Barnabe, C.

Dujardin, J.P.

Tibayrenc, Michel

Horizon / Pleins textes

Ann. Soc. belge Méd. 
1983. 63, 319-324 
trop. 
TRYPANOSOMA CRUZ/ : A PHARMACOLOGICAL COMPARISON 
OF SOME BOLIVIAN ISOENZYMIC STRAINS 
by 
C. BARNABE, M. TIBAYRENC & J. P. DUJARDIN 
IBBA-ORSTOM, Embajada de Francia, Casilla 824, La Paz, Bolivia 
Summary - The drug susceptibility of 3 isoenzymic strains of Trypanosoma cruzi was 
studied. Two drugs (SQ 18, 506 and Ro 7-1051) and 6 stocks were used. The drug test 
was carried out on epimastigote cultures. Significant susceptibility differences were found 
for Ro 7-1051 between the 3 strains. For SQ 18, 506, there were differences between strains 
1 and 2, and 2 and 3, but no differences between 1 and 3. Strain 2 seemed more sensitive 
to EQ 18,506 than to Ro 7-1051. For strain 1, the opposite was observed. There was no 
difference of susceptibility for the two drugs in the case of strain 3. A great intrastrain 
variability was observed, and the interstrain differences were not clearly proportional to the 
genetic distances Nevertheless, drug susceptibility was correlated in some degree to the 
biochemical classification. 
KEYWORDS : Trypanosoma cruzi; Isoenzymic; Pharmacological Comparison. 
Introduction 
Drug resistance variability among different Trypanosoma cruzi strains 
has been proved on several occasions (Brener & Chiari, 1967; Brener et al., 
1976; Andrade & Figueira, 1977; Avila el al., 1981; Cover & Gutteridge, 
1981). Howsver, these different strains were not related to any zoological 
classification. 
The presence of distinct isoenzymic strains of T. cruzi was established 
by Miles et al. (1977). Ready & Miles (1980) proposed a numerical taxo- 
nomy of T. cruzi zymodemes, based on an intuitive interpretation of the 
zymograms. Genetic interpretation of the zymograms (Tibayrenc et al., 
1981 a & b) allowed a classification based on the use of genetic distances : 
average number of codon differences per gene between two populations 
(Nei, 1972; Tibayrenc, 1980). On these theorical bases, two main strains 
were observed in Bolivia (Tibayrenc & Desjeux, 1983), as well as some 
lesser strains (Tibayrenc et al., 1983). 
In this work, we compared drug susczptibility for 3 isoenzymic strains 
encountered in Bolivia. 
Material and Methods 
- Origin of the stocks: 
We initially used at least two different stocks for each strain. But after 
the end of experiments, we observed by electrophoresis that stock C24 
(theorically strain 1) had been mixed accidentally with laboratory reference 
313 , 
strain Tulahuen, which is closely related to strain 2. So we did mot take - I - .  - 
in account the results concerning this stock and strain 1 was represented 
by only one stock. Table 1 shows the origin of the six T. cruzí stocks 
which were finally used. Matrix 1 indicates their classification by genetic 
distances (Tibayrenc et al., 1983). All the stocks, isolated from Triatoma 
infestans (Tibayrenc et al., 1982) were adapted by weekly passage in GLSH 
monophasic culture medium, at 28% (Le Ray, 1974), during at least two 
months prior to the experiments. We used only one cloned stock (SC43c11) 
but Tibayrenc & Miles (1983) showed the genetic homogeneity of the 
uncloned stocks used in the present work. 
MATRIX 1 
Genetic distances among the strains studied (Tibayrenc et al., 1983), estimated on 12 enzymic loci. 
Strain 1 Strain 2 
%rain 2 1.71 O 
Strain 3 1.77 0.41 
TABLE 1 
Origin of the stocks 
Isoenzymic strain : Stock (laboratory code) : Place : 
Strain 1 c37 
Strain 2 C50 
Y5 
SC43cll 
Strain 3 TU16 
TU18 
Chiwisivi 
Chiwisivi 
Yungas 
Santa Cruz 
Tupiza 
Tupiza 
Chiwisivi : a river valley at 60 km SE La Paz. Altitude 2700 m. 
Yungas : Subtropical valleys at 100 km E La Paz. Altitude 1600 m. 
Santa Cruz : (suburb Cotoca) Subtropical town at 400 km SE La Paz. Altitute 400 m. 
Tupiza : A river valley at 1000 k m S La Paz. Altitude 2700 m. 
- Drugs used: 
a) 5-nitrof uran (transd-am ino-3-(2-(5-nitro-2-f uryl) vinyl) 1,2,4 oxad iazole; 
SQ 18, 506), from the SQIBB Institute for Medical Research, Princston, New 
Jersey, U. S. A. 
b) 2-nitroimidazole (N-benzly-2-nitro-l-imidazole acetamine); Ro 7-1051 
benzimidazole; Rochagan@; Ragonil@; from ROCHE Laboratories, Buenos 
