rate of Necator americanus in both areas was much higher than that of Ancylostoma duodenale , and also that the prevalence rate of S. stercoralis of the IMIP ...
Rev. Inst. Med. trop. São Paulo 3 0 (4): 313-321, julho-agosto, 1988
PARASITOLOGICAL AND SEROLOGICAL STUDIES ON AMOEBIASIS AND OTHER INTESTINAL PARASITIC INFECTIONS IN RECIFE AND ITS SUBURBAN AREA, NORTHEAST BRAZIL
M i t s u O K A Z A K I (1), M a s a i c h i O K A Z A K I (1), P a u l o M I R A N D A ( 2 ) J o a n N E T O (3), V i l n e i d e D I E G U E S (3), J o a n A L V E S (3), M a c h a d o C A U A S (3), M a s a n o b u T A N A B E (2, 4), S e i k i K O B A Y A S H I (2, 4), N o b u a k i K A N E K O (2, 4 ) , K o u i c h i N A G A K U R A (2, 5), M a s a s h i K O B A Y A S H I (2, 6), S e v e r a M O T T A (2, 4, 7), S e i k i r
T A T E N O (2, 4 ) & T s u t o m u T A K E U C H I (4).
S U M M A R Y
P a r a s i t o l o g i c a l e x a m i n a t i o n s were c a r r i e d o u t d u r i n g A p r i l to A u g u s t , 1987, w i t h 187 o u t - p a t i e n t s of t h e I M I P h o s p i t a l , l o c a t e d i n t h e c e n t e r of R e c i f e C i t y , and 464 i n h a b i t a n t s of s e v e r a l v i l l a g e s a r o u n d C a b o C i t y , 50 K m s o u t h e a s t of R e c i f e , P e r n a m b u c o , B r a z i l . A p p r o x i m a t e l y 7 1 % of t h e I M I P p a t i e n t s a n d 92% of the C a b o i n h a b i t a n t s were i n f e c t e d w i t h at l e a s t o n e s p e c i e s of i n t e s t i n a l p a r a s i t e . T h e r e w a s m i n i m u m d i f f e r e n c e i n t h e p r e v a l e n c e r a t e of T r i c h u r i s t r i c h i u r a b e t w e e n two a r e a s , w h e r e a s the p r e v a l e n c e r a t e s of A s c a r i s l u m b r i c o i d e s , h o o k w o r m s , S t r o n g y l o i d e s s t e r c o r a l i s , S c h i s t o s o m a m a n s o n i and E n t a m o e b a h i s t o l y t i c a w e r e h i g h e r i n t h e i n h a b i t a n t s of t h e C a b o C i t y area. O n l y G i a r d i a l a m b l i a w a s m o r e p r e v a l e n t i n t h e o u t - p a t i e n t s of I M I P h o s p i t a l . T e s t t u b e c u l t i v a t i o n r e v e a l e d t h a t t h e p r e v a l e n c e rate of N e c a t o r a m e r i c a n u s i n b o t h areas w a s m u c h h i g h e r t h a n t h a t of A n c y l o s t o m a d u o d e n a l e , a n d a l s o t h a t t h e p r e v a l e n c e rate of S . s t e r c o r a l i s of t h e I M I P p a t i e n t s a n d C a b o i n h a b i t a n t s w e r e 4.5% and 9.6%, r e s p e c t i v e l y . S i x h u n d r e d a n d f i f t e e n s e r a w e r e s e r o l o g i c a l l y e x a m i n e d for a m o e b i a s i s b y t h e gel d i f f u s i o n p r e c i p i t a t i o n t e s t ( G D P ) and e n z y m e l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) u s i n g t h e a n t i g e n p r e p a r e d f r o m a x e n i c a l l y c u l t u r e d t r o p h o z o i t e of E . h i s t o l y t i c a ( s t r a i n H M - I T M S S ) . N o p o s i t i v e r e a c t i o n w a s o b s e r v e d i n all of the s e r a as e x a m i n e d b y G D P , w h i l e 32 o u t of 615 s e r a w e r e p o s i t i v e o n E L I S A .
