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Antimicrobial Agents and Chemotherapy, January 1998, p. 129-134, Vol. 42, No. 1
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Optimizing the Correlation between Results of Testing In Vitro
and Therapeutic Outcome In Vivo for Fluconazole by Testing Critical
Isolates in a Murine Model of Invasive Candidiasis
John H.
Rex,1,*
Page W.
Nelson,1
Victor L.
Paetznick,1
Mario
Lozano-Chiu,1
A.
Espinel-Ingroff,2 and
Elias J.
Anaissie3
Division of Infectious Diseases, Department
of Internal Medicine, Center for the Study of Emerging and Reemerging
Pathogens, University of Texas Medical School, Houston, Texas
770301;
Division of Infectious Diseases,
Medical College of Virginia-VCU, Richmond, Virginia
232983; and
Section of Oncologic
Emergencies, Division of Hematology/Oncology, University of
Arkansas for Medical Sciences, Little Rock, Arkansas
772052
Received 7 July 1997/Returned for modification 12 August
1997/Accepted 17 October 1997
 |
ABSTRACT |
The trailing growth phenomenon seen when determining the
susceptibilities of Candida isolates to the azole
antifungal agents makes consistent endpoint determination difficult,
and the M27-A method of the National Committee for Clinical Laboratory
Standards addresses this problem by requiring an 80% reduction in
growth after 48 h of incubation. For some isolates, however, minor
variations of this endpoint criterion can produce up to 128-fold
variations in the resulting MIC. To investigate the significance of
this effect, isolates of Candida that exhibited various
forms of trailing growth when tested against fluconazole were
identified. The isolates were examined in a murine model of invasive
candidiasis and were ranked by their relative response to fluconazole
by using both improvement in survival and reduction in fungal burden in
the kidney. The resulting rank order of in vivo response did not match the MICs obtained by using the M27-A criterion, and these MICs significantly overestimated the resistance of three of the six isolates
tested. However, if the MIC was determined after 24 h of
incubation and the endpoint required a less restrictive 50% reduction
in growth, MICs which better matched the in vivo response pattern could
be obtained. Minor variations in the M27-A endpoint criterion are thus
required to optimize the in vitro-in vivo correlation for isolates that
demonstrate significant trailing growth when tested against
fluconazole.
| |
INTRODUCTION |
The Subcommittee for Antifungal
Susceptibility Testing of the National Committee for Clinical
Laboratory Standards (NCCLS) has collaborated with many other
investigators to develop NCCLS document M27, entitled "Reference
Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts" (10). Now released in its approved level version
(10) as document M27-A, this document describes reproducible
macrodilution and microdilution methods for the susceptibility testing
of Candida species and Cryptococcus neoformans
against amphotericin B, flucytosine, ketoconazole, itraconazole, and
fluconazole. The most recent step in the method's evolution has been
the proposal of breakpoints for Candida when tested against
fluconazole, itraconazole, and flucytosine (10, 16). By
necessity, the primary goal of the M27 process was development of a
method with good interlaboratory reproducibility. Only with this
accomplished could the second, but actually more important, goal of
proving that the method has a good correlation with in vivo treatment
outcome fully proceed. In addition, the recent analysis of data sets
correlating the MICs obtained by the M27 method with the outcomes for
fluconazole and itraconazole (16) demonstrates that the
M27-A method produces results that are comparable in predictive power
to the results produced by antibacterial susceptibility testing
(12).
However, these successes do not preclude the possibility of further
improvements to the method. Like all susceptibility methods, the M27-A
method contains a number of features that significantly influence the
measured MIC. While the specific procedures used by the M27-A method
and their rationale have been reviewed (18), two features
are of particular interest with regard to this paper. First, the time
of reading of the results for Candida spp. was fixed at
48 h on the basis of an initial study of the macrodilution variant
of M27-A in which readings at 48 h improved interlaboratory reproducibility by ~20% over the reproducibility achieved by taking readings at 24 h for amphotericin B, flucytosine, and ketoconazole (9). Similar differences were also present but were somewhat less apparent in a parallel study that used both the macrodilution and
the microdilution methods for the testing of amphotericin B,
flucytosine, ketoconazole, and fluconazole (6). The second feature of the M27-A method that is of interest is the definition of
the endpoint for the azole antifungal agents. On the basis of data from
the two studies just mentioned (6, 9), the MICs of these
agents were defined as the lowest drug concentrations that produced
prominent reductions in growth. This endpoint, also known as MIC-2, was
used to handle the well-known trailing growth phenomenon seen with the
azole antifungal agents (18). The turbidity of this endpoint
has been shown to be less than or equal to the turbidity of a fivefold
dilution of the drug-free growth control from a macrodilution format
assay (6, 8), and this convenient 80% reduction rule was
rapidly incorporated into the M27-A method. This endpoint is obviously
arbitrary, and it has been suggested that a less restrictive 50%
reduction endpoint might be more relevant (1, 20). However,
proof of this assertion has been lacking.
