Rev. Soc. Bras. Med. Trop. vol.2 número5

E X P E R I M E N T A L C H E M O T H E R A P Y O F S C H I S T O S O M I A S I S . m — L A -

B O R A T O R Y A N D C L IN IC A L T R IA L S W I T H T R I C H L O R P H O N E . A N

O R G A N O P H O S P H O R U S C O M P O U N D *

N a f t a l e K a t z * * , J. P e l l e g r i n o a n d J. P e d r o P e r e i r a

O o g r a r n s t u ã i e s h a v e b e e n c a r r i e á o u t o n m i c e , h a m s t e r s , a n d Cebus

m o r i k e y s e x p e r i m e n t a l l y i n f e c t e d w i t h S ch isto so m a m a n so n i a n d t r e a t e d w i t h t r i c h l o r p h o n e ( 0 , 0 - ã i m e t h y l l - h y á r o x y - 2 , 2 , 2 - t r i c h l o r o e t h y l p h o s p h o n a t e ) . I n m i c e , d e s p i t e a s l i g h t h e p a t i c s h i f t o f s c h i s t o s o m e s , a l i a n i m a i s p r e s e n t e d o o g r a m c h a n g e s w h e n d o s e d , p er os, a t t h e s c h e d u l e s o f 2 0 0 , a n d 1 0 0 m g / k g / / d a y

x

I n 5 v o l u n t e e r s , a s h a r p á r o p i n c h o l i n e s t e r a s e p l a s m a l e v e i w a s o b s e r v e d 2 4 h o u r s a f t e r a s i n g l e o r a l d o s e o f 7 . 5 m g / k g . H o w e v e r , c h o l i n e s t e r a s e l e v e i s r e t u r n e d t o t h e i n i t i a l v a l u e s w i t h i n a p e r i o d o f 11 t o 2 7 d a y s . T r i c h l o r p h o n e w a s t h e n a d m i n i s t e r e d t o 1 2 s c h i s t o s o m e p a t i e n t s ( 7 . 5 m g / k g / d a y , e v e r y f o r t -n i g h t , x 5 ). O n e m o n t h a f t e r t h e r a p y , i n t e r r u p t i o n o f e g g l a y i n g w a s ó b s e r v e a i n 6 p a t i e n t s . L a t e p a r a s i t o l b g i c a l c o n t r o l s h o w e â t h a t a l i t r e a t e d p a t i e n t s c o n t i n u e d t o p a s s v i a b l e S . m a n so n i e g g s w i t h t h e i r s t o o l s .

In 1962, C erf a n d co llab ora to rs (2) in v e stig a te d th e p o ssib ility o f u sin g o rg a n o -p h o s-p h o ru s co m -p o u n d s lesa in ju rio u s to m a m m a ls fo r th e tr e a tm e n t o f h u m a n p a ra sitic h e lm in th s . It w as fo u n d th a t tr ic h lo r p h o n e gave en co u ra g in g resu lts in t h e tr e a tm e n t o f a n cy lo sto m ia sis, a s-ca r ia sis, trich u ria sis, creep in g eru p tion , a n d in te s tin a l s c h ito s o m ia s is . T h ese fin d -in g s w ere su b seq u en tly co n firm ed by T a la a t e t a l . (1 1 ). M ore recen tly , F cr -sy th & R a sh id (4, 5) rep orted t h a t sp aced a d m in istr a tio n of tr ich lo rp h o n e to p a t ­ ie n ts in fe c te d w ith S c h i s t o s o m a h a e m a t o

-b i u m is h ig h ly su cce ssfu l o n red u cin g th e

le v e is o f u rin ary eg g o u tp u ts. H owever, no a n tisc h isto so m a l a c tiv ity cou ld be d etected b y A b d alla e t a l . (1) on m ice, gerbils and

C e r c o p i t h e c u s a e t h í o p s ex p erim en ta lly ín-fe c te d w ith S c h i s t o s o m a m a n s o n i . In view of th e se c o n flic tin g resu lts it w as fe lt w o rth w h ile to co n d u ct a th o ro u g h stu d y on la b orato ry a n im a is as w ell as a lim it-ed c lin ic a i tr ia l w ith tric h lo rp h o n e (0,0--d im e th y l l-h y d r o x y -2 , 2, 2 -trich lo r o eth y l­ p h o sp h o n a te, fig. 1) in sch isto so m ia sis m a n so n i. T h e resu lts are h erein reported.

