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Antimicrobial Agents and Chemotherapy, April 1998, p. 818-820, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vitro Evaluation of Drug Susceptibilities of
Babesia divergens Isolates
Philippe
Brasseur,1,*
Sophie
Lecoublet,1
Nathalie
Kapel,2
Loic
Favennec,1 and
Jean J.
Ballet3
Laboratoire de Parasitologie, Centre
Hospitalier Universitaire, Hôpital Charles Nicolle, 76031 Rouen,1
Laboratoire de Parasitologie,
Faculté de Sciences Pharmaceutiques et Biologiques, 75006 Paris,2 and
Laboratoire d'Immunologie
et Immunopathologie, Centre Hospitalier Universitaire,
Hôpital Clémenceau, 14033 Caen,3
France
Received 18 August 1997/Returned for modification 31 October
1997/Accepted 27 January 1998
 |
ABSTRACT |
The susceptibilities of three bovine and two human Babesia
divergens isolates to antimicrobial agents were evaluated in
vitro by a tritiated hypoxanthine incorporation assay. The MICs at
which 50% of isolates are inhibited (MIC50s) for
mefloquine (chlorhydrate), chloroquine (sulfate), quinine
(chlorhydrate), clindamycin (phosphate), pentamidine (isethionate),
phenamidine (isethionate) plus oxomemazine (chlorhydrate), lincomycin
(chlorhydrate monohydrate), and imidocarb (dipropionate) were
determined. Except for imidocarb, the MIC50s observed for
the different isolates were close. Imidocarb and the combination of
phenamidine plus oxomemazine exhibited the highest in vitro activity,
while antimalarial agents such as mefloquine, choroquine, and quinine
were inactive. Other drugs had intermediate activities. The data
support further in vitro evaluation of antimicrobial agents active
against B. divergens for the improvement of therapeutic strategies.
| |
INTRODUCTION |
Babesiosis is due to an
intraerythrocytic protozoan parasite transmitted by tick bites to a
wide variety of wild and domestic animals, including cattle, horses,
dogs, and rodents. In Europe, Babesia divergens is
considered the most pathogenic agent of bovine babesiosis and is
responsible for important economic losses in beef- and milk-producing
breeds. Current therapy includes the use of imidocarb dipropionate, but
it remains debatable whether differences in outcomes among individual
animals are due to variations in the sensitivities of the isolates to
drugs or to host factors in animals which are not highly inbred
(11).
In humans, 19 of the 24 cases of babesiosis reported in Europe were due
to B. divergens, and all occurred in splenectomized patients. The initial therapeutic trials with antimalarial drugs for
the treatment of this condition, characterized by severe symptoms and
high mortality rates, have had limited success. More recently, complete
parasite clearance and recovery were obtained with the combination of
clindamycin and quinine in association with exchange transfusion
(6).
Limited information is available on the antimicrobial susceptibility of
Babesia spp. in vitro. The first reports of the use of
tritiated nucleic acid precursors for the screening of drugs (7) showed a positive correlation between in vitro and in
vivo activities in animals (10). More recently, in vitro
assays have been proposed for determination of the susceptibilities of
Babesia bovis and Babesia bigemina to drugs
(4, 12). The aim of this work was to compare the
antimicrobial susceptibilities of B. divergens isolates of
bovine and human origin by a triatiated hypoxanthine incorporation
assay.
| |
MATERIALS AND METHODS |
B. divergens isolates.
Five B. divergens isolates were tested. Three isolates were of bovine
origin and were obtained from different geographical areas in Europe:
Muenchen (Munich, Germany), Weybridge 8843 (Weybridge, United Kingdom),
and Taylor (Ireland). Two isolates were obtained in France from
patients admitted to Rouen University (isolate Rouen) and Le Mans
(isolate Le Mans) hospitals. Isolates were maintained in human
erythrocyte (RBC) cultures and infected 10 to 20% of the RBCs. The
isolates were then suspended in RPMI 1640 medium (Gibco, Cergy, France)
supplemented with 40% (vol/vol) fetal calf serum and 15% (vol/vol)
dimethyl sulfoxide and frozen in liquid nitrogen.
