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Antimicrobial Agents and Chemotherapy, January 1999, p. 25-28, Vol. 43, No. 1
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development of Rifapentine Susceptibility Tests for
Mycobacterium tuberculosis
L.
Heifets,*
T.
Sanchez,
J.
Vanderkolk, and
V.
Pham
National Jewish Medical and Research Center
and University of Colorado Health Sciences Center, Denver, Colorado
Received 15 June 1998/Returned for modification 29 September
1998/Accepted 25 October 1998
 |
ABSTRACT |
Two methods for testing the susceptibility of Mycobacterium
tuberculosis to rifapentine have been developed: the agar
proportion method and the radiometric BACTEC technique. A critical
concentration of 0.5 µg of rifapentine per ml is proposed for both
methods since it provides a reliable means of distinguishing between
susceptible and resistant M. tuberculosis isolates. It is
recommended that two quality control M. tuberculosis
strains be used at the introduction of these tests in a clinical
laboratory: one that is pansusceptible (H37Rv) and one that is
resistant to rifapentine. The resistant strain can be obtained from the
American Type Culture Collection, where it is deposited under the
number ATCC 700457.
| |
INTRODUCTION |
Rifapentine (RPT) is a long-lasting
rifamycin recently approved by the Food and Drug Administration as an
alternative to rifampin (RMP) in treatment regimens for patients with
tuberculosis. There were many reports on the antimicrobial activity of
RPT in vitro (1, 4, 7, 12, 13), in macrophages (3, 9,
10), and in mice (1, 2, 8, 11). The purpose of this
study was to develop for clinical laboratories a procedure(s) suitable for testing the susceptibility of Mycobacterium tuberculosis
isolates to RPT rather than a procedure that again ascertains the
well-known data on the activity of this drug against M. tuberculosis in vitro. The RPT susceptibility tests were developed
for two types of culture media: Middlebrook-Cohn 7H10 and 7H11 agars
and BACTEC 7H12 broth.
| |
MATERIALS AND METHODS |
Laboratory strains.
Eight pansusceptible M. tuberculosis strains from our collection were used for preliminary
studies with RPT. In addition, RPT-resistant mutants were developed
from three of these strains (H37Rv, Erdman, and Atencio).
QC strains.
Two quality control (QC) strains were used in
experiments with clinical isolates: one pansusceptible strain, M. tuberculosis H37Rv (ATCC 27294), and one strain that had
monoresistance to RPT and that was developed in our laboratory
(H37RPT-R; see below). The latter strain has been deposited at the
American Type Culture Collection (ATCC) as strain ATCC 700457.
Clinical isolates.
A total of 95 pansusceptible M. tuberculosis isolates from new patients who had not received any
prior antituberculosis treatment were included in this study. These
isolates were retrieved from cultures of specimens from 52 patients.
The 33 RMP-resistant isolates were selected from isolates submitted to
our laboratory for routine drug susceptibility testing. Most of these
isolates were resistant to two or more drugs, but four of them had
monoresistance to RMP.
Drugs.
RPT was obtained from Hoechst Marion Roussel, Inc.
(Kansas City, Mo.), and RMP was obtained from Sigma Chemical Co. (St.
Louis, Mo.). The stock solutions were made in methanol and were
subsequently diluted in distilled water before incorporation into
culture media.
Media.
Two commercially available media were used: (i) 7H12
broth in 12B vials (Becton-Dickinson, Sparks, Md.) and (ii) 7H10 and 7H11 agars made from Middlebrook-Cohn 7H10 agar base (Becton-Dickinson) with 10% oleic acid-albumin-dextrose-citrate (Remel, Lenexa, Kans.).
The agar medium was distributed in quadrant plastic plates (5 ml per
quadrant) so that three quadrants contained three different drug
concentrations and one quadrant in each plate was drug-free.
MIC determination.
On the basis of the definition of the MIC
as the lowest drug concentration that inhibited more than 99% of the
bacteria in a culture, the MIC on agar plates was determined by
comparing the number of colonies in the drug-free control sections of
the plate with the number of colonies growing in the presence of drug concentrations ranging from 0.03 to 8.0 µg/ml.
To determine the MIC in BACTEC 7H12 broth, a series of 12B vials were
supplemented with RPT or RMP (0.03 to 8.0 µg/ml), each
of which was
added in a volume of 0.1 ml. A culture from a seed
vial, in which the
radiometric growth index (GI) reached 999,
was diluted 1:10 to
inoculate 0.1 ml of it into all drug-containing
vials and one of two
control vials. Cultures that reached a GI
of 500 were used undiluted.