Aires, Argentina. 
- Drug test: 
Fresh media were inoculated with 8 days old epimastigote forms in 
order to obtain the same final parasite concentration for an endvolume 
of 10 ml. The drugs, sterilized by filtration (Millipore@, 0.22 pm pores) 
were immediately added in 50 pl of DMSO (dimethyl sulfoxide), to a final 
concentration of 1 pg.ml-l for SQ 18, 506, and 15 pg.ml-l for RO 7-1051 
according to Baker & Selden (1981). A control was made for each test : two 
inoculations were made at day O, one with the drug, one without the drug, 
with DMSO. Two trials at least (see table 2) were made for each stock and 
320 
each drug. Preliminary experiments had shown that the differences of drug 
susceptibility between the stocks were more pronounced from day 5 to day , 
7. So we used a comparison at day 6. The living parasites were diluted in 
Hanks Wallace isotonic solution and then counted in a haemocytometer at 
a 400x magnification. In order to estimate the drug effect, we used the 
control concentration of living epimastigotes 
following ratio (see table 2) : - 
test concentration of living epimastigotes 
It was homogeneous (mean of 0.09 k 0.07) for all the tests at day O. 
The initial concentration of living epimastigotes was 2.81 -I. 0.54.106 para- 
sites.ml-l for all the tests. 
TABLE 2 
Results obtained at day 6. Drug susceplibility for all stocks studied, with the two drugs used. 
With Ro 7-1051 at 15 pg.ml-1 With SQ 18,506 at 1 pg.mi-1 
Isoenzymic Stock Ratio (1) Stock mean Strain mean Ratio ( l )  Stock mean Strain mean 
strain 
56:;; 5.60 -F 0.74 5.60 f 0.74 Strain 1 C37 15.24 E:; i:::: 15.73 f 3.09 (2) 15.73 -I 3.09 - 
c37 19.00 - 
I Strain 2 c50 C 0 2:; 1 2.46 f 9.35 ;;::: 1 17.65 f 0.49 1 7.88 5 0.97 I (12.62 f 4.73, 1 2.20 5 0.30 3.13 f 1.26 t 8.50 Y5 Y5 - 
TU15 9.89 7.53 I 
5.48 1 5.63 f 1.85 I 33:; 3.51 f 0.03 1 4.77 t 1.74 8.23 t '1.87 3.84 TU15 9.80 9.85 f 0.06 Ti;: :::: 1 6.67 f 0.18 Strain 3 TU15 - 
. Control concentration of living epimastigotes 
( I )  Ratio : 
Test concentration of living epimastigotes 
(2) Standard deviation. 
, on day 6. 
Results and Discussion 
The results are summarized in tables 2 and 3. Table 2 shows the results 
obtained at day 6. 
1) Comparisons interstrain, infradrug 
For Ro 7-1051, we used the statistical distribution-free test of Mann 
8. Whitney : there were significant differences between the 3 strains. For 
SQ 18,506, we used the same test to compare strains 2 and 3, which were 
significantly different. To compare strain 1 with the 2 others, we had to use 
the T test, the variances being not significantly different, and supposing 
that the distributions were normal : strains 1 and 2 showed significant dif- 
ferences (T = 6.55, ddl = 7) but this was not the case for strains 1 and 
3 (T = 0.73; ddl = 5). These differences are correlated in some degree to 
the biochemical classification. However, they are not clearly proportional to 
the genetic distance. This may be explained by the fact that the two phe- 
321 
nomena have not the same genetic determinism : drug resistance (which 
is problably an adaptative character) is perhaps under the dependance of 
biochemical taxonomy is based on the study of protein loci, often consid- 
ered as being selectively neutral (Wilson et al., 1977). 
regulatory genes (Ninio, 1979;- Tibayrenc et al., -1983). On the _other.hand, _- 
TABLE 3 
Comparison for drug sensibility interstock, intradrug 
(= : no significant différence; # : significant difference) 
Ro 7-1051 (drug I) SQ 78.506 (drug 2) 
C37 C50 Y5 SC43 TU15 C37 C50 Y5 SC43 TU15 
C50 f -  C50 # -  
Y5 f =  - Y5 = # -  - - - cc43 # # = - sc43 +  
TU15 + # # # - TU15 = - 
TU18 f - 
1 
- r # -  
1 -   Tuia = f # # =  - - 
2) Comparisons interstock, intradrug 
We uszd the variance analysis, which showed that the whole population 
of the stocks was not homogeneous for drug resistance considering each 
drug. Then we used for individual comparisons the T test, the variance 
being estimated on the whole range of stocks. The results are summarized . 
in table 3. They show clearly in some cases an intrastrain variability. For 
example, within strain 2, there is a variability for drug 2. We did not 
