CLINICAL MICROBIOLOGY REVIEWS, Oct. 1990, p. 321-334

Vol. 3, No. 4

0893-8512/90/040321-14$02.00/0 Copyright © 1990, American Society for Microbiology

Candida albicans Strain Delineation WILLIAM G. MERZ

Department of Laboratory Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205 INTRODUCTION ......................................................................... 321 TYPING OF C. ALBICANS BASED ON FACTORS ASSOCIATED WITH VIRULENCE OR PATHOGENESIS ......................................................................... 322 TYPING SYSTEMS BASED ON PHENOTYPIC CHARACTERISTICS ...........................................322 322 Morphologic Typing .......................................................................... 323 Serotyping ......................................................................... 323 Antibiogram ......................................................................... 323 Resistogram Typing ......................................................................... 324 Biotyping ......................................................................... Biotyping Based on Commercial Carbon Assimilation Patterns and Enzyme Profiles ........................325 Typing by Sensitivity to Yeast Killer Toxins .......................................................................... 325 326 Typing Based on Protein Variability .......................................................................... STRAIN DELINEATION BASED ON GENOTYPIC (DNA) METHODS ..........................................327 327 Electrophoretic Karyotyping ......................................................................... RFLPs ......................................................................... 327 CONCLUSIONS AND FURTHER DIRECTIONS ........................................................................328 ACKNOWLEDGMENTS ......................................................................... 331 LITERATURE CITED ......................................................................... 331

INTRODUCTION

infections. These models have been reviewed by Guentzel et al. (23). Of the 500 or so species representing 60 genera of yeasts, only a few are capable of causing human infections. These species must possess specific factors or mechanisms of pathogenesis that enable them to cause infection. Frequently, the first mechanism is a species-specific ability of the organism to become established as a persistent member of the normal flora. Candida albicans is known to colonize humans more frequently than other Candida spp., becoming established as a part of the flora of the oral cavity, GI tract, and female genitourinary tract. C. tropicalis and C. glabrata can be found as normal flora of the oral cavity, GI tract, and vagina but less frequently than C. albicans. C. krusei, C. guilliermondii, and C. parapsilosis are found more frequently as part of the skin flora. Colonization rates increase dramatically for all species in patients receiving cytotoxic drugs that alter the GI mucosa and/or broad-spectrum antibacterial agents that suppress the normal bacterial flora, especially anaerobes. Factors that permit these fungi to become established as part of our normal flora are not completely understood. Specific cell surface receptors have been found which are associated with the ability of C. albicans to adhere to epithelial cells and artificial surfaces in vitro. As supported by data from clinical studies and experimental animal models, differences also exist in the ability of the different Candida sp. to invade and proliferate in deep tissues. Most infections are caused by C. albicans, although in compromised patients the incidence of disseminated infections caused by C. tropicalis, C. krusei, and C. parapsilosis has increased. Reproducible differences in pathogenicity among the Candida species have been demonstrated in animal studies (68). In decreasing order of virulence are C. albicans, C. tropicalis, C. stellatoidea, C. parapsilosis, C. kefyr (pseudotropicalis), C. krusei, and C. guilliermondii. However, specific fungal factors that correlate with the

A dynamic interaction exists between Candida spp. as a component of our normal flora on cutaneous or mucosal surfaces. Usually, conditions favor suppression of the fungus, and disease occurs when the ability of the microorganism to cause an infection exceeds the mechanisms of the host to prevent invasion or to eradicate the microorganism. Candida spp. have become a major cause of opportunistic infections producing a wide spectrum of superficial and invasive infections of mucosal, cutaneous, or ocular surfaces; single-organ infections including endophthalmitis, endocarditis, meningitis, pyelonephritis, septic arthritis; and disseminated infections with liver, kidney, spleen, and lung

involvement. Host factors are of major importance related to both the increase in infections and the changing spectrum of clinical disease. Evidence for the role of neutrophil activity as a main defense against systemic Candida infection has been supported by the significant incidence of disseminated infection during chemotherapy-induced granulocytopenia in patients with hematologic malignancies. On the other hand, the importance of the T cell in the prevention of mucocutaneous candidiasis has been shown by the development of chronic infections in children with altered cell-mediated immunity and in patients with acquired immunodeficiency syndrome. In addition, alterations in cutaneous or mucosal barriers, which provide a portal of entry, have also increased the incidence of infections, e.g., candida endocarditis and endophthalmitis in intravenous drug abusers and in patients with indwelling catheters or extensive destruction of the skin by deep bums. An increased incidence of disseminated candidiasis is also associated with an altered gastrointestinal (GI) mucosa resulting from drug-induced or tumor-induced GI damage in patients with hematologic malignancies. The role of specific host factors has been examined in experimental animal models that mimic specific types of candida 321

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ability of these species to cause infections have not been completely elucidated. With this background, the purpose of this review is to answer the following questions: (i) can strains of C. albicans be subdivided into subpopulations that reflect their pathogenic potential and (ii) can strains of C. albicans be subdivided reproducibly into types to permit epidemiologic and pathogenetic investigations? Systems for strain delineation, e.g., serotyping, biotyping, and bacteriophage typing, have been used as important tools for both pathogenetic and epidemiologic studies with many bacteria and viruses. Typing systems for the medically important fungi, however, have not received the same attention. Systems available for C. albicans have been reviewed (70, 79, 107, 113); most are based on phenotypic characteristics. More recently, DNA techniques have been developed that will aid in furthering our knowledge about this important human pathogen and also provide better methods for strain delineation. The ultimate typing system would reflect the genetic makeup of the strain (possibly entire DNA base composition and structure). Even though this seems unreasonable today, it is worth remembering that the entire human genome is presently being sequenced: perhaps C. albicans tomorrow! Typing systems based on genotypic differences have obvious advantages compared with systems based on phenotypic differences; however, problems in elucidating the genetic makeup of C. albicans have been hampered by its lack of a sexual cycle and the lack of molecular tools. Currently available molecular-genetic techniques have the potential to overcome this problem. This review of specific systems for typing C. albicans is divided into two main sections. The first is devoted to the delineation of strains or types based on factors associated with virulence of C. albicans. The second section reviews the literature on typing schemes which may or may not correlate with virulence but may be used for epidemiologic purposes. Details on the development and application of each system are provided to draw conclusions on the extent of variation within the species, as well as accuracy, reproducibility, and practical considerations of each typing system.