An alternative is to use as the inoculum
cultures that are grown in 7H9
broth, adjusted to the optical
density of a McFarland no. 1 standard,
and diluted 1:100. This
approach provided an inoculum in the range of
10
4 to 10
5 CFU/ml. One of two control vials was
inoculated with a 1:100
dilution of the bacterial suspension, providing
initial contents
of 10
2 to 10
3 CFU/ml (1:100
control). The MIC determined radiometrically in
7H12 broth was defined
as the lowest drug concentration in the
presence of which the daily GI
did not exceed 50 while in the
1:100 control the daily GI was above the
GI in the drug-containing
vial for a minimum of 3 consecutive days. The
validity of an experiment
was controlled by the intensity of growth in
the drug-free control
vial inoculated with the original suspension used
for the drug-containing
vials (undiluted control). If the inoculum was
in the desired
range of 10
4 to 10
5 CFU/ml, the
GI in this control should not be at the maximum GI
(GI999) within less
than 3 days of cultivation, but the growth
should reach a GI of 500 or
greater within a maximum of 10 days
of
cultivation.
Although this BACTEC technology for determining more than 99% growth
inhibition is now a standard procedure, including for
RMP
susceptibility testing, we felt it necessary to validate the
GI
radiometric assessment of growth inhibition by performing actual
viable
counts in experiments with RPT and to use RMP for comparison.
More
details on MIC determinations in BACTEC broth were given
in our
previous publications (
5,
6).
| |
RESULTS |
Validation of the radiometrically determined MIC of RPT.
To
validate the radiometrically determined MICs of RPT, the actual MICs
based on the viable bacterial counts were determined in experiments
with three pansusceptible strains (H37Rv, Atencio, Erdman). In each
experiment two drugs were tested: RMP and RPT. Similar extensive work
was done previously with RMP (7), and inclusion of this drug
in the current experiment provided a control for consistency. The
results of these experiments are presented in Table
1. The actual MICs of both drugs
determined by performing viable counts were only twofold higher than or
equal to the MICs determined radiometrically for the same cultures.
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TABLE 1.
Comparison of MICs determined in 7H12 broth
radiometrically (GI) and by the viable count (CFU per
milliliter) methoda
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Drug-resistant mutants.
Laboratory strains with monoresistance
to rifamycins were developed by cultivating each of the three
pansusceptible strains mentioned above on 7H11 agar plates that
contained 8 µg of RPT or RMP per ml and that were inoculated with a
heavy bacterial suspension of about 1010 CFU/ml. From a few
single colonies grown under these conditions, we subcultivated mutant
substrains that were resistant to both rifamycins at concentrations
above 8.0 µg/ml. Each of these substrains represented a pure 100%
resistant culture as determined by the agar proportion method. Both
types of these single-colony clones derived by selection with RMP and
RPT were equally resistant to both drugs. One of these mutant clones
derived from strain H37Rv in the presence of RPT was selected as a
potential RPT-resistant QC strain, along with the original H37Rv strain
serving as a susceptible QC strain.
QC strains.
Current standards of the Centers for Disease
Control and Prevention, the Council of American Pathologists, and the
National Committee for Clinical Laboratory Standards require only one
QC strain (pansusceptible strain H37Rv) to be included with each batch
of isolates tested in clinical laboratories. Nevertheless, for a test
with a new antituberculosis agent (RPT), we felt that it was necessary
to have two QC strains: one pansusceptible and one with monoresistance
to RPT (strain H37RPT-R). Therefore, the results obtained with two
strains, ATCC 27294 (pansusceptible) and ATCC 700457 (with
monoresistance to RPT), were tested for their reproducibilities to
justify their subsequent use as QC strains. Each experiment included
simultaneous determination of the MICs of RPT and RMP by both the agar
and the broth dilution methods. Such experiments for each of the two QC
strains were repeated 10 times.
The results of 20 experiments with the susceptible and resistant QC
strains are presented in Table
2. In 10 experiments with
the susceptible strain in the BACTEC system, the
results were
identical, with no variation between the experiments: the
MICs
of RMP were always 0.12 µg/ml, and the MICs of RPT were 0.03 µg/ml
or less. Some variations were identified in the agar dilution
test with both drugs, but the MICs of either drug usually did
not
exceed 0.12 µg/ml, and the MIC of RMP was 0.5 µg/ml in only
one
experiment.
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TABLE 2.