observe any variability within strain 3 for the 2 drugs. 
3) Comparisons intrastrain, interdrug 
For strains 2 and 3, we used the distribution-free test of Mann & 
Whitney. Strain 3 had the same comportment for the 2 drugs. Strain 2 was 
more sensible to SQ 18,506 than to Ro 7-1051. For strain 1, we used the 
T test under the same conditions than in paragraph 1 : this strain appeared 
more sensible to Ro 7-1051 than to SQ 18,506 (T = 4.32, ddl = 4). 
4) Comparisons intrastock, interdrug 
We had to use an individual T test under the same conditions than in 
paragraph 1. Nevertheless, the variances showed significant differences for 
stocks SC43c11 (FI1 = 12844) and TU15 (FI1 = 844). We observed differ- 
ences of resistance for both drugs in all the stocks, except in stock TU15 
For exemple, stock C50 was much more sensible to SQ 18,506 than to 
Ro 7-1051 (see tab!e 2). 
Conclusion 
Biochemical taxonomy is useful to be taken into account for a pharma- 
cological study, but it is not sufficient to foresee all the results, for the 
following reasons : 
- lack of clear proportionality between pharmacological sensitivity and 
- great intrastrain variability. 
genetic distances; 
322 
Nevertheless, we were able to see that drug resistance is correlated 
in some degree to the biochemical classification of T. cruzi : as a matter of 
fact, there were significant differences between the isoenzymic strains. 
Further data are required with a greater number of stocks for each 
strain, and above all, with animal experiments. 
The study of the genetic background of drug resistance constitutes in 
itself an original way of research, which should be given greater importance 
in the future. 
Acknocledgments - This work was supported by the French Ministry of Foreign Affairs 
and the Direction Générale B la Recherche Scientifique et Technique (Grant no PVDI81.L- 
1423). We are grateful to Doctor Michael A. Miles (London School of Tropical Medecine and 
Hygiene), who cloned kindly SC43 stock. 
Trypanosoma cruzi : une comparaison pharmacologique de quelques souches isoen- 
zymatiques boliviennes. 
Résumé - La sensibilité médicamenteuse de 3 souches isoenzymatiques de Trypano- 
soma cruzi a été étudiée. Deux drogues (SQ 18,506 et Ro 7-1051) et 6 isolats ont été 
utilisés. Le test pharmacologique a été effectué s u r  cultures d’epimastigotes. On a observé 
des différences significatives entre les 3 souches pour le RO-7-1051. Pour le SQ 18,506 des 
différences ont été vues entre les souches 1 et 2, et 2 et 3, mais pas entre les souches 
1 et 3. La souche 2 est apparue plus sensible au SQ 18,506 qu’au Ro-7-1051. La souche 1 
a montré la situation inverse. La souche 3 n’a pas montré de différence de susceptibilité 
pour les 2 drogues. On a observé une grande variabilité intrasouche, et les différences inter- 
souches n’ont pas montré de proportionalité nette avec les distances génétiques. Cepen- 
dant, la susceptibilité médicamenteuse était corrélée dans une certaine mesure à la classi- 
fication biochimique. 
Trypanosoma cruzi : een farmacologischo vergelijking van enkele Boliviaanse isoen- 
zymatische stammen. 
Samenvatting - De medicamenteuse gevoeligheid van drie isoenzymatische T. cruzi 
stammen werd bestudeerd. Twee preparaten (SQ 18,506 en Ro 7-1051) en zes isolaten wer- 
den daartoe aangewend. De farmacologische test werd uitgevoerd op epimastigote kulturen. 
Tussen de drie stammen werden significante verschillen vastgesteld voor Ro 7-1051. Voor 
SQ 18,506 werden verschillen waargenomen tussen stammen 1 en 2, en 2 en 3, doch niet 
tussen de stammen 1 en 3. Stam 2 bleek gevoeliger voor SQ 18,506 dan voor Ro 7-1051. 
Voor stam 1 was dit omgekeerd. Stam 3 vertoonde geen verschil in gevoeligheid voor 
beide preparaten. Een grole intrastam variabiliteit werd vastgesteld en de interstam ver- 
Schillen waren niet duidelijk proportioneel t.0.v. de genetische afstanden. Niettemin was 
de medicamenteuse gevoeligheid in een bepaalde mate gecorreleerd aan de biochemische 
klassifi katie. 
Received for publication on June 15, 1983. 
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Trypanosoma cruzi : a pharmacological comparison of some Bolivian isoenzymic strains

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