In the context of these two features of the M27-A method, we observed
that several Candida isolates had anomalous behavior with
respect to the endpoint and time of reading when tested against fluconazole. The isolates shared a common behavior in that the MICs for
the isolates were relatively low at 24 h but were much higher
after 48 h, with the difference in MICs sometimes spanning seven
doubling dilutions. Strict application of the 80% growth reduction
endpoint rule also strongly influenced the MIC. Such extreme variations
in MICs would significantly affect the interpretation of the MIC and
suggested that at least one measurement was likely in error. While
approaches to selecting conditions which optimize in vitro-in vivo
correlations have been reviewed (16), the strong effect of
technical factors on the MICs for these isolates seemed to provide an
additional avenue for validating or refuting some of the features of
the M27-A method, and we now report on detailed studies with a selected
set of such isolates.
(This work was presented in part at the 34th Annual Meeting of the
Infectious Diseases Society of America, 1996 [15a].)
| |
MATERIALS AND METHODS |
Isolates.
Three isolates each of Candida albicans
and Candida tropicalis were studied (see Table 1). Isolate
UTR-14 was obtained from the oral cavity of a human immunodeficiency
virus-infected adult with oropharyngeal candidiasis (7),
while all of the other isolates were isolates from the bloodstream of
patients enrolled in a trial of therapy for candidemia (14).
The isolates were stored at 
70°C and were subcultured at least
twice on Sabouraud dextrose agar (Becton Dickinson Microbiology
Systems, Cockeysville, Md.) at 35°C prior to testing. The isolates
were identified to the species level by using the API 20C system
(Analytab Products, Plainview, N.Y.).
Susceptibility testing.
Susceptibility testing was performed
by four different methods. First, the isolates were tested by using
both the macrodilution and the microdilution versions of the NCCLS
M27-A method in 0.165 M MOPS (morpholinepropanesulfonic acid)-buffered
(pH 7) RPMI 1640 medium (10). In addition, the isolates were
tested by using the macrodilution and microdilution methods of the
M27-A method, but in 0.165 M MOPS-buffered (pH 7) RPMI 1640 medium
supplemented with 20 g of D-glucose per liter, as
suggested by Rodriguez-Tudela and Martinez-Suarez (19). The
isolates were tested against fluconazole (supplied by Pfizer
Pharmaceuticals) at serial twofold dilutions ranging from 0.06 to 64 µg/ml. For all methods, the MIC was the lowest concentration of
fluconazole that produced an 80% reduction of turbidity when compared
visually to the turbidity of the drug-free control.
In selected experiments, growth reduction was estimated
spectrophotometrically: plates from the microdilution format assay were
agitated and the optical densities of the wells were determined at 530 nm (EIA Autoreader; model EL310; Bio-Tek Instruments, Burlington, Vt.).
The background optical density of the drug- and organism-free sterility
check well was subtracted from all the optical densities of all wells,
and the resulting optical density values were divided by the optical
density of the drug-free growth-control well to calculate the
percentage of growth relative to the growth in the growth control.
Animal model.
Four- to six-week-old healthy male CF1 mice
(Harlan Sprague Dawley, Inc., Indianapolis, Ind.) were used. After the
fashion of inoculum preparation for the MIC studies, fungi for
inoculation in the animal model were prepared by growing the yeasts on
Sabouraud dextrose agar at 35°C, suspending the organisms in saline,
and adjusting the inoculum to the desired optical density. The inoculum was quantitated by plating 0.1 ml of serial 10-fold dilutions onto
Sabouraud dextrose agar and counting the resulting colonies after
48 h of incubation. To produce infection, the mice were inoculated
via the tail vein with a suitable inoculum in a volume of 0.2 to 0.25 ml. After infection, the mice were observed twice daily, and animals
exhibiting profound inanition or an inability to reach food and water
were sacrificed. All surviving mice were killed at 21 days after
inoculation. In preliminary studies, groups of 10 mice each were
infected with various inocula of each strain to determine an inoculum
for each strain that produced approximately 80% mortality after 7 days. In the treatment experiments, groups of 15 to 16 mice were
inoculated on day zero with the appropriate inoculum for each isolate.