Reproducibility of the agar and broth dilution methods:
MICs of RPT and RMP for two QC strains, susceptible strain H37Rv and
strain H37RPT-R with monoresistance to rifamycins
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|
The actual MICs of RPT and RMP for the resistant QC strain were 32.0 µg/ml, but in the reproducibility study we limited for
practical
purposes the range of concentrations to 8.0 µg/ml. Ten
experiments
with the resistant QC strain had identical results:
the highest
concentration used in these reproducibility experiments,
8.0 µg of
either RMP or RPT per ml, did not inhibit growth in
either the broth or
the agar system. These data indicated very
substantial differences in
the MICs for the susceptible and resistant
strains, confirming their
suitability as QC
strains.
Sensitivity of the rapid BACTEC method for detection of low-level
resistance to RPT and RMP.
The important advantage of the BACTEC
indirect susceptibility test over the 7H10 and 7H11 agar proportion
method is that the results can be obtained within less than 10 days
instead of 3 weeks. Nevertheless, it is known from clinical experience,
including our experience at the National Jewish Mycobacteriology
Laboratory, that for a relatively small percentage of isolates the
BACTEC method may not detect the low level of resistance to RMP and
other conventional drugs at the early stages of emerging resistance. With the introduction of the new rifamycin, we conducted a study to
evaluate the actual sensitivity of the BACTEC method for determination of this low level of resistance to either RPT or RMP. For this purpose,
we prepared, using three strains (H37Rv, Erdman, Atencio), artificial
mixtures containing 1, 10, and 50% of their rifamycin-resistant mutants mentioned above and tested these mixtures by both methods along
with fully susceptible original strains and 100% resistant mutants.
Table 3 indicates the actual proportions
of resistant bacteria in the bacterial population (expressed as
percentages) detected in these cultures on agar plates containing
various drug concentrations, as well as the MICs derived from the
results of tests on agar plates and in the BACTEC system. These
experiments showed that resistance to either RPT or RMP may not be
detected by the BACTEC method if the culture contained less than 10%
resistant bacteria. This phenomenon explains the discrepancies between
the agar proportion and BACTEC methods sometimes observed previously for RMP in clinical laboratories. This deficiency of the BACTEC method
may not be of great practical importance, since the cultures consisting
of clinical isolates identified as "resistant" rarely contain fewer
than 10% resistant bacteria. For example, there were no discrepancies
between the agar proportion and BACTEC methods in detecting resistance
to RMP or RPT in the experiments with 33 rifamycin-resistant clinical
isolates described below.
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TABLE 3.
Evaluation of sensitivity of BACTEC broth method for
detection of resistance to RMP and RPT in strains containing small
proportions of resistant bacteria
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MICs of RPT for pansusceptible strains.
The MICs of RPT for
eight laboratory strains were determined by the agar and broth dilution
methods; these included repeat tests for three of the strains for which
MIC data are presented in Table 1. It should be mentioned that the
radiometric MICs in these experiments were only 1 dilution higher (0.06 versus 0.03 µg/ml) for two strains and the same (0.03 µg/ml) for
one of those tested in the preliminary experiments (Table 1). The results of these experiments showed that the MICs of RPT by either method did not exceed 0.12 µg/ml (Table
4). Among the large number of
pansusceptible isolates obtained from patients who had negative cultures following 60 days of therapy, the RPT MICs were also in the
range of 0.03 to 0.12 µg/ml for 93 of 95 isolates, and for only 2 isolates the MICs were 0.25 µg/ml (Table
5). These data indicated that 0.5 µg of
RPT per ml could be considered a breakpoint for susceptibility.
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TABLE 4.
MICs of RPT for eight pansusceptible M. tuberculosis strains determined on agar plates and in
BACTEC broth
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TABLE 5.
Distribution of 95 pansusceptible M. tuberculosis isolates obtained from 52 new patients by
determination of MICs of RPT by the broth method
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|
MICs of RPT and RMP for resistant strains.
Thirty-three
clinical M. tuberculosis isolates that were reported in our
clinical laboratory to be resistant to RMP by the conventional agar
proportion method were included in this study. The MICs of both RPT and
RMP were determined by two methods: on 7H10 and 7H11 agar plates and
radiometrically in 7H12 BACTEC broth. The results of these experiments
are presented in Table 6. In the agar
plate experiments, the MICs of RPT were 8.0 µg/ml or greater than 8.0 µg/ml for 32 of 33 isolates and 4.0 µg/ml for only 1 of them. The
MICs of RMP were 4.0 µg/ml for only 3 isolates, and for 30 isolates
the RMP MICs were 
8.0 µg/ml. Identical results were obtained on
7H10 and 7H11 agar plates. In the BACTEC system the MICs of both RMP
and RPT for all 33 isolates were 8.0 µg/ml or greater. These
experiments confirmed full cross-resistance between RMP and RPT and
that the RPT concentration of 0.5 µg/ml suggested above as the
breakpoint for susceptibility would not inhibit the growth of the
rifamycin-resistant clinical isolates.
