patients. Candida is now the fourth most common organism recovered from blood cultures in hospitalized patients (7). C. glabrata has recently emerged as an important nosocomial pathogen, yet little is known about its epidemiology. Although C. albicans is the most common fungal species isolated from blood, C. glabrata currently ranks fourth among Candida species (third in patients who have undergone surgery) and is associated with an equally high mortality rate (51, 90, 181, 184). C. glabrata is of special importance because of its innately increased resistance to antifungal agents, specifically the azoles (49, 61, 174, 181, 184). The current epidemiological data for C. glabrata is summarized in Table 1.

TABLE 1 Epidemiology of C.glabratainfection

A clear understanding of the epidemiology of Candida infection and colonization has been difficult because of a lack of reliable typing systems to evaluate strain homology. Previous typing systems have relied on phenotypic differences within a Candida species, which may not reflect true strain differences (26, 71, 106). However, recent advances in the use of molecular techniques have enabled investigators to develop a typing system with greater sensitivity (26, 34, 70, 71, 106, 169, 172). Molecular typing of Candida by DNA fingerprinting involving various molecular techniques (restriction fragment length polymorphism, CHEF, and randomly amplified polymorphic DNA), has the capability to differentiate closely related strains which may have phenotypic similarities (26, 70, 79, 161, 169, 172).

Based upon epidemiological studies, it is apparent that humans are exposed repeatedly to Candida in food and other sources. However, the natural history of this commensal “normal” colonization over weeks, months, and years is poorly understood. Nevertheless, one may reasonably conclude that Candida colonization is almost universal. A feature common to colonized individuals is that the most frequent species are still C. albicans, and so far no unique strains of C. albicans or any non-albicans Candida species with specific gastrointestinal tract tropism have been identified. DNA typing of Candida strains obtained from AIDS patients with oral and esophageal candidiasis indicate an identical distribution frequency to those of isolates present in healthy subjects (12). This suggests that AIDS-associated candidiasis is not caused by unique or particularly virulent strains but probably results from defects in host defense mechanisms.

Until recently, most reports describing the epidemiology of nosocomial C. glabrata have been retrospective, and few studies have evaluated independent risk factors associated with nosocomial C. glabrata acquisition and subsequent infection. Knowledge of the epidemiology of fungal nosocomial colonization and infection with C. glabrata is, however, essential for the prevention of further spread as well as of nosocomial infection. In a recent study by Vazquez and colleagues (170), multivariate prospective case-control analysis along with molecular analysis of C. glabrata demonstrated that patients with new acquisition of C. glabrata had a longer duration of hospitalization (18.8 and 7.6 days, respectively; P < 0.001) and more frequent prior antimicrobial use (100 and 65%, respectively; P < 0.001) compared to patients from whom Candida species were not recovered during the study. These results are similar to the findings noted in earlier epidemiological studies of C. albicans, C. lusitaniae, and C. parapsilosis (138, 139, 172). Little is known about the hospital reservoirs of C. glabrata, but, as with C. albicans, probable sources include a complex interaction of environmental and human reservoirs (72, 172). The unique role of the hospital environment as a potential reservoir for Candida species is further suggested by findings in a recent study in which identical strains of C. glabrata were isolated from the environment before being newly acquired by patients admitted into a Bone Marrow Transplant Unit (170). Fungal organisms isolated from the inanimate hospital environment were previously considered to contribute little to nosocomial fungal infection. Although infecting strains can be cultured from environmental surfaces, it is believed that the environment becomes passively contaminated by organisms from patients (170, 172). Two studies have implicated carriage on the hands of hospital personnel as a possible source of an outbreak (75, 172). Thus, C. glabrata may be similar to C. albicans and other nosocomial pathogens that are acquired directly or indirectly from contaminated environmental surfaces. Previous understanding of the pathogenesis of C. glabrata colonization and infection assumed that the organisms responsible for disease were endogenously acquired exclusively from the patients’ own flora.

The role of carriage by personnel in dissemination of C. glabrata remains to be clarified. Although C. glabrata is not frequently recovered from the hands of hospital personnel, transient carriage is suggested by its isolation on environmental surfaces in contact with hands (170). Perhaps more frequent culturing of the hands of personnel or the use of liquid media to recover yeasts may have improved the detection rates of C. glabrata. Proximity to a patient with infection or colonization increases the risk of nosocomial acquisition (170). As in earlier studies (124, 172), the results of longitudinal cultures showed that 75% of patients generally carried the same strain type of C. glabrata over time (170), with minimal strain diversity among individual patients. This finding is significantly different from the results described for the nosocomial acquisition of C. albicans, in which there was considerable strain diversity (172). Moreover, in this study, 71% of patients with positive C. glabrata cultures had more than one Candida species isolated. The most frequent combination was C. glabrata and C. albicans, which was found in approximately 70% of the patients. This again is in contrast to the findings previously described for C. albicans, which showed that only 39% of patients with C. albicans had more than one Candida species identified (175). Finally, unlike C. albicans, C. glabrata has not been recovered from the food provided to hospitalized patients, potentially contributing to the lack of identifiable C. glabrata strain diversity.

In conclusion, these studies suggest that nosocomial acquisition of C. glabrata is not uncommon and may be due to exogenous acquisition. In addition, two major risk factors associated with C. glabrata colonization are prolonged duration of hospitalization and prior antimicrobial use. Further prospective studies are sorely needed to define more clearly the reservoirs of infection, as well as the mode of transfer and measures for preventing the spread of infection.

In this section, although very little has been studied, we discuss what is currently known about virulence factors of C. glabrata, host defense against this organism, and established experimental animal models of C. glabrata infections.