B. divergens cultures.
After thawing and
washing, 0.2 ml of packed infected RBCs was injected intraperitoneally
into 2-month-old female gerbils (Meriones unguiculatus)
(Centre D'elevage R. Janvier, Le Genest-Saint-Isle, France) 2 days
after an intraperitoneal injection of 2 mg of dexamethasone (Soludecadron; Merck-Sharp-Dohme, Paris, France). When the level of
parasitemia reached 50% (i.e., 2 to 3 days later), parasitized RBCs
were collected by cardiac puncture. Human RBCs were collected from
healthy type O- and Rh-positive volunteer blood donors, placed in
preservative-free heparin (Heparine Leo, Saint-Quentin-Les-Yvelines, France), and washed by serial centrifugations in RPMI 1640 medium. The
buffy coat and the upper part of the RBC pellets were discarded to
avoid leukocyte contamination, and 0.2 ml of packed human RBCs was
poured into 50-ml plastic flasks (Nunc, Roskilde, Denmark) containing 5 ml of RPMI 1640 medium supplemented with HEPES (25 mM; pH 7.4; Sigma,
St. Louis, Mo.), gentamicin (12 µg/ml), and 10% (vol/vol)
decomplemented pooled human serum from type O- and Rh-positive donors.
A volume of 0.01 ml of parasitized packed gerbil RBCs was added. The
cultures were maintained at 37°C in a 5% CO2 atmosphere
and were supplemented with human RBCs twice a week to adjust the level
of parasitemia to between 1 and 2%.
In vitro assays for drug susceptibility.
Drug susceptibility
assays were performed in 96-well culture microplates (Falcon, Meylan,
France) in the presence of a final RBC concentration of 1.8% (vol/vol)
and serial dilutions of drugs. Each duplicate well received 200 µl of
an RBC suspension and 50 µl of each drug dilution in RPMI 1640 medium. For each experiment, controls consisted of four wells
containing infected RBCs without drug (i.e., wells to which 50 µl of
RPMI 1640 alone was added) and two wells containing 200 µl of
noninfected RBCs.
[3H]hypoxanthine (specific activity, 4 Ci/mmol; Amersham,
Les Ulis, France) was added (0.4 µCi/well). The plates were incubated at 37°C for 48 h in a 5% CO2 atmosphere, the cells
were harvested on glass fiber filters (Whatman GF/C) with a cell
harvester, and the incorporated radioactivity (in counts per minute)
was counted in a liquid scintillation spectrometer.
Five milliliters of RBCs from each well were stained with Giemsa stain,
and 5,000 RBCs were microscopically examined to determine morphologically the percentage of infected RBCs.
Antimicrobial agents.
From a stock solution in 70% ethanol,
nine dilutions of the following antimicrobial agents were prepared in
RPMI 1640 medium: mefloquine chlorhydrate (0.3 to 0.017 µg/ml;
Hoffmann-LaRoche-Basle, Basel, Switzerland), chloroquine sulfate (6 to
0.023 µg/ml; Rhône Poulenc, Paris, France), quinine
chlorhydrate (1.2 to 0.047 µg/ml; Assistance Publique, Paris,
France), clindamycin phosphate (250 to 0.98 µg/ml; Upjohn, Val de
Reuil, France), pentamidine isethionate (10 to 0.07 µg/ml;
Rhône Mérieux, Toulouse, France), phenamidine isethionate
and oxomemazine chlorhydrate (2 to 0.008 µg/ml; Oxopirvédine; Rhône Mérieux, Lyon, France), lincomycin chlorhydrate
monohydrate (4,000 to 15.6 µg/ml; Lincocine; Upjohn, Val de
Reuil, France), and imidocarb dipropionate (25 to 0.098 µg/ml;
Carbesia; Pitman-Moore, Meaux, France). A volume of 50 µl of each
dilution was added to the wells. Control wells were filled with 50 µl
of medium without drug.