K E Y W O R D S : Parasitic helminths; Parasitic protozoa; Amoebiasis; Northeast Brazil; S t o o l e x a m i n a t i o n ; S e r o - e p i d e m i o l o g y .
INTRODUCTION A n a n a l y s i s of t h e a n n u a l s t a t i s t i c s o n the population in northeast Brazil demonstrated
t h a t t h e r e h a s b e e n a h i g h l e v e l of i n f a n t i l e m o r t a l i t y . M a i n c a u s e s of t h i s h i g h m o r t a l i t y
were
(1) S e c r e t a r i a d e S a ú d e , P e r n a m b u c o , B r a z i l . (2) L a b o r a t ó r i o d e I m u n o p a t o l o g i a
Prof. K e i z o A s a m i , U n i v e r s i d a d e F e d e r a l de P e r n a m b u c o ( U F P E ) , P e r n a m b u c o , B r a z i l .
(3) H o s p i t a l o f I n s t i t u t o M a t e r n o I n f a n t i l d e P e r n a m b u c o , P e r n a m b u c o , B r a z i l . (4) D e p a r t m e n t o f P a r a s i t o l o g y , S c h o o l o f M e d i c i n e , K e i o U n i v e r s i t y ,
Japan.
(5) D e p a r t m e n t o f P a r a s i t o l o g y , S c h o o l o f M e d i c i n e , T o k a i U n i v e r s i t y , J a p a n (6) D e p a r t m e n t o f P a r a s i t o l o g y , S c h o o l o f M e d i c i n e , C h i b a U n i v e r s i t y ,
Japan.
(7) D e p a r t m e n t o f B i o p h y s i c s a n d R a d i o b i o l o g y , U F P E , P e r n a m b u c o , B r a s i l . A d d r e s s for c o r r e s p o n d e n c e : Prof. M a s a n o b u T a n a b e . D e p a r t m e n t of P a r a s i t o l o g y , S c h o o l of M e d i c i n e , K e i o S h i n a n o m a c h i 3 5 , S h i n j u k u - k u , T o k y o 160, J a p a n
University.
O K A Z A K I , M; O K A Z A K I . M; M I R A N D A , P ; NETO, J.; D I E G U E S . V ;A L V E S , J.; CAUAS. M ;T A NA B E S H I , S . ;K A N E K O , N., N A G A K U R A , K . . K O B A Y A S H I
M ; MOTTA
M
K O B A Y A
S.;T A T E N O , S. & T A K E U C H I , T. — Parasito
l o g i c a l a n d s e r o l o g i c a l s t u d i e s o n a m o e b i a s i s a n d o t h e r i n t e s t i n a l p a r a s i t i c i n f e c t i o n s in R e c i f e a n d its s u b u r b a n area. Northeast Brazil.
R e v . I n s t . M e d . t r o p . S ã o P a u l o 30 ( 4 ) : 3 1 3 - 3 2 1 , 1988.
a s s u m e d to b e m a l n u t r i t i o n a n d i n f e c t i o u s d i s e a ses b y b a c t e r i a , v i r u s a n d p a r a s i t e
30
A s a m i ( L I K A ) , F e d e r a l U n i v e r s i t y of P e r n a m -
. Parasitic
buco. F r e s h stools were collected w i t h i n a couple
infections do not necessarily cause serious disea
of d a y s after t h e f i r s t v i s i t . I n h a b i t a n t s i n s e v e r a l
se i n t h e h u m a n h o s t , b u t t h e y i n t e r f e r e
v i l l a g e s a r o u n d C a b o C i t y , l o c a t e d 50 K m s o u
with
the n u t r i t i o n a l s t a t u s a n d ultimately lead t h e m
t h e a s t of R e c i f e , a l s o p r i m a r i l y r e c e i v e d g e n e r a l
m o r e s u s c e p t i b l e to o t h e r d i s e a s e s . P r e v a l e n c e
m e d i c a l e x a m i n a t i o n , a n d their age, s e x , m e d i
r a t e of p a r a s i t i c i n f e c t i o n s c a n , t h e r e f o r e , be o n e
c a l h i s t o r y as well as s o c i o - e c o n o m i c state were
of t h e m a j o r p a r a m e t e r s of l o c a l p u b l i c h e a l t h
r e c o r d e d . B l o o d a n d s t o o l s were c o l l e c t e d a t t h i s
of t h e i n h a b i t a n t s a n y w h e r e .