Proteinase production by C. albicans is associated with pathogenicity (17, 135). For example, virulent C. albicans isolates often produce aspartyl proteinase. These isolates are more pathogenic in a variety of animal models of experimental Candida infections (17, 23). Although little is known of proteinase production by C. glabrata, a single study has shown that isolates of C. glabrata are at least capable of proteinase production, but the type of proteinase was not specified (19).

Adherence is an extremely important virulence factor, although the actual adherence property may be compounded by other virulence properties. For example, cell surface hydrophobicity (CSH), which is affected by environmental factors, can affect specific adherence based upon interaction of adhesin receptors. In a study with limited numbers of C. glabrata isolates tested, C. glabrata was shown to have comparable CSH to C. albicans (85). Interestingly, however, while the CSH of C. albicans was extremely sensitive to specific growth conditions, numerous isolates of C. glabrata were relatively insensitive to those same growth conditions (60), suggesting that C. glabrata is not as sensitive or as influenced by environmental factors. In comparative in vitro assays of adherence to vascular endothelium, while C. albicans was by far the most adherent species, C. glabrata was the least adherent, alone with C. parapsilosis and C. kefyr, behind C. tropicalis and C. krusei (84). Moreover, while C. albicans is recognized avidly by monoclonal antibodies to β₂ integrins (adhesin receptors), binding to C. glabrata by the same antibodies was undetectable, as was binding to C. parapsilosis and C. krusei. These results suggest that C. glabrata may not express these specific adhesins and thus would have a disadvantage in adherence (8). The presence of fibronectin, and laminin receptors, fibrinogen-binding proteins, and mannoprotein adhesins are also considered important means of adhesion to endothelial and/or epithelial cells (reviewed in reference 69). While extensive work has been performed on surface ligands of C. albicans, nothing is known about these receptors and proteins on C. glabrata. It will be important to reexamine many of the parameters from earlier studies, together with a number of new parameters, by using current clinical C. glabrata isolates obtained from patients with fulminant candidiasis.

Extracellular membrane-damaging phopholipases are considered virulence factors for C. albicans (5, 73). Although these enzymes have not been studied extensively, phospholipase A and B and lysophospholipase-transacylase are produced by virulent but not avirulent (commensal) strains of C. albicans. These phospholipase-producing strains also adhered most strongly to epithelial cells. Furthermore, the production of these phospholipases by clinical isolates correlated with pathogenicity and was predictive of mortality in animal models (5, 73). Phospholipase activity has not been studied in C. glabrata.

Another virulence factor of C. albicans is specific phenotypic instability, which allows strains to switch colony phenotype without affecting the identifiable genotype; this is termed “phenotypic switching” (155, 156). Although phenotypic switching was studied largely as an in vitro phenomenon, there is some evidence of in vivo phenotype switching and an association of switched phenotypes with virulence. Switching of phenotypes in clinical C. albicans isolates from women with recurrent C. albicans vaginitis has been reported (158). Recently, it was determined that phenotype switching does occur in C. glabrata (157). It is interesting that such a phenomenon would occur in nondimorphic organisms as well as in haploid organisms. Although the relationship of this C. glabrata phenotype switching to virulence is unknown, it may enhance virulence and play a role in causing symptomatic infection.

Since C. glabrata is a commensal organism similar to C. albicans, there are likely to be normal host mechanisms that effectively control C. glabrata, holding it in check and suppressing the expression of its pathogenic properties, thereby preventing infection. However, the relatively low pathogenicity of C. glabrata compared to C. albicans in animal models (reviewed below) suggests that control of C. glabrata may not require mechanisms that are as stringent as that required to hold C. albicans in check. Nevertheless, the increased prevalence of C. glabrata infections in immunocompromised individuals indicates that some level of host defense does indeed exist. The interaction of Candida species with endothelial and epithelial cells has recently taken an immunological twist in addition to a simple adherence phenomenon. We recently showed that epithelial cells inhibit the growth of C. albicans in vitro (160), and Filler et al. have shown that endothelial cells phagocytize C. albicans (47). Unfortunately, C. glabrata did not induce endothelial-cell phagocytosis (47), suggesting that this endothelial-cell activity may be species specific or restricted to C. albicans alone. However, it remains possible that both conventional and unconventional immune cells play some role in innate and/or acquired host defense against C. glabrata infection.

There has been only one formal clinical study that examined host defenses in patients with C. glabrata infections (105). In this German study, humoral and innate cellular defenses were examined in women with either C. glabrata or C. albicans vaginitis. A total of 14 women with C. glabrata vaginitis and 20 and 42 women with acute or chronic C. albicans vaginitis, respectively, were tested. The responses were compared to those in 77 control women. For each woman, secretory IgA (sIgA), IgA, and numbers of granulocytes and macrophages in vaginal secretions and IgA in blood were tested. For each parameter, few differences were detected with respect to the controls. In fact, the only difference in the entire study was in women with C. glabrata vaginitis, who showed a slight, but significantly lower level of sIgA in vaginal secretions (105). However, it is unclear what proportion of the IgA measured was C. glabrata or Candida specific. Also noted in the women with C. glabrata vaginitis was a lack of inflammation compared to those with C. albicans vaginitis. While no clear pattern of local or systemic innate or humoral immune deficiency was observed in women with C. glabrata vaginitis and although local or systemic T-cell function in response to C. glabrata was not tested, it would appear that identification of immunological deficiencies and dysfunctions in C. glabrata-infected women may prove to be as difficult as it has been for those with C. albicans vaginitis (42, 44, 48, 105).