Expression of results and statistical analysis.
The amount
of parasite-dependent tritiated hypoxanthine incorporated was obtained
by subtracting the amount of background incorporation observed in
noninfected cultures. For each drug, a
concentration-versus-[3H]hypoxanthine incorporation curve
was established, and the inhibitory activity was expressed as the
minimum concentration (wt/vol) of drug which either inhibited tritiated
hypoxanthine incorporation or decreased the proportion of
morphologically infected RBCs by 50% of the value in control cultures
without drug (the concentration at which 50% of isolates are inhibited
[MIC50]). MIC50s were expressed in micrograms
per liter of blood with a 50% hematocrit.
Variance analysis was performed by the Fisher and Yates chi-square test
or the Student
t test, thus assuming normal, like
distributions of values. The significance of
r values was
used
to study linear correlation.
| |
RESULTS |
In preliminary experiments, tritiated hypoxanthine incorporation
was measured in cultures maintained in the absence of antimicrobial agents (15 microplates, 24 wells/microplate). For the noninfected wells, the mean ± 1 standard deviation (SD) level of
incorporation was 191 ± 52 cpm/well, and for infected wells
without drug, the mean ± 1 SD level of incorporation was
11,707 ± 2,575 cpm/well. No significant difference was found
between isolates (P > 0.1; data not shown). One SD
accounted for less than 5 and 17% of the mean for each microplate and
for the pooled data for the 15 microplates, respectively. The
variations for the cultures maintained in the presence of the different
antimicrobial agents are summarized in Fig. 1 to 3. In the case of
clindamycin, it was verified with the Le Mans isolate that a good
correlation was found between MIC50s obtained with 18 independent microplates (r = 0.7; P < 0.01).
A detailed comparison of the MIC50s obtained by the
tritiated hypoxanthine incorporation assay and microscopic counting was performed for the Weybridge 8843 and Rouen isolates in the presence of
clindamycin or imidocarb. As indicated in Fig.
1, no significant difference in the
MIC50s was observed in 15 independent experiments (P > 0.1). This was confirmed for all isolates in the
presence of the other antimicrobial agents (P > 0.1;
data not shown).
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FIG. 1.
Comparison of MIC50s of clindamycin and
imidocarb for the Weybridge 8843 bovine isolate and the Rouen human
isolate obtained in the [3H]hypoxanthine incorporation
assay and by microscopic examination. Results are expressed as mean
values for 15 independent experiments (one bar indicates 1 SD).
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|
From these data, the MIC50s of clindamycin and imidocarb
varied from 2,200 to 3,400 µg/liter and from 27 to 34 µg/liter,
respectively. Imidocarb, a drug restricted to the treatment of
babesiosis in animals, was efficient against both human and bovine
B. divergens isolates (Fig. 1 and
2). The MIC50s of imidocarb
and clindamycin for bovine and human isolates were not significantly
different; however, MIC99s determined by using probit/log
concentration regression curves suggested that they had higher levels
of activity against human isolates than against bovine isolates (14,100 and 28,800 µg/liter, respectively). The combination of phenamidine
plus oxomemazine was more effective than imidocarb against the bovine
isolates Muenchen and Weybridge 8843 but not against the bovine isolate Taylor (Fig. 3). None of the B. divergens isolates tested was found to be sensitive to
antimalarial agents (Table 1).
Pentamidine isethionate inhibited B. divergens in vitro, and
lincomycin was active and had a high MIC50 (Table 1).
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FIG. 2.
MIC50s of phenamidine plus oxomemazine and
of imidocarb for bovine B. divergens isolates. Results are
expressed as mean values for eight independent experiments (one bar
indicates 1 SD).
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FIG. 3.
MIC50s of clindamycin and clindamycin plus
quinine for bovine and human isolates of B. divergens in 18 independent experiments. Results are expressed as mean values for 18 independent experiments (one bar indicates 1 SD).
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TABLE 1.