M ATERIALS AND METHODS

I n f e c t i o n o f a n i m a i s

M ice, h a m ste r s a n d C e b u s m onkeys w ere in fe c te d w ith S . m a n s o n i cercariae (LE str a in , m a in ta in e d in th e laboratory) as d escrib ed elsew h ere (9) .

23 8 R e v . S o e . B r a s . M e d . T r o p V o l . II — N* 5

T r e a t m e n t o f i n f e c t e d a n i m a i s

S ev en w eek s a fte r exp osu re to S . m a n ­

s o n i cerca ria e (10 o rg a n ism s p er a n i­

m a l), group s o f 12 m ice w ere tr ea te d w ith tr ich lorp h on e, p e r o s , fo r 7 co n sec u tiv e days, a t th e d ose le v e is o f 200, 100, 50 a n d 25 m g / k g /d a y . A grou p o f 8 u n trea ted m ice w as le f t a s co n tro l.

G roups o f 7 h a m ste r s (C r i c e t u s a u r a t u s )

w ere tr ea te d fo r 7 c o n sec u tiv e d ays, p e r

o s , a t th e d ose lev eis o f 200, 100, a n d 50

m g /k g /d a y , 8 w eek s a fte r exp osu re (80 cercariae p er a n im a l v ia th e ch ee k p o u c h ) . A group o f u n trea ted h a m ste r s w a s le f t as c o n tr o l.

Fou r C e b u s a p e l l a m a c r o c e p h a l u s w ere d osed p e r o s , 8 to 11 m o n th s a fte r e x ­ p osure (150 to 200 cerca ria e p er a n im a l), w ith 15, a n d 30 m g /k g /d a y x 10 (2 a n i­ m a is ), 30 m g /k g /d a y x 5 (1 a n im a l), a n d

30 m g /k g /d a y every fo r tn ig h t x 5 (1 a n i­ m a l) . O ne u n tr e a te d m o n k ey w a s le f t as c o n tr o l.

O o g r a m a n d d i s t r i b u t i o n o f s c h i s t o s o m e s

T h e m eth o d s a n d criteria u sed for oogram stu d ie s a n d w orm d istrib u tio n w ith in th e h e p a tic p o rta l sy ste m h a v e b een d escrib ed elsew h e re ( 9 ) .

A ltera tio n s in th e o ogram fr o m in t e s ­ tin a l fr a g m e n ts o f m ice a n d h a m ste r s w ere con sid ered s ig n ific a n t w h e n o n e or m ore d ev elo p m en ta l s ta g e s o f im m a tu r e eggs w ere a b se n t. In C e b u s m on k eys, th e a sse ssm e n t o f dru g a c tiv ity w a s b a sed on t h e d isa p p ea ra n ce o f im m a tu r e v ia b le eggs in re c ta l sn ip s o b ta in e d b y m u co sa l cu re tta g e (8) .

C l i n i c a i s t u d i e s

C h o lin estera se p la sm a le v e is w ere d ete r-m in ed in 5 r-m a le a d u lt v o lu n te ers b efo re a n d a t d iffe r e n t p erio d s Cl. 4, 7, 11, 14, 18, a n d 27 d ays) a fte r a sin g le ora l dose of tric h lo rp h o n e (7 .5 m g / k g ) . T h e m e th o d used w a s t h a t d ev elo p ed by R a p p a p o rt e t

a l . (G ra d w oh l (6) a n d is b a sed o n th e

ch a n g e in colou r o f m - n itr o p h e n o l. T w elve a d u lt m a le p a tie n ts w ith a ctiv e sch isto so m ia sis m a n so n i (h e p a to -in te sti-n a l fo rm ) w ere tr ea te d w ith tr ic h lo r p h o sti-n e (ta b lets o f 50 m g ) a t th e d ose le v e i of 7 .5 m g /k g /d a y , every fo r tn ig h t x 5.

