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Journal of Clinical Microbiology, March 2000, p. 971-976, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evaluation of a Capacitance Method for Direct Antifungal
Susceptibility Testing of Yeasts in Positive Blood Cultures
Hsein
Chang Chang,1
Jui
Jung Chang,1
Ay
Huey
Huang,2 and
Tsung
Chain Chang3,*
Institute of Medical Engineering, National
Cheng Kung University,1 Division of
Clinical Microbiology, Department of Pathology, National Cheng
Kung University Hospital,2 and
Department of Medical Technology, College of Medicine,
National Cheng Kung University,3 Tainan 701, Taiwan, Republic of China
Received 12 July 1999/Returned for modification 28 September
1999/Accepted 9 December 1999
 |
ABSTRACT |
The feasibility of using a capacitance method (CM) for direct
antifungal susceptibility testing of yeasts in positive blood cultures
was evaluated. The CM used the same test conditions as those
recommended by the National Committee for Clinical Laboratory Standards. After direct inoculation of positive culture broths into
module wells (Bactometer; bioMérieux, Inc., Hazelwood, Mo.), the
end-point determination was made by monitoring the capacitance change
in the culture broths with Bactometer. The MIC of amphotericin B was
the lowest concentration at which yeast growth was completely inhibited, while the MICs of ketoconazole, flucytosine, and
fluconazole were the concentrations at which a 
80% reduction in
capacitance change was observed. The MICs of the four drugs against
each blood isolate obtained on subculture plates were also determined
by the macrodilution method. For 51 positive blood cultures tested, the
percent agreement (±2 log2 dilutions) between the CM and
the macrodilution method were as follows: amphotericin B (98%),
ketoconazole (92%), flucytosine (84%), and fluconazole (96%). The CM
was further used for breakpoint susceptibility testing of fluconazole
(8 and 64 µg/ml) and flucytosine (4 and 32 µg/ml) against yeasts in
positive blood cultures. After testing of 74 specimens by the CM,
flucytosine and fluconazole produced one (1.4%) major error and two
(2.8%) minor errors, respectively. All yeasts that displayed
resistance to flucytosine or fluconazole were detected within 24 h
after direct inoculation of the positive broths into Bactometer. The CM
may be useful for the rapid detection of antifungal resistance in
positive blood cultures containing yeasts.
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INTRODUCTION |
In the past few years, there has
been a dramatic increase in the number of systemic fungal infections
reported around the world. The incidence of nosocomial candidemia was
estimated to rise fivefold in the past 10 years (3). In a
recent report from Taiwan (4), fungal pathogens accounted
for a higher proportion of nosocomial bloodstream infections than
any single bacterial species. At the same time, there has been
increasing concern about the emergence of resistance to antifungal
agents among a variety of yeast species (15, 16, 22, 23,
26-28). Therefore, the development of a standardized method for
antifungal susceptibility testing that can predict clinical outcome and
response to therapy has assumed greater importance (11). The
National Committee for Clinical Laboratory Standards (NCCLS)
has developed a reference broth macrodilution method for
susceptibility testing of yeasts (19). Several modifications
of the reference method have been proposed; these techniques include
flow cytofluorometric detection (21, 34), colorimetric
microdilution (6, 24, 30), Etest (5, 25, 29), and
a modified agar dilution method (37).
Candidemia has been shown to contribute to excess hospitalization stay
and to be an independent determinant for death (1, 35). The
mortality rates of fungemia are about two to three times those of
bacteremia (33). A positive blood culture bottle containing
yeasts, as revealed by the Gram stain, normally has been subcultured on
agar plates for colony isolation followed by species identification and
susceptibility testing, if necessary. Several studies (8, 17,
18) have demonstrated that direct antibacterial susceptibility
testing, that is, performance of susceptibility testing without a prior
isolation step, is feasible under most conditions for positive blood
cultures containing bacteria. The benefits of rapid antimicrobial
susceptibility testing for infectious disease outcome have been
studied, and several advantages are recognized (7, 9, 31).
The activities of microbial growth will cause electrical
changes (impedance, conductance, or capacitance) in the culture media. Measurement of an electrical signal is basically not prevented by the
color or turbidity of the clinical specimens. The purpose of this study
was to test the feasibility of direct antifungal susceptibility testing
of positive blood cultures by a capacitance method (CM).