TYPING OF C. ALBICANS BASED ON FACTORS ASSOCIATED WITH VIRULENCE OR PATHOGENESIS Specific factors associated with the ability of the fungus to cause infection include (i) cell wall receptors causing adherence to host cells or artificial surfaces, (ii) surface receptors that bind a complement component, (iii) hydrolytic enzymes capable of hydrolyzing complex host substrates, and (iv) hypha production. The discussion of these factors will be limited to a few selected studies that have examined variations among strains of C. albicans. A system to delineate a strain by virulence factors would have the potential to predict the virulence of a given strain. This assumes that these factors vary quantitatively among strains within this species. Variations among isolates of C. albicans to adhere to epithelial cells have been demonstrated (35, 58), and in one such study (58) a correlation was observed between the ability of a strain to adhere and virulence in an animal model. Production of extracellular hydrolytic enzymes has been demonstrated (15) among strains of C. albicans. A few have been associated with pathogenesis, including an acid phosphomonoesterase (76), phospholipase (2, 87-90, 120), and an acid protease (8, 21, 40, 48-50, 93, 105, 106). Isolates with greater enzyme

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activity in vitro were usually more virulent, or they were recovered more frequently from infected sites. However, the direct evidence linking an enzyme activity and virulence comes from two studies of induced proteinase-negative mutants of C. albicans (40, 50). In both studies, proteinasenegative mutants were less lethal in mice than parental strains. A revertant of one mutant was found (40) to be as pathogenic as the normal parental strain, further confirming the association. In only two studies have multiple factors associated with virulence been correlated with pathogenicity. Barrett-Bee et al. (2) refined assays for the detection of extracellular and intracellular phospholipase A and lysophospholyase and compared the activities of these enzymes with the ability of an isolate to adhere and to be pathogenic for mice. Of four C. albicans isolates studied, three showed elevated phospholipase A activity, a tendency to adhere better to human buccal epithelial cells, and increased lethality for mice when compared with single isolates of C. parapsilosis and Saccharomyces cerevisiae used as negative controls. Further studies including C. albicans strains that do not produce this enzyme are needed as negative controls to confirm these findings. Ghannoum and Elteen (21) examined proteinase activity, adherence to buccal epithelial cells, and pathogenicity for mice of resistogram-defined types (to be discussed later) of oral C. albicans isolates. Proteinase activity varied among the population of isolates and also among isolates with a given resistogram pattern. The isolates of one specific resistogram type tended to have elevated activity. In addition, variation in adherence abilities was noted among representative isolates of the resistogram types tested, with a tendency for those isolates with high proteinase activity to have a relatively greater ability to adhere to human buccal epithelial cells. The results from pathogenicity studies with a subset of isolates of different resistogram types are difficult to assess, because only single isolates of many of the resistogram-defined types were studied. Isolates that were more lethal to mice tended to have elevated proteinase activity and enhanced adherence abilities compared with less lethal isolates. Further studies with more isolates are necessary to support these findings. At present, no in vitro system based on known virulence factors of C. albicans can accurately predict the pathogenic potential of an isolate. However, if a panel of standardized methods were developed to assess these factors quantitatively, perhaps a strain could be characterized in a clinical laboratory and its pathogenic potential could be accurately predicted. TYPING SYSTEMS BASED ON PHENOTYPIC CHARACTERISTICS C. albicans strains can be distinguished by typing systems based on phenotypic characteristics that vary within this species. Applications of these systems permit epidemiologic and pathogenetic investigations.

Morphologic Typing Many C. albicans strains produce colonies that are obviously different from the typical, smooth, white colony; frequently, a fringed border or a wrinkled surface is present. Brown-Thomsen (6) in 1968 characterized 14 types of C. albicans based on variable colony morphologies of 314 isolates. The organisms were compared on malt-extract agar

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and incubated at 250C for 10 days. The morphotypes appeared to change during subculturing and during various storage methods, demonstrating the unstable nature of this typing system. This variation in colony morphology has recently been explained on the basis of high-frequency switching by Slutsky et al. (99). In a more recent, similar study, Phongpaichit et al. (80) generated a six-digit code based on differences in the nature and extent of marginal fringing and the surface topography of streak colonies. Theoretically, over 100 morphotypes could be distinguished with this procedure. Reproducibility was 84% for the identical type and 96% for a morphotype differing by one character. When this same typing system was tested by Hunter et al. (29), only 50 different morphologies were distinguished within 446 clinical isolates of C. albicans. The smooth colony type was predominant and represented approximately 25% of the isolates. No significant morphotype differences were noted among isolates from distinct geographical locations. Of strains recovered from patients with fatal infections, however, 67% had the discontinuous colony fringe type, whereas this morphotype was seen in only 11% of strains isolated from other types of infections. Serotyping Serotyping has proven to be an important tool for strain delineation among many medically important bacteria and viruses. In 1961, Hasenclever and Mitchell first demonstrated the two serotypes of C. albicans based on whole-cell agglutination reactions with rabbit antisera (24). The two types were distinguished by absorption studies; the cells of one type (type B) would not remove all antibodies directed against the cells of the second type (type A) which possessed additional antigen(s). An initial investigation (26) of 653 C. albicans isolates recovered from 242 individuals revealed that 68% of the yeasts were serotype A and 32% were serotype B. No major differences in the recovery of serotypes from different anatomic sites was noted; however, more serotype B isolates were recovered from women than from men. When five or more isolates recovered from the same patient over time were examined, 95% of the isolates from an individual were of the same serotype. Pathogenicity studies in murine model did not show major correlation of serotype with virulence (25). However, serotype A tended to be more frequently associated with human infections (55). An association of serotype with susceptibility to 5-fluorocytosine (5-FC) has also been noted (1, 19). In one study (1) in which 74% of the C. albicans isolates were serotype A and 26% were serotype B, resistance to 5-FC was more common with serotype B than with serotype A, 50 and 11%, respec-

tively. Other C. albicans antigens and/or variations of antigens have been described and reviewed by Poulain et al. (84). Tests to detect surface antigens may allow species-specific and, possibly, strain-specific markers to be distinguished. A panel of specific polyclonal antisera that can accurately identify the common medically important species of Candida has been developed (112). In addition, current investigations indicate that specific monoclonal antibodies detecting surface antigens may permit assessment of virulence or epidemiology among strains of C. albicans (3-5, 14, 65, 110). For example, Brawner and Cutler (5) demonstrated quantitative surface antigen differences among isolates of C. albicans that were recovered from infected compromised patients compared with isolates from normal individuals.