In the absence of other formal studies, there have been clinical observations that provide some indication of what may be important for host defense against mucosal or systemic C. glabrata infections. The incidence of C. glabrata mucosal or systemic infections in cancer patients (182), transplant recipients (184), and AIDS patients (37, 140, 179), in whom T-cell function is impaired, suggests that T cells may be important for protection of at least some tissues against C. glabrata infection. Additionally, histological examination of tissues infected with C. glabrata has shown relatively mild infiltrates of lymphocytes, macrophages, and neutrophils (61) compared to that observed in C. albicans infection. In contrast, there are no known reports of increased C. glabrata infections in patients with B-cell deficiencies, again suggesting that antibodies are not critical to protection against C. glabrata infections.

In studies comparing antigens of C. glabrata to those found in other Candida species, specific antigens appear to be common across several Candida species (13, 109). Certain antibodies produced against C. albicans recognize C. glabrata as well as other Candida species. Specifically, antibodies reacting with antigen 6 of C. albicans serotype A react with C. glabrata as well, suggesting that antigen 6 is conserved between the two species (109). Additionally, Cutler and coworkers (13) have reported an antibody produced against C. glabrata that also cross-reacts with other Candida species. These results suggest that protective immunity against Candida species, specifically C. albicans, may be capable of providing a level of protection against C. glabrata infections as well. This could potentially include any form of innate resistance (polymorphonuclear leukocytes, macrophages, and natural killer cells) or acquired CMI (T cells) in addition to humoral responses (B cells and antibodies).

Our laboratory has performed a limited number of experiments involving immune system reactivity to C. glabrata. In a limited number of tests performed with human peripheral blood lymphocytes, we recently found that human peripheral blood lymphocytes respond in vitro to heat-killed C. glabrata in a manner similar (approximately 80 to 85% in magnitude) to that observed for C. albicans (38). Thus, normal healthy adults appear to be sensitized to C. glabrata with demonstrable cell-mediated responsiveness, although we recognize that such responses may be the result of cross-reactive antigens on C. glabrata recognized by C. albicans-specific cells. In an animal model, we found that nonobese diabetic (NOD) mice infected vaginally with C. glabrata did not respond by developing delayed-type hypersensitivity to C. albicans culture filtrate antigen (38) whereas mice used in the experimental C. albicans vaginitis model (CBA/J mice) readily respond to C. albicans culture filtrate antigen by developing delayed-type hypersensitivity (39). This data suggests that a vaginal C. glabrata infection does not induce a systemic CMI response that is cross-reactive or responsive to C. albicans antigen. However, it is not known whether this is due to the lack of cross-reactivity between C. glabrata and C. albicans, the lack of induction of C. glabrata-specific CMI, or the inability of NOD mice to mount an effective T-cell response. There have been inconsistent results with the NOD mice regarding in vitro T-cell reactivity. In one study, draining lymph node cells from NOD mice infected vaginally with C. glabrata responded to both heat-killed C. glabrata and heat-killed C. albicans as detected by lymphocyte proliferation, whereas in another study, the lymph node cells did not respond to either particulate antigen (38). Although additional studies should be performed, if indeed C. glabrata-infected mice do generate Candida-specific T-cell responses in the draining lymph nodes, there appears to be some level of cross-reactivity between the responses to C. glabrata and C. albicans. However, the critical experiments involving the lymph node responses to C. glabrata in C. albicans-infected mice have not been performed. The predominant response of draining lymph node cells in such infected mice to C. albicans antigen is a Th1-type response characterized by the production of IL-2 and gamma interferon (110). Finally, understanding the important host defenses against C. glabrata will require controlled studies conducted in animal models of systemic and mucosal C. glabrata infections.

A recent report describing an increase in C. glabrata vaginal infections (151) emphasized the need to develop a vaginal model of C. glabrata infection. In particular, models of C. glabrata mucosal infections had been difficult to establish. In one report, an oral C. glabrata infection in rats could not be achieved (145). Our laboratory attempted to develop an experimental model of vaginal C. glabrata infection to complement our model of vaginal C. albicans infection (39). This also proved difficult. Preliminary experiments with the mouse strain used for C. albicans vaginal infection (immunocompetent CBA/J mice) showed no detectable C. glabrata vaginal burden as early as 6 days following an intravaginal inoculum in spite of using multiple clinical C. glabrata isolates and pseudoestrus conditions (required to achieve a vaginal C. albicans infection) (41). Similarly, a low detectable vaginal fungal burden was observed in DBA/2 mice, which are highly susceptible to systemic C. albicans infection (99). In contrast, nonobese diabetic (NOD/Lt) mice were susceptible to C. glabrata vaginal infection (Fig. 3) (41). In comparison to C. albicans infections, although a higher inoculum of C. glabrata was routinely used (1 × 10⁷ blastoconidia) than that of C. albicans (5 × 10⁵ blastoconidia), inocula as low as 5 × 10⁵ blastoconidia were capable of establishing C. glabrata infections. The infection was sustained for 14 days at high titers and became resolved in most animals by 21 days. The vaginal titers of C. glabrata at 6 to 14 days postinoculum (>10⁶ CFU) were higher than those commonly observed in C. albicans-infected mice (10⁴ to 10⁵ CFU) (39) and persist in pseudoestrus-treated mice for 8 weeks or more (39). We next examined how NOD mice could support vaginal infections caused by other fungal species, namely, C. albicans (highly virulent) and Saccharomyces cerevisiae (low virulence). Intravaginal inoculation with C. albicans resulted in extremely high titers of C. albicans (>10⁶ CFU) and a surprising 20% mortality rate, although no dissemination of the organism could be detected (kidney dysfunction was suspected as the cause of death). Animals inoculated with S. cerevisiae had low but detectable titers of vaginal fungal burden (<10³ CFU) early postinoculum (days 6 to 10), with the majority of animals resolving the infection by 14 days (Fig. 4). Another interesting feature of the C. glabrata vaginal infection in NOD mice was the relative lack of a requirement for pseudoestrus to acquire a sustained vaginal infection with either C. glabrata or C. albicans. Although the rates of infection were generally greater in pseudoestrus-treated mice, the vaginal fungal burdens were comparable in pseudoestrus-treated or and nontreated mice. This observation is in keeping with a clinical observation of C. glabrata being frequent in postmenopausal women developing Candida vaginitis (150).