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MATERIALS AND METHODS |
Study design.
The objectives of this study were (i) to
evaluate the reproducibility and correlation of the CM with the
NCCLS macrodilution method for the determination of MICs for pure
yeast strains, (ii) to evaluate the feasibility of the CM for direct
measurement of the MICs of antifungal agents against yeasts in positive
blood cultures, and (iii) to perform the CM in a breakpoint broth
dilution format for direct determination of interpretive
susceptibilities (resistant, susceptible-dose dependent [S-DD] or
intermediate, or susceptible) of yeasts in positive blood cultures.
Yeast stock cultures.
A panel of 10 yeast strains was used
to test the reliability of the CM for the determination of MICs. These
yeasts included Candida albicans ATCC 18804, 24433, and
10231; C. tropicalis ATCC 750; C. parapsilosis
C4-13 (a clinical isolate); C. glabrata ATCC 2001 and C3-2
(a clinical isolate); C. krusei ATCC 6258; and C. guilliermondii ATCC 9058. Among these 10 strains, C. krusei ATCC 6258 was a quality control strain, while C. albicans ATCC 24433 and C. tropicalis ATCC 750 were reference strains, as recommended by the NCCLS
(19). All strains were subcultured at least twice on
Sabouraud dextrose agar (Difco, Detroit, Mich.) and incubated at 35°C
to ensure purity and viability.
Antifungal agents.
Amphotericin B (Sigma Chemical Co., St.
Louis, Mo.), ketoconazole (U.S. Pharmacopoeia, Rockville, Md.),
flucytosine (U.S. Pharmacopoeia), and fluconazole (Pfizer, New York,
N.Y.) were used for MIC determinations. Stock solutions were prepared
with the following solvents: dimethyl sulfoxide for amphotericin B and
ketoconazole and sterile deionized water for flucytosine and fluconazole. The weight of each antifungal agent was adjusted according
to the potency of each drug.
Clinical specimens.
The blood specimens were collected from
the National Cheng Kung University Hospital (an 800-bed teaching
hospital) and from the Kaoshung Chang Gung Memorial Hospital. BACTEC
blood culture bottles (Becton Dickinson Microbiology Systems,
Cockeysville, Md.) were normally inoculated with 3 to 10 ml of blood
from patients, inserted into the BACTEC NR9240 instrument (Becton
Dickinson Microbiology Systems), and incubated at 35°C. Positive
bottles were automatically detected by the instrument, and smears were
prepared to check the presence of yeasts in the positive bottles. Data
for mixed cultures containing more than one strain of yeasts or
containing yeasts and bacteria, as revealed on subculture plates, were
not used for the calculation of agreement values.
Numbers of yeast cells in positive blood culture bottles.
To
enumerate the numbers of yeast cells in positive bottles, serial
10-fold dilutions of the culture broths were made in sterile saline.
The numbers of cells (CFU per milliliter) in the diluted suspensions
were determined by the plate count method (10) with Sabouraud dextrose agar as the culture medium. Plates were incubated at
35°C and enumerated after 48 h of incubation. Fourteen randomly selected positive blood culture bottles were analyzed.
Determination of MICs.
For pure yeast strains, the CM used
the same test conditions (medium, inoculum size, and incubation
temperature) as those recommended by the NCCLS (19) for
MIC determinations, except that the end point was determined by
monitoring the capacitance change in the culture broth. To each module
well (Bactometer; bioMérieux, Inc., Hazelwood, Mo.) containing
0.9 ml of the test organism (0.5 × 103 to 2.5 × 103 cells/ml), 0.1 ml of a dilution series of antifungal
agent was added and mixed. The modules were incubated at 35°C for
48 h, and the capacitance change (i.e., capacitance growth curve)
in each well was continuously monitored with Bactometer. Each strain was tested five times against each of the four drugs on different experimental days. A growth control (no antifungal agent in the well)
and a negative control (culture broth only) were included for each
strain tested. The MIC of amphotericin B was the lowest concentration
at which yeast growth was completely inhibited; i.e., no change in
capacitance was observed compared to the negative control. The MICs of
flucytosine, fluconazole, and ketoconazole were the concentrations at
which a 80% reduction in capacitance change (compared with the
growth control) was observed. Detection time in hours for each module
well was automatically determined by the instrument software when three
consecutive readings of the signal change exceeded the default value in
the instrument. At the detection time, the slope of the capacitance
growth curve had an accelerating increase.
