Susceptibilities of Candida spp. to Antifungal Agents Visualized by Two-Dimensional Scatterplots of Relative Growth

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1996, p Vol. 40, No /96/$ Copyright 1996, American Society for Microbiology Susceptibilities of Candida spp. to Antifungal Agents Visualized by Two-Dimensional Scatterplots of Relative Growth F. C. ODDS, 1 * G. DAMS, 1 G. JUST, 2 AND P. LEWI 3 Department of Bacteriology and Mycology 1 and Department of Information Science, 3 Janssen Research Foundation, B-2340 Beerse, Belgium, and Klinikum der J. W. Goethe-Universität, Abteilung für Infektiologie, Frankfurt, Germany 2 Received 2 October 1995/Returned for modification 26 October 1995/Accepted 11 December 1995 The growth of 811 clinical yeast isolates in the presence of single concentrations of antifungal agents was measured spectrophotometrically and expressed as a percentage of growth in inhibitor-free control cultures. Two-dimensional scatterplots of the relative growth data allowed for the simple visual determination of some susceptibility trends, including correlations in relative growth between different agents and in relative susceptibilities between different yeast species. A positive susceptibility correlation was found for relative growth results with the azole antifungal agents fluconazole, itraconazole, and ketoconazole for 504 Candida albicans isolates. The relative growth scatterplots for fluconazole versus itraconazole showed that 50 (9.9%) of 504 C. albicans isolates were outliers with respect to the 95% confidence limits for a line of correlated relative growth established with an initial test panel of 59 isolates of this species. The outlying isolates were relatively less susceptible to fluconazole than to itraconazole under the conditions of the test. Most of the outliers were received in 1993 and 1994; only 3.9% of the isolates received in 1991 and 1992 and 1.7% of the isolates received before 1991 showed this differential susceptibility. In addition, most of the outliers came from patients with human immunodeficiency virus infections. The relative growth scatterplots confirmed the known high susceptibility of most Candida parapsilosis isolates to both fluconazole and itraconazole and the specifically low susceptibility of Candida krusei isolates to fluconazole. The scatterplots also illustrated a tendency towards lower (and correlative) relative growth among oral isolates obtained from AIDS patients who responded to azole antifungal treatment than among isolates from clinical nonresponders. Interest in determining the susceptibilities of clinical yeast isolates to antifungal agents in vitro is high, partly because of a rising incidence of fungal bloodstream infections (1, 33) and partly because of reports of fluconazole resistance in vivo and in vitro among some oral Candida albicans isolates from patients with human immunodeficiency virus (HIV) infection (3, 4, 11, 15, 23, 24, 26, 32). Changes in the prevalences of Candida spp. with time and with usage of antifungal agents have been observed in some institutions (5, 19, 20). A National Committee for Clinical Laboratory Standards subcommittee on antifungal agents is working to establish a reference broth dilution method for yeast susceptibility testing (9), and commercial systems for susceptibility testing are becoming available (7, 8, 17, 18, 21). Nevertheless, monitoring and surveillance of susceptibility trends among clinical yeast isolates by means of MIC determinations are expensive and time-consuming, so simpler approaches to the characterization of yeast susceptibility phenotypes could greatly facilitate epidemiologic studies. Several antifungal agents, particularly antifungal azoles and flucytosine, characteristically show trailing, i.e., partial inhibition of yeast growth with increasing drug concentration, rather than sharp transitions from uninhibited to fully inhibited growth (12, 22). This phenomenon leads to diagonal slopes in dose-response curves, which permit the determination of relative susceptibilities to such agents by measurement of growth as a percentage of control growth at a single concentration of the antifungal agents (13). If that single concentration is well chosen, different yeast isolates can be compared in terms of their relative growth in the presence of that agent, with a * Corresponding author. Mailing address: Dept. of Bacteriology and Mycology, Janssen Research Foundation, B-2340 Beerse, Belgium. Phone: Fax: spread of relative growth distributions between 0 and 100% of the control value (13). Isolates showing low relative growth may be judged more susceptible to an agent in vitro