FIG. 3 Experimental C. glabrata infections in mice with intermediate (CBA/J) and high (DBA/2) susceptibilities to C. albicans systemic infection and in NOD/Lt mice. Data points represent mean CFU ± standard errors of the mean (SEM) in animals with positive (more ...)

FIG. 4 Comparative analysis of C. glabrata, C. albicans, and S. cerevisiae vaginal fungal burden in NOD mice. Data points represent mean CFU ± SEM for animals with positive cultures (the percentage of animals with positive cultures is shown) following (more ...)

Since it is difficult in animal models of vaginitis to determine whether a state of colonization or infectivity is achieved in the absence of measurable signs and symptoms of inflammation (and more difficult for the non-hypha-producing C. glabrata), there is nevertheless considerable evidence that the C. glabrata-inoculated animals were indeed infected. First, NOD mice had high titers of vaginal fungal burden whereas other murine strains did not. Second, there was a lymphoid cell-like cellular infiltrate in the lavage fluid of C. glabrata-infected similar to that observed in C. albicans-infected mice. Third, histopathologic sections of vaginal tissue showed the presence of C. glabrata blastospores in epithelial vacuole-like vesicles and not simply lying at the epithelium (Fig. 5). Figure 5 also shows how C. albicans presents as predominantly hyphae superficially associated with the epithelium during an infection. Thus, these results show that NOD mice will be useful in studying the pathogenesis and host response during vaginal C. glabrata infections, as well as in developing strategies to treat the infection pharmacologically.

FIG. 5 Histopathology of vaginal tissue from estrogen-treated NOD mice inoculated with C. glabrata (A) and C. albicans (B). Arrows represent blastoconidia or hyphae. Magnification, ×100. Reprinted from reference 41 with permission of the publisher.

The use of NOD mice as a diabetic model was an interesting caveat to these studies. The model was originally conceived based on the high susceptibility of women with diabetes mellitus to C. glabrata and C. albicans vaginitis (53, 125, 149, 151). However, the NOD mice that were susceptible to the vaginal C. glabrata infection were not yet hyperglycemic. In fact, these mice do not achieve hyperglycemia until at least 12 weeks of age (80). The animals used in the named study were 7 to 10 weeks of age, and in our hands the NOD mice did not become hyperglycemic until 22 weeks of age. This susceptibility of NOD mice to C. glabrata vaginal infections before the onset of hyperglycemia prompted us to test the congenic insulitis-resistant strain of mice (NOR/Lt) for susceptibility to vaginal C. glabrata infection. Interestingly, the NOR mice were resistant to the vaginal infection (41). These results suggested that NOD mice may be susceptible to C. glabrata vaginal infection, not by virtue of a state of hyperglycemia but simply by their genetic susceptibility to diabetes mellitus. On the other hand, CBA/J mice made diabetic by exogenous treatment with streptozocin became susceptible to C. glabrata vaginal infection and NOD mice similarly treated had higher rates of infectivity (38). Certainly, more studies are required to better understand the factors that contribute to the susceptibility to C. glabrata vaginal infection. However, this experimental model of C. glabrata vaginitis provides an opportunity to study the pathogenesis of C. glabrata vaginal infections, as well as to dissect the genetic issues related to susceptibility to infection.

There continues to be considerable controversy about whether C. glabrata, as part of a mixed fungal culture with coexistent C. albicans, actually contributes to the development of OPC. Many investigators consider that C. glabrata functions as an innocent bystander only and that therapy should be based upon susceptibility of the coexistent C. albicans (140). While C. albicans is undoubtedly the more virulent, frequent, and dominant pathogen, C. glabrata is occasionally found as the single and only clinical species isolated in AIDS patients with OPC. Accordingly, while directing therapy against C. albicans in mixed infections, especially those not responding to appropriate therapy, it is prudent not to ignore C. glabrata in mixed infections.

Oropharyngeal.

(i) Clinical manifestations. Several clinical forms of OPC exist; the most common and widely recognized is acute pseudomembranous candidiasis, commonly referred to as thrush. OPC can also occur in an erythematous form that is often asymptomatic. OPC is often the first manifestation of HIV infection (21, 56, 147), with approximately 80 to 90% of patients with AIDS ultimately developing OPC at some stage during their disease progression (28).

(ii) Management. Numerous antifungal agents are available for the treatment of OPC, esophageal and vaginal candidiasis (Table 2). Since the comparative efficacy of the antifungal agents has not been established in infections due to C. glabrata, the choice, dosage, and duration of treatment have not been well established in patients and remain somewhat controversial. Antimycotic efficacy and response time are inferior in the HIV-positive population to those in cancer patients. To date, most of the clinical trials contained few patients with OPC caused by C. glabrata alone. Thus, the efficacy of these antifungal agents for OPC due to C. glabrata is largely unknown. Azoles have replaced the topical polyene agents for the treatment of oral candidiasis in most circumstances. Accordingly, azole therapy for OPC due to C. glabrata is also extrapolated from the data accumulated from the numerous studies performed on OPC due primarily to C. albicans (Table 2).