For direct determination of MICs of the four drugs against yeasts in
positive blood cultures, serial log2 dilutions of each drug
in 1 ml of RPMI 1640 broth were constructed in the module wells of
Bactometer. Positive culture broths containing yeasts were diluted 1:10
with sterile saline, and 10 µl of the sample was inoculated into each
of the module wells. The modules were incubated at 35°C for 48 h, and MICs were read by using the same criteria for end-point
determination as those used with pure yeast cultures. A total of 51 positive blood cultures were analyzed by the CM for MIC determinations.
Each blood isolate obtained on subculture plates was identified by
conventional procedures (32), and the MICs of the four drugs
against the blood yeast isolate were determined by the macrodilution
method (19). Discrepancies (±1 log2 and ±2
log2 dilutions) in the MICs obtained by the CM and the
macrodilution method were used for calculation of the percent agreement values.
Breakpoint susceptibility testing of yeasts in positive blood
cultures.
Fluconazole and flucytosine were used for direct
susceptibility testing, with each agent being tested at the two
interpretive breakpoint concentrations (fluconazole, 8 and 64 µg/ml;
flucytosine, 4 and 32 µg/ml), as defined by the NCCLS
(19). Positive culture broths containing yeasts were diluted
1:10 with sterile saline, and 10 µl of the diluted sample was
inoculated into each module well containing 1 ml of RPMI 1640 broth
supplemented with an antifungal agent. The inoculated modules were
incubated at 35°C, and the capacitance change in each module was
monitored for 48 h. A growth control and a negative control were
included for each blood specimen tested. A total of 75 positive blood
cultures containing yeasts were analyzed. Interpretive categorization
of the blood isolate by the direct method was based on the inhibition
of the microorganism at the two breakpoint concentrations
(19). All yeast isolates obtained on subculture plates were
identified (32), and the MICs of fluconazole and flucytosine
against each isolate were determined by the macrodilution method. The
MIC data for each isolate were used for categorization of the
interpretive susceptibilities (19).
Definitions of test errors.
The results of breakpoint
susceptibility testing by the CM were compared with those obtained from
the macrodilution method, and discrepancies were classified as very
major, major, or minor errors (8). A very major error was a
susceptible result by the CM and a resistant result by the standard
method. A major error was a resistant result by the CM and a
susceptible result by the macrodilution method. A minor error was any
change involving an S-DD or intermediate result.
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RESULTS |
Reliability of the CM for MIC determination.
The MICs for 10 pure yeast cultures were determined by using four antifungal agents,
with each strain being tested five times against each drug on different
days. Figure 1 shows typical capacitance growth curves for C. krusei ATCC 6258 in the presence of
different concentrations of fluconazole. The MIC of fluconazole was
determined to be 64 µg/ml (Fig. 1, curve E); at this drug
concentration, the reduction in capacitance change was 80% when a
comparison of capacitance change against the growth control was made.
The detection time of the growth control was about 10 h (Fig. 1). Figure 2 shows the capacitance growth
curves for C. parapsilosis C4-13 in the presence of
amphotericin B. The MIC was 0.5 µg/ml; at this drug concentration,
yeast growth was completely inhibited and almost no capacitance change
was observed when a comparison of capacitance change against the
negative control was made. The detection time of C. parapsilosis C4-13 was longer (15 h) than that to detection of
C. krusei ATCC 6258.
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FIG. 1.
Capacitance growth curves for C. krusei ATCC
6258 in the presence of different concentrations of fluconazole. Curve
A, growth control; curves B, C, D, and E, 8, 16, 32, and 64 µg/ml,
respectively; curve F, negative control. The MIC was determined to be
64 µg/ml.
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FIG. 2.
