Critical Reviews in Microbiology

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A Critical Review of Typing Methods for Candida albicans and Their Applications Paul R Hunter To cite this article: Paul R Hunter (1991) A Critical Review of Typing Methods for Candida albicans and Their Applications, Critical Reviews in Microbiology, 17:6, 417-434 To link to this article: http://dx.doi.org/10.3109/10408419109115206

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A Critical Review of Typing Methods for Candida albicans and Their Applications Paul R. Hunter, M.D.

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ABSTRACT During the 1980s, a large number of typing methods for the strain differentiation of Candidu albicans were described in the literature. Although these methods have been based on a variety of physiological and genetic markers, none is ideal. This review discusses the characteristics of an ideal typing method in terms of its typability, reproducibility, and discriminatory power. Ways of determining these characteristics are presented so that the available typing methods for Candidu afbicanscan be objectively compared. Available typing methods for C. albicam include serotyping, morphotyping, resistotyping, biotyping, and killer yeast typing. Electrophoretic methods include immunoblotting,isoenzyme analysis, analysis of DNA restriction fragment length polymorphism, karyotyping, and the use of DNA probes. The application of these methods to epidemiological research, the investigation of outbreaks of disease, and the study of virulence is described. The potential impact of the phenomenon of phenotypic switching on the reproducibility of these typing methods is discussed. It is concluded that many of the available typing methods have not been adequately assessed by their developers and that several have only poor discriminatory power or reproducibility.

1. INTRODUCTION After more than 20 years work by many different scientists, it is still not possible to type Candidu afbicans below the species level with a high degree of certainty. The lack of a suitable typing scheme has undoubtedly interfered with the pace of research into the epidemiology and virulence of this pathogenic yeast. This does not mean to say that there are no typing methods available to distinguish between distinct strains of C. afbicans. Indeed, there are actually too many typing methods from which the prospective research worker can choose. Despite the large diversity of typing methods that have been described for C. albicans, and perhaps because of it, no single procedure has become the accepted standard. There are many typing methods but no typing scheme. This article reviews the various typing methods that have been described for C. afbicum and djscusses the use to which some of them have been put to answer questions of epidemiology and virulence. Before describing the available typing methods, we must f i t consider what distinguishes a typing scheme from a typing method and

what characteristics define a good typing scheme. A typing method is able only to distinguish between two unrelated strains. A typing scheme is able to describe a strain in such a way that workers in different centers can have a high degree of certainty that their strains are similar or distinct from each other when comparing their typing results. When assessing the value of any typing method or scheme, the three main characteristics that need to be considered are typability, reproducibility, and discriminatory In addition, three secondary characteristics of cost, ease of use, and time taken to obtain a result should be considered. The typability of a method is the proportion of a population of isolates that can be typed by that method. For example, Stuphyfococcus aureus phage typing is not able to assign a type to about 20% of strains; thus, this typing method has a typability of 80%.3 All existing typing methods for C. albicans have 100% typability. The reproducibility of a typing method covers three concepts, that of in vim0 and in vivo reproducibility and reproducibility between centers. In vitro reproducibility is the proportion of strains that are typed the same on repeat testing after aperiod of a few months. Thus, if 100 strains were retyped and the assigned type of five of those strains differed from the initial typing, such that if the strain history was unknown they would be presumed to be different strains, the in v i m reproducibility would be 95%. In vivo reproducibility is a rather more difficult concept that reflects whether strain populations on a given host are stable. Studies of in vivo reproducibility usually require repeat testing of multiple strains from volunteers or patients over a period of time. In vitro reproducibility reflects the reliability of the typing method, while in vivo reproducibility reflects the stability of the organism under study. Clearly, poor in vivo reproducibility may be due to transient colonization by different strains or to phenotypic instability of a single resident strain. This latter point of phenotypic instability or phenotypic switching is a potentially serious problem in C. albicans and its implications are discussed later in more detail. Studies of reproducibility between centers are seldom undertaken; still, unless we can be sure that one center’s results are identical to another, then we cannot assume that,experimental results from one center could be repeated in another. The discrimination of a method is an estimate of its ability to differentiate between two unrelated strains. Until recently this was usually described in terms of the number of types discriminated and the frequency of the most common type.

Paul R. Hunter, M.D., Public Health Laboratory, City Hospital, Hoole Lane, Chester, CH2 3EG, England.

1991

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Critical Reviews In This was unsatisfactory as can be seen if we consider a population of 100 strains divided into 1 1 groups, with the most frequent group containing 50% of the population. If the remaining 50 strains were distributed as 5 in each of the remaining 10 groups, that typing method would be manifestly more discriminatory than if 41 strains were allocated to the second group with 1 strain in each of the remaining 9 groups. The problem was to define more accurately what is meant by discriminatory power. The underlying aim of typing microbial pathogens is to distinguish between unrelated strains. Hunter and Gaston4suggested that discriminatorypower can be defined mathematically as the probability that two strains, chosen at random from a population of unrelated strains, will be distinguished by that typing method. Once this definition was accepted, this naturally led to a numerical index of discriminatory power (0). This probability is given by:

and the value D,, read from the graph (Figure 1). Alternately, and more accurately, linear regression analysis can be used. Wherever possible, this D,, value will be used to compare typing methods.

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Discrimination

where s = the number of types, xj = the proportion of the population falling into jth type, and N is the size of the poplat ti on.^ This initial equation, which is identical to Simpson’s diversity index, is applicable only to situations where all strains can be placed into mutually exclusive groups., To overcome this limitation, a generalized version was developed: N

D = 1 - N(N I- 1) jC aj -1

where aj = the number of strains in the population that are indistinguishable from the jth strain and N = number of strains in the populati~n.~ These two indices of discriminatory power can also form the basis of automatic test selection procedures when developing new typing methods.6 Many typing methods characterize strains by the use of a battery of tests. When these typing methods are used to type strains, such strains may be identical or may differ by one, two, or more tests. If the method is not 100% reproducible, one may decide that two strains will be deemed to be distinct only if they differ by. two, three, or more test differences. Although the reproducibility increases when more test differences are required to distinguish between strains, the discriminatory power declines. A method has been described for the standardization of the discriminatory indices of typing methods that use multiple tests to characterize strain^.^ Essentially, the index of discriminatory power and reproducibility are determined for the typing method - if one test difference is sufficient to distinguish between strains, if two test differences are required, and so on until the reproducibility is 100%. Using these two sets of data, the discriminatory power for a typing method assuming a 95% reproducibility is then calculated. Discriminatory power can be plotted against reproducibility 41 8

FIGURE 1. Plot of reproducibility against the indices of discriminatory power for carbon source assimilation reaction typing of C a d i & ulbicuns for one through five test differences. (From Hunter P. R., J . Clin. Microbiol., 28, 1903, 1990. With permission.)

The secondary factors of cost, ease of use, and time to undertake the test are reasonably self-explanatory. However, additional factors such as delay and cost of postage should be considered for those methods that are generally undertaken by reference laboratories. For example, a test that takes several days to perform in a peripheral laboratory may still give a more rapid result than one only taking 48 h to perform if it has to be sent away.

