Response to chemotherapy with benznidazole of clones
isolated from the 2 1 SF strain of Trypan osoma cru zi
( biodeme Type II,
Trypan osoma cru zi II)
Resposta à quimioterapia com benzonidazol de clones isolados da cepa
21SF do
Trypanosom a cruzi
( biodema Tipo II,
Trypanosom a cruzi II)
Rozália Figueira Campos
1,2, Marcos Lázaro S. Guerreiro
1,2, Karina de Souza Castro Sobral
1,
Rita de Cássia P. Cunha Lima
1and Sonia G. Andrade
1ABSTRACT
Su sc e p ti b i li ty to c h e m o th e ra p y wi th b e n zn i d a zo le wa s i n ve sti ga te d o f 5 c lo n e s i so la te d f ro m th e 2 1 SF stra i n ( b i o d e m e typ e II, Tr ypano so ma c r uzi II) . Sw i ss m i c e w e re i n f e c te d w i th th e p a re n ta l stra i n f o r e a c h c lo n e a n d su b m i tte d to c h e m o th e ra p y wi th b e n zn i d a zo le ( 1 0 0 m g/k g/d a y d u ri n g 9 0 d a ys) . Tre a tm e n t d e te rm i n e d n e ga ti vi ty o f th e p a ra si te m i a . Cu re ra te s w e re e va lu a te d b y p a ra si to lo gi c a l c u re te sts. Se ro lo gy w a s e va lu a te d f o r tre a te d a n i m a ls ( ti te rs f ro m n e ga ti ve to 1 :6 4 0 ) a n d u n tre a te d c o n tro ls ( 1 :1 6 0 to 1 :6 4 0 ) . Cu re ra te s va ri e d f ro m 3 0 to 1 0 0 % f o r th e 5 c lo n e s, a n d we re 2 5 % f o r th e p a re n ta l stra i n . Re su lts su gge ste d th a t th e va ri a b i li ty o f re sp o n se to tre a tm e n t o f th e c lo n a l p o p u la ti o n s o f Tr ypano so m a c r uzi II stra i n s i s re sp o n si b le f o r th e h i gh va ri a ti o n i n th e re sp o n se to c h e m o th e ra p y wi th b e n zn i d a zo le a n d n i f u rti m o x b y stra i n s o f th i s b i o d e m e .
Ke y-words: Tr ypano so ma c r uzi. Tr ypano so ma c r uzi II. Bi o d e m e Typ e II. Clo n e s. Be n zn i d a zo le . Ch e m o th e ra p y.
RESUMO
A su sc e ti b i li d a d e à q u i m i o te ra p i a c o m o b e n zo n i d a zo l, d e 5 c lo n e s i so la d o s d a c e p a 2 1 SF ( b i o d e m a Ti p o II, T. c r uzi II) , f o i i n ve sti ga d a . Ca m u n d o n go s su í ç o s f o ra m i n f e c ta d o s c o m a c e p a p a re n ta l e c o m c a d a c lo n e e su b m e ti d o s à q u i m i o te ra p i a c o m b e n zo n i d a zo l ( 1 0 0 m g/k /d i a d u ra n te 9 0 d i a s) . Os í n d i c e s d e c u ra f o ra m a va li a d o s p e lo s te ste s d e c u ra p a ra si to ló gi c o s. A so ro lo gi a f o i a va li a d a p a ra o s a n i m a i s tra ta d o s e ( d e n e ga ti vo a 1 : 6 4 0 ) e p a ra o s c o n tro le s n ã o tra ta d o s( 1 :1 6 0 a 1 :6 4 0 ) . Os í n d i c e s d e c u ra va ri a ra m d e 3 0 % a 1 0 0 % p a ra o s 5 c lo n e s se n d o d e 2 5 % p a ra a c e p a p a re n ta l. Os re su lta d o s su ge re m q u e a va ri a b i li d a d e d e re sp o sta a o tra ta m e n to d a s p o p u la ç õ e s c lo n a i s d a s c e p a s
Tr ypano so ma c r uzi II é re sp o n sá ve l p e la gra n d e va ri a ç ã o n a re sp o sta à q u i m i o te ra p i a c o m b e n zo n i d a zo l e n i f u rti m o x d a s c e p a s d e ste b i o d e m a .
