In vitro susceptibility of clinical yeast isolates to fluconazole and terconazole

In vitro susceptibility of clinical yeast isolates to fluconazole and terconazole Chester R. Cooper, Jr., PhD, and Michael R. MeGinnis, PhD Galveston, Texas OBJECTIVE: Fifty clinical yeast isolates, representing equally Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, and Torulopsis glabrata, were tested in vitro for their susceptibility to terconazole and fluconazole. STUDY DESIGN: The minimal inhibitory concentrations of terconazole and fluconazole were determined by use of a proposed standardized broth macrodilution assay. Also, the response of selected yeast isolates to 25 I~g of either drug was measured by agarose disk diffusion experiments. RESULTS: For all species the minimum inhibitory concentrations for terconazole were significantly lower than those for fluconazole (/3 < 0.05). In fact, for each individual isolate the minimum inhibitory concentration of terconazole was consistently lower than that of fluconazole. Differences in the geometric mean of terconazole and fluconazole minimum inhibitory concentrations were largest among C. krusei and -F. glabrata, followed by C. parapsilosis, C. tropicalis, and C. albicans, in order of decreasing difference. Disk diffusion experiments suggested that terconazole is a more effective fungistatic agent than fluconazole is. CONCLUSION: Terconazole may be more effective than fluconazole against yeast species other than C. albicans. (Am J Obstet Gynecol 1996;175:1626-31 .)

Key words: Fluconazole, susceptibility testing, terconazole, vulvovaginitis

The triazole antifungal agents terconazole and fluconazole are highly effective against yeasts capable of causing vulvovaginal candidiasis. These agents act against the ergosterol biosynthetic pathway of yeasts by inhibiting the cytochrome P 4 5 0 - d e p e n d e n t 14-c~-demethylation of lanosterol. 1'2 These triazoles are more active than the imidazole antifungal agents because they are highly selective for the yeast cytochrome P450 rather than for mammalian microsomal cytochrome P450. In the treatment of vulvovaginal candidiasis the triazoles generally show greater efficacy, shorter treatment regimens, lower relapse rates, and better mycologic and clinical cure rates in comparison with other antifungal agents, s-6 Published studies have suggested that fluconazole is effective in the treatment of vulvovaginal candidiasis. 7' s However, the widespread use of fluconazole, especially in patients with acquired immunodeficiency syndrome, has resulted in the emergence of resistant isolates of Candida albicans. 9-12 Prophylactic or excessive use of fluconazole From the Medical Mycology Research Center, University of Texas Medical Branch. Support in part by an educational grant from Ortho-McNeil Pharmaceutical, Raritan, New Jersey. Receivedfor publication March 19, 1996; revisedJuly 3, 1996; accepted July 18, 1996. Reprint requests: Chester 1L Cooper, Jr., PhD, Medical Mycology Research Center, Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0609. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00 + 0 6/1/76699

may also give rise to infections by species of yeasts other than C. albicans. 12-a4 The emergence of antifnngal resistance may be of particular significance in choosing a therapy for vulvovaginal candidiasis because a wider spectrum o f yeasts in addition to C. albicans is increasingly associated with vulvovaginitis?5 A tool that can detect potential for resistance is in vitro susceptibility testing. Often these tests are predictive of the clinical responses of yeasts to antifungal agents. 9' 10,16,17 In addition to an agarose disk diffusion assay, a standardized method for in vitro antifungal agent susceptibility testing was used to study the effectiveness of terconazole and fluconazole against several species of yeasts that are major etiologic agents of vulvovaginitis. This method may have some applicability for assessing the potential occurrences of resistance to antifungal agents. Material and methods

Antifungal agents. Terconazole powder was obtained from the R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey. Fluconazole powder was obtained from Roerig-Pfizer, Groton, Connecticut. Both drugs and the solutions containing them were stored at - 2 0 ° C until needed. Media and reagents. A single lot of RPMI 1640 medium used in the broth macrodilution assay was obtained from Life Technologies (Gaithersburg, Md.) and prepared as previously described. TM For the agarose disk diffusion assays, HR medium (Oxoid, Basingstoke, 1626