II. BlOTYPlNE METHODS A. Serotyping Sero-agglutination reactions were first applied to C. albicans (then known as Monilia albicans) by Benham.’ She examined only a few strains, but found cross-reactions between all strains of C. albicans and between strains of C. albicans and other “Monilia” species. Furthermore, all strains of C. albicam completely absorbed out all agglutinating activity of sera raised to other strains of the same species. It was not until the early 1960s that Hasenclever and Mitchells produced a serum which did not lose its agglutinating activity for the immunizing strain after complete absorption by another strain of C. albicans. This serum showed differential agglutinating ability between other strains of C. albicans. The strains that were agglutinated by the antiserum were designated group A, those that were not, group B. In a population of 71 strains, 38 (54%) were group A and 33 (46%) were group B (0=

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0.505). It was further shown that group A strains could completely absorb agglutinating activity from sera raised to group B strains, but that the reverse did not occur, suggesting that group A strains possessed both common and specific antigens while group B strains possessed only common antigens. This suggestion was confirmed some years later.g A third minor serogroup was described by Miiller and Kirchhoff O and called group c. Despite the simplicity of a method based on slide agglutination reactions, the presence of only two major and one minor serogroups limits the usefulness of serotyping for C. albicuns. Furthermore, subsequent studies of serotyping found even poorer discriminatory power as group A strains predominate in clinical samples. In a later study, Hasenclever and Mitchell1]found that of 653 strains examined, 68%were group A (D = 0.438). Studies performed in France found group A even more frequently, 615 (90.4%)of 680 strains in one study (D = 0.186),12 and 547 (93.8%)of 583 in another (D = 0.116).13Such extremely low discriminatory power severely limits the value of serotyping to distinguish strains of C. albicuns. Interestingly, 5-fluorocytosine resistance nearly always has been found associated with group B ~trains.’~-’~ More recently, Brawner and CutlerIs described a study serotyping C. albicuns using monoclonal antibody H9.lS They were able to divide 128 strains into 74 weakly reactive and 54 strongly reactive strains (0= 0.492). This antibody produced results that correlated very well with results obtained by Hasenclever’s original

Table 1 Coding - of Streak Features in the Full Morphotyping c~de’~.’~ Feature Fringe Distribution

Code

0 1

2 3 5

7

Width

0

2 3 5

Texture

0 1

2 3 4

Absent Discontinuous; 6 mm Absent Very coarse

coarse Intermediate Fine

Streak surface

Topography

0 1

2 4

5

6

a Quality

0

2 3

8. Typing Based on Colonial Morphology Variation in colonial morphology of C. albicuns was first reported by Negroni16in 1935, who described a rough colonial variant that was not pathogenic for rabbits. In 1968, BrownThomsen” characterized the morphological forms of a greater variety of strains after streak culture, incubated on a malt extract agar at 25°C over 10 d. The malt extract agar was composed of 6% Oxoid malt-extract agar with 2% Difco special agar. Brown-Thomsen described 15 morphological forms of C. albicuns, designated A1 to A13, B1 and B2. Nearly all freshly isolated strains were type Al, giving smooth, featureless colonies. After storage these strains developed characteristics, enabling him to reclassify them. Brown-Thomsen regarded the smooth A1 form as the wild type with the other forms derivatives from this type. Brown-Thomsen’s observations were developed into a more formal typing method by Phongpaichit and co-workers. These authors developed a more detailed system that was able to code far more morphological features. This code was based on two sets of three digits, with occasional ancillary codes to describe other features (Table 1). The first set (termed primary features) described the colonial fringe (distribution, width, and texture), the second set (secondary features) described the streak surface (topography, quality, and depth and abundance). The occa-

Description

4

Depth and abundance

0 2 4

6

a

Smqoth Nodular Pitted Cmteriform Crateriform plus wrinkles or folds Wrinkles or folds Hairy Absent Coarse or broad Intermediate Fine or narrow Absent Shallow and sparse Shallow and abundant Deep and sparse Deep and abundant

sional digit in parenthesis describes various ancillary features such as the presence of hairs over the streak or fringe surface. The quoted reproducibility of the method, over 2.5 years, was 84% for those giving an identical morphotype and 96% for those differing by one or less adjacent characters. However, it was noted that the colonial morphology was sensitive to changes in the malt agar or the conditions of incubation. For example, even slight overheating of the malt agar during preparation could abolish fringe formation. This last point may explain why earlier workers had not commonly detected fringe formation.17*19 In a subsequent paper, the morphotyping method was able to identify 50 types in a population of 446 strains, the most frequent type accounting for 22.9%of the strains (0= 0.905).m As discussed earlier, the reproducibility for the method is 84%. If reproducibility was increased to 96%, by assuming that a 1991

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Critical Reviews In single strain could not be distinguished if it differed by only a difference of one or less in a single digit, then discrimination fell to D = 0.824. A problem with this code is that the very diversity of possible types make it difficult to compare populations.” This led to these codes being regrouped, for the purposes of certain studies, into six fringe states (no fringe, discontinuous fringe, widefine fringe, wide-coarse fringe, narrow-fine fringe, and narrowcoarse fringe) and two surface states (featureless and featured). In further studies, using this simplified code, it was shown that the reproducibility of surface features was 82% and the reproducibility of fringe features was 89%, while the discriminatory power of the method using just the six fringe states was D = 0.615, though this is a marked underestimate of the discriminatory power achieved by the full code.5 Figure 2 shows a selection of morphotypes and their codes. The main advantage of morphotyping is that it requires little expensive equipment and is suitable for occasional use in laboratories where other methods are impractical. In addition, as discussed later, one morphotype seems to correlate with strains of increased virulence.zo*21 C. Resistotyping Resistotyping was first applied to C. albicans by Wamock et al.” This method is based on the ability of differing strains to grow on solid media containing known concentrations of various inhibitor chemicals. In the original method six dilutions of each inhibitor chemical (malachite green, boric acid, sodium arsenate, copper sulfate, acrylamide, and Cchlororesorcinol) were used. Each plate was inoculated with up to 25 strains and the sensitivity or resistance of each strain determined in relation to that of known control strains. The six dilutions enabled a titration of the effect of each inhibitor on the control strains, the point at which growth of the control strains ceased could then be used as a breakpoint level for determining susceptibility of the test strains. The authors commented that there were discrepancies between certain test results that they considered to be due to variations in the inoculum density. Insufficient unrelated strains were examined at the time to allow an estimate of the discriminatory power. Resistotyping was modified by McCreight and Warnock to include four dilutions of just five inhibitors: sodium selenite, boric acid, cetrimide, sodium periodate, and silver nitrate.23.24 The authors reported 96% in v i m reproducibility as well as a very good in vivo reproducibility. By studying 60 oral strains from 22 normal subjects 16 types were identified, the most frequently isolated type accounting for 25% of the population (D = 0.882). This method was further modified by Medcraft” who added two further inhibitors, malachite green and copper sulfate. In our hands, reproducibility was poorer than that quoted by McCreight and Wamock, especially for silver nitrate which was omitted.26 Figure 3 shows a typical resistotype plate. Resistotyping was also developed independently by Kash-