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Antigen strain characterization may be possible in the future. Currently, serotyping is limited to the two serotypes, and this characteristic has been incorporated into typing systems. Antibiogram Bacterial strains have been delineated based on their susceptibility to a panel of antibacterial agents, referred to as an antibiogram. Use of antifungal agents to delineate strains of C. albicans is not feasible because (i) only a limited number of antifungal agents is available, (ii) essentially all isolates of C. albicans are susceptible to amphotericin B within a very narrow range of concentrations, and (iii) methods have not been standardized to perform antifungal testing. Therefore, only resistance to the antifungal drug 5-FC is used as a characteristic in some biotyping systems (described in other sections). Resistogram Typing Resistogram typing was first developed for bacteria in the early 1970s. Bacterial species are delineated into strains based on their resistance or susceptibility to selected organic or inorganic compounds or both. In 1979, Warnock et al. developed a resistogram typing method for distinguishing strains or types of C. albicans (114). Originally, six chemicals (malachite green, boric acid, sodium arsenate, copper sulfate, acrylamide, and 4-chlororesorcinol) were tested at six concentrations. Initial data revealed that most patients harbored isolates of identical resistogram type, although a few patients harbored two types. Problems encountered with this procedure included (i) designating specific endpoints and (ii) standardizing the inoculum concentration for reproducibility. Warnock et al. (115) characterized 420 C. albicans isolates recovered over multiple visits from 30 patients with vulvovaginitis. Sixteen women were colonized or infected with a single resistogram strain. The same strain was recovered over multiple sampling times, and the vaginal tract was recolonized or infected with the same strain isolated from the intestinal tract when women were analyzed over time. Similarly, sexual partners were usually colonized by the same resistogram-defined strain. Fourteen women harbored more than one resistogram-type strain. In 1982, McCreight and Warnock screened 75 additional organic and inorganic compounds to make the procedure easier to perform and growth endpoints easier to interpret (59). Sodium selenite, boric acid, cetrimide, sodium periodate, and silver nitrate tested at four concentrations were selected; theoretically, 32 potential resistograms were possible. Initial results demonstrated improved reproducibility over the original procedure: 61 of 66 isolates tested twice yielded the same results; the pattern for the other 5 isolates differed by only one chemical. Of 198 oral isolates recovered from 22 healthy individuals, 16 of the 32 possible resistograms were found. One resistogram type was represented in 15 of 22 individuals, 9 additional types were found in multiple individuals, and 6 types were represented once. Related individuals and married couples tended to have identical resistogram-defined strains. The modified resistogram method has also been used in oral microbiology investigations. The distribution of C. albicans resistograms in patients with denture-induced stomatitis was determined by McCreight et al. (61). When cultures from 10 oral sites of 22 patients were analyzed, 18 different resistogram strains were detected, 10 types in single

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patients and 8 types in multiple individuals. Of 12 patients recultured 2 weeks after treatment, all were colonized by the same pretreatment strains. Similarly, the resistogram types of C. albicans recovered from oral and cutaneous sites of 27 inpatients and outpatients who received radiation therapy for oral and laryngeal cancer were compared (60). A total of 13 resistogram-defined types were recovered from this population; one major type was found among both patient groups. Of the 27 patients, 25 were colonized by the same type or types at both the oral and cutaneous sites of irradiation. Ghannoum et al. (22) also used the resistogram method to investigate whether specific strains of C. albicans are more frequently associated with cancer patients undergoing chemotherapy or radiotherapy than healthy control individuals. Forty-four isolates recovered from cancer patients (25 from outpatients and 19 from inpatients) were compared with 10 isolates from healthy controls. Two strains predominated, but they were equally distributed among the three populations. The two strains predominating in this study and the primary oral strain distinguished by McCreight et al. (61) were different. These findings support the conclusions that no particular strains delineated by resistogram typing were associated with the oral cavity, although there may be geographical differences in their frequencies. Modified resistogram methods have been used in epidemiologic investigations. The epidemiology of vaginal candidiasis was studied by Meinhof (62) with a resistogram system supplemented by proteinase activity, lipase activity, and 5-FC susceptibility. Oral, fecal, anal, and vaginal isolates of C. albicans recovered from 134 women were typed. More than 70% of the vaginal strains were identical to the oral or intestinal strains recovered from the same individual. In similar studies with a modified resistogram method, Hunter and Fraser (27) compared strains of C. albicans recovered from the GI tract with strains recovered from patients with vaginitis. No particular resistogram type(s) was associated more with vaginal infections than any other strains. Resistogram methods can be used for strain delineation, pathogenesis, or epidemiologic studies. The resistograms, however, do not correlate with the pathogenic potential of the strain. This method does not require any special equipment and the procedures are similar to those used in clinical microbiology laboratories; however, some of the chemicals and media may not be routinely available. Even though improvements in methods have been made, the growth/no growth endpoints often present problems with inoculum, interpretation, and reproducibility. Addition of more tests to expand the variation and development of more objective endpoints may improve the value of this method. Biotyping In 1980, Odds and Abbott developed a nine-test scheme to biotype isolates of C. albicans (72). The tests were based on growth or no growth endpoints under a variety of conditions and the generation of a three-digit code that theoretically could distinguish 512 strains. The nine tests, grouped in threes, were growth at pH 1.4, proteinase activity, and 5-FC resistance; urea assimilation, sorbose assimilation, and NaCl tolerance; and citrate assimilation, glycine assimilation, and safranin resistance. Multiple isolates (up to 60 per plate) were inoculated with a replicator system. Plates were incubated at 370C and read up to 8 days for some tests. In the initial study, 45 biotypes were distinguished among oral and vaginal isolates of C. albicans recovered from 85 patients.

CLIN. MICROBIOL. REV.