than isolates showing high relative growth with that agent. Relative susceptibility measurements cannot be done with amphotericin B, because this agent yields sharp transitions from uninhibited to inhibited growth. Relative growth measurement permits the testing of large numbers of yeast isolates in an inexpensive manner. It also lends itself to a method of graphical visualization of the data, by means of two-dimensional scatterplots, that provides a simple and effective means for determining susceptibility phenotypes of different yeast species and strains and for stratifying these phenotypes by clinical and other variables. Correlations between susceptibilities to any two agents are easily visualized, and so too are group differences in susceptibility between species. This article describes relative growth determinations with itraconazole, fluconazole, ketoconazole, and flucytosine against a panel of clinical yeast isolates and illustrates the use of scatterplots for visualization and analysis of the results. MATERIALS AND METHODS Yeast isolates. The test material comprised a total of 811 yeast isolates representing the five most common Candida spp. isolated from clinical material. The yeasts were all identified by standard morphologic and physiologic criteria. The isolates represented single anatomical sites and patients; i.e., each site in each patient is represented only once among the isolates studied, except in instances in which more than one species was grown from the same site, when both species were included. Most of the yeast isolates sent to this laboratory between 1991 and 1994 were routinely tested within 6 weeks of receipt for relative growth with single concentrations of fluconazole, itraconazole, ketoconazole, and flucytosine. Yeast isolates received before 1991 were obtained from our culture collection, and these were used initially for establishing the methodology. The isolates tested were not obtained systematically or prospectively except for a group of 76 isolates (including 49 C. albicans isolates) that were recovered prospectively from AIDS patients whose oropharyngeal Candida infections had 588

2 VOL. 40, 1996 TESTING OF CANDIDA SUSCEPTIBILITIES BY RELATIVE GROWTH 589 FIG. 1. Relative growth of 504 C. albicans isolates in the presence of pairs of antifungal agents at single concentrations. (a) Fluconazole, 15 g/ml (y axis), versus flucytosine, 12.5 g/ml (x axis). (b) Fluconazole, 15 g/ml (y axis), versus ketoconazole, 0.53 g/ml (x axis). (c) Ketoconazole, 0.53 g/ml (y axis), versus itraconazole, 0.71 g/ml (x axis). not responded clinically to 7 days or more of treatment with fluconazole at 400 mg/day. The 811 yeast isolates comprised 504 isolates of C. albicans, 112 of Candida glabrata, 93 of Candida krusei, 65 of Candida parapsilosis, and 37 of Candida tropicalis. The mouth was the most common anatomical site of isolation (n 409), followed by the vagina (n 138) and other superficial sites (n 82). For 181 isolates the anatomical site of isolation was unknown. Among the 811 yeast isolates, 255 were obtained from patients with HIV infection and 435 were from patients without HIV infection. The HIV statuses of the patients for the remaining 120 isolates were unknown. The yeasts were obtained from many different countries, but the majority were isolated in Germany, Belgium, France, the United Kingdom, and the United States. Preparation, inoculation, and reading of test plates. Microdilution plates containing single concentrations of test substances were prepared as described previously (13). The four agents and final concentrations tested were as follows: fluconazole (Pfizer UK, Sandwich, United Kingdom), 15 g/ml ( M); flucytosine (Roche, Basel, Switzerland), 12.5 g/ml ( M); itraconazole (Janssen Research Foundation), 0.71 g/ml ( M); and ketoconazole (Janssen Research Foundation), 0.53 g/ml ( M). These concentrations were chosen on the basis of pilot experiments which showed they gave the widest distribution of relative growth data for C. albicans isolates among several concentrations tested. The methods for preparation of stock solutions and growth medium (casein digest-yeast extract-glucose) have been detailed before (13). Yeast inocula were prepared in 5-ml volumes of 10-fold-diluted casein digestyeast extract-glucose broth (13) incubated at 30 C for 18 to 24 h with rotation at 20 rpm at an angle of 5 from the horizontal (13). This procedure yielded suspensions containing yeast cells per ml without the need for further standardization (14, 16). The suspensions were diluted 400-fold in sterile water, and 200- l volumes were added to the wells of the test plates. The plates were