420

whose set of inhibitor chemicals differed markedly from the McCreight and Warnock set. The chemicals used by Kashbur were chlorhexidine, benzalkonium chloride, mercurochrome, ferric nitrate, Irgasan DP 300, cetrimide, hexachlorophene, amphotericin, and domiphen bromide. ’Hunter and FraserS used a resistotyping set composed of selenite, boric acid, cemmide, malachite green, copper sulfate, benzalkonium chloride, chlorhexidine, mercurochrome, arsenate, 5-fluorocytosine, salt, and MacConkey agar. Instead of using several dilutions of each inhibitor, they used just two dilutions. The strain was stated to be resistant to an inhibitor if there was growth on both dilutions, sensitive if there was no growth on either or weak growth on one dilution, and equivocal if there was growth on one or weak growth on both dilutions. The in v i m reproducibility and discriminatory power of this method varies depending on the number of differences required to distinguish strains (see Table 2). The standardized index of discriminatory power (D,) is 0.564. In vivu reproducibility was good.21.28 Resistotyping appears to offer a discriminatory and reproducible method for typing C . albicans. However, it is one that requires meticulous attention to media and strain preparation, otherwise reproducibility can deteriorate markedly. It is probably not a suitable method for those laboratories that only occasionally type strains of C. albicans. D. Biotyping Included in the category of biotyping are methods that either are based on assimilation of carbon sources or on the detection of preformed extracellular enzymes. The biotyping method of Odds and Abbott is in fact a hybrid of resistotyping and carbon source assimilation reactions, but is also discussed under this heading. The first biotyping method described was based on lipase and proteinase production and on susceptibility to nystatin and p i m a r i ~ i n . ~ ~ Using 28 carbon sources, Brown-Thomsen” demonstrated 6 auxanographic types of C. albicans due to variation in the assimilation of D-melezitose, L-arabinose, adonitol, and citric acid. The most common type accounted for 68% of all strains (D = 0.490). No reproducibility studies were reported. Syversonso reported a study of assimilation of 11 carbon compounds by 215 strains. Despite all the carbon sources being listed by other authors as giving variable reactions, Syverson found variability in assimilation of only five of these compounds. Furthermore, those that did give variable reactions did not give consistent results for each strain and changes in the test conditions gave differing results. In 130 strains, 13 biotypes were distinguished by the API 20C (API-Bio Merieux (U.K.) Ltd., Basingstoke, England) due to variation in 11 assimilation reaction^.^' However, 75% of all strains fell within a single biotype (D = 0.431, reproducibility = 97%). Hunter and Frasef used 64 carbon sources by combining

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Microbiology

A

B

C

D

E

FIGURE 2. Some example morphotypes giving the fringe state” and the full six digit code.” (A) = Fringeless, OOO OOO, (B) = discontinuous fringe, 123 432; (C) = discontinuous fringe, 533 444;(D)= narrow coarse fringe, 722 OOo; and (E) = wide fine fringe, 754 244.

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Critical Reviews In later,36 and recently reduced to 10 again.37 The most recent ten tests include tolerance of pH 1.40 and 1.55; the capacity to grow in the presence of 5-fluorocytosine, salt, borate, and safronin; an ability to utilize urea, sorbose, and citrate; and the production of extracellular proteinase production. Although the in vitro reproducibility of the method as a whole is not given by the authors, this can be estimated from the probability of error in each character. The discrimination of the original set of 10 test, standardized to 95% reproducibility was D,, = 0.657. More recently, it was shown that the results obtained in one laboratory are not directly applicable to those obtained in another, this being one of the few occasions when betweencenter reproducibility studies have been undertaken for candidal typing methods3' Odds et al.37commented that the method of Odds and Abbott is not suitable for routine use but for research purposes only. Nevertheless, the Odds and Abbott methods have been used quite extensively during the past 10 years and they have made several contributions to our knowledge of the epidemiology of candidal infections.

E. Killer Yeast Typing FIGURE 3. An example resistogram plate showing selective inhibition of some strains.%

two kits, the API 50CH and the ATB 32GN (both API-Bio Merieux (U.K.) Ltd.). Esculin was excluded from analysis as it was found to be difficult to read. Variable reactions were given by 3 1 of the carbon sources. Although the discriminatory power of this large number of carbon sources was high, reproducibility was very poor, and the standardized discriminatory power was only 0.155 (Table 2). Generally, it would appear that typing based on carbon source assimilation reactions are unreliable, either because of poor discriminatory power or poor reproducibility. Although discrimination can be increased by increasing the number of carbon sources, this gain is offset by the decline in reproducibility. The commercial kit API ZYM (API-Bio Merieux (U.K.) Ltd.) enables a rapid analysis of a strain's profile to be made for 19 preformed enzymes. Casal and LinaresSZdemonstrated four biovars (A-D) due to variability in four enzymes: valine arylamidase, cystine qlamidase, a-glucosidase, and N-acetyl-P-glucosaminidase. They reported 100% reproducibility, though discrimination was poor (D = 0.549), the most common biovar accounting for 64% of all strains. Variability in a further enzyme, P-glucosidase, was identified later.33 Other workers found even poorer discrimination: D = 0.471, reproducibility = 100%;" D = 0.487, reproducibility = 97%;3' and D = 0.308, reproducibility = 93%.*Biotyping by API ZYM is easy and rapid, taking just 4 h to perform. However, in spite of its good reproducibility, poor discriminatory power limits its potential. Odds and Abbott described a biotyping method based on 10 biochemical reaction^,'^ which were increased to 14 a few years '

422

This method, first described by Polonelli et al.,38 is based on the ability of nine killer yeasts to selectively inhibit the growth of different strains of C. albicans. The original method was able to differentiate 25 different types, though the most frequent type accounted for 52%of a population of 100 strains (D = 0.724). The authors made no mention of any reproducibility studies. Caprilli et al.39used the same set of 9 killer strains and found a much poorer discriminatory power identifying only 7 biotypes with 78%contained in one type (D = 0.376). This discrepancy is probably related to the populations of C. albicans used to validate the method. Caprilli used only clinical isolates from human sources while Polonelli used a mixture of human and animal strains. When the number of killer strains was increased to 21, 27 biotypes were distinguished in 100 strains, the most frequently isolated type accounting for 48% of them (D = 0.756). These authors quoted 99% reproducibility, but they retyped the strains after only 10 d. Lehmann et al." were able to increase the discrimination of the method even further by using 30 killer strains. They described 19 types with the most common type containing 21% of strains (D = 0.899). At that time no reproducibility rate was given, however, in a later paper Lehmann's group4' commented on the poor reproducibility of the killer system over several months. Until formal studies of reproducibility are undertaken the usefulness of killer yeast typing will remain uncertain. An alternate method to performing killer typing looks at susceptibility to the partly purified killer toxin, rather than to the killer yeasts t h e m ~ e l v e sIn . ~this ~ approach, a purified fraction of the broth supernatant was simply spotted onto a lawn of the strain under test. The zone of inhibition was recorded

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Microbiology Table 2 Reproducibilities and Discriminatory Powers of Typing Methods when Differing Numbers of Test Differences are Required to Distinguish between Strains Number of test differences required to distinguish between&S

I

III

II

Iv

V

D,

Ref.