Capacitance growth curves for C. parapsilosis
C4-13 in the presence of different concentrations of amphotericin B. Curve A, growth control; curves B, C, D, and E, 0.06, 0.12, 0.25, and
0.5 µg/ml, respectively; curve F, negative control. The MIC was
determined to be 0.5 µg/ml.
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Table
1 summarizes the median MICs of
amphotericin B, ketoconazole, flucytosine, and fluconazole against each
of the 10 yeast
strains, as determined by the CM and the broth
macrodilution technique.
For the quality control strain (
C. krusei ATCC 6258) tested by
the CM, the median MICs of each of the
four antifungal agents
all fell within the reference ranges established
by the NCCLS
(
19). The MICs for the two reference
strains (
C. albicans ATCC
24433 and
C. tropicalis
ATCC 750) also fell within the established
ranges. In Table
1, a total
of 40 pairs of data were obtained
for method comparison. If a
discrepancy of median MICs no greater
than 2 log
2 dilutions
was allowed, the agreement rates between
the CM and the
reference method were 90% for ketoconazole and
100% for amphotericin
B, flucytosine, and fluconazole.
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TABLE 1.
Comparison of the MICs of amphotericin B, ketoconazole,
flucytosine, and fluconazole determined by the CM and the broth
macrodilution method (BMM) for 10 yeast strains
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Yeast cell numbers in positive blood cultures.
The counts of
yeast cells in 14 randomly selected positive bottles ranged from
105 to 107 CFU/ml, with 13 bottles (93%) being
in the range of 106 to 107 CFU/ml.
Direct MIC determination for yeasts in positive blood cultures.
Fifty-two positive blood culture bottles containing yeasts
were used
for the direct determination of MICs of amphotericin
B, ketonconazole,
flucytosine, and fluconazole by the CM. The
MICs for the blood isolates
obtained on subculture plates were
also determined by the reference
macrodilution method. With one
bottle being a mixed culture (
C. albicans and
C. pelliculosa),
a total of 53 yeast
strains were recovered from the 52 blood samples.
The remaining 51 strains included
C. albicans (30 strains),
C. parapsilosis (8 strains),
C. tropicalis (7 strains),
C. glabrata (4 strains),
C. guilliermondii (1 strain), and one unidentified
species. For
the mixed culture containing two strains of yeast,
the CM detected the
more resistant side of the mixed flora. For
example, the respective
MICs (in micrograms per milliliter) for
the mixed culture (
C. albicans and
C. pelliculosa) determined
by the
reference method were as follows: amphotericin B (0.5 and
1),
ketoconazole (0.06 and 2), flucytosine (0.12 and 0.25), and
fluconazole
(0.06 and 0.25). However, the MICs (in micrograms
per milliliter)
obtained by the direct CM were as follows: amphotericin
B
(1.0), ketoconazole (1.0), flucytosine (0.25), and fluconazole
(0.25).
Table
2 shows the correlation of MICs
obtained by the two methods. If a discrepancy of 1 log
2
dilution was allowed, the percent
agreement values between the CM
and the macrodilution method ranged
from 61% (flucytosine) to 84%
(fluconazole). However, if a discrepancy
of 2 log
2
dilutions was allowed, the values increased to 84%
(flucytosine),
92% (ketoconazole), 96% (fluconazole), and 98%
(amphotericin B).
Direct breakpoint susceptibility testing of yeasts in positive
blood cultures.
Fluconazole (8 and 64 µg/ml) and flucytosine (4 and 32 µg/ml) were used to perform the breakpoint susceptibility
testing of yeasts in positive blood cultures by direct inoculation into
Bactometer. After testing of 75 specimens, the CM produced one (1.4%)
major error when a strain of C. tropicalis was tested
against flucytosine. However, two minor errors (2.8%) were observed
when fluconazole was tested against one strain each of C. albicans and C. glabrata. Therefore, the rates of
agreement between the two methods for interpretive susceptibility
testing were 98.6 and 97.2%, respectively, for flucytosine and
fluconazole. Sixty-eight strains (92%) were susceptible to both
flucytosine and fluconazole. The MIC range and MICs at which 10, 50, and 90% of these yeast blood isolates were inhibited by amphotericin
B, flucytosine, fluconazole, and ketoconazole, as determined by the
macrodilution method, are shown in Table
3.