TABLE 2 Agents available for treatment of OPC and esophagealcandidiasis

The newer triazoles, itraconazole and fluconazole, which have markedly improved efficacy and safety profiles, have become extremely popular, especially for HIV and AIDS patients with severe OPC (29, 59, 87, 108, 111). Fluconazole (50 to 100 mg daily) has been studied in several open, placebo-controlled and double-blinded comparative studies versus clotrimazole or ketoconazole (59, 87, 108). Studies indicate that while clinical recovery is achievable in most patients treated, mycological cure is more difficult to attain. Additionally, most of the isolates recovered from study patients were C. albicans, with only a few isolates being identified as C. glabrata.

Itraconazole is a newer triazole antifungal with a broad spectrum of activity. Like the other azoles, it has a similar mechanism of action, acting by inhibiting the synthesis of fungal ergosterol. However, unlike fluconazole, it has in vitro activity against many of the non-albicans Candida species, specifically C. glabrata and C. krusei. In a recently completed prospective randomized trial involving HIV-positive or AIDS patients with OPC, itraconazole solution (200 mg/day) was compared to fluconazole (100 mg/day), both given for 14 days. The results revealed that the oral solutions of itraconazole and fluconazole were equivalent for most efficacy parameters. The clinical response rate was 97% for itraconazole and 87% for fluconazole, with few adverse events in both groups. Unfortunately, even anecdotally there is little data on OPC due to C. glabrata as a single pathogen or with C. albicans functioning as a contributory pathogen in a mixed infection in which specific anti-C. glabrata therapy was found to be effective when the anti-C. albicans regimens have failed.

Esophageal.

(i) Clinical manifestations. Candida species are the most common cause of esophagitis, and after the oropharynx, the esophagus is the most common site of gastrointestinal candidiasis. The prevalence of Candida esophagitis has increased mainly because of the increased frequency during AIDS. Approximately 10 to 15% of AIDS patients will suffer from Candida esophagitis during their disease progression (28).

Candida is frequently cultured from the esophageal surface and reaches the esophagus in oral secretions. C. albicans is the species implicated in the majority of patients with esophagitis; rarely is C. glabrata or any other Candida species recovered from esophageal samples. As with OPC, any C. glabrata strain recovered from esophageal surfaces is generally coisolated with C. albicans. However, in contrast to oral candidiasis, even less is known about host and yeast factors operative in the pathogenesis of esophageal candidiasis, and experimental models have not been established. Esophageal candidiasis in HIV-positive patients may be the first manifestation of frank AIDS.

(ii) Management. As stated above, all of the clinical efficacy studies evaluating antifungal agents for esophageal infection were performed on C. albicans. Therefore, as with most strategies used to treat infections due to C. glabrata, we tend to extrapolate the data acquired from studies involving C. albicans.

Oral and intravenous fluconazole treatments have now become an integral part of the management of Candida esophagitis. Oral fluconazole enjoys a superior safety profile compared to ketoconazole and has superior gastric absorption; when necessary, fluconazole can be given intravenously.

In a recently published trial by Wilcox et al., patients treated with oral itraconazole solution at a dose of 200 mg/day had a rate of clinical response comparable to that of patients treated with 100 mg of fluconazole per day (94 and 91%, respectively) (178). The mycological cure rates for this study was also similar, 92% for itraconazole and 78% for fluconazole.

Although used extensively in the pre-azole era for the more severe forms of esophagitis, therapy with an intravenous solution of amphotericin B is now used primarily in the azole-refractory cases. Low-dose intravenous amphotericin B, using either 0.15 to 0.3 mg/kg/day or 10 to 20 mg/day for 10 days, is often sufficient for moderate disease caused by C. albicans (9, 104), but with azole-refractory esophagitis, higher doses (0.5 to 0.7 mg/kg/day) are necessary.

Vulvovaginal.

(i) Clinical manifestations. The majority of women with Candida vaginitis suffer from uncomplicated vaginitis characterized by sporadic attacks of mild to moderate severity due to C. albicans, and these attacks occur in healthy adult women without any predisposing factors (152). In contrast, approximately 10% of women suffer from complicated Candida vaginitis, in which attacks either are more severe, occur on a recurrent basis, or are due to non-albicans Candida species. Patients with complicated Candida vaginitis frequently have predisposing factors in the form of uncontrolled diabetes or other immunosuppressive conditions. Accordingly, vaginitis caused by C. glabrata represents a complicated form of disease.

Most clinical series have found that C. albicans is responsible for approximately 90% of episodes of Candida vaginitis. In the last decade, there have been increasing reports of vaginitis due to non-albicans Candida species. In these patients, C. glabrata is the most common organism isolated (18, 151). Whether there is a real, absolute increase in vaginitis episodes caused by C. glabrata or whether the reported incidents reflect an increased awareness resulting in more frequent cultures taken, as opposed to routine microscopy, is unclear. Unfortunately, epidemiological studies do not include sentinel screening sites, but depend on data obtained from tertiary-care centers, which reflect a major acquisition bias in the overall prevalence and distribution of Candida species. The apparent increase in vaginitis caused by non-albicans Candida species is thought to reflect the increased use of short courses of both topical and oral azole antimycotic regimens. Other theories include the widespread use and abuse of topical over-the-counter antifungal agents. Finally, some investigators have postulated that C. glabrata infections emerge as breakthrough vaginal infections in women receiving long-term maintenance low-dose fluconazole prophylactic regimens. In prospective longitudinal studies performed by Fidel and coworkers, the emergence of non-albicans Candida species causing breakthrough Candida vaginitis in women already receiving maintenance azole therapy was not apparent in studies performed over many years (97). In contrast, HIV-positive women treated with fluconazole (200 mg) once weekly as long-term suppressive maintenance chemoprophylaxis showed a moderate shift in vaginal mycoflora while demonstrating effective reduction in episodes of Candida vaginitis (141). The vaginal flora in women receiving fluconazole shifted to an increase in absolute isolation rates of C. glabrata, but with a low attack rate of clinical vaginitis.