T h e r a p e u tic a l a c tiv ity w a s a sse sse d b y o n e r e c ta l b iop sy p erfo rm ed 1-2 m o n th s a fte r co m p letio n o f tr e a tm e n t a n d sto o l e x a m -in a tio n s 150 d a y s a fte r th er a p y .

RE SUL TS

M i c e

T h e d a ta co n ce rn in g th e th e r a p e u tic a l a c tiv ity o f tr ic h lo r p h o n e on m ice e x -p e r im e n ta lly in fe c te d w ith S . m a n s o n i are

su m m a rized in T a b le I . A li m ice d osed w ith 200, a n d 100 m g /k g p r ese n ted oo g ra m c h a n g e s . A t th e d ose le v e is o f 50,, a n d 25 m g /k g , th e p e r c e n ta g e s o f a n im a is w ith a lter ed oogram w ere 3 7 .5 a n d 11.1, r e s-p e c tiv e ly . O n ly a s lig h t h e s-p a tic s h if t w a s ob served a t th e h ig h e s t d ose le v e i e m -p lo y e d .

H a m s t e r s

T ab le II sh o w s th e resu lts o b ta in e d in h a m ste r s. O ogram c h a n g e s w ere o b serv ­ ed o n ly in a n im a is d osed a t to x ic lev eis

(200, a n d 100 m g /k g /d a y x 7 ) . N o s ig ­ n if ic a n t h e p a tic s h if t o ccu rred w ith th e sch ed u le s u se d . D e a d w orm s w ere fo u n d in th e liv er o f th e o n ly su rv iv in g a n im a l o f th e grou p tr e a te d w ith 200 m g /k g .

C e b u s m o n k e y s

A s c a n b e see n fr o m T a b le III, a s lig h t b u t tr a n s ie n t a n tisc h isto so m a l a c tiv ity , a s ju d g ed by o ogram s p ro vided by m u co sa l cu retta g es, cou ld be d e te c te d in C e b u s n .°

1, w ith in th e p erio d o f tr e a tm e n t, a n d in

C e b u s n .° 3, ju s t a fte r th e la s t d o se.

C h o l i n e s t e r a s e p l a s m a l e v e i

A sh a rp drop in p la sm a ch o lin e ste r a se lev ei occu rred in a li v o lu n te ers o n e d a y a fte r tr ic h lo r p h o n e a d m in is tr a tio n . T h e re tu rn to p r e tr e a tm e n t lev eis w a s o b serv ­ ed a fte r 11 to 27 d a y s (F ig . 2) .

C l i n i c a i t r i a l s

-S e t e m b r o - O u t u b r o , 1 9 6 8 R e v . S o c . B r o s . M e d T r o p . 2 3 9

( 1 ) . In fa c t, th e se a u th o rs did n o t disclose a n y a n tisc h isto so m a l a c tiv ity in m ice in ­ fe c te d w ith S . m a n s o n i a n d trea ted w ith tr ic h lo r p h o n e a t th e d aily d ose lev ei of 100 m g /k g fo r 3, 4, a n d 6 co n secu tiv e days. T h e criteria u sed by A b d alla a n d cow or-ker§ to a sse ss th e r a p e u tic a ctiv ity w ere a» com p arison o f th e a v era g e n um ber o f ex -creted eg g s a n d th e ir v ia b ility , b) w orm b urden, c) sep a ra tio n o f sex es, an d d)

h e p a t i c s h i f t o f sc h isto so m e s. B y u sfo g th e o ogram m e th o d , w h ic h is very sen sitiv e to d e te c t ev e n s lig h t d istu rb a n ces o n eg g --la y in g (7 ), w e w ere ab le to d em o n stra te a n u n eq u ivocal a c tio n o f trich lo rp h o n e a g a in st S . m a n s o n i in fe c tio n s in m ice an d h a m ste r s. Our poor re su lts in m on k ey s are in a cco rd a n ce w ith th o se rep orted by Ab­ d a lla e t a l . (1) a n d T h o m p so n (1 2 ).