sealed with transparent adhesive stickers and incubated for 48 h at 37 C (13). Growth was measured by reading the optical densities (ODs) of the cultures at 405 nm, and the ODs of drug-containing cultures were expressed as percentages of the mean control OD for each isolate tested (13). All of the test isolates gave control OD readings of greater than Statistical methods. Two-dimensional scatterplots of the percent relative growth data were generated with the aid of the StatView computer program. To determine the regression line for C. albicans scatterplots of fluconazole versus itraconazole, an orthogonal least-squares regression line (10) was fitted to the data for isolates of this species received prior to One manifestly outlying point was discarded from the calculation. The slope and intercept of the regression line were not significantly different from 1 and 0, respectively. A line perpendicular to the regression line was constructed from these data, and the datum points were projected upon it. The projections can be regarded as being proportional to the difference between the percent relative growths in fluconazole and itraconazole. Parallel 95% confidence bands around the regression lines were determined at twice the standard deviation (the standard deviation was 6.12) of the projections (10). The diagram was produced by the SAS computer program JMP. Rectangular contour plots indicating frequency densities in the scatterplots were obtained by the SAS procedure GCONTOUR (27). FIG. 2. Analysis of relative growth scatterplots for isolates of C. albicans. (a) Scatterplot for 59 isolates received before 1991, with regression line and confidence limits at twice the standard deviation. (b) Scatterplot for 504 isolates superimposed on the pre-1991 regression line and confidence limits. (c) Contour plot of the point density from the relative growth data for 504 C. albicans isolates. The outside contour boundary contains a density of two points per unit area, and each successive contour line demarcates a region of increasing point density on a doubling scale (4, 8, and 16 points per unit, and so on). The point density at the peak of region I is 128 per unit, and that at region II is 64 per unit. Two regions of lower point density (4 per unit) appear as broad clusters at the top right of the correlation line (region III) and away from the correlation line (region IV).

3 590 ODDS ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Scatterplots of percent relative growth with fluconazole versus itraconazole for six yeast isolates in replicate experiments. Each datum point represents one experiment. E, C. albicans 6F2C (n 54);, C. albicans B59630 (n 60)., C. albicans B63195 (n 64); {, C. krusei B68404 (n 7); Ç, C. glabrata 4046 (n 24); ç, C. albicans JDE (n 26). RESULTS Scatterplots of relative growth. Two-dimensional scatterplots of percent relative growth data for 504 isolates of C. albicans were generated for pairs of antifungal agents. The scatterplots given as examples in Fig. 1 are for fluconazole versus flucytosine (Fig. 1a), fluconazole versus ketoconazole (Fig. 1b), and ketoconazole versus itraconazole (Fig. 1c). Each point on these plots represents the susceptibility phenotype of a single yeast isolate for the two agents concerned, under the conditions of the test. The results indicated only small correlations between relative growths with flucytosine and fluconazole (r 0.11), flucytosine and itraconazole (r 0.12), and flucytosine and ketoconazole (r 0.12); some association between relative growths with fluconazole and ketoconazole (r 0.65) or fluconazole and itraconazole (r 0.66); and a high level of correlation between relative growth data for ketoconazole and itraconazole (r 0.91). Scatterplots for fluconazole versus itraconazole were used to study further the nature of the correlation between relative growth data for these two triazole antifungal agents. For isolates received in the laboratory prior to 1991 and maintained in our stock collection, a strong correlation was found (r 0.90), and only a single isolate gave a point outside the confidence intervals established at twice the standard deviation around the regression line (Fig. 2a). This isolate was strain AD, which was previously described as azole resistant (25). When data for all 504 C. albicans isolates for relative growth in fluconazole versus itraconazole were plotted, 50 isolates gave points that lay outside the original correlation confidence limits (Fig. 2b). A contour plot (Fig. 2c) showing the densities of the points per unit area in the C. albicans scatterplot indicated a major region (region I) of high point density coinciding