Volume 175, Number 6 AmJ Obstet Gynecol

United Kingdom) was generously provided by the Fungus Testing Laboratory, University of Texas Health Science Center (San Antonio). Sabouraud glucose broth (Difco, Detroit) and Bacto-agar (Difco) were obtained from commercial sources. Agarose (SeaKem LE) was purchased from FMC Bioproducts (Rockland, Me.). All other chemical reagents were purchased from Amresco (Solon, Ohio). Yeast isolates. Yeast strains used in this study consisted of 10 isolates each of C. albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, and Torulopsis glabrata. All were obtained from patients with candidiasis of some form, including systemic, dermatologic, and vaginal infections. Yeast strains C. krusei ATCC 6258 and C. parapsilosis.ATCC 22019 were used as internal controls for each assay. Response ranges for these strains to fluconazole have been previously established for the macrobroth dilution susceptibility assay. 19 Broth macrodilution susceptibility testing. The minim u m inhibitory c o n c e n tr a ti o n of fluconazole or terconazole for each yeast isolate was determined with a standardized susceptibility testing method, is with the following modifications. Stock solutions of both terconazole and fluconazole were prepared in dimethylsulfoxide at a concentration of 12,800 txg/ml. All stock solutions were stored at - 2 0 ° C until needed. Serial dilutions of the drugs in RPMI 1640 were prepared so that after addition of inoculum the final concentration of dimethyl sulfoxide in each tube was 1% (vol/vol). Growth controls consisted of dimethyl sulfoxide-supplemented medium with neither fluconazole nor terconazole. Results were validated with use of yeast strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 as internal controls, for which response ranges to fluconazole have been previously established. 19 I n accord with the published standard, is the minimum inhibitory concentration was defined as the lowest concentration of either fluconazole or terconazole that substantially inhibited growth of a yeast isolate as detected by visual comparison with the amount of growth in the assay tubes containing no drug. In Cases of trailing end points (i.e., significantly decreased yet persistent turbidity), the minimum inhibitory concentration was estimated as the lowest drug concentration producing ->80% growth inhibition. Agarose diffusion assay. Susceptibilities of selected yeast strains to a single concentration of either terconazole or fluconazole were determined with an agarose disk diffusion assay similar to that previously described. 2° Briefly, a 2.0% weight/volume ratio mixture of agarose in 0.1 m o l / L phosphate buffer (pH 7.5) was sterilized by autoclaving at 115 ° C for 10 minutes and then cooled to 56 ° C in a water bath. A double-strength solution of HR medium was filter sterilized after dissolution of 14.65 gm of HR powder and 1.0 gm of sodium bicarbonate in 500 ml of distilled water. Inoculum for this assay was pre-

Cooper and MeGinnis

1627

pared by suspending five or more isolated colonies (->1 m m diameter) of a yeast strain, grown on a Sabouraud glucose agar plate incubated at 35 ° C for 18 to 24 hours, in sterile saline solution (0.85% wt/vol)i The concentration of cells, determined with a Neubauer-ruled counting chamber, was adjusted to 4.2 × 107 cells/ml. Inoculum (100 pA) was added to 7.0 ml of the double-strength HR medium prewarmed to 38 ° C. Next, 7.0 ml of buffered agarose was added and the entire mixture was mixed with a vortex. Immediately, 12.0 ml of this mixture was pipetted into a 100 × 15 m m sterile plastic petri dish and allowed to solidify at room temperature. A single sterile p ap er filter disk (6 m m diameter, Schleicher and Schuell, Keene, N.H.) impregnated with 15 Ixl of dimethyl sulfoxide containing 25 p,g of either fluconazole or terconazole Was then placed in the center of each plate. The plates were incubated at 28 ° C. At 24 and 48 hours of incubation, the diameter of the zone of growth inhibition produced by a drug was taken as the average of two measurements made at right angles. Measurements were made to the nearest 0.1 mm with a metric caliper. Results for the response of a particular yeast strain were determined from the average of three assay plates prepared for each drug. Statistical analyses. Statistical analyses were performed as described by Wardlaw. 21 The sign test was used to compare differences between the minimum inhibitory concentrations for terconazole and fluconazole for a given yeast species~ Statistical differences in the response of selected yeast isolates to fluconazole and terconazole, as measured by the zones of inhibition produced in agarose diffusion assays, were determined with the Student t test.