Serotyping 0.505 (NS) 0.438 0.186 0.116 0.492

8 11

12 13 15

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Morphotyping (Full Code)

0.824 (96)

0.905 (84)

18, 20

Morphotyping (Short Code)

0.615 (89)

5 Resistotyping

0.882 (96) 0.903 (77)

0.694 (93)

0.438 (97)

0.221 (100)

0.564

24 5

0.155

17 31 5

0.226

32 34 31 5

0.657

35

Carbon Source Assimilation

0.490 (NS) 0.431 (97) 0.891 (53)

0.607(60)

0.331 (80)

0.174(93)

0.109 (100)

API ZYM

0.549 (100) 0.471 (100) 0.487 (97) 0.308 (93)

0.068(100) Biotyping

0.956 (55)

0.800(89)

0.564 (94) Killer Yeast Typing

0.724 (NS)- 9 killers 0.376 ( N S ) - 9 killers 0.756 (NS)- 21 killers 0.899 (NS) - 30 killers,

38 39 39 40

reproducibility poor

Combined Killer and Resistotyping

0.961 (NS)

40 Combined API 2OC, API ZYM and Boric Acld Resistance

0.855 (90)

31

1991

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Critical Reviews In Table 2 (continued) Reproducibilitiesand Discriminatory Powers of Typing Methods when Differing Numbers of Test Differences are Required to Distinguish between Strains Number of test differences requited to distinguish between strains'

I

m

II

V

IV

D,

Ref.

0.673

5

0.655

5

Combined Resistotyping and Morphotyping 0.957 (67)

0.814 (92)

0.593 (97)

0.362 (100)

Combined Resistotyping, Morphotyping, and Carbon Assimilation

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0.993 (40)

0.957 (53)

0.861 (80)

0.718 (93)

0.52 (100)

Combined Resistotyping, Morphotyping, Carbon Assimilation, and API ZYM 0.996 (40)

0.970 (47)

0.899 (80)

0.774 (93)

0.617 (100)

0.716

5

Note: ( ) = Reproducibility. (NS) = Reproducibility not stated or inadequate studies reported.

For example, if number of test differences required to distinguish strains is one, then if the results of just one of the'set of tests differ between two strains those two strains are deemed to be distinct. See text for further discussion.

for each of the killer toxins. The authors found that discriminatory power was improved, although they did not give sufficient data to enable calculation. The authors also reported that by use of a suitable mathematical adjustment, the in vitro reproducibility could be increased to 95%.

F. Combined Methods Although the first recorded outbreak of candidal infections to be typed was characterized by the use of three typing methods, morphotyping, biotyping, and serotyping,lg very few studies have attempted to show whether combining methods can increase discriminatory power. The first such study used a combination of killer yeast typing with a limited resistotyping set consisting of 5-fluorocytosine, cupric sulfate, and sodium arsenate.40By using 30 killer strains alone, it was possible to distinguish 19 types (D = 0.899). When the resistotyping set was added, the number of types distinguished increased to 33 with the most common set accounting for 1.5% of the population (D = 0.961), though there was no comment on reproducibility. In a later paper, Lehmann et al.41 commented that reproducibility over a 6-month period was poor. A further study combined carbon source assimilation reactions using the API 20C, which detected 13 groups, with the most common group contaiing 75% of strains (D = 0.431, reproducibility = 97%; extracellular enzyme production using the API 20ZYM, which detected 9 groups, with the most common group containing 70% of strains (D = 0.487, reproduc-

424

ibility = 97%; and resistance to boric acid, which split the population into two (D = 0.506, reproducibility = 97%).3' The combined typing schemes detected 33 biotypes, with the most common type representing 28.5% of strains (D = 0.855, reproducibility = 90%). Hunter and FraseP compared the reproducibility and discriminatory power of four typing methods alone and in combination. They found that the use of both resistotyping and morphotyping gave the best combination of discriminatory power and reproducibility D,, = 0.673 (Table 2). This combination was shown to have good in vivo reproducibility as If carbon source assimilation reactions were added to this combination, reproducibility declined to such an extent that the discriminatory power was impaired, D,, = 0.655. While the API ZYM did add to the discriminatory power of the combinations (Dg5 = 0.716), the improvement was not sufficient to justify its use on a routine basis.

111. ELECTROPHORETIC TYPING METHODS A. Protein Electrophoresis Electrophoresis polymorphisms in whole cell protein profiles were first noted by Shechter et al.43In a study of nine isolates of C. albicans, including C . albicans var. stellatoidea, three types were identified by variation in the positions of just two bands in the acidic protein profiles. Ibrahim-Granet et al." were able to detect polymorphisms in 20 polypeptide bands by

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Microbiology polyacrylamide gel electrophoresis (PAGE) of whole cell proteins. However, this method only differentiated between serogroups A and B. More recently, Bruneau and G ~ i n e talso ,~~ using PAGE, found identical protein profiles in 14 strains of C. albicans obtained from a wide variety of sources. It would appear that PAGE does not offer a method of typing C. albicans superior to that of serotyphg. Immunoblottinghas offered a more promising approach. Lee and co-workers* described a method using polyclonal hyperimmune rabbit serum. PAGE of a protein extract was performed and the separated proteins and glycoproteins were blotted onto a nitrocellulose membrane. The membrane was then incubated with the rabbit serum and the sites of antibody binding visualized by one of several methods. The discriminatingpower of the method is poor (D = 0.633). Although 16 types were distinguished, the most frequently isolated types accounted for 80% of the colonizing strains. No reproducibility rate was quoted, although the authors noted that gel-to-gel variation in the relative position of the antigens was a problem. Polonelli and Morace4’ used immunoblotting to demonstrate the binding of a monoclonal antibody to several polypeptides of differing molecular weights. They demonstrated five types in ten strains of C. albicans, the most common type accounting for 60% of strains. The same monoclonal antibody gave characteristic patterns for a wide range of other yeasts, including other species of Candida, Cryptococcus neoformuns, and Malassesia species. No reproducibility rates were given by the authors.