T h e a d m in istr a tio n o f tr ich lo rp h o n e a t t h e sch ed u le o f 5 m g /k g /d a y x 1 2 t o sc h is-to so m o se p a tie n ts w a s fo llow ed by a very m ark ed d ecrea se o f p la sm a a n d re d -cell c h o lin e ste r a se lev eis w ith a slow recovery

Fig. 2 — C h o l i n e s t e r a s e p l a s m a l e v e i i n 5 v o l u n t e e r s b e f o r e a n d a f t e r a d o s e o f t h i c h l o r p h o n e (7,5 ' m g / k g ) .

/ P- CHOHCC^

CH3-O

C h e m i c a l s t r u c t u r e o f t r i c h l o r p h o n e

terru p tio n o f e g g l a y i n g ( a b s e n c e o f v i a b l e

eggs) in 6 o u t o f 12 p a tie n ts (T ab le T V ) .

L a te p a r a sito lo g ic a l co n tro l sh o w ed v ia b le

S . m a n s o n i eg g s in a li tr e a te d p a tie n ts

(T a b le I V ) .

D ISC U SSIO N

I t is k n o w n t h a t tr ic h lo r p h o n e is a ctiv e a g a in s t a v a riety o f g a str o -in te s tin a l n em a to d e s in sh eep a n d c a ttle (3, 10) a n d ch o lin e stera se in h ib itio n is prob ab ly in -volved in its m e c h a n ism o f a c tio n (1 2 ) .

T á b l e I — A n tis c h is to s o m a l a c t iv it y o f T r ic h lo r p h o n e o n m ic e e x p e r im e n ta lly in f e c t e d w ith S . m a n s o n i . T h e a n im a is w ere s a c r ific e d 3 d a y s a f t e r t h e e n d o f t r e a t m e n t .

S ch ed u le o f tr ea tm e n t (m g /k g /d a y x 7)

p e r o s

N um ber o f m ice

A n im ais dead

M ean w orm burden

D istrib u tio n of sch isto so m es (% )

P er cen ta g e of a n im a is w ith oogram c h a n g e s

Liver P ortal

v ein

M esen teric v esse ls

200 12 3 16 .2

...

35 .6 16.4 4 8 .0 100.0

100 12 2 18.4 22 .8 15.8 61 .4 100.0

50 12 4 28.1 26.2 15.1 58.7 37.5

25 12 3 24 .5 19.0 19 .4

>

6 1 .6 11.1

T a b l e I I — A n tis c h is to s o m a l a c t iv it y o f T r ic h lo r p h o n e o n h a m s te r s e x p e r im e n ta lly in f e c t e d w it h S . m a n s o n i . T h e a n im a is w ere s a c r ific e 1 3 d a y s a fte r t h e e n d o f t r e a t m e n t .

S ch ed u le of tr ea tm e n t ( m g /k g /d a y x 7)

p e r o s

N um b er o f m ice

A n im ais dead

M ean w orm burden

D istrib u tio n o f sch isto so m es (% )

P er cen ta g e o f a n im a is w ith o ogram c h a n g es

L iver Portal

v ein

M esen teric v essels

200 7 6 10.0* 10.0 2 0.0 7 0 .0 100.0

100 7 6 4 1 .0 2 6 .8 12.2 61 .0 100.0

50 7 1 25.1 13.2 33 .8 5 3 .0 0 .0

C ontrol 7 0 3 7 .5 18.2 29.3 52 .5 0 .0

ea<

ggs

10

24 48

8

11

31 48 18

32 11

11

84 42 52 7

2 0 15 29 4

6

21

13 4 24 13

44 26 22 50 34

17 10 44 33 sch isto so m a l a c tiv ity o f T rich lo rp h o n e o n C e b u s m on k eys

e n ta lly in fe c te d w ith S . m a n s o n i .