time, a n d sera were s e p a r a t e d in L I K A
T h e p r e v a l e n c e of E n t a m o e b a h i s t o l y t i c a i n d e v e l o p i n g c o u n t r i e s i s o f t e n a s s u m e d to b e high, frequently without supporting data. Major r e a s o n s for t h i s m a y be t h a t p r o p e r i d e n t i f i c a t i o n of E . h i s t o l y t i c a c y s t i n s t o o l is d i f f i c u l t w i t h o u t professional experience, and that reliable diag n o s i s of a m o e b i a s i s i s o f t e n n o t a t t a i n a b l e b y stool e x a m i n a t i o n alone. Now, serologic proce d u r e s h a v e b e e n c o m m o n l y u t i l i z e d for t h e d i a g n o s i s of a m o e b i a s i s i n m a n y c o u n t r i e s b e c a u s e these tests are h i g h l y specific a n d h a v e a suffi 9
cient sensitivity '
1 0
*
1 3 , 1 6
19
2 5
. In northeast Brazil,
t h e p r e v a l e n c e of E . h i s t o l y t i c a h a s b e e n e s t i m a t e d to b e h i g h , b u t t h e i n c i d e n c e of a m o e b i c l i v e r 20
a b s c e s s a t a u t o p s y w a s r e p o r t e d to b e v e r y l o w .
within
4 h o u r s after b l e e d i n g . A single stool specimen collected as above 1 1
w a s e x a m i n e d b y t h e d i r e c t s m e a r , b y t h e form a l i n ether centrifugation technique ( M G L m e 1
t h o d r , a n d b y test tube c u l t i v a t i o n m e t h o d
8
to
d i s t i n g u i s h f i l a r i f o r m l a r v e of h o o k w o r m s a n d S t r o n g y l o i d e s . N i n e t y e i g h t s t o o l s , i n c l u d i n g 16 p o s i t i v e s t o o l s for E . h i s t o l y t i c a c y s t s , w e r e ino3
c u l a t e d i n t o t h e B a l a m u t h m e d i u m a n d t h e trop h o z o i t e g r o w t h w e r e e x a m i n e d after c u l t i v a t i o n for 72 h o u r s a t 2 7 ° C . A l l p a r a s i t o l o g i c a l e x a m i n a t i o n of s t o o l s w e r e d o n e b y a s i n g l e m e d i c a l parasitologist, who h a d sufficient experience on parasitological diagnosis.
S u c h d i s c r e p a n c i e s a r e a p p a r e n t l y d u e to l a c k T h e a n t i g e n u s e d for s e r o l o g i c t e s t of a m o e -
in reliable d a t a on the e p i d e m i o l o g y a n d e s t i m a tion on a n n u a l morbidity by this parasite, since
b i a s i s w a s p r e p a r e d b y t h e m e t h o d of T A K E U -
s e r o l o g i c a l e x a m i n a t i o n for a m o e b i a s i s i s s t i l l
CHI & KOBAYASHI
not c o m m o n in this area.
h i s t o l y t i c a ( H M 1 ; I M S S ) , c u l t u r e d i n B I S 33
2 5
f r o m a x e n i c s t r a i n of E .