Eleven types were detected more than once, whereas single isolates of 34 types were detected. Three prevalent biotypes were recovered. In addition, the biotypes of an oral isolate and vaginal isolate from the same patient were the same in seven of eight patients. The medium preparation, inoculum size, and definition of endpoints needed to be controlled strictly to ensure reproducibility of results. Even with careful attention, variability in test results occurred frequently; therefore, three or more test differences were required to delineate reproducibly between strains. This biotyping method has been applied to investigate several epidemiologic and virulence questions. In two studies (69, 74), epidemiologic aspects of genital candidiasis were investigated. A study of 25 women confirmed that in 88% the same biotype was recovered from different anatomic sites (69). In 11 of 12 women, the biotype of the vaginal isolates was the same as isolates recovered from the mouth, rectum, or urethra. These data support the conclusion that an individual usually harbors one strain of C. albicans and indirectly support the conclusion that the GI tract may be an endogenous reservoir for development of recurrent vaginal candidiasis. In the second study (74), analysis of the biotypes of 282 isolates of C. albicans recovered from 50 males and 181 females seen in a genitourinary clinic revealed that no specific biotype(s) was associated with Candida-infected individuals when compared with healthy control individuals. These data indicate that the biotyping scheme may be useful only for epidemiologic studies and not for comparing virulence among different biotypes. Variability of C. albicans biotypes from different geographic areas was investigated by Odds et al. (75). The three-code biotype, serotype, and 5-FC resistance of 247 isolates of C. albicans from the United Kingdom were compared with 330 isolates recovered from five different geographic locations in the United States. Analysis required computer support to test differences and to perform cluster analyses. Only 160 biotypes were distinguished among all isolates tested; therefore, all 512 possible strains may not occur in nature. Ten major clusters of related types were more prevalent in both populations; however, differences were noted between the isolates from the United States and those from the United Kingdom. Isolates from the United States tended to be more pH tolerant and more resistant to 5-FC or safranin and assimilated urea, sorbose, or glycine more often than isolates from the United Kingdom. Overall, no biotypes or cluster of biotypes could be associated with any anatomic site(s) nor was any association found between biotypes from infected versus colonized individuals. A simplified Odds and Abbott system using only acid tolerance, proteinase activity, and 5-FC susceptibility tests was used to study 69 C. albicans isolates from 22 patients (98). Four biotypes were detected: one type was found in 18 patients, another was found in 6, and 2 were found in single patients. Eight of the 22 patients harbored only one type. Even with this abbreviated system, limited epidemiologic studies might be possible. The original nine-test biotyping scheme was subsequently refined by Odds and Abbott in 1983 (73) to improve strain delineation of C. albicans and to allow species identification and strain delineation of other Candida species. Since glycine was not discriminatory (>90% of isolates assimilated glycine) and endpoints for this test were difficult to determine, it was replaced with boric acid resistance (see reference 114). In addition, resistance to pH 1.55 and four inhibition tests cetrimidee, tetrazolium, sodium periodate, and MacConkey agar) permitted species identification and

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strain delineation among other medically important Candida including Torulopsis (Candida) glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. (keyfr) pseudotropicalis, and C. tropicalis. The epidemiology of recurrent vulvovaginal candidiasis was studied by O'Connor and Sobel (67), using the Odds and Abbott biotyping system. Of 415 women diagnosed with recurrent infections, C. albicans was recovered from 91. Biotype analysis revealed 54 distinct strains, with 7 strains comprising ca. 40%; 15 strains were detected more than once and 39 were detected only once. When infection recurred early, the same strain of C. albicans was recovered ca. 60% of the time. Second biotypes were encountered in ca. 40% of early recurrences, and an even higher percentage was seen in later recurrences of infection. When rectal (73%) or oral (40%) cultures were also positive, ca. 80% were the same biotype as vaginal isolates. All sexual partners (6 of 23; 26%) with positive penile cultures had the identical biotype as their partner. This study suggests that different strains may be responsible for recurring vulvovaginal infections. Burnie et al. (10) used biotyping to confirm an outbreak of systemic candidiasis in an intensive care unit. Strain delineation was determined by serotyping, morphotyping (6), and the modified biotyping system. Isolates from the 13 cases of systemic candidiasis that occurred over a 9-month interval as well as from 44% of superficial infections in this same hospital unit typed as the same strain, although variation in tolerance to pH 1.4 and resistance to boric acid were noted. At the same time, this strain was associated with only 17% of candidiasis outside of this unit. Four of 65 staff members spp.

within the unit were also colonized by the outbreak strain. This strain was more persistent in hand-washing experiments possibly due to increased resistance to antiseptics when compared with other strains of C. albicans. The yeast species and biotypes from patients with oral leukoplakia and lichen planus were determined by Krogh et al. (39), using the modified biotyping system. No specific species or biotype association was noted among these patients. One biotype was recovered from six patients, another from four, a third from two patients, and 14 biotypes were recovered from single patients. In addition, multiple biotypes were recovered from the same individual. Korting and Dorsch (37) investigated whether there is an association of antifungal susceptibility patterns among specific biotypes of C. albicans. Forty-two biotypes were detected among the 52 isolates recovered from mucocutaneous sites. However, no significant correlation of specific biotypes and susceptibilities to 5-FC or ketoconazole or both was noted. Biotyping schemes have the potential to be useful for epidemiologic studies of C. albicans and potentially for other medically important yeast species, but not to assess pathogenic potential. The procedures are relatively simple and require only special equipment for inoculation. Childress et al. (16) demonstrated that inoculum standardization for the Odds and Abbott biotyping system could be done either visually with McFarland standards or with a Klett colorimeter. In addition, a multipronged replicate inoculation system is commercially available that is accurate and reproducible. The number of detectable biotypes should permit a range of variation appropriate for epidemiologic studies (170 biotypes of the 512 possible have been detected). Problems with the system are that endpoints are difficult to interpret so that a single test variation may not be significant and laboratories would need to make at least 12 different media. Recently, Odds et al. (71) evaluated this biotype system in

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four laboratories with 18 coded yeast isolates. The results were highly reproducible within a laboratory, but reproducibility among laboratories was poor. Unfortunately, comparisons of biotypes from different laboratories cannot be assessed accurately. Therefore, use of this method should be limited to epidemiologic studies when testing is performed in one laboratory or when this method is used for research purposes. Biotyping Based on Commercial Carbon Assimilation Patterns and Enzyme Profiles The API 20C Yeast Identification Kit (Analytab, Inc., Plainview, N.Y.) with 19 carbon assimilation reactions was used to biotype 61 isolates of C. albicans recovered from human immunodeficiency virus-infected patients (38). One biotype was dominant (64%) and eight other biotypes were detected. No correlation between these biotypes and antifungal susceptibility results was revealed. Unfortunately, problems in interpreting growth/no growth endpoints, the lack of reproducibility, and limited strain variation restrict this test to delineate strains of C. albicans. Biotyping based on enzyme profiles obtained with a commercially available product, the API ZYM system (API Laboratory Products Limited, Farmborough, Hampshire, United Kingdom), has also been investigated. The kit includes 19 hydrolytic enzyme reactions and a negative control, requires less than 8 h for results, and has colored endpoints after the addition of appropriate reagents. Roman and Sicilia (92) typed 126 isolates of C. albicans and found that five enzymatic reactions, i.e., valine, leucine and cystine arylamidases, a-glucosidase, and N-acetyl-p-glucosaminidase, were discriminatory. Only four biotypes (biovars) were distinguished among the 126 isolates, with one major type representing 64% of the isolates. Williamson et al. (119) used the same enzyme system to type 213 oral isolates of C. albicans. Valine arylamidase, phosphoamidase, a-glucosamine, and N-acetyl-p-glucosaminidase were discriminatory, but only eight types were detected. One major type represented 72% of the isolates and the other seven minor types each represented U), O..bo n