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TABLE 3.
MICs of amphotericin B, ketoconazole, flucytosine, and
fluconazole for 74 blood yeast isolates as determined by the
macrodilution method
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Among the 75 positive blood specimens, one was found to be a mixed
culture containing
C. albicans and
Serratia
marscens. Since
the bacterium was resistant to the
antifungal agents, the mixed
culture produced false-resistant results
(fluconazole MIC, >64
µg/ml; flucytosine MIC, >32 µg/ml) in the
CM method. The sample
was excluded from data analysis. Of the
remaining 74 yeast strains,
2 strains of
C. tropicalis were
resistant to flucytosine, but
other strains were susceptible to the
drug. For fluconazole, one
strain of
C. albicans was
resistant and three strains of
C. glabrata were S-DD (Table
4). Table
4 shows the incubation time
elapsed
when resistance or S-DD was detected by the CM; in all
situations,
the time needed was less than 24 h after
direct inoculation of
the positive culture broths. Figure
3 shows the capacitance growth
curves for
a fluconazole-resistant blood isolate (
C. albicans 5364524)
grown with fluconazole concentrations of 8 and 64 µg/ml.
The
detection time (21 h) was determined with Bactometer when
the increase
in capacitance of three consecutive readings exceeded
the default value
of the instrument.
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TABLE 4.
Incubation time elapsed when resistant or S-DD yeast
strains in positive blood cultures were detected by the direct CM
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FIG. 3.
Capacitance growth curves for a positive blood culture
(C. albicans 5364524) directly inoculated into Bactometer
and grown at the two breakpoint concentrations of fluconazole. Curve A,
growth control; curves B and C, 8 and 64 µg/ml, respectively; curve
D, negative control. The detection time for curve C was determined to
be 21 h by Bactometer.
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The species isolated from the 74 blood culture bottles were
C. albicans (43 strains),
C. tropicalis (12 strains),
C. parapsilosis (8 strains),
C. glabrata (6 strains),
C. guilliermondii (1 strain),
Cryptococcus
neoformans (1 strain),
C. famata (1 strain), and
two
unidentified yeast species. The
C. neoformans strain failed
to grow in RPMI 1640 broth after 48 to 72 h of incubation, and
susceptibility results were not obtained by both the CM and the
macrodilution
method.
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DISCUSSION |
A direct antifungal susceptibility testing method based on the
measurement of capacitance change was investigated for positive blood
cultures containing yeasts. For the determination of MICs of four
antifungal agents against yeasts in 51 positive blood culture bottles,
the agreement rates (±2 log2 dilutions) between the CM and
the NCCLS reference method were as follows: amphotericin B (98%),
ketoconazole (92%), flucytosine (84%), and fluconazole (96%) (Table
2).
The CM was also performed in a format of breakpoint susceptibility
testing of flucytosine and fluconazole against yeasts in positive blood
cultures, with results being expressed as resistant, S-DD or
intermediate, or susceptible. After testing of 74 blood specimens, a
major error rate of 1.4% was found for flucytosine; however,
fluconazole had a minor error rate of 2.8%. Under most conditions, the
interpretive susceptibility results were available within 24 h
(Table 4) with the direct CM and 72 h with routine procedures
encompassing yeast isolation followed by susceptibility testing. That
all resistant and S-DD strains were detected within 24 h after
inoculation does not mean that strains causing no change in
capacitance within 24 h should be susceptible to a test drug. Delayed resistance patterns may be encountered with slowly
growing yeasts (e.g., C. parapsilosis). However, all
eight strains of C. parapsilosis isolated from the 74 blood
samples were susceptible to both flucytosine and fluconazole.
Although isolates of C. krusei are considered to be
intrinsically resistant to fluconazole, the rate of isolation of this species in blood cultures is relatively low (4, 20), and strains of C. krusei were not isolated in this study.
Although the breakpoints for itraconazole also have been defined
(19, 27), the data for itraconazole were completely from
studies of oropharyngeal candidiasis. For this reason, itraconazole was not included for direct interpretive susceptibility testing. For testing of pure yeast strains, the CM produced reproducible results (Table 1) and was comparable to the broth macrodilution method (19). The cost of one module of Bactometer was about $8
(U.S. dollars), and a test for one drug was estimated to cost $10.