6. lsoenzyme Typing Methods Isoenzyme typing of C. albicans relies on the extraction of intracellular proteins and their analysis by PAGE. After electrophoresis, the location of specific enzyme activity within the gel is detected by applying enzyme substrates. Lehmann et a1.40reported variation in six enzyme systems: a-glucosidase (aG), alkaline phosphatase (ALP),glucose-6-phosphate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH), and superoxide dismutase (SOD). A total of 37 strains of C. albicans were analyzed by these enzymes. There were four patterns of aG, the most common type accounting for 84%(D = 0.296). There were two patterns of ALP, the most common accounting for 86% (D = 0.240). For GDH, there were three patterns, the most common accounting for 75% (D = 0.419). For MDH, there were three patterns, the most common accounting for 89%(D = 0.204). For ICDH, there were two patterns, the most common accounting for 54% (D = 0.511). For SOD, there were four patterns, the most common accounting for 68% (D = 0.503). When all the enzymes were combined, 23 types were detected, of which the most common represented only 27% of strains (D = 0.923). This level of discriminatorypower is high compared to virtually all typing methods thus far described. However, no reproducibility studies were reported by Lehrnann and co-workers.

C. DNA Restriction Fragment Length Polymorphisms Scherer and Stevens48 were the first to report the use of restriction fragment length polymorphisms in C. albicanr as epidemiological markers. In their original paper they used an ’ EcoRl restriction digest and revealed 10 subgroups in 17 strains of C. albicans, the largest subgroup accounting for six (35%) of the strains, giving an acceptable discriminatory power (D = 0.805). Formal reproducibility studies were not undertaken, however, the authors demonstrated that six different strains run on the same gel could be accurately paired. Comparing strains from one gel to another was felt to be difficult and strains had to be run together to absolutely ensure they were the same, a major disadvantage when large numbers of strains need to be compared. The restriction pattern of a single strain subcultured 30 times in yeast nitrogen base broth remained stable. In a further paper, Stevens et al.49reported the presence of 29 different types in 91 strains of C . albicans. However, 37 (41%)fell into a single type and discriminatory power was still only moderate (D = 0.814). The authors showed that restriction enzyme digests were stable after passage through mice, suggesting good in vivo reproducibility. Other workers5’O’’ have used restriction fragment length polymorphism to type C. albicans and found the method to be of value. However, Mathews and Burnie5’ reported only 16 groups in 94 strains, of which the most common group accounted for 63% of the population (D = 0.599), much lower than that reported by other authors. Furthermore, Stevens et reported that the isolates originally deemed to be a single strain in a presumed outbreak at the London HospitalIg were in fact five different strains. That Matthews and Bumie found that the restriction digest patterns supported their hypothesis that this was a single strain outbreak casts doubt on the reliability of this method between lab~ratories.~’ Why these two groups of workers should obtain such different results using virtually the sarne method on the same isolates is unclear. The London Hospital “outbreak” is discussed in more detail below, but these discordant results must raise some concern for the reliability of typing based on restriction fragment length polymorphisms. Smith and colleagues50reported that the restriction enzyme Hinfl was more discriminatory than EcoRl ,but did not provide sufficient data for the relative discriminatory powers to be determined.

D. Karyotyping Orthogonal field alternating gel electrophoresis (OFAGE) and field inversion gel electrophoresis (FIGE) are techniques enabling separations to be made of very large pieces of DNA. Several groups have investigated the application of these rnethods to epidemiological typing of C. albicans. Using OFAGE Magee and Magee52noted that the electrophoretic karyotypes of four strains of C. albicans were all quite distinct from one

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Critical Reviews In another. Using FIGE Snell et al.’3 demonstrated four distinct patterns in five strains of C. albicans. They further reported that a strain, ATCC 10261, that had been in laboratory culture for 15 years gave an identical banding pattern to ATCC 10261 which had been freeze-dried in 1960. Contrary to these findings, Lott et al.54 did not find any variability in eight strains using FIGE. Merz et al.55 used OFAGE to examine multiple strains isolated from 17 different patients. They found 14 different patterns, of which three were represented in two patients each. Insufficient unrelated strains were analyzed in this study to enable calculation of the discriminatory power, but if results from these 17 strains can be extrapolated to the general population, it would then appear that discriminatory power would be at least 0.950. Multiple isolates were examined from 12 patients, the patterns being identical in all but two, suggesting that the method has good in vivo reproducibility. No in vitro reproducibility studies were reported. A further variation was reported by Mahrous et al.56who used a transverse alternating-field electrophoresis (TAFE)system. These authors examined 12 typical and atypical strains and found variation between strains; particularly between C. albicuns and C . ulbicans var. stellatoidea. The disadvantages of karyotyping methods are that rather expensive equipment is required and run times may take several days limiting the number of strains that can be tested. That karyotypes may change dramatically in a single strain after phenotypic switching is discussed later.

E. Application of Nucleic Acid Probes Several attempts have been made to type C. albicuns using nucleic acid probes to improve the discrimination of Southern blots of restriction fragment digest electrophoresis. One of the fmt probes to be described was a 2-kb DNA fragment containing the single C. albicuns actin gene.57 Although some differences between species were noted, there was no variation between six strains of C. ulbicans isolated from widely separate geographical sources. Using labeled ribosomal DNA to probe an EcoR1 digest Magee and colleagues5*found six different types in 12 strains, though 58% of these strains fell into a single class. A further approach was to use a labeled mixture of fragments of cloned C. albicans mitochondrial DNA to probe whole cell DNA dige~ts.’~ When the whole cell DNA was digested with EcoRI, PvuII, or HindIII restriction enzymes, no variation was detected; however, with HueIII or TaqI four distinct patterns were noted in 26 isolates. Repeat isolates from the same patient were always identical. Poor discriminatory power of mitochondrial DNA probes was also reported by other workem60 Scherer and Stevens6*developed a DNA probe (27A) to a dispersed, repeated gene family. They found that the use of the probe provided much greater discrimination than the use of restriction fragment length polymorphism alone. However, 426

they reported that polymorphisms can arise during laboratory subculture. Furthermore, multiple isolates from the same patient differed slightly from each other. Using the same probe, another group found that of 63 patients examined only three patients had an identical strain type, while the remaining 60 patients had unique types (D = 0.998).@’ These workers found that in vivo reproducibility was excellent as all patients showed identical patterns. Soll and co-workers62-64 have used DNA midrepeat sequence probes to distinguish between strains of C. albicuns for several years. It was recently shown that the power of these methods could be improved by the use of computer-assisted analytical technique^.^^ The Ca3 probe was shown to be highly discriminatory (D = 0.986). Furthermore, this probe gave highly reproducible patterns over many laboratory subcultures.