with the correlation line at the bottom left of the scatterplot and three other clusters of point concentrations (II, III, and IV). Points for the majority of the C. albicans isolates tested fell in region I, with relative growth below 50% of control growth for both fluconazole and itraconazole, which indicated putative cross-susceptibility to both agents. Isolates giving points around region II were also cross-susceptible to both agents under the conditions of the test, but, with relative growth in the approximate range of 50 to 75%, these isolates showed susceptibilities to fluconazole and itraconazole lower than those of isolates with points in region I. Isolates with points in region III were the least susceptible of all the isolates tested to both agents. Their relative growth was generally 80% of control growth or greater with both itraconazole and fluconazole. Isolates whose scatterplot points fell well outside the general trend of correlation were found entirely to the left in Fig. 2c, and their greatest density is indicated by region IV. These isolates grew to a higher percentage of the drug-free control OD in the presence of fluconazole (15 g/ml) than in itraconazole (0.71 g/ml), suggesting a differential susceptibility in favor of itraconazole. Two of the C. albicans isolates tested gave scatterplot points that fell just outside the right boundary of the confidence limits established with the earliest isolates tested (Fig. 2b). Reproducibility of relative growth data. To demonstrate the level of reproducibility obtainable with relative growth scatterplots, data from replicate tests with fluconazole versus itraconazole and six isolates of Candida spp. are shown in Fig. 3. The results, which in three cases were from more than 50 repeated tests with the same isolates in the course of 4 years, suggest a generally fair level of reproducibility of the positions of points on the scatterplot. Five of the six yeast isolates illustrated in Fig. 3 showed a relative growth variation range of less than 30% on the scatterplot for fluconazole versus itraconazole; only C. albicans 6F2C exceeded this level of variation.

4 VOL. 40, 1996 TESTING OF CANDIDA SUSCEPTIBILITIES BY RELATIVE GROWTH 591 Downloaded from FIG. 4. Scatterplots of percent relative growth with fluconazole and itraconazole for C. albicans isolates received before 1991 (n 59) (a), in 1991 and 1992 (n 155) (b), and in 1993 and 1994 (n 289) (c and d). In each plot the points are shown as closed circles for isolates from HIV-positive patients and as open circles for isolates from patients without HIV infection and with unknown HIV status. The scatterplot in panel c excludes results for 49 isolates collected prospectively from patients whose oropharyngeal Candida infections did not respond clinically within 7 days of treatment with fluconazole (400 mg/day). Changes in the C. albicans scatterplots for fluconazole versus itraconazole with time. Figure 4 illustrates changes in distributions of scatterplot points for C. albicans isolates stratified by patient HIV status and by year of receipt in the laboratory. The results indicate an increase in the prevalence of C. albicans phenotypes with differential fluconazole-itraconazole susceptibilities among the most recently received isolates. Among the 59 isolates received prior to 1991 (Fig. 4a), only 1 (1.7%) was an outlier from the regression line confidence limits already established for these isolates (Fig. 2a). Among these 59 isolates, 16 were from HIV-positive patients. Among 155 isolates received in the calendar years 1991 and 1992 (Fig. 4b), 59 (38%) were from HIV-positive individuals, and 6 (3.9%) of the isolates (4 from patients with HIV infection) were outliers to the left of the confidence intervals established in Fig. 2, with a single outlier (from an HIV-negative patient) to the right of those confidence intervals. The group of 289 C. albicans isolates received in the laboratory in 1993 and 1994 was biased by the inclusion of 49 isolates that were isolated prospectively from AIDS patients with oropharyngeal Candida infections resistant to treatment with fluconazole (400 mg/day). The scatterplot in Fig. 4c shows the data for the 240 isolates remaining after the exclusion of this selected group. Sixty-three of them (26%) were from HIVpositive patients, and a total of 17 isolates (14 from HIVinfected patients) were definite outliers from the correlation confidence limits (Fig. 2). In Fig. 4d, results for the 49 isolates from patients with Candida infections resistant to fluconazole treatment are added to the scatterplot for isolates received in 1993 and 1994, raising the number of points outside the correlation zone to 43 (40 from patients with HIV infection). A single isolate received in 1993 or 1994 was an outlier to the right of the correlation confidence limits. Relative growth of other Candida spp. with fluconazole and itraconazole. The scatterplots in Fig. 5 show relative growth data for isolates of Candida species other than C. albicans superimposed on a background of the scatterplot for 504 C. albicans isolates (Fig. 2b). Isolates of C. glabrata (n 112) on January 12, 2019 by guest