| |
DISCUSSION |
A critical concentration of 0.5 µg of RPT per ml was developed
for tests of the susceptibility of clinical M. tuberculosis isolates by two methods: the 7H10 and 7H11 agar proportion method and a
radiometric method in BACTEC 7H12 broth (in 12B vials). Two QC strains
were evaluated: one strain that was susceptible to RPT and one strain
that had monoresistance to RPT. We would suggest that with the
introduction of the susceptibility test for this new drug in clinical
laboratories both QC strains be used and, subsequently, that only the
susceptible QC strain alone be used as required.
The proposal of the critical concentration of 0.5 µg/ml is based on
determination of the MICs of RPT for 95 pansusceptible and 33 rifamycin-resistant isolates. The MICs for susceptible strains ranged
from 0.03 to 0.12 µg/ml for most strains and were 0.25 µg/ml for
only two of them. The MICs for most resistant strains were 8.0 µg/ml
or greater, and the MIC was 4.0 µg/ml for only one isolate. In other
words, the concentration of 0.5 µg/ml was greater than the highest
MIC of this drug in tests with pansusceptible isolates and it was
substantially lower than the lowest MIC found in tests with the
rifamycin-resistant isolates. Therefore, the concentration of 0.5 µg/ml served well in making a clear distinction between susceptible
and resistant isolates by both methods. Nevertheless, it is well known
from the experience in clinical laboratories that in some cases the
drug susceptibility test in the BACTEC system with conventional drugs,
particularly with RMP, may not detect resistance, causing a discrepancy
with the result of a test performed on solid medium. We speculated that
such a discrepancy may have been related to the isolates with low
levels of drug resistance. For some drugs, for example, isoniazid,
differences in the level of resistance may reflect the presence of
mutants with different genetics on the basis of their low and high
levels of resistance. Although the stepwise development of resistance of M. tuberculosis to rifamycins cannot be excluded, the
fact that its mechanism of resistance to RMP and RPT is limited to the
genetic alterations in an 81-bp region of the rpoB gene may suggest that the differences in the degree of resistance to these drugs
most likely reflects the differences in the proportions of resistant
bacteria in a culture of the isolate. Practically, from among all
rifamycin-resistant strains isolated in our laboratory (including 33 strains used for this study), we could not find a culture which would
have a low level of resistance that was not related to the low
proportion of resistant bacteria in the population. Therefore, to
investigate the reason for relatively rare false-susceptible test
results in the BACTEC system compared with the rate of occurrence of
such results by the agar proportion method, we tested cultures of
strains containing various proportions of rifamycin-resistant bacteria.
We found that the test in the BACTEC system inconsistently detected low
levels of resistance to either RMP or RPT in cultures in which the
proportions of resistant bacteria were less than 10%. It may not have
great practical importance, since clinical isolates with such low
levels of resistance are quite rare, probably because in patients the
emerging resistance to rifamycins is usually progressing very rapidly.
Nevertheless, this low probability of false-susceptible results should
be taken into account when the BACTEC susceptibility test is the method of choice. This does not diminish the advantage of this method as a
rapid indirect susceptibility test with a turnaround time of less than
10 days and as a means of obtaining reliable results for the vast
majority of M. tuberculosis isolates. On the other hand, the
indirect test by the agar proportion method, with a turnaround time of
3 to 4 weeks, has other advantages that must be taken into account: it
is less expensive, it is more accurate in detecting low levels of
resistance, and it provides the data on the actual proportion of
resistant bacteria in the population. Beside this, the agar proportion
method can be used as a direct test for smear-positive specimens, in
which case the total turnaround time from the moment that the raw
sputum sample is obtained can be almost the same as that for isolation
plus an indirect test in the BACTEC system. The most rational approach
is probably the use of a combination of both techniques in laboratory
protocols, depending on the patient's condition, the type of specimens
to be tested, laboratory capabilities, etc.
| |
ACKNOWLEDGMENTS |
This study was supported in part by Public Health Services grant
U19-AI40972 from the National Institutes of Allergy and Infectious Diseases and in part by a grant from Hoescht Marion Roussel, Inc.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National Jewish
Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Phone: (303) 398-1384. Fax: (303) 398-1953. E-mail:
heifetsl{at}njc.org.
| |
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Antimicrobial Agents and Chemotherapy, January 1999, p. 25-28, Vol. 43, No. 1
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