IV. SOME APPLICATIONS OF TYPING METHODS TO EPIDEMIOLOGY A. The Epidemiology of Vulvovaginal Candidiasis Although often thought of as a mild disease, vulvovaginal candidiasis is extremely common and can be the cause of much misery to patients and their families.& It is generally accepted that host factors play a major part in the etiology of vaginal candidiasis and that most cases are due to endogenous infect i ~ n . In ~ the ~ , main, ~ studies using biotyping methods have supported these accepted conclusions. Strains of C. ulbicans causing vaginal infection are usually of a type similar to those Nevertheless, sexual transcolonizing the patient’s mission does seem to occur as identical biotypes are frequently Odds73 isolated from the genitalia of sexual has also made the suggestion that although strain carriage is usually stable in most women, promiscuous women tend to carry a variety of strain types. Whether the epidemiology of severe recurrent vaginal candidiasis differs from nonrecurrent vaginal candidiasis is unclear. As with nonrecument vaginitis, the feces of women with recurrent vaginitis are usually colonized with C. albicuns of the same strain as that causing their ~aginitis.’~ O’Connor and Sobe170reported that in 56.3% of patients and HunteP reported that in 85.7% of patients, the isolates obtained from first relapses were the same as the initial infecting strain. In a study that questions the value of colony morphology as a stable strain characteristic, Soll et a1.- studied a patient with recurrent vaginitis for three episodes of infection. While Southern blot hybridization of total cellular DNA with the Ca3 probe revealed that the same strain was responsible for all three episodes of infection, the colony phenotype of all isolates from the second infection were markedly different from isolates from the first and third infections. Thus, previous studies of recurrent vaginiti~~’.’~ should be evaluated cautiously as apparent strain differences may reflect solely phenotypic switching rather than true differences in strain genotypes.

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Microbiology 6. Studies of Nosocomial lnfections There is no doubt that nosocomial candidiasis causes a great deal of morbidity and mortality. C. albicam is a particular problem in certain susceptible patients such as neonates, patients in intensive care units, and those suffering from severe bums or malignant d i s e a ~ e . Even ~ ~ . ~with ~ the use of prophylaxis and aggressive antifungal therapy, 4.1 % of burn patients develop candidemia and about 50% of these die.77Reports of invasive candidiasis in low birth weight babies range from 0.9 to 3.8% and are associated with a high m ~ r t a l i t y . ~In~ -leu~’ kemics, the reported incidence of invasive candidiasis is even higher, around 15%, and the associated mortality is about 92%.82-84 Probably the most important use of typing methods for C. albicuns is in the investigation of outbreaks of invasive candidiasis. Outbreaks of candidiasis have been described for many year^.^^.^^ However, the first outbreak to be investigated using typing methods occurred in an intensive care unit at the London Hospital. l9 In this unit 13 patients developed invasive candidiasis due to C . albicuns over a 9-month period. Initially, the strains were characterized by a combination of serotyping, Brown-Thomsen’s morphotyping method, and Odds and Abbott’s biotyping method.’9 Burnie et al. concluded that all these invasive infections were caused by a single strain of C. albicans and that the same strain caused 44% of the superficial candidal infections on the unit and only 17% of candidal infections elsewhere in the hospital. The outbreak strain was isolated from nurses on two occasions and the authors concluded that the strain was being spread via nurses’ hands. This conclusion was supported by the observation that the outbreak ceased when adequate hand washing techniques were enforced. Further work by the same authors using immunoblotting,46and DNA restriction enzyme digest patterns appeared to confirm the singlestrain nature of the o ~ t b r e a k . ~ * ~ ’ However, one of the original authors has now cast doubt on the conclusion that this was a single-strain outbreak.49Using DNA restriction enzyme digest patterns as the typing method, Stevens and c o - w o r k e r ~found ~ ~ a total of five different strain types among 1 1 strains previously reported as being identical. Furthermore, using the morphotyping method of Phongpaichit et al., Hunter and F~aser’~’ also found strains from this outbreak to be distinct when they had previously been reported as indistinguishable. It would appear that this temporal cluster of cases was not a single-strain outbreak after all. This raises questions about the discriminatory value of the morphotyping, biotyping, and immunoblotting studies reported by Burnie and colleagues.19,46 Using a combination of imunoblotting and morphoryping, Burnie et a P 7reported four more single-strain outbreaks. Three of these were in intensive care units and one Occurred in a special care baby unit. However, in view of the doubts raised about the first London outbreak, it would seem wise to entertain

some skepticism about the “single-strain” nature of these outbreaks. Resistotyping was used to investigate a cluster of cases of candidiasis over a 5-rnOnth period in a special care baby unit in Birmingham, England.BBAt least four different strains of C. albicans were identified. A fuaher presumed outbreak of fatal invasive C . albicans infections was shown by killer yeast typing to be due to more than one strain.89 One apparent outbreak affected babies in a neonatal intensive care unit in Alberta, Canada, when seven cases of invasive candidiasis occurred during a %month period.g0DNA restriction enzyme digest patterns and SDS-PAGE were used to examine the five available strains from these cases. Two distinct types were identified in these five strains, although two cases that occurred on the same day were due to identical strain types. It would appear that although clusters of invasive candidiasis occur quite regularly, as yet no unequivocal singlestrain outbreak has been described. Several studies have investigated the epidemiology of candidal infections in hospital patients in the absence of any obvious outbreak. Superficial candidal infections of the oral cavity and of the skin cause a considerable degree of discomfort in patients undergoing radiotherapy for head and neck carcin ~ m a . ~McCreight ’ - ~ ~ et al.%resistotyped strains of C. albicans isolated from 27 patients before, during, and after radiotherapy. In 25 of the 27 cases, strains colonizing the skin were indistinguishable from those colonizing the mouth, suggesting that in this group infection was probably endogenous. However, there was one, generally uncommon, resistotype that was isolated from four patients on one ward and not from patients on other wards. This suggested that cross infection may indeed play some part in the epidemiology of C. albicans infection in these patients. A further study, also using resistotyping, failed to find any association of particular strain types with strains of C . albicans isolated from cancer patients or from healthy controls.95 Odds et al.% reported a study performed over a 9-month period on 153 patients undergoing chemotherapy or bone marrow transplantation. Using the Odds and Abbott biotyping method, they found that there was a remarkable stability in the biotype of C. albicans carried by individual patients, even over prolonged periods of time. They concluded that cross infection was an uncommon Occurrence on such a unit. That cross infection is unusual in immunocompromised patients was further suggested in a study by Fox and co-workers.60Different DNA probe fingerprint patterns were seen in strains of C . albicans from 60 of the 63 patients studied. Furthermore, for the 20 patients from whom multiple isolates were obtained, the fingerprint patterns of C. albicans remained identical even over prolonged periods of time. The most frequent reports of temporal clustering of invasive candidiasis come from intensive care units. However, there