6
medium . T h e lyophilized antigen was dissolved P a r a s i t o l o g i c a l e x a m i n a t i o n of
intestinal
i n 20 raM s o d i u m p h o s p h a t e buffer, p H 7.2. A f t e r
helminths and protozoa, and serological exami-
s t i r r i n g for 30 m i n u n d e r i c e c o l d c o n d i t i o n , the
n a t i o n for a m o e b i a s i s w e r e c a r r i e d o u t
s o l u t i o n w a s c e n t r i f u g e d a t 15,000 g for 30 m i n
during
A p r i l to A u g u s t , 1987, i n R e c i f e a n d i t s s u b u r b a n
at 4
a r e a . T h i s r e p o r t p r e s e n t s t h e r e s u l t s of t h e s e
M i l l e x - G S filter ( M i l l i p o r e C o . B e d f o r d , M A , U S A )
s t u d i e s a s a n a t t e m p t to d e f i n e a r e l i a b l e p r e v a
a t 0.2ptm p o r o s i t y a n d k e p t a t — 70°C u n t i l u s e .
l ,
C . T h e s u p e r n a t a n t fluid w a s filtered by
lence rate in intestinal h e l m i n t h s a n d protozoa, i n p a r t i c u l a r t h a t of a m o e b i a s i s i n t h i s a r e a .
A l l s e r a w e r e t e s t e d for a n t i b o d i e s to E . h i s t o l y t i c a b y the gel diffusion p r e c i p i t a t i o n (GDP)
MATERIALS AND METHODS
1 3
s a y ( E L I S A ) a c c o r d i n g to t h e m o d i f i e d F r o m A p r i l to A u g u s t , 1987, t h e o u t - p a t i e n t s
test
a n d e n z y m e linked i m m u n o s o r b e n t as method
1 5
of M A T S U D A et a l . . E L I S A w a s p e r f o r m e d i n
of t h e h o s p i t a l of I n s t i t u t o M a t e r n o I n f a n t i l d e
96 w e l l m i c r o l i t e r
P e r n a m b u c o ( I M I P ) , l o c a t e d i n t h e c e n t e r of R e -
Co. Denmark). T h e optimum antigen concentra-
flat b o t t o m e d plate
(NUNC
cife, w e r e s e l e c t e d a t r a n d o m (10 to 30 p a t i e n t s
tion was determined by checker board titration
per d a y ) a n d their g e n e r a l h e a l t h c o n d i t i o n s were
b e t w e e n s e r i a l d i l u t i o n of a n t i g e n a g a i n s t p o s i -
checked. O n this occasion, age,
tive a n d negative reference sera. H a l f n u m b e r
s e x , t h e p l a c e of r e s i d e n c e a s w e l l a s s o c i o - e c o -
of w e l l s i n a p l a t e w e r e s e n s i t i z e d w i t h lOOpd of
preliminarily
were
the a n t i g e n prepared as a b o v e at concentration
recorded, a n d the p e r i p h e r a l blood w a s collec-
of 10/xg p r o t e i n p e r m l i n 50 m M c a r b o n a t e buffer,
ted. S e r a were s e p a r a t e d by centrifugation
p H 8.15, i n a m o i s t c h a m b e r a t 3 7 ' C for 2 h o u r s .
n o m i c s t a t e o f t h e f a m i l y of t h e p a t i e n t
at
the L a b o r a t ó r i o de I m u n o p a t o l o g i a Prof. K e i z o
N o n - s e n s i t i z e d w e l l s w e r e f i l l e d w i t h 100/xl of c a r
O K A Z A K I , M ; O K A Z A K I , M.; M I R A N D A , P . ; N E T O , J . , D I E G U E S , V . ; A L V E S , J . ; C A U A S . M . ; T A N ; > H E M.: S H I , S . ; K A N E K O , N . : N A G A K U R A , K ; K O B A Y A S H I , M.: M O T T A
K O B A Y A
S..T A T E N O , S. & T A K E U C H I , T. — Parasito
l o g i c a l a n d s e r o l o g i c a l s t u d i e s o n a m o e b i a s i s a n d other i n t e s t i n a l p a r a s i t i c infections in R e c i f e a n d its area, Northeast Brazil.
suburban
R e v . I n s t . M e d . t r o p . S a o P a u l o , 30 ( 4 ) : 3 1 3 3 2 1 , 1 9 8 8 .