Pal avr as-chave s: Tr ypano so ma c r uzi. Tr ypano so ma c r uzi II. Bi o d e m a Ti p o II. Clo n e s. Be n zo n i d a zo l. Qu i m i o te ra p i a .
1 . La b o r a tó r io de Cha ga s Expe r im e nta l, Auto im unida de e I m unida de Ce lula rdo Ce ntr o de Pe s q uis a s Go nç a lo Mo niz da Funda ç ã o Os wa ldo Cr uz, Sa lva do r, B A. 2 . De par tame nto de Ciê nc ias B io ló gic as da Unive r sidade Estadual de Fe ir a de Santana, Fe ir a de Santana, B A, B r asil.
Addr e ss to: Dr a So nia G. Andr ade . Ce ntr o de Pe sq uisas Go nç alo Mo niz/FIOCRUZ. R. Walde mar Falc ão 1 2 1 , B r o tas, 4 0 2 9 5 - 0 0 1 Salvado r, B A, B r asil. e -mail: sgandr ade @ c pq gm. fio c r uz. b r
Re c e b ido par a pub lic aç ão e m 7 /5 /2 0 0 4 Ac e ito e m 3 0 /1 2 /2 0 0 4
Strains of Trypa no so m a c ruzi c harac terized as different Types or biodemes1 3 have exhibited well-marked differenc es in
the ir r e spo nse to c he mo the r apy, e ithe r with nifur timo x o r benznidazole2. A variability in the susc eptibility between the
str ains o f b io de m e Type I I has also b e e n o b se r ve d2 and,
c onsidering the mean c ure rates obtained by the evaluation of 1 5 str ains o f the r efer r ed bio deme, they wer e c lassified as presenting medium to high susc eptibility. A wide individual variability of the c ure rates between them has been detec ted. These results differ from those obtained for the biodeme Type I
strains ( Z2 b) , that showed high susceptibility and for the biodeme Type III strains ( T. cruzi I), with a high degree of resistanc e2.
In the present study we aimed to investigate the susc eptibility of c lones obtained from the 2 1 SF strain, c lassified as biodeme Type II (T. cruzi II). Biologic al and isoenzymic c harac ters of the isolated clones reproduced the patterns of the parental strain7.
Molec ular c harac terization of the c lones isolated from the 2 1 SF strain has been performed by the restric tion fragment length polymorphism ( RFLP) of the kDNA8. A high perc entage of genetic
Ca mpo s RF e t al
c lones ( 6 7 to 1 0 0 % , for restric tion endonuc lease Rsa I and 8 3 to 1 0 0 % for Hinf I) , although a low degree of divergenc e has been observed. Considering the above results, it is important to investigate the responses to c hemotherapy of c lonal populations of the 2 1 SF strain, to c larify whether the variable response to c hemotherapy, already demonstrated between the various strains c lassified as biodeme Type II, c orresponds to differenc es in the susc eptibility of its c lones. It is important to investigate if there is any relation between resistanc e to c hemotherapy and c lonal c o nstitutio n, tak ing into ac c o unt no t o nly the sim ilar itie s observed, but also the dissimilarities that have been detec ted in some c lo ne s8.
MATERIAL AND METHODS
Tr ypan osom a cr u zi s tr a in a nd c lo ne s . The 2 1 SF str ain o f T. c ru zi was iso late d fr o m a patie nt in the ac ute phase o f Chagas’ dise ase , fr o m São Fe lipe , B ahia State and c lassifie d as b io de me Type II, b y Ro c ha Filho1 5.