Results Table I gives the range and geometric mean minimum inhibitory concentrations for the 10 isolates of each yeast species tested with the broth macrodilution assay. For all species the minimum inhibitory concentrations of terconazole were significantly lower than those of fluconazole on the basis of the sign test (p < 0.05). In fact, for each individual isolate the minimum inhibitory concentration of terconazole was consistently lower than the corresponding one for fluconazole (data not shown). The differences in the geometric mean of minimum inhibitory concentrations of terconazole and fluconazole were largest among C. krusei and T. glabrata isolates, followed by C. parapsilosis, C. tropicalis, and C. albicans, in order of decreasing difference. The minimum inhibitory concentration for the control isolates C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 to fluconazole were always 64 ~ g / m l and 2.0 ~ g / m l , respectively. These values are within the range previously established for these strains, I9 thereby validating the assay results. One interesting aspect of this assay is that many yeast

1628

Cooper and McGinnis

December 1996 AmJ Obstet GynecoI

Fig. 1. Growth response of C. kruseiATCC 6258 to 25 Ixg offluconazole (left) or terconazole (right) after 48 hours of incubation at 28° C. T a b l e I. Range and geometric mean of m i n i m u m inhibitory concentrations of 50 clinical yeast isolates, 10 from each species, and internal control strains to fluconazole and terconazole as determined by standard broth macrodilution assay MIC (~g/ml) Terconazole

Fluconazole Yeast species

Range

Geometric mean

Range

Geometric mean

C. albicans C. krusei Control (ATCC 6258) C. parapsilosis Control (ATCC 22019) C. tropicalis 7". glabrata

0.25-1.0 64->128 64 0.5-32 2.0 0.25-32 4.0->128

0.35 78.8 64.0 1.4 2.0 0.71 19.7

--<0.125-0.5 1.0-4.0 0,5-1.0 0.125-0.5 --<0.125 -<0.125-1.0 0.25-4.0

0.14 1.5 0.79 0.18 -<0.125 0.16 0.93

MIC, Minimum inhibitory concentration.

isolates exhibited a trailing end point (i.e., persistent yet slight turbidity) indicating the growth of the isolate beyond the estimated m i n i m u m inhibitory concentration. In the current study all C. albicans and C. tropicalis isolates exhibited trailing end points up to and including those assay tubes containing 128 btg/ml fluconazole. In contrast, the same C. albicans isolates and 7 of the 10 C. tropicalis isolates did not exhibit trailing end points in any assay tubes containing ->16 b~g/ml terconazole. This p h e n o m e n o n was not observed for isolates of T. glabrata or C. parapsilosis with either antifungal agent. Only three isolates of C. krusei exhibited trailing end points in terconazole-containing assay tubes. Interestingly, these same C. krusei isolates all possessed the highest m i n i m u m inhibitory concentration of terconazole among those tested (4.0 ~g/ml; data not shown). These observations strongly suggest that fluconazole is

less fungistatic than terconazole. Supporting data for this suggestion were obtained from agarose disk diffusion assays in which the growth response of selected yeast isolates was measured against a single concentration of either fluconazole or terconazole. The isolates chosen represent a range of m i n i m u m inhibitory concentration responses within a given species. Typical responses are shown in Fig. 1 and the results for selected isolates are given in Table II. For each isolate assayed the average zone of inhibition produced by terconazole was statistically greater in size than that produced by fluconazole (p < 0.05). For isolates of C. albicans and C. krusei the zone of inhibition produced by fluconazole disappeared after 48 hours, whereas the zones produced by terconazole in these same isolates persisted -->2 weeks, albeit slightly smaller in size than those observed after 24 hours. For isolates of C. parapsilosis and C. tropicalis both

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Volume 175, Number 6 Am J Obstet Gynecol

T a b l e II. Average size o f growth i n h i b i t i o n z o n e s in r e s p o n s e to 25 ~g o f f l u c o n a z o l e a n d t e r c o n a z o l e as m e a s u r e d by agarose disk diffusion assay

MIC determined by macrobroth assay (p~g/ml)