S c : t h e c o r r e c t e d a b s o r b a n c e of s a m p l e s e rum
b o n a t e buffer alone. T h e plate w a s w a s h e d three t i m e s w i t h 150 m M s o d i u m p h o s p h a t e - b u f f e r e d s a l i n e , p H 7.2, c o n t a i n i n g 0.05% T w e e n 20 ( P B S / T ) . A f t e r w a s h i n g t h e w e l l s , 200 /ml of P B S / T c o n
Protein concentration was determined
taining 2% bovine serum albumin ( B S A , S i g m a
t h e m e t h o d of L O W R Y et a l . , w i t h b o v i n e se
C h e m . , S t L o i s , M O , U S A ) w a s a d d e d to a l l w e l l s
r u m a l b u m i n (fraction V) as a s t a n d a r d .
to b l o c k n o n - s p e c i f i c i m m u n o g l o b u l i n
by
1 2
binding, RESULTS
a n d t h e p l a t e w a s i n c u b a t e d a t 37 ° C for 60 m i n . After w a s h i n g the wells w i t h P B S / T , test sera 1%
S e x , age distribution and socio-economic
B S A were p l a c e d in a s e n s i t i z e d a n d n o n sen
c o n d i t i o n of t h e o u t - p a t i e n t s of I M I P h o s p i t a l
d i l u t e d 200 t i m e s w i t h P B S / T c o n t a i n i n g
sitized wells a n d the plate further i n c u b a t e d at
a n d the i n h a b i t a n t s i n C a b o C i t y area were s u m
37 C for 60 m i n . A f t e r w a s h i n g w i t h P B S / T , 100
m a r i z e d i n T a b l e 1. V i r t u a l l y a l l of the I M I P p a
/ x l of h o r s e r a d i s h p e r o x i d a s e ( H R P ) l a b e l l e d a n
t i e n t s w e r e u n d e r 9 y e a r s o l d , a n d o n l y 4% of
ti-human I g G rabbit immunoglobulin
(Miles
t h e m w e r e f r o m 10 to 12 y e a r s o l d . M o s t of t h e
S c i e n t i f i c , N a p e r v i l l e , I L , U S A ) , 10 fold d i l u t e d ,
I M I P p a t i e n t s i n h a b i t e d i n t h e d o w n t o w n or ur
was added a n d i n c u b a t i o n was carried out
b a n a r e a of R e c i f e . A p p r o x i m a t e l y o n e - t h i r d of
4
at
37°C for 60 m i n . A f t e r w a s h i n g t h e w e l l s 3 t i m e s
the C a b o i n h a b i t a n t s who took our h e a l t h e x a
w i t h P B S / T , 200 /ml of t h e s u b s t r a t e s o l u t i o n , i.e.,
m i n a t i o n were u n d e r 9 y e a r s old, another one-
30 m g d i a m m o n i u m s a l t of 2 , 2 ' a z i n o - b i s ( 3 - e t h y l benzthiazoline-sulfonic acid) ( S i g m a Chem.) dis
TABLE 1
s o l v e d i n a m i x t u r e of 100 m M c i t r i c a c i d a n d
S e x , a g e d i s t r i b u t i o n a n d s o c i o - e c o n o m i c f e a t u r e of t h e o u t p a
200 m M s o d i u m p h o s p h a t e d i b a s i c , p r i 4 . 6 , w i t h 10
of H 0 2
2
t i e n t s o f I M I P h o s p i t a l a n d t h e i n h a b i t a n t s of C a b o C i t y a r e a
( 3 0 % ) , w a s a d d e d to e a c h w e l l a n d
t h e n t h e p l a t e w a s k e p t a t r o o m t e m p e r a t u r e for 60 m i n . S u b s e q u e n t l y , t h e a b s o r b a n c e of r e a c t i o n m i x t u r e w a s m e a s u r e d b y E I A R e a d e r (Mo
Sex
IMIP
Cabo
patients
inhabitants
Male
100 ( 5 3 . 5 % )
178 (38.4% )
Female
87 ( 4 6 . 5 % )
286 (61.6% )
0 9
179 ( 9 6 . 0 % )
156 (33.6% )
d e l 2550, B i o R a d L a b . , T o k y o ) a t 405 n m . T o c o r r e c t d a i l y v a r i a t i o n of a b s o r b a n c e , t r i p l e t of positive reference s e r u m (isolated from a J a p a
Age
nese patient with confirmed amoebic liver abs
(4.0%)
153 ( 3 3 . 0 % )
10-19
8
20-39
0
81 ( 1 7 . 5 % )
40
: 3 1 3 3 2 1 . 1988.