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somewhat lower than the theoretical maximum for each of these systems. ods, protein differences detected by immunoblotting or electrophoretic karyotyping, and RFLP systems has not been quantitated. These procedures depend on separation of proteins or nucleic acids, and the number of molecules or fragments is limited by the reproducible resolution of size differences. Computerized analysis that uses strict controls and size markers will be necessary for both discrimination and reproducibility of these procedures. The time required for results with the different typing systems varies from 1 to 8 days. The most rapid method, enzyme profile, requires less than 1 day; however, the test is limited by lack of extensive variation among strains. Most systems require 2 to 4 days, whereas some systems, i.e., biotyping and morphotyping systems, may require up to 8 days of incubation. The availability of equipment and reagents is also important. The four systems based on growth/no growth endpoints or a color reaction do not require specialized equipment; however, three require specialized media not commercially available, and the fourth requires the purchase of the commercial enzyme kits. The killer sensitivity assay requires a panel of killer toxin-producing strains and control sensitive strains. The other systems require special electrophoretic equipment that may not be available in all clinical laboratories. Radionuclides are currently used in the immunoblotting and some of the DNA-RFLP analyses; however, biotinylated or other chromogenic labels may soon replace the need for radionuclides. Specific DNA probes must be standardized and made available for wide use. Last, it is difficult to compare results from these diverse methods since very few comparative studies have been conducted. Data from 100 isolates of C. albicans sent blindly to the investigators for each of these systems would provide data to assess the abilities of these systems to delineate C. albicans strains accurately and reproducibly. At this time, strains of C. albicans with pathogenic potential cannot be distinguished; however, a number of previously unavailable systems now permit strain delineation for use in epidemiologic and pathogenetic studies.

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The help of Alan O'Neill and Cindy Kaufmann in preparing the manuscript is appreciated.

activity. Hereditas 60:355-398. 7. Bruneau, S. M., and R. M. F. Guinet. 1987. Candida albicans and Candida tropicalis antigens studied by SDS polyacrylamide gel electrophoresis and western blot. Mykosen 30:271280. 8. Budtz-Jorgensen, E. 1971. Proteolytic activity of Candida spp. as related to the pathogenesis of denture stomatitis. Sabouraudia 12:266-271. 9. Burnie, J. P., R. Matthews, W. Lee, J. Philpott-Howard, R. Brown, N. Damani, J. Breuer, K. Honeywell, and Z. Jordans. 1987. Four outbreaks of nosocomial systemic candidiasis. Epidemiol. Infect. 99:201-211. 10. Burnie, J. P., F. C. Odds, W. Lee, C. Webster, and J. D. Williams. 1985. Outbreak of systemic Candida albicans in intensive care unit caused by cross infection. Br. Med. J. 290:746-748. 11. Caprilli, F., G. Prignano, C. Latella, and S. Tavarozzi. 1985. Amplification of the killer system for differentiation of Candida albicans. Mykosen 28:569-573. 12. Carle, G. F., and M. V. Olson. 1984. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 12:5647-5663. 13. Carle, G. F., and M. V. Olson. 1986. An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA 82:3756-3760. 14. Chaffin, W. L., J. Skudlarek, and K. J. Morrow. 1988. Variable expression of a surface determinant during proliferation of Candida albicans. Infect. Immun. 56:302-309. 15. Chattaway, F. W., F. C. Odds, and A. J. E. Barlow. 1971. An examination of the production of hydrolytic enzymes and toxins by pathogenic strains of Candida albicans. J. Gen. Microbiol. 67:255-263. 16. Childress, C. M., I. A. Holder, and A. N. Neely. 1989. Modifications of a Candida albicans biotyping system. J. Clin. Microbiol. 27:1392-1394. 17. Cutler, J. E., P. M. Glee, and H. L. Horn. 1988. Candida albicans- and Candida stellatoidea-specific DNA fragment. J. Clin. Microbiol. 26:1720-1724. 18. DeJonge, P., F. C. M. dejonge, R. Meijers, H. Y. Steensma, and W. A. Scheffers. 1986. Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts. Yeast 2:193204. 19. Drouhet, E., L. Mercier-Soucy, and S. Montplasir. 1975. Sensibilite et resistance des levures pathogenes aux 5-fluoropyrimidines. I. Relation entre les phenotypes de resistance A la 5-fluorocytosine, le serotype de Candida albicans et l'ecologie de differentes especes de Candida d'origine humaine. Ann. Microbiol. (Paris) 126B:25-39. 20. Fox, B. C., H. L. T. Mobley, and J. C. Wade. 1989. The use of a DNA probe for epidemiological studies of candidiasis in immunocompromised hosts. J. Infect. Dis. 159:488-494.

LITERATURE CITED

21. Ghannoum, M., and K. A. Elteen. 1986. Correlative relationship between proteinase production, adherence and pathoge-

ACKNOWLEDGMENTS

1. Auger, P., C. Dumas, and J. Joly. 1979. A study of 666 strains of Candida albicans: correlation between serotype and susceptibility to 5-fluorocytosine. J. Infect. Dis. 139:590-594. 2. Barrett-Bee, K., Y. Hayes, R. G. Wilson, and J. F. Riley. 1985. A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts. J. Gen. Microbiol. 131:1217-1221. 3. Brawner, D. L., and J. E. Cutler. 1984. Variability in expression of a cell surface determinant on Candida albicans as evidenced by an agglutinating monoclonal antibody. Infect. Immun. 43:966-972. 4. Brawner, D. L., and J. E. Cutler. 1986. Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans. Infect. Immun. 51:327-336. 5. Brawner, D. L., and J. E. Cutler. 1989. Oral Candida albicans isolates from nonhospitalized normal carriers, immunocompetent hospitalized patients, and immunocompromised patients with or without acquired immunodeficiency syndrome. J. Clin. Microbiol. 27:1335-1341. 6. Brown-Thomsen, J. 1968. Variability in Candida albicans (Robin) Berkhout. I. Studies on morphology and biochemical

22. 23.