For the groups of treated mice, 1 or 5 mg of fluconazole per kg of body
weight in 0.2 ml of saline was given intraperitoneally daily starting
1 h after intravenous infection, and treatment was continued for 5 days. On day 4, five mice from each group were sacrificed for
determination of the numbers of CFU of Candida per gram from
the kidney. This determination was made by removing and weighing both
kidneys, homogenizing kidneys in 5 to 10 ml of saline with a Stomacher
80 (A. J. Seward, UAC House, London, England), and plating
suitable dilutions on Sabouraud dextrose agar. The plates were
incubated at 35°C, and the number of colonies was enumerated after
48 h of growth. All animal care procedures were supervised and
approved by the University of Texas-Houston Animal Welfare Committee.
Statistical methods.
Survival times were estimated by using
the Kaplan-Meier method and were compared by the log-rank technique.
Organ clearance data were compared by using analysis of variance and/or
the t test, as appropriate. All calculations were performed
by using SPSS for Windows, version 7.5.1 (SPSS, Inc., Chicago, Ill.).
| |
RESULTS |
In vitro susceptibility testing results.
The results
of repeated testing of the six isolates by the four variations of the
M27-A method are presented in Table 1. All isolates were tested at least once by each method. For isolates for
which multiple MIC estimates differed widely (e.g., the 48-h readings
for C. albicans 707-15), high MICs were somewhat more likely
when testing was performed by the microdilution format. Glucose
supplementation had no effect on the MIC distributions (data not
shown). On the basis of these observations, the MICs for the isolates
were classified as being relatively low at both 24 and 48 h
(low/low), low at 24 h but higher at 48 h (low/high), or high
at both time points (high/high).
The basis for the variability in the MIC estimates was clearly
illustrated when the growth as a percentage of the growth of
the growth
control was determined by measuring the optical densities
of the wells
from the microdilution format assays (Fig.
1). As
can be seen, both isolates for
which the MICs were low/low had
growth curves that broke sharply and
fell to <10% of that for
the growth control at 0.125 to 0.5 µg of
fluconazole per ml. Both
isolates for which the MICs were low/high had
a break in their
growth curves with fluconazole at 0.25 to 0.5 µg/ml,
but the trailing
growth was <20% of the growth for the growth control
at 24 h and
>20% of the growth for the growth control at 48 h. Finally, the
two isolates for which the MICs were high/high were
different
in character. The
C. albicans isolate for which
MICs were high/high
showed no response to fluconazole until the
fluconazole concentration
reached 8 to 16 µg/ml, but thereafter, its
growth was sharply
reduced to <5% of the growth for the growth
control. On the other
hand, the
C. tropicalis isolate for
which the MICs were high/high
showed a break in its growth curve at
24 h (although not a value
consistently <20% of the growth for
the growth control) but demonstrated
essentially no response to
fluconazole at 48 h.
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|
FIG. 1.
Effect of fluconazole. Each isolate was tested by the
microdilution version of the M27-A method, and the reduction in growth
is expressed as a percentage of the growth in the drug-free growth
control well (y axis) versus fluconazole concentration (in
micrograms per milliliter, x axis) for the isolates for
which the MICs were low/low (thin lines), the isolates for which the
MICs were low/high (thick lines), and the isolates for which the MICs
were high/high (dotted lines). Results for tests in 0.165 M
MOPS-buffered (pH 7) RPMI 1640 medium are presented. GC, growth
control. Similar results were obtained if testing was performed in
medium supplemented with glucose at 20 g/liter (data not shown).
|
|
To aid in the interpretation of these disparate MIC estimates, the
morphological effect of fluconazole on the fungi was assessed
visually
(Fig.