Although it was postulated that HIV infection would be associated with an increased prevalence of vaginal non-albicans Candida species in a manner similar to the emergence of OPC caused by non-albicans Candida species, no such data have emerged to date in HIV seropositive women. In a women’s cohort study (142) (HIV Epidemiological Research Study [HERS]), both baseline and follow-up studies failed to identify an increased colonization rate as well as vaginitis caused by non-albicans Candida species in HIV-positive women. Similarly, in contrast to OPC, non-albicans Candida species as well as C. albicans did not emerge with increased frequency in women with low CD4 counts (142).

In small clinical studies, a variety of risk factors have emerged for C. glabrata vaginitis. These include older patients, underlying medical conditions such as uncontrolled diabetes mellitus, and douching (53). Given the small number of patients with C. glabrata vaginitis, no large-scale studies have described the clinical characteristics of vaginitis caused by C. glabrata. It is widely assumed that clinical symptoms would be identical. Geiger et al., however, have reported subtle differences in the clinical presentation of C. glabrata vaginitis (53). In a study of 80 patients, an abnormal discharge was less frequently reported in women with symptomatic vaginitis due to C. glabrata in comparison to C. albicans. This may reflect the effects of lack of hypha formation by the C. glabrata blastoconidia. In general, vaginitis due to C. glabrata was reported to be more indolent with reduced inflammation and hence less dyspareunia. In addition, patients with C. glabrata vaginitis frequently reported a burning sensation as an alternative to itch. Clinical findings of the inflammatory reaction in the vulva and vestibule were similar to those associated with C. albicans. In contrast, speculum examination of the vagina, although revealing diffuse erythema, rarely revealed a caseous discharge in the presence of C. glabrata.

Diagnosis of C. glabrata vaginitis is more difficult than that of typical Candida vaginitis. This is because of the failure of the C. glabrata organisms to form pseudohyphae and hyphae in vivo. Accordingly, on saline and KOH microscopy, numerous budding yeasts are seen but hypha elements are absent. There is some evidence that vaginitis with C. glabrata often occurs at a somewhat higher vaginal pH, usually at the upper limit of normal. Not infrequently, C. glabrata vaginitis coexists with bacterial vaginosis, and the higher pH of the latter may represent the link between the two entities.

(ii) Management. There is scant information on guidelines for management of vaginitis due to C. glabrata. In virtually all clinical studies of yeast vaginitis, patients with vaginitis due to C. glabrata were excluded or the numbers were not large enough that any variable response rate was detectable, even in large studies. Accordingly, the clinical response of patients with C. glabrata vaginitis to conventional topical or oral therapy is largely unknown. Published experience in the management of C. glabrata patients reflects a biased view of patients referred to specialized clinics only after they have failed to respond to a large number of topical and oral azole agents (53, 151, 159). The percentage of patients with C. glabrata vaginitis, seen by primary care practitioners, who respond to initial courses of azole therapy is therefore unknown.

In vitro studies reveal that the MICs of all available azoles for C. glabrata are higher than that for most C. albicans isolates (96). The increase in MICs varies, however, with the specific azole. Butoconazole shows excellent in vitro activity, as do miconazole and clotrimazole. Terconazole, itraconazole, and ketoconazole show moderate activity. Fluconazole shows relatively poor in vitro activity, and, not infrequently, there is frank resistance. Published studies, of which there are few, reveal that in spite of in vitro activity, azole therapy does not predictably eradicate C. glabrata in vivo (125, 151). If an attempt is to be made to treat C. glabrata with either oral or topical azole therapy, fluconazole should not be the drug of choice, and all the other azoles agents should not be prescribed as short course regimens, i.e., single-dose or 1- to 3-day regimens. Accordingly, in a previously untreated patient, it is not unreasonable to use nonfluconazole azoles for 7 to 14 days.

Sobel et al. recently reported on the successful use of boric acid vaginal capsules in the treatment of C. glabrata vaginitis in women who had failed several courses of azole therapy (151). Boric acid, 600 mg in gelatin capsules, was administered intravaginally once a day for 14 days. In uncontrolled studies, the success rate measured by mycological eradication of the organism approximated 70%. Approximately 30% of the patients remained culture positive, and many of these returned within a short period with recurrence of vulvovaginal symptoms. These patients were then retreated with boric acid and given a maintenance regimen of boric acid prescribed several times a week for an additional period. However, the safety of the latter regimen is unknown, and, given the potential systemic toxicity of boric acid, it should not be undertaken lightly. As an alternative to boric acid maintenance therapy, nystatin vaginal suppositories (100,000 U daily) can be used as a maintenance regimen following the initial clinical and mycological successful therapy with boric acid. For patients who fail to respond to boric acid or for whom the boric acid or nystatin maintenance therapy becomes ineffective, topical flucytosine prescribed once a day for 14 days is generally recommended. A maintenance regimen with flucytosine is not available because of local toxicity, expense, and the potential for development of resistance. Most patients who receive flucytosine do extremely well, since C. glabrata is highly sensitive to this drug. For patients who fail to respond to both boric acid and flucytosine regimens, combination regimens including a topical antifungal such as boric acid, flucytosine, and nystatin can be coadministered with oral itraconazole. Although the value of oral itraconazole as definitive therapy is largely unknown, itraconazole demonstrates considerable in vitro activity (96). Based on disk agar diffusion susceptibility testing, terconazole has been considered to be highly active against C. glabrata; however, clinical experience with terconazole does not indicate any advantage over any of the other topical agents (151).