Average zone size* Fluconazole

Yeast species and isolate

Terconazole

24 hr

48 hr

24 hr

48 hr

20.2 + 0.7 24.6 ± 1.0 20.7 ± 1.4

0 0 0

25.8 _+ 1.0 28.8 _+ 0.1 23.8 _+ 0.6

15.4 ± 0.2 17.2 -+ 1.3 17.7 ± 1.1

4.0 1.0 4.0 0.5

8.9 15.0 12.6 12.5

_+ 0.2 -+ 3.1 + 2.6 -- 0.6

0 0 0 0

31.5 42.9 41.1 42.0

-+ 1.0 ± 0.6 _+ 0.9 -+ 1.3

30.7 42.5 34.7 40.6

± 0.3 ± 0.0 -+ 0.2 -+ 1.4

32 1.0 1.0 2.0

0.5 0.125 -<0.125 --<0.125

26.3 42.6 46.7 38.4

± 2.7 + 0.3 _+ 0.5 _+ 0.5

_+ 5.7 _+ 1.3 _+ 0.3 ± 1.4

49.5 49.1 52.6 46.7

-+ 0.5 ± 0.6 -+ 0.3 -+ 1.1

49.5 51.3 53.1 44.6

± ± + ±

32 2.0 0.5

1.0 0.25 -<0.125

29.9 + 0.4 30.4 _+ 0.5 25.3 _+ 0.2

Fluconazole

Terconazole

C. albicans 685-94 701-94 704-94

1.0 1.0 0.25

--<0.125 0.5 -<0.125

C. krusei 418-91 717-91 699-91 ATCC 6258

128 64 >128 64

C. parapsilosis 549-94 649-94 Ev-2 ATCC 22019

15.5 43.1 47.1 32.8

1.2 1.0 6.3 0.4

C. tropicalis 2149 2253 Ja-1

23.7 + 0.1 28.8 _+ 0.7 24.22 1.4

47.2 _+ 0.3 42.1 _+ 0.6 34.7 ± 0.1

45.7 -+ 0.2 41.9 -+ 0.9 35.3 -+ 1.7

T. glabratat 240-0921 Hi-7 Sm-8

16 4.0 >128

0.5 0.25 4.0

25.9 -+ 1.3 33.0 _+ 1.8 0

48.5 -+ 0.5 36.9 -+ 0.7 38.2 ± 0.6

MIC, Minimum inhibitory concentration. * Diameter in millimeters. J- Zone-of-inhibition measurements were taken at 72 hours for these isolates.

t h e fluconazole- a n d t e r c o n a z o l e - p r o d u c e d z o n e s o f in-

recalcitrant to t r e a t m e n t . Such testing m u s t b e r e p r o d u c -

h i b i t i o n r e m a i n e d for > 4 8 hours. Isolates o f 7". glabrata,

ible a n d lead to satisfactory in v i t r o - i n vivo correlations.

however, grew m u c h m o r e slowly in the solidified H R

T h e National C o m m i t t e e o f Clinical L a b o r a t o r y Stan-

m e d i u m u s e d in this assay. Z o n e s o f i n h i b i t i o n c o u l d only

d a r d s has recently p r o p o s e d a m a c r o b r o t h dilution

b e a d e q u a t e l y m e a s u r e d n o earlier t h a n 72 h o u r s after

m e t h o d for testing yeasts against azoles a n d o t h e r anti-

inoculation. O n e o f the t h r e e isolates o f T. glabrata

fungal agents, la Evidence is a c c u m u l a t i n g t h a t shows t h a t

s h o w e d n o growth i n h i b i t i o n to f l u c o n a z o l e at this time,

m i n i m u m inhibitory c o n c e n t r a t i o n s o b t a i n e d with this

w h e r e a s all t h r e e isolates were i n h i b i t e d by terconazole.

m e t h o d can b e c o r r e l a t e d to clinical r e s p o n s e . 16 A m o r e

Finally, for all isolates o f e a c h species tested, n o g e n e r a l

r e c e n t study r e s u l t e d in C. parapsilosis ATCC 22019 a n d

correlation of broth macrodilution generated minimum

C. krusei ATCC 6258 b e i n g d e s i g n a t e d as the quality

i n h i b i t o r y c o n c e n t r a t i o n s a n d zone-of-inhibition sizes

c o n t r o l isolates w h e n testing a m p h o t e r i c i n B, flucytosine, a n d fluconazole. 19 T h e s e same strains were u s e d in the

was a p p a r e n t .

Comment

study r e p o r t e d h e r e . By use o f a similar m e t h o d Lynch a n d Sobe122 f o u n d

In vitro resistance o f isolates to t e r c o n a z o l e o r flucon-

that the m i n i m u m inhibitory c o n c e n t r a t i o n s for flucon-

azole f r o m cases o f vaginitis has yet to b e r e p o r t e d .