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Critical Reviews In have been few studies of the transmission of C. afbicans on these units in the absence of any apparent outbreak. Using just three biotyping tests, lipase production, proteinase production, and resistance to 5-fluorocytosine, Huben and showed that there was a positive relationship between length of stay and carriage of 5-fluorocytosine-resistantstrains of C. albicuns. Using a combination of resistotyping and morphotyping, Hunter et aLZ8studied the colonization of patients and nursing staff in an intensive care unit over a Cmonth period. During the study period there were no obvious clinically relevant candidal infections. All but one patient were colonized by a single strain throughout their stay on the unit, whereas nurses were often colonized by more than one strain type. Strains isolated from nurses's hands were indistinguishable from strains colonizing their patient, while strains isolated from the nurses' mouths were usually distinct from those of their patients. The probability that a C. afbicans culture-positive nurse carried an indistinguishable strain from that of her culture-positive patient was about twice the probability that a randomly chosen culturepositive nurse would carry a strain indistinguishable from that of a randomly chosen culture-positive patient. Strain diversity in patients' strains was lower than that of nurses' strains, suggesting that cross infection by C. afbicans is a common occurrence in intensive care units. C. Other Studies Blas~hke-Hellmessen~~ reported the first-ever epidemiological study of candidal infection, using a typing method. He used lipase and proteinase production along with susceptibility to nystatin and pimaricin to examine strains of C. ufbicans taken from mothers and their babies. He found that all culturepositive newborns and 95% of older babies carried strains indistinguishable from those carried by their mothers, thus confirming the long-held belief that babies acquired their strains of C. afbicans from their mothers. Systemic candidal infections are frequently reported from i.v. heroin abusers.98-'" It has been postulated that the source of infection was contaminated lemon juice, frequently used to dissolve the heroin. lol~loz Indeed, C. albicans has been isolated from such lemon juice. '02 Using the Odds and Abbott biotyping method, it was found.that 14 of 21 heroin abusers with disseminated candidiasis in Spain were infected with a single biotype.Io3The isolates grew particularly well in lemon juice when compared to other strains of C. albicuns, further suggesting that lemon juice may be a common source for some of these infections. Also using the Odds and Abbott biotyping method, Shankland and Richardson1@' typed strains of C. a f bicans isolated during a cluster of Candida endophthalmitis in Glasgow, Scotland. They found that isolates from heroin addicts and their paraphernalia were predominantly of the same biotype as the Spanish cases. These two studies strongly suggested that a single strain of C. afbicans was responsible for most cases of candidal infection in heroin addicts. However, 428

when the Glasgow strains were typed by DNA restriction fragment length polymorphisms, it was found that these strains were genetically diverse. Io5 Oral candidiasis due to C. afbicans is extremely common in human immunodeficiency virus (H1V)-infected pat i e n t ~ . ' ~Despite . ' ~ ~ the very great importance of this disease, there have been relatively few reports of the use of typing methods to analyze strains of C. afbicans from such patients. Korting et al.'08 used the poorly discriminatory API 20C carbon source assimilation reactions and found that not surprisingly, 63.9% of isolates fell within a single biotype and that the reproducibility was only 72.7%. Brawner and Cutlert5 used the monoclonal H9 antibody and Hasenclever's original typing serum to characterize oral isolates from 89 immunocompetent and 39 immunocompromised patients, 20 of whom were suffering from AIDS. They reported 7 1.8%of the immunocompromised were colonized with weakly reacting strains, compared to 5 1.7% of the immunocompetent. Using Hasenclever's serum, 66.7% of the immunocompromised were colonized with serogroup B strains, compared to 52.7% of the immunocompetent. Using both immunoblotting and DNA restriction fragment lengthpolymorphisms Matthews et al. '09 found that strains isolated from AIDS cases were similar to strains isolated from other cases. Odds et a l . " O used the moderately repetitive sequence probe Ca3 to examine strains from nine AIDS patients resident in Leicester, U.K. They found that the similarity between these nine strains was much greater than expected. This is particularly interesting as there was no obvious route of infection between the four patients who carried the most similar strains. Whether selection pressures in AIDS patients are such that only a restricted range of strains can predominate remains to be proven by application of this technique to a larger number of patients. One novel approach to the study of the epidemiology of C. afbicunswas based on the theory of adaptive polymorphism.'" This theory predicts that species occupying broad ecological niches will be phenotypically and genotypically more varied than those occupying narrow niches. Carbon source assimilation reactions with the API 50 CH, AF'I 32 GN, and the API ZYM kits were performed on five species of pathogenic yeasts. Each reaction was coded as zero or one and the mean intraspecies variance calculated. It was found that C. glubrara and C . albicuns, two obligate commensals, were less variable than Cryptococcus neoformans, an environmental pathogen. Two facultative commensals, C. purapsifosis and C . tropicalis, were of intemediate variance. This study provided additional support for the widely held view that C. albicans was indeed an obligate commensal.

V. APPLICATION OF TYPING METHODS TO STUDIES OF VIRULENCE There have been many studies of strain variation of virulence

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Microbiology of a more virulent "switched phenotype" was not clear. In in C. albicam. Most of the studies that have shown differences view of the negative results obtained by whole cell DNA rein virulence have been of small numbers of strains, often instriction fragment polymorphism typing,49it may well be the cluding laboratory-derived mutants, in animal models.'12-"8 latter. Indeed, it has recently been shown that different switched This article is concerned only with those few studies that have phenotypes of the same strain can exhibit different attempted to identify strain markers associated with increased ' virulence. virulence by typing strains from cases of infection and from asymptomatic controls. VI. THE PROBLEM WITH PHENOTYPIC Martin and Larnb'l9 showed that 29 of 30 patients with denture-induced stomatitis were infected with serotype A strains SWITCH1NG compared to all control patients who tended to have mixtures That various physiological characters of C. albicans can of serotypes A and B. In contrast, McCreight and col1eaguesl2' change under certain circumstances has been known for many found no particular resistotype associated with denture-induced years. Negroni16 was the first (as early as 1935) to note the stomatitis. "rough-smooth" colony variation. Bro~n-Thomsen'~ showed Cassone et al.12' demonstrated higher secretion of extracelthat the colonial morphology of various strains frequently lular proteinase by strains isolated from patients with vulvochanged after storage for 4 months. That these morphological vaginal candidiasis compared to strains isolated from healthy changes are associated with changes in carbohydrate assimicarriers. However, no differences have been found between lation reactions was reported as early as 1952,'" and again in the types of strains isolated from cases of vulvovaginitis and strains from the vagina or feces of control c ~ s ~ s . ~ ~ * ~ 1968.128 ~ * ' ~SyversonS0 ~ ~ ' ~ also ~ reported that for some strains carbon assimilation patterns were not a stable characteristic. NeverStudies attempting to identlfy strain types associated with theless, it was not until Slutsky and c o - ~ o r k e r s 'reported ~~ invasive disease include that of Odds et al.,'24 who used biohigh-frequency switching of colony morphology in 1985 that typing and found no significant differences in the distribution the phenomenon of ''phenotypic switching" was investigated of phenotypes between deep and superficial sites. Using DNA in more detail. In this initial paper, it was reported that a single fragment length polymorphisms, no differences in the districlone of C. albicans was able to switch its colonial morphology bution of DNA types between deep and superficial infections to one of seven phenotypes at a frequency of 1.4 X 10-4.'29 were observed." However, on the basis that there were no A second switching system was soon described, that of the fatalities associated with the one cluster of cases due to imwhite-opaque transition, and it was shown that changes in munoblot type 1 strains, Burnie et aLX7suggested this type is colonial morphology was accompanied by major changes in less virulent. the cellular physiology. 130~131 Morphotypic differences between strains isolated from deep and superficial sites were first demonstrated by Hunter and Several articles have since suggested that such phenotypic collaborators.2oIn a retrospective study of the morphotypes of switching can have significant effects on the results of biotyp446 strains of C. albicans, 20% of strains from deep infections ing methods. Such phenotypic switching is probably the cause had discontinuous fringes compared to only 4.36% of strains of the poor reproducibility of typing methods based on carisolated from superficial sites. Furthermore, of 12 strains from bohydrate assimilation reactions.s+30-'27J28 That morphotyping fatal infections, 67% had discontinuous fringes compared to may be adversely affected is suggested by the fact that phenonly 11% of other deep infections. This would suggest that otypic switching was first described based on colonial morpatients with a deep candidal infection due to a discontinuously p h ~ l o g y . ' ~It* 'has ~ ~ also been shown that results obtained by fringed strain have a relative risk of death of 8, compared with the biotyping method of Odds and Abbott can change as a patients having a deep infection caused by another strain. result of phenotypic switching. IS2 Immunoblotting can suffer This association of discontinuous fringe with fatal invasive poor reproducibility due to the effects of the phenotypic switchinfections was c o k m e d in a subsequent study.2' Here, 52 ing as there can be variation in the expression of surface ansystemic and 54 superficial strains of C. albicam were typed tigens. " ' ~ ~ Even ~ 4 ~karyotyping ~ may be adversely affected by a combination of morphotyping, resistotyping, and carbon as quite major changes in chromosomal numbers occur during source assimilation reactions. 21 A highly significant difference phenotypic switching.'36J37These major changes in karyotype was found between the two populations (p cO.01). Strains can occur at frequencies as high as l.4%.137 from systemic infections were more likely to be susceptible to What is clear from the work on phenotypic switching of C. orthoboric acid and 5-fluomytosine, were less likely to be albicans is that for any typing method to gain general acceptsusceptibleto arsenate, were more likely to assimilate L-serine, ance, rigorous studies of its reproducibility must be performed. and less likely to assimilate valerate. Whether these differences Such studies must cover both in vivo and in vitro reproducwere due to a more virulent phylogenetic group or to the seibility. As already discussed, reproducibility studies on most lection of a more virulent phylogenetic group or to the selection typing methods have been inadequate.