24. 25.

nicity of various strains of Candida albicans. J. Med. Vet. Mycol. 24:407-413. Ghannoum, M. A., M. S. Motawy, A. L. Al-Mubarek, and H. A. Al-Awadhi. 1985. Candida albicans strain differentiation in cancer patients undergoing therapy. Mykosen 28:388-393. Guentzel, M. N., G. T. Cole, and L. M. Pope. 1985. Animal models for candidiasis, p. 57-116. In M. R. McGinnis (ed.), Current topics in medical mycology, vol. 1. Springer-Verlag, New York. Hasenclever, H. F., and W. 0. Mitchell. 1961. Antigenic studies of Candida. I. Observation of two antigenic groups in Candida albicans. J. Bacteriol. 82:570-573. Hasenclever, H. F., and W. 0. Mitchell. 1961. Antigenic studies of Candida. III. Comparative pathogenicity of Candida albi-

cans group A, group B, and Candida stellatoidea. J. Bacteriol. 82:578-581. 26. Hasenclever, H. F., and W. 0. Mitchell. 1963. Antigenic studies of Candida. IV. The relationship of the antigenic groups of Candida albicans to their isolation from various clinical specimens. Sabouraudia 2:201-204. 27. Hunter, P. R., and C. Fraser. 1987. Use of modified resisto-

332

MERZ gram to type Candida albicans isolated from cases of vaginitis and from faeces in same geographical area. J. Clin. Pathol.

40:1159-1161. 28. Hunter, P. R., and C. A. M. Fraser. 1989. Application of a numerical index of discriminatory power to a comparison of four physiochemical typing methods for Candida albicans. J. Clin. Microbiol. 27:2156-2160. 29. Hunter, P. R., C. A. M. Fraser, and D. W. R. Mackenzie. 1989. Morphotype markers of virulence in human candidal infections. J. Med. Microbiol. 28:85-91. 30. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466. 31. Ibrahim-Granet, O., J. M. Delga, C. Granet, and E. Drouhet. 1986. A program in BASIC for the determination of molecular weights and linear graphic representation of an electrophoretic protein pattern: application to serotypes A and B of Candida albicans studied by polyacrylamide gel electrophoresis and immunoblotting. Electrophoresis 7:316-323. 32. Kakar, S. N., R. M. Partridge, and P. T. Magee. 1983. A genetic analysis of Candida albicans: isolation of a wide variety of auxotrophs and demonstration of linkage and complementation. Genetics 104:241-255. 33. Kandel, J. S., and T. A. Stern. 1979. Killer phenomenon in pathogenic yeast. Antimicrob. Agents Chemother. 15:568-571. 34. Kaufmann, C. S., and W. G. Merz. 1989. Electrophoretic karyotypes of Torulopsis glabrata. J. Clin. Microbiol. 27:21652168. 35. Kearns, M. J., P. Davies, and H. Smith. 1983. Variability of the adherence of Candida albicans strains to human buccal epithelial cells: inconsistency of differences between strains related to virulence. Sabouraudia 21:93-98. 36. Kelly, R., S. M. Miller, M. B. Kurtz, and D. R. Kirsch. 1987. Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants. Mol. Cell. Biol. 7:199-207. 37. Korting, H. C., and R. Dorsch. 1987. On the correlation of biotype and antimicrobial susceptibility in Candida albicans.

Mykosen 30:512-519. 38. Korting, H. C., M. Ollert, A. Georgii, and M. Froschl. 1988. In vitro susceptibilities and biotypes of Candida albicans isolates from the oral cavities of patients infected with human immunodeficiency virus. J. Clin. Microbiol. 26:2626-2631. 39. Krogh, P., P. Holmstrup, J. J. Thorn, P. Vedtofte, and J. J. Pindborg. 1987. Yeast species and biotypes associated with oral leukoplakia and lichen planus. Oral Surg. Oral Med. Oral Pathol. 63:48-54. 40. Kwon-Chung, K. J., D. Lehman, C. Good, and P. T. Magee. 1985. Genetic evidence for role of extracellular proteinase in virulence of Candida albicans. Infect. Immun. 49:571-575. 41. Kwon-Chung, K. J., B. L. Wickes, and W. G. Merz. 1988. Association of electrophoretic karyotype of Candida stellatoidea with virulence for mice. Infect. Immun. 56:1814-1819. 42. Lasker, B. A., G. F. Care, G. S. Kobayashi, and G. Medoff. 1989. Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques. Nucleic Acids Res. 17:3783-3793. 43. Lee, W., J. Burnie, and R. Matthews. 1986. Fingerprinting Candida albicans. J. Immunol. Methods 93:177-182. 44. Lehmann, P. F., L. E. Cowan, R. M. Jones, and W. J. Ferencak. 1987. Use of killer fungi and antifungal chemicals in characterization of yeast species and biotypes. Trans. Br. Mycol. Soc. 88:199-206. 45. Lehmann, P. F., C. B. Hsiao, and I. F. Salkin. 1989. Protein and enzyme electrophoresis profiles of selected Candida species. J. Clin. Microbiol. 27:400-404. 46. Lehmann, P. F., B. J. Kemker, C. Hsiao, and S. Dev. 1989. Isoenzyme biotypes of Candida species. J. Clin. Microbiol. 27:2514-2521. 47. Lott, T. J., P. Boiron, and E. Reiss. 1987. An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples. Mol. Gen. Genet. 209:170-174. 48. MacDonald, F. 1984. Secretion of inducible proteinase by pathogenic Candida species. J. Med. Vet. Mycol. 22:79-82.