5 592 ODDS ET AL. ANTIMICROB. AGENTS CHEMOTHER. Downloaded from FIG. 5. Scatterplots of percent relative growth with fluconazole versus itraconazole for 112 isolates of C. glabrata (a), 93 isolates of C. krusei (b), 65 isolates of C. parapsilosis (c), and 37 isolates of C. tropicalis (d). The small grey points indicate the scatterplot for 504 C. albicans isolates (taken from Fig. 2b). (Fig. 5a) showed an association (r 0.83) between the relative growths with fluconazole and itraconazole, although the apparent correlation curve did not correspond with that for the C. albicans isolates. All of 93 C. krusei isolates were outliers well to the left of the C. albicans correlation line, suggesting a differentially high susceptibility to itraconazole compared with fluconazole. Most of 65 C. parapsilosis isolates gave relative growth points well to the bottom left of the scatterplot for fluconazole versus itraconazole (Fig. 5c), although 3 isolates appeared to be differentially susceptible outliers and 1 isolate lay in region II of the C. albicans scatterplot (Fig. 2c). All except one of the 37 C. tropicalis isolates tested (Fig. 5d) gave points within the C. albicans correlation confidence zone. The relative growth point for a single C. tropicalis isolate lay close to region III on the C. albicans scatterplot (Fig. 2c). Scatterplots for C. albicans isolates from patients for whom the outcome of fluconazole or itraconazole treatment was known. For 90 oral C. albicans isolates received from HIVpositive patients with oropharyngeal Candida infection, the clinical status of the patient and the outcome of any antifungal treatment given within 7 days before or after isolation was known. For 19 of these patients, no prior azole antifungal treatment at all had been given; a further 7 patients had been treated with itraconazole (100 mg/day); and the remaining 64 patients had received fluconazole. Of the last group, 4 patients received 50 mg/day, 6 patients received 100 mg/day, 40 patients received 400 mg/day, and for 5 patients the fluconazole dosage was not recorded. Figure 6 shows a scatterplot of the percent relative growth with fluconazole versus itraconazole for these isolates, stratified by antifungal agent and treatment outcome. By comparison with Fig. 2c it is evident that the points for most of the isolates from patients who responded to treatment with fluconazole or itraconazole appeared in the scatterplot in contour regions I and II, while the points for most of the isolates from patients who failed to respond to treatment appeared in regions III and IV. Indeed, only 2 isolates (3.6%) from the group of 36 patients who either had no prior exposure to azole antifungal treatment or responded clinically to fluconazole or itraconazole treatment were outliers from the correlation confidence limits (Fig. 2b), compared with 22 isolates (40.7%) from the group of 54 patients who failed to respond clinically to azole therapy. on January 12, 2019 by guest

6 VOL. 40, 1996 TESTING OF CANDIDA SUSCEPTIBILITIES BY RELATIVE GROWTH 593 FIG. 6. Scatterplot of percent relative growth with fluconazole versus itraconazole for 19 HIV-positive patients with no prior exposure to azole antifungal agents (å), 13 patients who responded clinically to fluconazole treatment (F), 4 patients who responded to itraconazole treatment ( ), 51 patients who failed to respond to fluconazole treatment (E), and 3 patients who failed to respond to itraconazole treatment ( ). DISCUSSION The measurement of relative growth at single concentrations of antifungal agents as a percentage of the control value is not intended as a substitute for MIC determination. For any individual isolate there is no clear cutoff point in a relative growth determination that can be used as a susceptibility breakpoint. However, for large-scale epidemiologic surveys the approach offers a cost-effective method for the detection of clusters of isolates with similar susceptibilities, and the visualization of results in the form of two-dimensional scatterplots facilitates analysis of large numbers of data. Figure 6 confirms that the positions for isolates on the scatterplots are related