2). By comparison with the yeast
cells of the growth
control, all strains except the
C. albicans strain for which the
MICs were high/high demonstrated
aberrant morphology (enlarged,
swollen cells and/or clustering due to
failure of cell separation)
by 1 µg of fluconazole per ml. Such
morphological effects are
similar to those reported for azole
antifungal agents (
4,
5).
In addition, a qualitative
decrease in the number of fungi could
be detected. By contrast, such
effects were not clearly noted
for the
C. albicans isolate
for which the MICs were high/high
until the concentration of
fluconazole reached 8 to 16 µg/ml.
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|
FIG. 2.
Morphological effect of fluconazole on the fungi. After
24 h of incubation, samples of the material in the susceptibility
assays were removed and examined microscopically. Note the aberrant
morphology (clustering and/or ballooning of cells) that was readily
seen by 0.5 to 1 µg/ml for all isolates except the C. albicans isolate for which the MICs were high/high. Such changes
were not evident until a concentration of 8 to 16 µg/ml was reached
for the C. albicans isolate for which the MICs were
high/high. Magnification, ×500.
|
|
Response to therapy in vivo.
After establishing suitable
inoculum sizes for each organism, three treatment trials were performed
with the C. tropicalis isolate for which MICs were high/high
and two trials were performed with the other isolates. For each
isolate, the survival times of the untreated controls did not differ
between treatment trials (P > 0.1), and thus, the data from
the replicate treatment trials were pooled to provide a single estimate
of survival for each isolate with each dose of fluconazole (Table
2). At 1 mg/kg/day, fluconazole prolonged
survival significantly for all isolates except the C. albicans isolate for which the MICs were high/high. At 5 mg/kg/day, fluconazole prolonged the survival times significantly for
all isolates, but the least prolongation was again noted for the
C. albicans isolate for which the MICs were high/high.
The data on the reduction in the numbers of CFU per gram in the kidney
by fluconazole therapy showed a pattern comparable
to that obtained
with the survival data (Table
3). Because
statistically
significant two- to fivefold differences in the numbers
of CFU
per gram of kidney tissue were noted for the untreated controls
between replicate treatment trials, the mean numbers of CFU per
gram of
kidney tissue was computed for the untreated controls
in each
experiment, and all values from each experiment were then
divided by
this value and aggregated across experiments to produce
a single
estimate of the percent reduction in the numbers of CFU
per gram of
kidney tissue relative to the numbers of CFU per gram
for the untreated
controls for each dose of drug.
Aggregate analysis of response from in vitro and in vivo data.
Considering the overall pattern of response based on prolongation of
survival and the reduction in the numbers of CFU per gram of kidney
tissue, the six isolates can be loosely classified as having good,
fair, or poor responses to fluconazole therapy (Table
4). This classification is clearly
arbitrary, but it is helpful in generating a summary of the results and
comparing them with the MIC data. Neither of the mouse model-based
response rank orders were well captured by the MICs obtained at 48 h by the M27 method with an endpoint of 80% reduction of growth. Two
problems are apparent. First the MICs obtained at 48 h by the
M27-A method with an endpoint of 80% reduction of growth would make
both isolates for which the MICs were low/high appear to be more
resistant than the C. albicans isolate for which the MICs
were high/high. This clearly contradicts the animal model data, and for
these isolates the MICs obtained at 24 h by the M27-A method with
an endpoint of an 80% reduction of growth best matched the response in
vivo. Although this effect was seen for the MICs obtained by both the macrodilution and the microdilution methods, it was somewhat more prominent for readings determined in the microdilution format (Table
1). Second, the C. tropicalis isolate for which the MICs were high/high presents an additional problem. The MIC at neither 24 nor 48 h consistently correctly matched the response in vivo if
an 80% reduction in growth was the endpoint criterion. Indeed, when
the MIC was determined at 48 h, this isolate was the most resistant of the six isolates when any possible endpoint criterion was
used. However, the growth of the isolate was clearly reduced at a
fluconazole concentration of 0.5 µg/ml after 24 h, but capturing this result required use of a less restrictive endpoint of 50% reduction of growth relative to the control.
| |
DISCUSSION |
The striking behavior of the test isolates in the assays described
here provides yet another demonstration of the methodology dependence
of all susceptibility test approaches. While these results validate the
fundamental principles of the M27-A method and demonstrate that the
method can produce results that correlate with clinical outcome, the
results also indicate that minor variations in the M27-A method are
required to optimize the in vitro-in vivo correlation for isolates that
demonstrate significant trailing growth.