CLIN. MICROBIOL. REV. 49. MacDonald, F., and F. C. Odds. 1980. Inducible proteinase of Candida albicans in diagnostic serology and in the pathogenesis of systemic candidosis. J. Med. Microbiol. 13:423-435. 50. MacDonald, F., and F. C. Odds. 1983. Virulence for mice of a proteinase-secreting strain of Candida albicans and a proteinase-deficient mutant. J. Gen. Microbiol. 129:431-438. 51. Magee, B. B., T. M. D'Souza, and P. T. Magee. 1987. Strain and species identification by restriction fragment length polymorphisms in the ribosomal DNA repeat of Candida species. J. Bacteriol. 169:1639-1643. 52. Magee, B. B., Y. Koltin, J. A. Gorman, and P. T. Magee. 1988. Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes. Mol. Cell. Biol. 8:4721-4726. 53. Magee, B. B., and P. T. Magee. 1987. Electrophoretic karyotypes and chromosome numbers in Candida species. J. Gen. Microbiol. 133:425-430. 54. Manning, M., and T. G. Mitchell. 1980. Analysis of cytoplasmic antigens of yeast and mycelial phases of Candida albicans by two-dimensional electrophoresis. Infect. Immun. 30:484495. 55. Martin, M. V., and D. J. Lamb. 1982. Frequency of Candida albicans serotypes in patients with denture-induced stomatitis and in normal denture wearers. J. Clin. Pathol. 35:888-891. 56. Mason, M. M., B. A. Lasker, and W. S. Riggsby. 1987. Molecular probe for identification of medically important Candida species and Torulopsis glabrata. J. Clin. Microbiol. 25:563-566. 57. Matthews, R., and J. Burnie. 1989. Assessment of DNA fingerprinting for rapid identification of outbreaks of systemic candidiasis. Br. Med. J. 298:354-357. 58. McCourtie, J., and L. J. Douglas. 1984. Relationship between cell surface composition, adherence, and virulence of Candida albicans. Infect. Immun. 45:6-12. 59. McCreight, M. C., and D. W. Warnock. 1982. Enhanced differentiation of isolates of Candida albicans using a modified resistogram method. Mykosen 25:589-598. 60. McCrieght, M. C., D. W. Warnock, and M. V. Martin. 1985. Resistogram typing of Candida albicans isolates from oral and cutaneous sites in irradiated patients. J. Med. Vet. Mycol. 23:403-406. 61. McCreight, M. C., D. W. Warnock, and A. C. Watkinson. 1984. Prevalence of different strains of Candida albicans in patients with denture-induced stomatitis. J. Med. Vet. Mycol. 22:8385. 62. Meinhof, W. 1982. Demonstration of typical features of individual Candida albicans strains as a means of studying sources of infection. Chemotherapy (Basel) 28(Suppl. 1):51-55. 63. Merz, W. G., C. Connelly, and P. Hieter. 1988. Variation of electrophoretic karyotypes among clinical isolates of Candida albicans. J. Clin. Microbiol. 26:842-845. 64. Middlebeek, E. J., J. M. H. Hermans, C. Stumm, and H. L. Muytjens. 1980. High incidence of sensitivity to yeast killer toxins among Candida and Torulopsis isolates of human origin. Antimicrob. Agents Chemother. 17:350-354. 65. Miyakawa, Y., K. Kagaya, Y. Fukazawa, and G. Soe. 1986. Production and characterization of agglutinating monoclonal antibodies against predominant antigenic factors for Candida albicans. J. Clin. Microbiol. 23:881-886. 66. Morace, G., C. Archibusacci, M. Sestito, and L. Polonelli. 1984. Strain differentiation of pathogenic yeasts by the killer system. Mycopathologia 84:81-85. 67. O'Connor, M. I., and J. D. Sobel. 1986. Epidemiology of recurrent vulvovaginal candidiasis: infection and strain differentiation of Candida albicans. J. Infect. Dis. 154:358-363. 68. Odds, F. C. 1979. Candida and candidosis. University Park Press, Baltimore. 69. Odds, F. C. 1982. Genital candidosis. Clin. Exp. Dermatol. 7:345-354. 70. Odds, F. C. 1985. Biotyping of medically important fungi, p. 155-171. In M. R. McGinnis (ed.), Current topics in medical mycology, vol. 1. Springer-Verlag, New York. 71. Odds, F. C. 1989. Biotyping of Candida albicans: results of an

VOL. 3, 1990

72. 73. 74.

75.

76. 77.

international collaborative survey. J. Clin. Microbiol. 27:15061509. Odds, F. C., and A. B. Abbott. 1980. A simple system for the presumptive identification of Candida albicans and differentiation of strains within the species. Sabouraudia 18:301-317. Odds, F. C., and A. B. Abbott. 1983. Modification and extension of tests for differentiation of Candida species and strains. Sabouraudia 21:79-81. Odds, F. C., A. B. Abbott, T. A. G. Reed, and F. E. Willmott. 1983. Candida albicans strain types from the genitalia of patients with and without Candida infection. Eur. J. Obstet. Gynecol. Reprod. Biol. 15:37-43. Odds, F. C., A. B. Abbott, R. L. Stiller, H. J. Scholer, A. Polak, and D. A. Stevens. 1983. Analysis of Candida albicans phenotypes from different geographical and anatomical sources. J. Clin. Microbiol. 18:849-857. Odds, F. C., and J. C. Hierholzer. 1973. Purification and properties of a glycoprotein acid phosphatase from Candida albicans. J. Bacteriol. 114:257-266. Olaiya, A. F., and S. J. Sogin. 1979. Ploidy determination of

Candida albicans. J. Bacteriol. 140:1043-1049. 78. Olivo, P. D., E. J. McManus, W. S. Riggsby, and J. M. Jones. 1987. Mitochondrial DNA polymorphisms in Candida albicans. J. Infect. Dis. 156:214-215. 79. Pfaller, M. A. 1987. Strain variation among Candida species: application of various typing methods to study the epidemiology and pathogenesis of candidiasis in hospitalized patients. Infect. Control 8:273-276. 80. Phongpaichit, S., D. W. R. Mackenzie, and C. Fraser. 1987. Strain differentiation of Candida albicans by morphotyping. Epidemiol. Infect. 99:421-428. 81. Polachek, I., and G. A. Lebens. 1989. Electrophoretic karyotype of the pathogenic yeast Cryptococcus neoformans. J. Gen. Microbiol. 135:65-71. 82. Polonelli, L., C. Archibusacci, M. Sestito, and G. Morace. 1983. Killer system: a simple method for differentiating Candida albicans strains. J. Clin. Microbiol. 17:774-780. 83. Polonelli, L., M. Castagnola, D. V. Rosetti, and G. Morace. 1985. Use of killer toxins for computer-aided differentiation of Candida albicans strains. Mycopathologia 91:175-179. 84. Poulain, D., V. Hopwood, and A. Vernes. 1985. Antigenic variability of Candida albicans. Crit. Rev. Microbiol. 12:223270. 85. Poulter, R., K. Hanrahan, K. Jeffery, D. Markie, M. G. Shepherd, and P. A. Sullivan. 1982. Recombination of naturally diploid Candida albicans. J. Bacteriol. 152:969-975. 86. Poulter, R. T. M., and E. H. A. Rikkerink. 1983. Genetic analysis of red, adenine-requiring mutants of Candida albicans. J. Bacteriol. 156:1066-1077. 87. Price, M. F., and R. A. Cawson. 1977. Phospholipase activity in Candida albicans. Sabouraudia 15:179-185. 88. Price, M. F., I. D. Wilkinson, and L. 0. Gentry. 1982. Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia 20:7-14. 89. Pugh, D., and R. A. Cawson. 1975. The cytochemical localization of phospholipase A and lysophospholipase in Candida albicans. Sabouraudia 13:110-115. 90. Pugh, D., and R. A. Cawson. 1977. The cytochemical localization of phospholipase in Candida albicans infecting the chick chorioallantoic membrane. Sabouraudia 15:29-35. 91. Riggsby, W. S., L. J. Torres-Bauza, J. W. Wills, and T. W. Townes. 1982. DNA content, kinetic complexity, and the ploidy question in Candida albicans. Mol. Cell. Biol. 2:853862. 92. Roman, M. C., and M. J. L. Sicilia. 1983. Preliminary investigation of Candida albicans biovars. J. Clin. Microbiol. 18:430431. 93. Ruchel, R., R. Tegeler, and M. Trost. 1982. A comparison of secretary proteinases from different strains of Candida albicans. Sabouraudia 20:233-244. 94. Sarachek, A., D. D. Rhoads, and R. H. Schwarzhoff. 1981. Hybridization of Candida albicans through fusion of protoplasts. Arch. Microbiol. 129:1-8.