To date, it is unclear whether recurrent vaginitis due to C. glabrata is due to the same pathogenic mechanisms as recurrent vaginitis due to C. albicans. With C. albicans, a host factor rather than the lack of susceptibility of a microorganism to therapy is postulated to be responsible for recurrent disease (40, 149). In contrast, the additional element contributing to recurrence of C. glabrata infection is likely to be the resistance of the organisms to antifungal agents rather than a host factor. Nevertheless, in some patients, both components may be active. The treatment of C. glabrata vaginitis in HIV-positive women follows the same principles, and there is no evidence of higher failure rates.

Urinary tract.

(i) Clinical manifestations. Urinary tract infections due to Candida species have markedly increased in the last two decades (132). Candida species are now responsible for approximately 10% of urinary tract infections in hospitalized patients (185). In contrast to OPC and vaginal candidiasis, approximately 50% of urinary isolates of Candida are due to non-albicans Candida species, the most common of which is C. glabrata. In a recent large multicenter study, C. glabrata was responsible for 20% of the Candida urinary tract infections (153). Not infrequently, C. glabrata is part of a polymicrobial infection, including either bacterial uropathogens or a second Candida species, usually C. albicans.

No unique epidemiological risk factors for C. glabrata urinary tract infections have been reported, although underlying diabetes mellitus is by no means an infrequently associated factor. Similar to C. albicans urinary tract infections, the majority of C. glabrata urinary tract infections occur in elderly hospitalized, debilitated, and catheterized patients who have recently received antibacterial agents.

The clinical spectrum of C. glabrata urinary tract infections appears identical to that caused by other species of Candida. The majority of patients are asymptomatic. Rarely do lower urinary tract symptoms develop, especially in catheterized patients. The risk of an ascending infection with involvement of the kidneys is rare and occurs mostly in patients with foreign bodies or stents and in the presence of obstruction. Rarely does C. glabrata fungemia complicate ascending Candida pyelonephritis. To complete the picture, candiduria caused by C. glabrata rarely complicates hematogenous candidiasis, in which renal candidiasis occurs with subsequent seeding of the urine. The diagnosis of C. glabrata urinary tract infection, although confirmed on culture, is usually suggested by the presence of budding yeast without hypha formation on microscopy of urine samples. The finding of C. glabrata, even in large numbers, in the urine, while indicative of urinary tract infection, does not localize the anatomical site of infection, which requires clinical correlation. Identifying the site of infection forms the basis for successful management.

(ii) Management. Asymptomatic candiduria is generally not treated. The natural history of asymptomatic candiduria is such that the candiduria often resolves spontaneously, especially when catheterization is changed or discontinued. Moreover, ascending infections resulting in sepsis are infrequent. Asymptomatic candiduria should be treated following renal transplantation, in neutropenic patients, and before attempting elective instrumentation or surgery of the urinary tract.

Symptomatic urinary tract infection caused by C. glabrata, although often successfully treated with amphotericin B blood irrigation or washout, may be effectively treated by systemic therapy with either amphotericin B or fluconazole. In a recent study of a large number of patients with asymptomatic candiduria, C. glabrata urinary tract infection appeared to respond to fluconazole therapy (200 mg/day) for 14 days at the same rate as did C. albicans infection. In a logistic regression analysis, C. glabrata species did not emerge as a factor influencing the outcome of antifungal therapy (2).

Clinical manifestations. Candida may involve any organ system, and candidemia has a diverse clinical picture, ranging from low-grade fever to fulminant septic shock. There are no characteristic signs and symptoms in disseminated candidiasis. Similarly, no unique clinical features are associated with C. glabrata. Often, the only manifestation is persistent fever in a patient whose condition is deteriorating and who is unresponsive to antimicrobial agents and has negative blood cultures. C. glabrata fungemia has been associated with a higher mortality rate than C. albicans. In fact, Komshian et al. reported a 100% mortality in 12 patients with C. glabrata fungemia (90). The higher mortality rate described by some investigators may not signify increased virulence but may reflect the more advanced state of debilitation in patients who acquire C. glabrata infection. In a more recent study, Fraser et al. found no difference in mortality rates between C. albicans and C. glabrata (51).

Management. Amphotericin B has been the “gold standard” in systemic fungal infections including candidemia, despite having a high adverse effect profile. Recently, prospective randomized clinical studies concluded that fluconazole, at a minimum dose of 400 mg/day, is as effective as amphotericin B in the management of candidemia in neutropenic and nonneutropenic patients. In addition, fluconazole is better tolerated and has fewer adverse effects (2, 130). Unfortunately, as in previous clinical antifungal trials, the majority of patients treated in these studies were infected with C. albicans and few had non-albicans Candida species, including C. glabrata. Accordingly, a Candida species-specific subanalysis and conclusion was not possible. Physicians are left to extrapolate the data obtained from the clinical trials treating C. albicans to managing infections due to C. glabrata. All antifungal agents have higher MICs for C. glabrata strains than for C. albicans (129). Thus, until more data are available, many clinicians treat C. glabrata fungemia with high-dose amphotericin B (0.6 to 1.0 mg/kg/day) or fluconazole (10 to 15 mg/kg/day) until the in vitro susceptibility data indicate that the clinical isolate is susceptible to fluconazole. After resolution of fungemia, the treatment course may be completed with oral fluconazole (30). Amphotericin B is more likely to be chosen to treat the hemodynamically unstable and septic patient.