TABLE 3 P r e v a l e n c e of h e l m i n t h s in t h e out p a t i e n t s of I M I P h o s p i t a l a n d the i n h a b i t a n t s of C a b o C i t y area Parasites
Cabo inhabitants
I M I P patients 0 12 y r s
0-12 y r s
13 y r s -
Total
Ascaris lumbricoides
42 ( 2 6 . 9 ' , )
121 (71.1% )
164 (56.7% )
285 (62.1% )
Trichuris trichiura
75 (48.1% ) 10 (6.4% )
107 (62.9% )
169 (58.5%)
276 (60.1% )
110 (64.7% )
196 (67.8% )
306 (66.7% )
95 (55.9% )
184 (63.7% )
279 (60.8% )
Hookworm Neeator a m e r i c a n u s
7
(4.5% )
Ancylostoma duodenale unknown
1
(0.6% )
2
(1.2%)
4
(1.4%)
6
(1.3%)
2
(1.3% ) (4.5% )
14
(8.2%)
7
(2.4%)
21
(4.6%)
18 (10.6% )
26
(9.0% )
44
(9.6%)
2
(0.4%)
Strongyloides stercoralis
7
Enterobius vermicularis
0
Schistosoma mansoni
2
Hymenolepis diminuta
0
2 (1.3% )
(1.2%)
0
19 (11.2% )
25
1
(0.6% )
(8.7% )
0
44 (11.2% ) 1
(0.2%)
V a l u e s i n p a r e n t h e s e s r e p r e s e n t t h e p r e v a l e n c e r a t e (% ) of e a c h r e g i o n .
TABLE 4 P r e v a l e n c e of i n t e s t i n a l p r o t o z o a s i n the o u t p a t i e n t s of I M I P h o s p i t a l a n d t h e i n h a b i t a n t s of C a b o C i t y area Parasites
I M I P patients
Cabo inhabitants
0 12 y r s
0-12 y r s
Giardia lamblia
41 (26.3% )
24 (14.1% )
23
(8.0%)
47 (10.2% )
E n t a m o e b a coli
27 (17.3% )
74 (43.5% )
91 (31.5% )
165 (35.9% )
Entamoeba histolytica
12
18 (10.6% ) 7 (4.1%)
36 (12.5% )
54 (11.8% )
35 (20.6% )
57 (19.7% )
Entamoeba
hartmanni
(7.7% )
0
E n d o l i m a x nana
21 (13.5% )
2
13 y r s -
8
(2.8% )
Total
15
(3.3%)
92 (20.0%)
(1.2%)
10
(3.5%)
12
(2.6%)
17 (10.0% )
26
(9.0%)
43
(9.4%)
14
(4.8%)
21
(4.6%)
Chilomastix mesnili
1
(0.6% )
Iodamoeba buetschlii
2
(1.3%)
Trichomonas hominis
4
(2.6% )
7
Isospora belli
2
(1.3% )
0
0
B a l a n t i d i u m coli
0
0
2
(4.1%)
0 (0.7%)
2
(0.4% )
V a l u e s i n p a r e n t h e s e s r e p r e s e n t t h e p r e v a l e n c e r a t e (% ) of e a c h r e g i o n .
ca (7.7% ), a n d T r i c h o m o n a s h o m i n i s (2.6% ). C h i -
T r o p h o z o i t e of B a l a n t i d i u m c o l i w a s a l s o f o u n d
lomastix
i n two p r i m a r y s c h o o l p u p i l s i n C a b o .