In vitro activities of mefloquine, chloroquine, quinine,
pentamidine, and lincomycin against bovine and human B. divergens isolates
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| |
DISCUSSION |
In this report a 48-h in vitro assay was used to evaluate the
susceptibilities of human and bovine B. divergens isolates
to antimicrobial agents. Continuous cultures of B. divergens
were maintained as described previously (7), and the level
of incorporation of tritiated hypoxanthine was measured in the presence
of different concentrations of drugs. This assay was easier to perform
than microscopic counting and was found to be reliable and sensitive. The MIC50s did not differ significantly from the values
obtained by microscopic examination. Uptake of tritiated purines such
as hypoxanthine was previously proposed for the screening of drug activities against Babesia spp. with short-term cultures of
blood from experimentally infected animals (8, 9). In the
present culture assay, which uses human instead of bovine RBCs, higher levels of tritiated hypoxanthine incorporation were obtained.
Bovine babesiosis is generally treated with imidocarb dipropionate. For
bovine isolates, the MIC50 of this compound was
significantly lower than the maximum (Cmax)
concentration in serum of 6.6 mg/liter observed in cattle
receiving a dose of 17 mg/100 kg of body weight. This is
consistent with the high level of activity of imidocarb against bovine
babesiosis. Similar MIC50s, i.e., 0.87 µg/ml of medium, have been reported for B. bovis
(15). In addition, the comparable results obtained for human
and bovine isolates suggest that imidocarb is possibly a candidate
agent for the treatment of human B. divergens babesiosis.
The combination of phenamidine isethionate plus oxomemazine
chlorhydrate, which is currently restricted for use in the treatment of
canine babesiosis, was found to be more active than imidocarb in vitro.
The treatment of human B. divergens babesiosis with
antimalarial drugs has had limited success. Of the seven patients
treated with chloroquine for several weeks, four died and three
recovered (1, 5, 10, 14). A treatment based on blood
exchange and the administration of clindamycin plus quinine has been
successfully used in six patients. Our results confirm the
susceptibility of B. divergens to clindamycin in vitro. The
in vivo efficacy of quinine is controversial (3), since some
of the patients treated with quinine alone died and others recovered
after several weeks. The very high MIC50s of quinine found
in this study suggest that this compound is not useful for treatment.
The MIC50s of antimalarial drugs found in this study were
higher than the cutoff limits of sensitivity for Plasmodium
falciparum (8.6, 2.7, and 1 µg/liter of blood for quinine,
chloroquine, and mefloquine, respectively). Interestingly, it has
been shown in a recent study that atovaquone, which has
antimalarial activity, was more active than imidocarb for the
treatment of bovine B. divergens babesiosis (13).
All isolates were susceptible to pentamidine isethionate in vitro. For
the Rouen isolate, the MIC50 (2.2 ± 0.08 µg/liter
of blood) was higher than the Cmax (0.2 to 0.5 µg/liter of blood) resulting from an injection of 4 mg/kg, which is
the maximum dose that can be used in humans due to the renal toxicity
of this compound (16). This may explain the treatment
failures reported for patients treated with pentamidine alone
(17) or with pentamidine and chloroquine (2). No
data on the pharmacokinetics of pentamidine isethionate in cattle are
available, and it cannot be stated whether pentamidine isethionate can
be proposed for use in the treatment of bovine babesiosis. The
MIC50 of lincomycin found in this study is higher than the
Cmax values obtained in humans (2 to 7 µg/liter of blood), which suggests that lincomycin probably has
limited use for the treatment of this disease.
Data suggest that tritiated hypoxanthine incorporation by B. divergens in human RBC cultures provides an accurate and
convenient means of evaluating the antibabesial activities of drugs,
further drug screening, detecting resistant isolates, and comparing in vitro activities with in vivo activities.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Parasitologie, Hôpital Charles Nicolle, 1, rue de Germont, 76031 Rouen, France. Phone: (33) 2 32-88-80-15. Fax: (33) 2 32-88-80-17. E-mail: philippe.Brasseur{at}wanadoo.fr.
| |
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Antimicrobial Agents and Chemotherapy, April 1998, p. 818-820, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