O ogram s from r e c ta l sn ip s o b ta in e d by c u r e tta g e .

D a y s b efo re (— ) or a fte r ( + ) th e b eg in n in g

o f tr e a tm e n t

V iab le e g g s. S ta g e s

l s t 2n d 3rd 4 th M atu re

_ _{5 16 2 3 1 141}

— 3 22 17 16 3 11

+ 2 45 52 63 49 86

+ 8 0 0 0 0 26

+ 11 5 5 5 0 11

+ 16 12 54 18 60 76

+ 36 14 4 52 34 119

+ 160 4 10 19 15 72

— _{19 8 31 95 163 94}

— 6 10 15 33 48 109

0 8 3 17 17 19

+ 9 0 44 47 0 98

+ 17 7 0 3 12 101

+ 25 35 30 3 14 142

+ 145 32 10 34 27 51

_ _{30 17 16 14 18 47}

— 18 7 2 0 5 38

+ 3 51 36 3 5 20

9 5 0 103 0 21

+ 25 2 4 52 0 2

+ 56 1 55 7 4 72

+ 64 0 0 68 30 45

+ 77 0 0 0 0 23

+ 104 10 14 54 50 77

+ 158 2 0 11 22 8

_ _{63 36 39 58 43 83}

— 1 40 4 38 6 105

+ 13 20 29 26 0 57

+ 70 36 15 68 9 260

+ 82 35 45 73 27 209

+ 82* 4 10 36 4 80

+ 88 29 9 7 5 17

+ 108 14 17 46 20 100

+ 258 2 4 41 5 70

T a b l e I V — R e su lts o f th e tr e a tm e n t o f 12 a d u lt p a tie n ts su ffe r in g fr o m sch isto so m

i-a sis m i-a n so n i w ith T rich lorp h orm e

S c h e d u le : 7 .5 m g /k g , every fo r tn ig h t x 5 (T o ta l d ose = 3 7 .5 m g / k g ) .

P a tie n ts D a y s b efore (— ) or a fte r ( + ) th e co m -p le tio n o f tr e a tm e n t