C. ALBICANS STRAIN DELINEATION

333

95. Scherer, S., and D. A. Stevens. 1987. Application of DNA typing methods to epidemiology and taxonomy of Candida species. J. Clin. Microbiol. 25:675-679. 96. Scherer, S., and D. A. Stevens. 1988. A Candida albicans dispersed, repeated gene family and its epidemiologic applications. Proc. Natl. Acad. Sci. USA 85:1452-1456. 97. Schwartz, D. C., and C. R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37:67-75. 98. Skorepova, M., and H. Hauck. 1985. Differentiation of Candida albicans biotypes by the method of Odds and Abbott. Mykosen 28:323-331. 99. Slutsky, B., J. Buffo, and D. R. Soil. 1985. High-frequency switching of colony morphology in Candida albicans. Science 230:666-669. 100. Snell, R. G., I. F. Hermans, R. J. Wilkins, and B. E. Corner. 1987. Chromosomal variation in Candida albicans. Nucleic Acids Res. 15:3625. 101. Snell, R. G., and R. J. Wilkins. 1986. Separation of chromosomal DNA molecules from C. albicans by pulsed field gel electrophoresis. Nucleic Acids Res. 14:4401-4406. 102. Soil, D. R., R. Galask, S. Isley, T. V. Gopala Rao, D. Stone, J. Hicks, J. Schmid, K. Mac, and C. Hanna. 1989. Switching of Candida albicans during successive episodes of recurrent vaginitis. J. Clin. Microbiol. 27:681-690. 103. Soll, D. R., C. J. Langtimm, J. McDowell, J. Hicks, and R. Galask. 1987. High-frequency switching in Candida strains isolated from vaginitis patients. J. Clin. Microbiol. 25:16111622. 104. Soil, D. R., M. Staebell, C. Langtimm, M. Pfaller, J. Hicks, and T. V. G. Rao. 1988. Multiple Candida strains in the course of a single systemic infection. J. Clin. Microbiol. 26:1448-1459. 105. Staib, F. 1965. Serum proteins as nitrogen source for yeastlike fungi. Sabouraudia 4:187-193. 106. Staib, F. 1969. Proteolysis and pathogenicity of Candida albicans strains. Mycopathol. Mycol. Apple. 37:345-348. 107. Stenderup, A. 1986. Ecology of yeast and epidemiology of yeast infections. Acta Derm. Venereol. Supply. 121:27-37. 108. Strockbine, N. A., M. T. Largen, and H. R. Buckley. 1984. Production and characterization of three monoclonal antibodies to Candida albicans proteins. Infect. Immun. 43:10121018. 109. Strockbine, N. A., M. T. Largen, S. M. Zweibel, and H. R. Buckley. 1984. Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera. Infect. Immun. 43:715-721. 110. Sundstrom, P. M., M. R. Tam, E. J. Nichols, and G. E. Kenny. 1988. Antigenic differences in the surface mannoproteins of Candida albicans as revealed by monoclonal antibodies. Infect. Immun. 56:601-606. 111. Suzuki, T., T. Kanbe, T. Kuroiwa, and K. Tanaka. 1986. Occurrence of ploidy shift in a strain of the imperfect yeast Candida albicans. J. Gen. Microbiol. 132:443-453. 112. Tsuchiya, T., Y. Fukazawa, M. Taguchi, T. Kase, and T. Shinoda. 1974. Serologic aspects on yeast classification. Mycopathol. Mycol. Appl. 53:77-91. 113. Warnock, D. W. 1984. Typing of Candida albicans. J. Hosp. Infect. 5:244-252. 114. Warnock, D. W., D. C. E. Speller, J. K. Day, and A. J. Farrell. 1979. Resistogram method for differentiation of strains of Candida albicans. J. Appl. Bacteriol. 46:571-578. 115. Warnock, D. W., D. C. E. Speller, J. D. Milne, A. L. Hilton, and P. I. Kershaw. 1979. Epidemiological investigation of patients with vulvovaginal candidiosis. Br. J. Vener. Dis. 55:357-361. 116. Whelan, W. L., D. M. Markie, K. G. Simpkin, and R. M. Poulter. 1985. Instability of Candida albicans hybrids. J. Bacteriol. 161:1131-1136. 117. Whelan, W. L., R. M. Partridge, and P. T. Magee. 1980. Heterozygosity and segregation in Candida albicans. Mol. Gen. Genet. 180:107-113. 118. Wickner, R. B. 1985. Killer yeasts, p. 286-305. In M. R.

334

MERZ

McGinnis (ed.), Current topics in medical mycology, vol. 1. Springer-Verlag, New York. 119. Williamson, M. I., L. P. Samaranayake, and T. W. MacFarlane. 1986. Biotypes of oral Candida albicans and Candida tropicalis isolates. J. Med. Vet. Mycol. 24:81-84. 120. Williamson, M. I., L. P. Samaranayake, and T. W. MacFarlane. 1986. Phospholipase activity as a criterion for biotyping

CLIN. MICROBIOL. REV.

Candida albicans. J. Med. Vet. Mycol. 24:415-417. 121. Williamson, M. I., L. P. Samaranayake, and T. W. MacFarlane. 1987. A new simple method for biotyping Candida albicans. Microbios 51:159-167. 122. Wills, J. W., W. B. Troutman, and W. S. Riggsby. 1985. Circular mitochondrial genome of Candida albicans contains a large inverted duplication. J. Bacteriol. 164:7-13.