The data suggest two possible refinements of the M27-A method. First,
the time of reading could be shortened from 48 to 24 h. Such
reading times have previously been suggested on a practical basis
(1, 2, 20), but these results demonstrate that the MIC at
24 h is superior to the MIC at 48 h, at least for some isolates. Although these data apply to fluconazole, it has also been
shown that the meaning of amphotericin B MICs in a broth-based system
is improved by reading the MIC at 24 h rather than at 48 h
(15). It thus seems possible that these changes would be
helpful for other drugs as well. Such reading time is also consistent with work now being reported for agar-based testing of fluconazole (3) and amphotericin B (21). The second
improvement suggested by these data is that the endpoint criterion, at
least for fluconazole, could be loosened so that the MIC is read as the
lowest drug concentration that produces a significant effect on growth.
The precise definition of "significant effect" may require
additional work, but a 50% reduction would appear to be a reasonable
starting point and has been conveniently used in prior work (1,
20). Adoption of these suggestions would also require that
reference ranges for the quality control strains defined by the M27-A
method (11, 17) be determined.
These data also illustrate the inherent limitation of using a single
number (an MIC) to try to predict the global response to an infection.
None of the MIC rank orders completely matches the responses obtained
in vivo (Table 4). In particular, the C. tropicalis isolates
tended to be more difficult to clear from the kidney and also to have
less clear-cut morphologic responses to fluconazole (Fig. 2). This is
most apparent with the C. tropicalis isolates for which the
MICs were low/high and high/high, and this raises the possibility that
the differences in the growth inhibition patterns seen in Fig. 1 are
indicative of small, but real differences in fluconazole
susceptibility. As can be seen, capturing these differences in a single
measure of in vitro susceptibility is difficult.
This study has two principal limitations. First, animal models are a
valuable model for investigating drug efficacy, but they do not
necessarily mimic every aspect of the infection in humans (16). Second, it is difficult to prove that the results
obtained with the various isolates are completely comparable. These
isolates were from widely scattered geographic locations, and five of
the six isolates have been shown to be unrelated by a DNA typing method (13) (the C. albicans isolate for which the MICs
were high/high, isolate UTR-14, was not included in this survey). Thus,
there is always the potential that the isolates might differ in more ways than just their MIC. In addition, production of exactly the same
mortality in the untreated control mice infected with each isolate was
not possible, but the mean survival times were similar. When a
difference exists (the survival times of the untreated mice infected
with the C. tropicalis isolates for which the MICs were
low/low and low/high are shorter than those of mice infected with the
other isolates), this bias actually places the treatment at an
additional disadvantage with respect to that agent and thus serves to
strengthen the results. In addition, the results obtained in the animal
studies are also supported by microscopic inspection of the
fluconazole-exposed yeast. Thus, despite the limitations of studies of
this type, the aggregate data suggest that it is reasonable to conclude
that the rank orders for response derived from the animal studies
(Table 4) are a true estimate of the relative susceptibilities of the
isolates to fluconazole in vivo.
The implications of these results for users of the M27-A method are
clear. For compliance with the M27-A method, results should be reported
on the basis of the MICs at 48 h and the corresponding published
breakpoints. While initial data from our laboratory suggest that
isolates with discordant MICs at 24 and 48 h are only a minority
of tested isolates, the MIC should ideally be determined at both time
points, and special care in reporting of results should be taken with
any isolate that produces strongly discrepant results. Likewise,
interpretation of the results for isolates with significant trailing is
difficult, and the results obtained for such isolates should be
carefully reviewed. Such caution is especially warranted when testing
is done by the more convenient microdilution version of the M27-A
method. It might, for example, be appropriate to provide a supplemental
report indicating that the MIC for the isolate shows substantial
variation with the technique used to determine the MIC and that the
predictive significance of the reported MIC is less certain than usual.
| |
ACKNOWLEDGMENT |
This work was supported by a grant from Roerig/Pfizer
Pharmaceuticals.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 6431 Fannin,
1728 JFB, Houston, TX 77030. Phone: (713) 500-6738. Fax: (713)
500-5495. E-mail: jrex{at}heart.med.uth.tmc.edu.
| |
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0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