R e c ta l biop sy V iab le e g g s / D ead eggs

S to ol e x a m in a tio n for v ia b le S . m a n s o n i

eggs

* 1 - 80+ 32

+ 150

300/93 0/56

Po sitive

- 84

2 + 48 58/34 Po sitive

+ 150 0 /0

— 90

3 + 43 55/26 Po sitive

+ 90 82/12

,

4 + 34 169/106 P ositive

+ 150 0/26

— 83

5 + 50 264/0 P ositive

+ 150 106/0

- 82

6 _{+ 55} 148/61 P o sitiv e

+ 150 127/0

- 80 131/45

7 _{+ 60 150/0 N o t done}

- 80 137/50

8 _{+ 57 139/0 N ot done}

- 77

9 _{+ 41 107/171 P o sitiv e}

+ 150 107/60

- 77

10 _{+ 38} 170/41 P o sitiv e

+ 150 0 /0

— 77

11 + 41 158/126 P ositive

+ 150 0/239

— 71

12 + 35 113/112 P o sitiv e

2 4 4 R e v . S o c . B r a s . M e d . T r o p . V o l . n — N ? 5

w h ich did n o t re a ch its n o rm a l v a lu es ev en a t 45 d a y s a fte r t h e en d o f tr e a t­ m e n t (1) . S in c e in th e p r e se n t stu d y, co n firm in g early re p orts by F o rsy th a n d R a sh id (4, 5 ), th e reco very o f c h o lin e s ­ tera se p la sm a le v ei w a s ob served to occur ab ou t a fo r tn ig h t a fte r a sin g le ora l doso o f 7.5 m g /k g o f trich lo rp h o n e, it w a s d ecid -ed to tr e a t sch isto so m e p a tie n ts w ith a 2 -w eek in terv a l b etw e en c o n sec u tiv e doses. A lth ou gh sid e e ffe c ts w ere o f m in or c lin ­ ica i sig n ific a n c e , o n ly a te m p orary in te r -ru p tion o í eg g la y in g co u ld be d e te c te d . A li tr ea te d p a tie n ts co n tin u e d to p ass viable eggs a fte r th e co m p letio n o f t r e a t ­ m e n t.

A ccord ing to F o rsy th a n d R a sh id (5),

th e c o n flic tin g re p orts fr o m w ork ers w h o h a v e u sed tr ic h lo r p h o n e fo r tr e a tin g s c h is ­ to so m ia sis are in p a rt e x p a in e d b y th e in c lu sio n o f b o th S . m a n s o n i a n d S . h a e

-m a t o b i u -m p a tie n ts in th e ir se r ie s. W h en

tr ich lo rp h o n e w as g iv en by m o u th in s i n ­ g le sp a ced doses o f 7 .5 m g /k g to 73 p a tie n ts in fe c te d w ith S . h a e m a t d b m m , ali cea sed v o id in g eg g s o f th e p a ra site in th e u rin e (5) . H ow ever, a s fa r a s S . m a n s o n i

in fe c tio n s are co n cern ed , th e ç o o r th e r -a p eu tic-a l -a c tiv ity o f tr ic h lo r p h o n e -a n d th e w ell k n o w n to x ic p co p ertie s o f h ig h e r d o sa -g es o f th is o r-g a n o p h o sp h o ru s co m p cu n d for m a n are very d isco u ra g in g fa c to rs to su p -p ort th e co n tin u a tio n o f fu rth e r clin ic a i tr ia ls.

R E S U M O

E n s a i o s p r é - c l i n i c o s f o r a m r e a l i z a d o s e m c a m u n ã o n g o s , h a m s t e r s e m a ­ c a c o s C ebus e x p e r i m e n t a l m e n t e i n f e c t a d o s c o m S ch isto so m a m a n so n i e t r a ­ t a d o s c o m t r i c h l o r p h o n e (0 - 0 - d i m e t i l l - h i ã r o x i - 2 , 2 , 2 - t r i c l o r o e t i l f o s f o n a t o ) . E m c a m u n ã o n g o s , a p e s a r d e t e r s i d o d i s c r e t o o d e s l o c a m e n t o d o s v e r m e s p a r a o f í g a d o , e m t o d o s o s a n i m a i s h o u v e a l t e r a ç õ e s d o o o g r a m a , q u & n d o f o r a m a d m i n i s t r a d o s 2 0 0 e 1 0 0 m g / k g / d i a x 7 , p er o s . E m h a m s t e r s , a a t i v i d a d e s ô b r e o s e s q u i s t o s s o m o s , s ó p ô d e s e r d e t e c t a d a c o m d o s e s t ó x i c a s . E m m a c a c o s , o t r i c h l o r p h o n e m o s t r o u a ç ã o i n s i g n i f i c a n t e , m e s m o a p ó s a d m i n i s t r a ç ã o o r a l d e 3 0 m g / k g / d i a d u r a n t e 1 0 d i a s c o n s e c u t i v o s .