Bactometer was not designed for susceptibility testing but for the
determination of total counts. Simpler equipment with the same function
would be cost-effective for routine use.
There are three electrical signals (conductance, impedance, and
capacitance) available for measurement of microbial growth in
Bactometer. Our preliminary data showed that capacitance measurements had a greater response (data not shown) than the signals of impedance and conductance; therefore, this parameter was used throughout this study.
The rate of nosocomial candidemia increased by almost 500% from
1981 through 1989 (2, 3), particularly in large teaching hospitals. Therefore, rapid antifungal susceptibility testing of yeasts
in blood cultures may have clinical importance. Through years of study,
some alternative methods, including the colorimetric broth
microdilution technique (6, 24, 30), flow
cytometry (21, 34), and Etest (5, 25, 29), have
been proposed for antifungal susceptibility testing. However, all of
these procedures require isolated pure colonies for testing and do not
seem feasible for direct susceptibility tests with positive culture broths.
Direct antimicrobial susceptibility testing, either by agar disk
diffusion (8, 13, 17) or broth dilution
(14), of positive blood cultures containing bacteria was
found to be feasible under most conditions. In addition, an
impedimetric method has been developed for direct antimicrobial
susceptibility testing of gram-negative bacilli (12) and for
detection of oxacillin-resistant Staphylococcus aureus in
blood cultures (36). These results prompted us to use the CM
for direct antifungal susceptibility testing of yeasts in positive
blood cultures. Compared with the occurrence of bacteremia, the
occurrence of fungemia caused by multiple yeast strains is very rare.
Therefore, the difficulty of susceptibility interpretation in
situations of polymicrobial infections is seldom encountered. Mixed
cultures of bacteria and yeasts may be occasionally observed in
positive blood culture bottles; however, the presence of the two
completely different organisms normally can be detected by the Gram
stain, which is a routine step when a positive blood culture bottle is
found. The CM tended to detect the more resistant side if a mixed
culture containing two strains of yeasts was encountered. In case of a mixed culture containing yeasts and bacteria, the CM might produce false-resistant results due to the resistance of bacteria to antifungal agents.
The numbers of cells in positive blood culture bottles containing
yeasts were about 106 to 107 (CFU/ml), close to
the cell density of a yeast cell suspension with a McFarland turbidity
of 0.5. Therefore, an inoculum of 1 µl (i.e., 10 µl of a
1:10-diluted sample) of positive broth in 1 ml of RPMI 1640 medium
would achieve a final cell density of about 1 × 103
to 10 × 103 CFU/ml. Although the inocula were
somewhat larger than those (0.5 × 103 to 2.5 × 103 CFU/ml) recommended by the NCCLS (19),
inoculum densities seem not to have caused significant deviations in
the end-point determinations of the MICs (10). Direct
susceptibility testing can test a broader representation of the yeast
population present in blood cultures. Theoretically, about
103 cells were inoculated into each module well of
Bactometer, whereas only several colonies on subculture plates were
sampled for inoculum preparation in the conventional testing protocol
(19).
In conclusion, the CM seems to be capable of earlier detection of
resistant (or S-DD or intermediate) yeast isolates in positive blood
cultures. The method would be simpler especially if performed in a
format of breakpoint susceptibility testing. The signal
detection in Bactometer is a continuous, real-time process, and
susceptibility patterns can be obtained by a real-time comparison
with the growth curve for a growth control.
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ACKNOWLEDGMENTS |
This project was supported by a grant (NSC 88-2314-B006-078) from
the National Science Council, Taipei, Taiwan.
We thank Kaoshung Chang Gung Memorial Hospital for supplying some of
the positive blood cultures.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Technology, College of Medicine, National Cheng Kung
University, 1 University Rd., Tainan 701, Taiwan, Republic of China.
Phone: 886-6-2353535, ext. 5790. Fax: 886-6-2363956. E-mail:
tsungcha{at}mail.ncku.edu.tw.
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Journal of Clinical Microbiology, March 2000, p. 971-976, Vol. 38, No. 3
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Abstract
Introduction