Fi ve c l o n e s o f th i s s tr a i n , p r e vi o u s l y o b ta i n e d b y mic romanipulation and isolation investigation. These c lones were identified as 2 1 SF-C1 , 2 1 SF-C2 , 2 1 SF-C3 , 2 1 SF-C4 and 2 1 SF-C5 and were c harac terized ac c ording to the biologic al and iso enzymic c harac teristic s7 and also submitted to mo lec ular
c harac terization8.
Pe r c e ntile s o f similar ity r e sulting fr o m the analysis o f the RFLP o f the se ve r al sc hizo de me s o b taine d fr o m the par e ntal str ain and its c lo ne s8 ar e sho wn in Tab le 1 .
Mo rta li ty: the c umulative mo r tality was e valuate d fr o m the initial day o f tr e atme nt until 3 0 days afte r the e nd o f tr e atm e nt, whe n the c ur e te sts we r e pe r fo r m e d. Anim als s u r vi vi n g a t th e e n d o f e x p e r i m e n t we r e s a c r i fi c e d b y e xsanguinatio n afte r ane sthe sia, and b lo o d was c o lle c te d fo r sub ino c ulatio n, se r o lo gy and he mo c ultur e .
Cure rates were evaluated through the direct study of parasites in peripheral blood and by cure tests: hemoculture, subinoculation of the blood into newborn mice and xenodiagnosis.
He m o culture: 0 .5 ml of blood from the treated mic e was cultivated using Warren medium: Hemoculture examination was done on 1 0 , 2 0 , 3 0 and 4 5 days after day 1 .
Subino cula tio n was performed by intraperitoneal injection of 0 .2 ml of blood from each treated mouse in 5 newborn mice and tests of parasitemia in these animals from the 1 2th to the 2 0th day
after inoculation. Xenodiagnosis was done using five 1st stage nymphs
of Dipetalogaster maximus, and examined on the 3 0th and 6 0th days. Se r o lo gy. Tite r s o f spe c ific anti-Tryp a n o so m a c ru z i
antibodies in the serum c ollec ted from eac h mouse, treated and untreated, were evaluated by indirec t immunofluoresc enc e test, using as antigens c ulture forms of the parasite and goat anti-mouse fluoresc einated IgG for the sec ondary antibody.
RESULTS
Pa r a s ite mia . Figur e 1 sho ws the par asite m ic c ur ve s o btained fr o m mic e infec ted with the 2 1 SF-P ( par ental str ain) and the c lo ne s 2 1 SF- C1 and 2 1 SF-C5 , ( tr e ate d and untr e ate d c o ntr o ls) . The o the r c lo ne s sho we d ir r e gular pr o file s with p a r a s i te m i c p e a k s fr o m 1 4 to 2 4 da ys p o s t- i n fe c ti o n , c har ac ter istic s o f b io deme Type II. Tr eatment has deter mined pr o gr e ssive de c r e ase in the par asite mia, with ne gativatio n b y dir e c t e xaminatio n o f the pe r iphe r al b lo o d, fr o m the 2 0th day
afte r initiatio n o f tr e atme nt.
Mo r tality. Table 2 sho ws the mo r tality, evaluated between the 9 0th to the 1 2 0th day afte r the e nd o f tr e atme nt, fo r tr e ate d
and untr e ate d animals in the var io us e xpe r ime ntal gr o ups. Mo r tality var ie d fr o m 3 0 to 6 6 % in the tr e ate d gr o ups and fr o m 6 0 to 9 0 % in the untr e ate d c o ntr o ls.
Cur e te sts. Re sults o f the par asito lo gic al c ur e te sts ar e e xpr e sse d in Tab le 3. Mic e infe c te d with the par e ntal str ain ( 2 1 SF-P) sho we d a 2 5 % par asito lo gic al c ur e . Mic e infe c te d
Table 1 - Percent of similarity from RFLP analysis of clones of the 21SF strain*.