E m 5 v o l u n t á r i o s , u m a a c e n t u ã a ã a b a i x a d a c o l i n e s t e r a s e p l a s m á t i c a , f o i o b s e r v a d a 2 4 h o r a s a p ó s u m a ú n i c a d o s e o r a l d e 7 ,5 m g / k g . T o d a v i a , o n í v e l d a c o l i n e s t e r a s e v o l t o u a o s v a l o r e s i n i c i a i s n o p e r í o d o d e 11 a 2 7 d i a s . O t r i ­

c h l o r p h o n e f o i a d m i n i s t r a d o a 1 2 p a c i e n t e s c o m e s q u i s t o s s o m o s e m a n s o n i (7 .5 m g / k g / ã i a , q u i n z e n a l m e n t e , x 5 ) . U m m ê s a p ó s o f i m d o t r a t a m e n t o , a i n ­ t e r r u p ç ã o d a p o s t u r a d o s v e r m e s f o i o b s e r v a d a e m 6 p a c i e n t e s . O c o n t r o l e p a r a s i t o l ó g i c o t a r d i o r e v e l o u a i n e f i c á c i a d o t r a t a m e n t o e m t o d o s o s p a c i e n t e s .

REFERENCES

1 — ABDALLA, A ., SAIF, M ., TAHA, A ., ASHMAWY, H ., T AW FIK J„ A B

-D EL-FATTAH , F . , SABET, S . &

A BDEL-M EG UID, M . — E v a lu a tio n o f a n o rg a n o -p h o sp h o ru s com pou d, d ip terex, in th e tr e a tm e n t o f b ilh a r -z ia sis. J . E g y p t. M ed . A s s ., 48: .. 262-273, 1965.

2 — CERF, J . , LEBRUN, A . & D IE -R ICH X, J . — A n e w a p p r o a ch to h e lm in th ia s is con tr ol: th e u se o f organ op h osp h orou s co m p o u n d . A m . J . T rop . M ep . H y g ., 11: 514-517, 1962.

3 — ELSLAGER, E . F . & THOM PSON,

P . E . — P a ra site C h e m o th e r a p y . A n n . R ev . P h a r m a c o l., 2: 193-214, 1962.

4 — FO RSYT H, D . M . & R ASH ID, C . — T r e a tm e n t o f u rin a ry s c h isto so m ia ­ s is . P r a c tice a n d th e o r y . L a n cet, 1: 130-133, 1967.

5 — FO RSYT H, D . M . & R ASH ID, C . — T r e a tm e n t o f u rin a ry s c h isto so m ia ­ sis w ith tr ic h lo r p h o n e . L a n cet, 2: 909-912, 1967.

6 — GRADW OHL, R . B . H . — C lin ica i

L ab oratory M eth o d s a n d D ia g n o sis. V o l. 1. p p . 143. 6 th E d itio n . T h e

C . V . M osby C o. 1963. ,

7 — PELLEGRINO, J . & FARIA, J . — E arly e ffe c t o f T ris (p a m in o p h e -n y l) ca rb o n iu m p a m o a te o n th e e g g -la y in g o f S c h i s t o s o m a m a n s o n i .

J . P a r a s it., 50: 587, 1964.

I-4

S e t e m b r o - O u t u b r o , 1 9 6 8 R e v . S o c . B r a s . M e d . T r o p 24 5

VEIRA, C . A . & OKABE, K . — R e c ­ ta l biop sy a n d m u co sa l cu re tta g e in

C e b u s m o n k ey s ex p er im en ta lly i n ­

fe c te d w ith S c h i s t o s o m a m a n s o n i

a n d S c h i s t o s o m a j a p o n i c u m . J . P a r a s it., 51: 617-621, 1965.

9 — PELLEGRINO, J . KATZ, N . &

SCHERRER, J . F . — C ogram stu d ie s w ith H iy ca n th o n e , a n e w a n tis c h is -to so m a l a g e n t. J . P a r a s it., 53: . . . . 55-59, 1967.

10 — STANDEN, O ..D. — C h em oth erap y o f H elm in th ic I n fe c tio n s . I n “E x p e­

rim e n ta l C h em o th era p y ” . V ol. I. E d . by S ch n itzer, R . J . a n d H awk-in g, F . C h ap ter 20, p p . 701-892. A cad em ic Press I n c . , L ondon, 1963 11 — TALAAT, S . M . , AMIN, N . & EL