azole were h i g h e r t h a n those for t e r c o n a z o l e w h e n they

However, a c o n c e r n d o e s exist r e g a r d i n g w h e t h e r the use

tested a n u m b e r o f yeast isolates r e c o v e r e d f r o m p a t i e n t s

o f f l u c o n a z o l e m i g h t result i n resistance such as t h a t o c c u r r i n g in i m m u n o c o m p r o m i s e d patients. 9-14 Given

with Candida vaginitis. Isolates o f C. parapsilosis, C. tropi-

the w i d e r s p e c t r u m o f yeasts o t h e r t h a n C. albicans n o w

tially h i g h e r m i n i m u m inhibitory c o n c e n t r a t i o n s o f flu-

associated with vulvovaginitis, ~5 it is possible that o n e or

c o n a z o l e t h a n o f terconazole. We have o b s e r v e d a similar

m o r e species that are resistant to the antifungal a g e n t

r e s p o n s e (Table I) w h e n we tested f l u c o n a z o l e a n d

may b e c o m e m o r e p r o m i n e n t . O n e a p p r o a c h to assess-

t e r c o n a z o l e against C. parapsilosis,

calls, Saccharomyces cerevisiae, a n d T. glabrata h a d substan-

ing e m e r g i n g resistance is the use o f in vitro susceptibility

C. tropicalis, a n d T. glabrata with the p r o p o s e d s t a n d a r d i z e d m e t h o d . Simi-

testing, especially in cases o f vulvovaginitis t h a t are

larly, the average z o n e sizes for fluconazole a n d t e r c o n -

1630 Cooper and McGinnis

azole measured in an agarose diffusion assay (Table II) showed larger zone sizes for terconazole. However, caution must be exercised in interpreting agar disk diffusion assay data because this method tends to have poor interlaboratory agreement. 2° In using the minimtim inhibitory concentration data, care must be taken not to make clinical predictions solely on the basis of the m i n i m u m inhibitory concentration; tissue levels achieved by the agent should also be considered. In studies by Houang et al. 2~ and Dellenbach, 24 mean concentrations of fluconazole in plasma, vaginal tissues, and vaginal secretions were measured after oral administration of a single 150 mg dose. Specimens of vaginal secretions collected after 24 hours after drug administration contained a mean concentration of fluconazole ranging from approximately 1.19 to 2.93 Ixg/ml. After 72 hours apparently effective levels of fluconazole persisted in the secretions at yet lower levels (range 0.6 to 1.0 p~g/ml), in contrast, data provided from a 1989 clinical study shows that terconazole levels in vaginal fluids 24 hours after use of an 80 mg suppository for 3 days or a 0.4% topical cream for 7 days ranged from 500 to 750 ~ g / m l (R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ., 1989. Unpublished data.). Significant levels of terconazole ranging from 36 to 58 t~g/ml were reported to persist 72 hours after treatment. When these data are compared with the geometric mean minimum inhibitory concentrations for the yeast Strains reported here, fluconazole would be expected to be very effective against most isolates of C. albicans and C. tropicalis. Some yeast isolates, though, including some C. parapsilosis strains and all C. krusei and T. glabrata strains, would be expected to be resistant even to peak levels of fluconazole achieved 24 hours after treatment because these levels do not reach the minimum inhibitory concentration reported here for the organism. In contrast, terconazole would be expected to be very effective against all the yeast isolates examined in this study because the lowest 72-hour in vivo level is 24 to 36 times greater than the mean minimum inhibitory concentrations reported here and at least nine times greater than any of a given isolate. These suggestions are further supported by the results of the agarose disk diffusion assay, in which the zones of inhibition were larger and remained viable for a longer period with terconazole. One curious and general distinction between terconazole and fluconazole was provided by the response of C. albicans and C. tropicalis to these agents in the broth macrodilution assay. T h e extensive trailing end points exhibited by these species to fluconazole, c o m p a r e d with the response to terconazole, suggests that a portion of the inoculum population is affected less by. fluconazole than by terconazole. Complete fungistasis, then, would appear to require significantly higher levels of fluconazole (>-128 ~ g / m l ) beyond the esti-

December 1996 AmJ Obstet Gynecol

mated m i n i m u m inhibitory concentration for C. albicans and C. tropicalis. This seems not to be the case with terconazole, where i n f r e q u e n t trailing end points ( 3 / 2 0 isolates tested) a p p r o a c h e d concentrations at least eightfold lower than those noted above. Comparative in vivo responses of t h e s e two drugs might require increased dosage or prophylactic use of fluconazole, both of which are suspected in the emergence of resistance2 -14 In summary, our data indicate that terconazole may be more effective than fluconazole in treating infections caused by different yeast species. The limited use of fluconazole thus far for vulv0vaginal candidiasis has not resulted in published reports of resistance; however, collective data suggest that resistance to oral fluconazole may be a concern, z5 Although in vitro results do not necessarily predict in vivo activity, it is possible that terconazole may be more effective than fluconazole in treating vulvovaginal infections caused by yeast species other than C. albicans. Clinical studies are needed to correlate in vitro and in vivo results. REFERENCES

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