Clones 2 1 SF - P Rsa I Hinf I
2 1 SF – C1 85 87
2 1 SF – C2 93 83
2 1 SF – C3 1 0 0 92
2 1 SF – C4 82 1 0 0
2 1 SF – C5 67 85
* In: Campos et al. Mem. Inst. Oswaldo Cruz 9 4 : 2 3 -2 9 , 1 9 9 9 . 2 1 SF- P = 2 1 SF parental strain
Exper imental gr oups. For the present investigation, six experimental groups of 40 swiss mice weighing 15 to 20g were used. Each group was inoculated with 5x104 trypomastigotes obtained from
the blood of mice infected with the parental strain and each clone, respectively. Parasitemia of the infected mice was evaluated daily during the acute phase, from the 7th day post-infection.
Tr eatment with benznidazole. From eac h experimental group, 3 0 mic e were submitted to treatment with benznidazole and 1 0 mic e were maintained untreated as c ontrols. Treatment was initiated on the 1 4th day of infec tion for all the c lones and
the parental strain. B enznidazole was administered orally, by gavage, at the doses of 1 0 0 mg/kg body weight/day; five doses were administered per week, during 9 0 days. Pa ra site m ia of the treated and untreated groups were evaluated weekly. Means of parasitemia were expressed by evaluation in 5 animals of gr o up.
Table 2 - Mortality of mice in fected with the 21SF strain of Tr ypanosoma cruzi an d 5 clon esa.
Mortality
Experimental groups treated ( %) untreated ( %) 2 1 SF – P 6 6 .0 9 0 .0 2 1 SF – C1 4 3 .0 8 0 .0 2 1 SF – C2 5 0 .0 8 0 .0 2 1 SF – C3 3 0 .0 6 0 .0 2 1 SF – C4 4 3 .0 7 0 .0 2 1 SF – C5 4 0 .0 8 0 .0
with the various c lones presented c ure rates that varied from 3 0 to 1 0 0 % . Clone 2 1 SF-C5 presented the lowest c ure rates ( 3 0 % ) , similar to that obtained for the parental strain ( 2 5 % ) .
Ser o lo gy. Sero lo gic al tests revealed a variatio n in the titers o f anti-T. c ru zi antib o die s in the diffe r e nt gr o ups as sho wn in Tab le 4 . The highe st tite r s fo r tr e ate d mic e we r e o b se r ve d in tho se infe c te d e ithe r with the 2 1 SF-P ( par e ntal str ain) o r with c lo ne 2 1 SF-C5 ( 1 :8 0 to 1 :6 4 0 ) . Titer s fo r tr eated mic e infec ted with c lo nes 2 1 SF - C1 , C2 , C3 , C4 varied fro m negative to 1 :1 6 0 . Untr e ate d c o ntr o ls sho we d tite r s fr o m 1 :1 6 0 to 1 :6 4 0 .
DISCUSSION
The data obtained in the present study revealed a variability of responses to c hemotherapy of the 5 c lones obtained from the 2 1 SF strain of T. cruzi. These data mimic the behavior registered fo r 1 5 str ains o f b io de m e Type I I , iso late d fr o m diffe r e nt geographic al areas ( inc luding São Felipe – Bahia State) ; strains of São Felipe present c ure rates of 2 8 .5 to 1 0 0 % of the infec ted and treated mic e, similar to those obtained with the c lones, as shown in the present study.