to clinical responses of patients to treatment but that there are nevertheless regions of overlap between points from treatment successes and failures as well as a few maverick isolates whose points are located in the wrong zone of the scatterplot. Similar overlaps and maverick points are also often found in susceptibility studies for clinical correlations based on conventional MIC testing (29). The reproducibility of the relative growth test system was good (Fig. 3), as has been previously demonstrated (13), although one of six yeast isolates tested repeatedly showed particularly high replicate variation over the course of 4 years of repeated testing. Similarly susceptibility-variable isolates have also been observed in MIC tests (6). Since most yeast isolates seem to give fair replicate variability in relative growth tests, the possibility that the variation seen with some isolates is a strain-dependent phenomenon, perhaps associated with phenotypic switching (28), is worthy of further investigation by testing of individual colonies obtained from such strains. The method described in this paper offers a particular advantage when it is desired to test susceptibilities of multiple colonies from individual yeast isolations, something that presents serious logistical difficulties with conventional MIC determinations if it is to be done on a large scale. In the present study it was self-evident that little or no correlation existed between susceptibilities of C. albicans isolates to flucytosine and azole antifungal agents (r 0.11 to 0.12). No such correlation has ever been noted before in studies based on MIC determinations, nor is any association predictable, since the two agents have entirely different mechanisms of action (31). On the other hand, the strong correlations seen between the relative growth data obtained with fluconazole, ketoconazole, and itraconazole confirm the common mechanism of action of these imidazole and triazole compounds (31). Comparisons between fluconazole and itraconazole have been made in previous MIC-based studies of yeast susceptibilities to antifungal agents, and the occurrence of C. albicans isolates, particularly from HIV-positive patients, with differentially high fluconazole MICs and low itraconazole MICs has been reported (2). It is therefore noteworthy that 50 of 504 C. albicans isolates (9.9%) in the present study showed a similar tendency to have differential relative growths with fluconazole and itraconazole, appearing as outliers with respect to the correlation line and confidence limits established with the earliest batch of such isolates. The points for 2 of the 504 isolates fell outside these limits in the direction of higher susceptibility to fluconazole than to itraconazole, but their distance from the correlation zone was far less than those of many of the 50 outliers in the opposite direction (Fig. 2b). For 89.7% of the C. albicans isolates, relative cross-susceptibility (and relative cross-resistance) to fluconazole and itraconazole was evident from the positions of their points within the correlation con-

7 594 ODDS ET AL. ANTIMICROB. AGENTS CHEMOTHER. fidence bounds (Fig. 2b). The scatterplot presentation of relative growth data for fluconazole and itraconazole suggested a growing incidence of C. albicans isolates with differential fluconazole-itraconazole susceptibilities, since more outlying points were seen with the most recent isolates (1993 and 1994) than with the earliest isolates (pre-1991). The scatterplot presentation of relative growth data facilitates visualization of relative yeast susceptibilities by stratification with respect to species (Fig. 5), clinical outcome (Fig. 6), and patient characteristics such as the presence and absence of underlying HIV infection (Fig. 4). The species-specific nonsusceptibility of C. krusei isolates to fluconazole is already well described (14, 23, 32), and the normally high susceptibility of C. parapsilosis isolates to both drugs has also been recorded (30), so the relative growth data, at least for these species, match their known susceptibilities in vitro. Relative growth determinations were originally designed as a means of phenotypic typing of Candida isolates by the positions of results for colonies on two-dimensional scatterplots. This study has indicated that clinically and epidemiologically relevant information on yeast susceptibilities may also be derived from these tests. We continue to measure relative growth prospectively on all yeast isolates we receive and to select isolates with unusual scatterplot positions for typing at the DNA level. 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