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VII. CONCLUSIONS The reproducibility and discriminatory powers of several of the typing methods are given in Tables 2 and 3. It is still not clear which is the most appropriate typing method for studying the epidemiology of C. albicuns.

Table 3 Summary of Discrimination and Reproducibility of Some Electrophoretic Typing Methods

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Discriminatory power and ?41 reproduability (in parenthesis)

Ref.

lmmunoblotting

0.633 (NS)

46 lsoenzyme Typing

0.923 (NS)6 different enzymes

41

Restriction Fragment Length Polymorphism

0.805 (100if strains run on same gel) 0.814 (lo0 if strains run on same gel) 0.599 (NS)

48 49 51

Karyotyping Insufficient strains examined in any of the

reports Nucleic Acid Probes

0.998 (NS) 0.986 (100% even between gels with computer analysis)

60 65

Note: (NS) = Reproducibility not stated or inadequate studies reported.

Biotyping methods will probably remain in use, as they are relatively inexpensive and require no special equipment. Of the biotyping methods, it is suggested that typing methods based on killer yeasts could be discounted as being poorly discriminatory and of uncertain reproducibility. API ZYM and serotyping can also be discounted on the basis of poor discrimination and typing based on carbon source assimilation reactions due to poor reproducibility. If a laboratory regularly types C.albicans,then of the biotyping methods, resistotyping or Odds and Abbott biotyping is probably the method of choice. Neither of these methods is particularly superior to the other and the prospective worker should choose the one he finds works best. Morphotyping is probably the easiest discriminatory typing method to establish and is ideal for laboratories that type C. albicans infrequently and cannot afford electro-

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phoretic equipment (always assuming that the laboratory has a good quality autoclave). The association of one particular morphotype With fatal infections should lead to the more widespread use of morphotyping until such time as a more rapid marker Of discontinuous fringe formation is recognized. MOTPhotyping is also useful in combination with other typing methods. However, there is an urgent need to compare and validate the results gained from the use of physiological and biotyping methods with those gained using methods that detect genetic markers. This need is made even more acute by the observation that results gained by biotyping methods were later disproved when restriction fragment length polymorphism analysis to the same isolates was used. In particular, it would appear that the London ~ u t b r e a k was ’ ~ not a single-strain outbreaP9 and that most heroin addicts are not infected by a single strain.’04J05 The fact that distinct strains taken from the same environment had very similar biotypes raises extremely interesting questions about the ability of strains of C. ulbicans to adapt to their environment. It is the author’s opinion that attempts to answer these questions would prove to be a very profitable line of research. Of the electrophoretic typing methods, those based on immunoblotting can be discounted as they are of low discriminatory power and uncertain reproducibility. Restriction fragment length polymorphism of whole cell DNA has only moderate discrimination, although it will probably continue to be used because it is the easiest electrophoretic method to perform. Furthermore, restriction fragment length polymorphism typing is easily adapted to many species of fungi where other methods have not been developed. Isoenzyme typing, karyotyping, and DNA probe typing are three approaches that appear to offer much greater discrimination. A disadvantage of isoenzyme typing is that it requires several gels to be run for a batch of strains. Karyotyping has yet to be fully assessed, although its complexity and requirement for expensive equipment may limit its general appeal. A disadvantage of the currently available nucleic acid probes is their labeling with radioactive tracers. This limits their shelf life and causes additional problems with their handling and disposal. However, it is likely that nonradioactive forms of the midrepeat sequence probes will be developed. A major problem with electrophoretic typing methods conh u e s to be gel-to-gel variation. Most methods require test strain patterns to be compared with other strains on the same gel, and preferably in adjacent lanes. The exception is DNA probe patterns analyzed with the aid of a c o m p ~ t e r . ~B*Y~ ’ suitable numerical analysis, strain patterns from several gels can now be compared reliably. DNA probe methods with computer analysis must become the preferred typing methods for C. albicans, where this is practical. In this author’s view, subject to reservations about their associated radioactivity, the best available typing method for

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Microbiology C. albicans is one of those based on the nucleic acid probes

for the midrepeat sequence^.^^@'.^^ With such a good electrophoretic typing method available, it could be argued that physiological typing methods are now no longer needed. However, if very large numbers of strains are regularly typed, then on cost grounds alone physiological typing methods will still be useful. In our hands, the best physiological typing method is the combination of resistotyping and morphotyping.

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Volume 17,Issue 6

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Microbiology 79. Bdey, J. E., Klieg~~~an, R. M., Boxerbaum, B., and Fanaroff, A. A,, Fungal colonization in the very low birth-weight infant, Pediumcs, 78, 225, 1986. 80. J O ~ S O DD., E., Thompson, T. R., Green, T. P., and Femeri, P., Systemic candidiasis in very low birth-weight infants (

A critical review of typing methods for Candida albicans and their applications.

During the 1980s, a large number of typing methods for the strain differentiation of Candida albicans were described in the literature. Although these...
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