Investigatio ns into the c lo nal c o nstitutio n o f the 2 1 SF str ain ( b io de me Type II) , in pr e vio us studie s b y Campo s & Andr ade7,
have sho wn a ho mo lo gy b e twe e n the iso late d c lo ne s. It has
Number
of
tr
ypomastigotes
per
50
microscopic
fields
300 250 200
100 50 0 150
6 8 10 12 14 16 18 20 22 24 26 28 30 Days of infection
30 Untreated Treated 21 SF - P
Number
of
tr
ypomastigotes
per
50
m
icroscopic
fields
(400x)
140 120 100
60 40 20 80
Days of infection Clone 21SF - C1
Untreated Treated
0
8 10 12 14 16 18 20 22 24 26 28 30
Number
of
tr
ypomastigotes
per
50
m
icroscopic
fields
(400x)
300
6
8 10 12 14 16 18 20 22 24
26
28
Days of infection
30
Clone 21SF - C5
Untreated
Treated
200
100
0
4
Table 3 - Cu re rates in mice in fected with Trypanosoma cruzi ( 21SF strain an d 5 clon es) treated with ben zn idazole, based on parasitological tests.
Identification Inocula Mice # Positive Parasitological Cure cases tests ( %) rates ª ( %) 2 1 SF – P 5 x 1 04
08 6 7 5 .0 2 5 .0 2 1 SF – C1 5 x 1 04 17 6 3 5 .0 6 5 .0
2 1 SF – C2 5 x 1 04 15 6 4 0 .0 6 0 .0
2 1 SF – C3 5 x 1 04
17 0 0 .0 1 0 0 .0 2 1 SF – C4 5 x 1 04
12 0 0 .0 1 0 0 .0 2 1 SF – C5 5 x 1 04 10 7 7 0 .0 3 0 .0
Total 79 25
a: Combined data of parasitological ( directed blood examination, subinoculation into newborn mice, xenodiagnosis) results.
2 1 SF- P = 2 1 SF parental strain
Fi gu r e 1 - Par asi te m i c pr of i l e s cor r e spon di n g to: A - the par e n tal str ai n ( 2 1 SF-P) ; B - cl on e 2 1 SF- C1 , hi ghl y su sce pti b l e to che m othe r apy, an d C – cl on e 2 1 SF-C5 wi th l ow su sce pti b i l i ty to tr e atm e n t, showi n g the pr of i l e s i n the u n tr e ate d con tr ol s i n com par i son wi th the m i ce tr e ate d wi th b e n zn i dazol e . Ei the r the par e n tal str ai n an d cl on e s 2 1 SF- C1 an d 2 1 SF-C5 , showe d n e gati vati on of di r e ct par asi te m i a f r om the 5th day of tr e atm e n t.
A
B
C
Table 4 - Serological titers in mice in fected with 21SF strain an d clon es, su bmitted to chemotherapy*.
Antibody titers
Identification Untreated controls Treated mice 2 1 SF – P 1 : 3 2 0 to 1 : 6 4 0 1 : 8 0 to 1 : 6 4 0 2 1 SF – C1 1 : 1 6 0 to 1 : 3 2 0 Neg to 1 : 1 6 0 2 1 SF – C2 1 : 3 2 0 to 1 : 6 4 0 Neg to 1 : 8 0 2 1 SF – C3 1 : 3 2 0 to 1 : 6 4 0 Neg to 1 : 2 0 2 1 SF – C4 1 : 3 2 0 to 1 : 6 4 0 1 : 4 0 to 1 : 1 6 0 2 1 SF – C5 1 : 3 2 0 to 1 : 6 4 0 1 : 8 0 to 1 : 6 4 0
b e e n de m o nstr a te d tha t the pr o file s fo r the iso e nzym e s5
pho spho gluc o mutase ( PGM) , gluc o se pho sphate iso mer ase ( GPI ) , a la n in e a m in o tr a n s fe r a s e ( ALAT) a n d a s p a r ta te aminotransferase ( ASAT) , expressed the zymodeme Z21 1, that
corresponds to the biodeme Type II, suggesting the predominance of a principa l clo ne in this strain. The differenc es in response to c hemotherapy between the various c lones of the 2 1 SF strain, c ould be responsible for the behavior of the parental strain that, in the present study, showed a 2 5 % c ure rate, despite presenting highly susc eptible c lones in its c omposition.