In the past, many patients with life-threatening candidiasis died without receiving antifungal therapy. Clinicians are frequently required to act definitively and early on the basis of a high index of suspicion. To be effective, any therapy must be given early and, regrettably empirically, in the febrile high-risk patient. Empirical therapy with amphotericin B is especially indicated in the granulocytopenic patient with persistent fever after 3 to 7 days of antibiotic therapy, even in the absence of microbiological confirmation. Amphotericin B has been the drug of choice in this setting. This choice is especially justified since several investigators have documented the increase in the isolation of C. glabrata and C. krusei in neutropenic patients (181–184). There are no data on the role of the new lipid formulation of amphotericin B in treating C. glabrata fungemia. Although these new formulations result in higher-dose amphotericin B administration, superior success rates have not been determined.

In vitro resistance can be subdivided into primary resistance and secondary resistance. Primary resistance is also known as intrinsic or innate resistance and occurs when the organism is naturally resistant to the antifungal agent (e.g., C. krusei, which is known to be universally resistant to fluconazole) (183). On the other hand, secondary or acquired resistance is described when the isolate producing infection becomes resistant to the antifungal agent. This form of resistance, which was rare in the past, is now the most frequently reported form in AIDS patients who suffer from recurrent azole-resistant oropharyngeal or esophageal candidiasis (36, 49, 76, 83).

An emerging dilemma is the development of multi-azole cross-resistance in Candida isolates recovered from AIDS patients with fluconazole-refractory OPC (112, 170). In one study, 45 isolates from 41 patients who failed to respond to at least 400 mg of fluconazole per day underwent in vitro testing. Twenty-seven C. albicans isolates had fluconazole MICs of >20 μg/ml; 41% of these isolates were also cross-resistant to clotrimazole, while another 11% were cross-resistant to itraconazole and ketoconazole (112). In another recent study, the authors evaluated 25 C. glabrata isolates recovered from patients with fluconazole-refractory OPC. Of these C. glabrata isolates, 68% were fluconazole resistant, and of these, 94% were also cross-resistant to ketoconazole and 88% were also cross-resistant to clotrimazole (170). In contrast, in the same study, 60 C. albicans isolates were recovered from similar patients, and while 78% were resistant to fluconazole, only 7% were cross-resistant to itraconazole, 11% were cross-resistant to ketoconazole, and 41% were cross-resistant to clotrimazole.

Fortunately, clinically significant amphotericin B resistance is still very uncommon among most Candida species except for C. lusitaniae and C. guilliermondii. Similarly, amphotericin B resistance has not been described in C. glabrata (52), although the MICs are higher than those seen for C. albicans.

Flucytosine resistance has been described extensively in C. albicans. Primary resistance rates vary from 5 to 50% depending on the species of Candida and the technique used to perform the susceptibility studies (54, 144, 173). Flucytosine resistance is also very common in C. tropicalis, C. krusei, and C. parapsilosis, many isolates of which have greater primary resistance rates than do C. albicans isolates (50, 118, 119, 144). In contrast, the majority of C. glabrata isolates are exquisitely susceptible to flucytosine. Flucytosine has not been widely used in C. glabrata infections but may be useful in the future.

The management of fluconazole-resistant mucosal candidiasis is frequently unsatisfactory or short-lived, with periodic and rapid recurrences. Some patients will respond to a doubling of the dose of fluconazole. For example, if they fail to respond to 200 mg/day, an increase to 400 mg/day will frequently produce a clinical response for a while. However, the improvement is generally transient, and the infection recurs rapidly once this stage of the disease is reached. Several recent studies of the itraconazole oral solution have demonstrated promising results in AIDS patients who have not responded to fluconazole at 200 mg/day (22, 33, 121). These studies have demonstrated clinical cure and improvement in 55 to 70% of patients entered into the study. As expected, mycological cure rates were very low (<30%) and relapses were rapid (usually within 14 days) once the itraconazole solution was terminated. The recent approval of amphotericin B oral suspension is a new therapeutic option in these patients with azole-unresponsive mucosal candidiasis (113). In several small studies, the clinical improvement rates varied from 50 to 75%, but as with all these patients, the relapse rate is high and usually occurred within 4 weeks (113, 173).

Several new antifungal compounds are currently in various phases of development, and the results appear encouraging in early in vitro trials. Two new azoles, voriconazole and SCH 56592, have excellent in vitro activity against fluconazole-resistant C. albicans and C. glabrata isolates (91, 93, 136). In addition to the azoles, a new group of antifungal agents, the pneumocandins, are being evaluated in clinical trials. MK-911, a new parenteral pneumocandin, is currently in clinical trials in the United States. In vitro results with this new antifungal drug are very promising for many Candida species, including fluconazole-resistant C. albicans. In addition, the in vitro activity against C. glabrata and C. krusei is excellent (173).

Candida glabrata is emerging as a major pathogen in the 1990s. Previously largely ignored, this organism received little attention; therefore, not surprisingly, our knowledge of it is not only incomplete but also significantly lacking. We now have the molecular tools to study the epidemiology of C. glabrata, and investigations are needed. With C. glabrata increasingly being recognized as a problem pathogen in superficial and systemic candidiasis, host risk factors need additional study, as do important virulence factors. A major issue is the management of C. glabrata infections. Symptomatic infection is more difficult to eradicate with all of the available antifungal drugs. The azole antifungal agents that have proven so successful against C. albicans have been woefully inadequate against C. glabrata vaginitis, although these azoles appear adequate for C. glabrata fungemia. Understanding the mechanism of innate and acquired resistance may facilitate the development of new targets for novel antifungal agents. In any event, if C. glabrata infections are to be adequately controlled in the future, comprehensive studies of their epidemiology, pathogenesis, and resistance must be performed.