Clones resistant to benznidazole have been obtained by Murta & Romanha1 3 from the Y strain, based on the selection of naturally
o c c ur r ing r e sistanc e to b e nznidazo le . Clo ne s sub mitte d to treatment with one single high dose of benznidazole for 9 , 1 2 or 2 5 passages in mic e, bec ame highly resistant as c ompared with the Y wild strain that was very sensitive. The authors suggest that the resistanc e of T. c ru zi strains seems to be related to the sensitive/resistant c lone ratio in the population. In a previous study by our laboratory1 0, an attempt was made to selec t, by
prolonged treatment, populations resistant to benznidazole and to investigate their isoenzymic c harac teristic s, as c ompared with the original strain. With this objec tive, prototypes of the three diffe r e n t b io de m e s ( Pe r uvia n , Type I ; 2 1 SF, Type I I a n d Colombian, Type III) were tested. No differences in the isoenzymic patterns of populations isolated from treated and untreated mic e c ould be detec ted, whic h indic ates absenc e of c lonal selec tion or genetic marker alterations in the isoenzymic profiles1 0.
Differences in the responses to chemotherapy with nifurtimox or benznidazole, between genetically characterized clonal isolates was demonstrated by Toledo et al1 7, in infec ted Balb/c mic e, with
high resistanc e of c lones 1 9 and 2 0 ( Group I - T. cruzi I) in c omparison with c lones 3 2 and 3 9 ( Group II - T. cruzi II) that were partially resistant to both drugs.
Furthermore, the susceptibility of clonal populations Z2 b1 2,
described as biodeme Type I by Andrade e t a l5, has been tested by
Toledo e t a l17, confirming its high susceptibility. In addition, these
authors detec ted a variability of response in the resistanc e to c hemotherapy, of isolates from T. cruzi II c lassified into two different genotypes ( 3 2 and 3 9 ) . Considering its response to chemotherapy, genotype 3 9 isolates presented a dual pattern of response, being either partially resistant or susceptible. According to these authors, this dual behavior has also been observed among other biological properties of stocks of genotype 3 91 7. These results
c onfirmed the assoc iation between phylogenetic diversity with biological characters9 1 5 and chemotherapeutic responses2 3 4 .
The perc entile of c lonal similarity, as defined by Solari et al1 6
by c omparing restric tion fragment length polymorphism ( RFLP) of the 3 3 0 bp fragment of variable regions of kDNA minic irc les in the sc hizo demes o f parental strain and c lo nes, has been previously determined by Campos et al8 as shown in Table 1 .No
c orrelation has been detec ted between the perc entile of similarity and susc eptibility to c hemotherapy. The c lone with the lowest similarity ( 2 1 SF-C5 ) has shown identical susceptibility ( 3 0 % cure rate) to the parental strain ( 2 5 % ) . Apparently this c lone is responsible for the lower responses to c hemotherapy observed in mic e infec ted with the parental strain, when c ompared to those
infec ted with other c omponents of the c lonal population of the 2 1 SF strain. This c ould suggest that phenotypic c harac teristic s, suc h as the response to c hemotherapy might differ even in the c lones with a high degree of genetic similarity, evaluated by the sc hizodeme profiles.
Pr o b ab ly, this var iatio n in susc e ptib ility is r e spo nsib le fo r failure o f c hemo therapy in a perc entage o f patients in endemic areas, even when infec ted with strains o f T. c ru zi that revealed a me dium to high susc e ptib ility.
The c hallenge for the c hemotherapy of Chagas’ disease is to detec t drugs that c o uld eliminate the mo re resistant c lo nes present in the strains with high or medium susc eptibility, as represented by biodeme Type II (T. cruzi II) and for the overall resistant c lones of the biodeme Type III (T. cruzi I) strains, as rec ently demonstrated by Camandaroba e t a l6.
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