mesnili
(one
case),
Iodamoeba
b u e t s c h l i i (2 c a s e s ) a n d I s o s p o r a b e l l i (2 c a s e s ) were also f o u n d . I n the C a b o i n h a b i t a n t s , E. coli i n f e c t i o n (35.9% ) w a s t h e m o s t p r e v a l e n t , f o l i o w e d b y E . n a n a (20.0% ), E . h i s t o l y t i c a (11.8% ),
M u l t i p l e i n f e c t i o n w a s o b s e r v e d a t 75.8% of the I M I P p a t i e n t s w i t h p a r a s i t i c i n f e c t i o n , w h i l e
G . l a m b l i a (10 2% ), I. b u e t s c h l i i (9 4% ), T . h o m i -
a h i g h e r r a t e of m u l t i p l e i n f e c t i o n (84.6%) w a s
n i s (4.6%) a n d E n t a m o e b a h a r t m a n n i
d e m o n s t r a t e d i n t h e C a b o i n h a b i t a n t s ( F i g . 1).
(3.3%).
O K A Z A K I , M., O K A Z A K I , M.; M I R A N D A , P.; N E T O , J . ; D I E G U E S , V.; A L V E S , J . ; C A U A S , M.; T A N A B E , S H I , S.; K A N E K O ,
N.;N A G A K U R A ,
K . ; K O B A Y A S H I , M.; M O T T A , S . ; T A T E N O ,
M
K O B A Y A
S. & T A K E U C H I , T , — Parasito
logical a n d serological studies o n amoebiasis a n d other intestinal parasitic infections in Recife a n d its s u b u r b a n area, Northeast Brazil.
R e v . I n s t . M e d . t r o p . S ã o P a u l o , 30 (4):
50
IMIP H o s p i t a l
40 30 20
k
10 -
o L 50
Cabo Area
40 9i
30 20 10 0 «3
4 5 6 7 8 9
Degree of m u l t i p l e i n f e c t i o n (Number of d i f f e r e n t kind of parasites/person) F i g . 1 — P r e v a l e n c e r a t e of m u l t i p l e i n f e c t i o n of i n t e s t i n a l p a r a sites i n the o u t p a t i e n t s of I M I P h o s p i t a l a n d i n h a b i t a n t s of
313 321, 1988.
b i t a n t s as m e n t i o n e d a b o v e ; t h u s , stool e x a m i n a t i o n i n d i c a t e d t h a t the o v e r a l l p r e v a l e n c e rate was 10.7%. A s s h o w n i n T a b l e 5, m o r e o v e r , all sera (615 s a m p l e s ) o b t a i n e d f r o m b o t h I M I P pat i e n t s and C a b o i n h a b i t a n t s were e x a m i n e d ser o l o g i c a l l y b y G D P . N o p o s i t i v e r e a c t i o n w a s obs e r v e d i n all of the sera e x a m i n e d , a l t h o u g h t h r e e positive control sera, obtained from J a p a n e s e patients with confirmed amoebic liver abscess, f o r m e d 3 to 5 c l e a r p r e c i p i t a t i o n l i n e s . I n a d d i t i o n to G D P , w e t r i e d t o e s t a b l i s h an E L I S A to d e t e c t a n t i b o d y a g a i n s t E . h i s t o l y t i c a a n t i g e n . I n t h e p r i m a r y e x p e r i m e n t , as s h o w n i n F i g . 2, the s p e c i f i c i t y and s e n s i t i v i t y of our E L I S A was evaluated. S e r u m s a m p l e s from 17 c a s e s w i t h c o n f i r m e d a m o e b i a s i s and 30 healt h y J a p a n e s e s t u d e n t s w e r e d i l u t e d 200 t i m e s and e x a m i n e d b y our E L I S A m e t h o d . S i n c e m e a n a b s o r b a n c e v a l u e of the n e g a t i v e c o n t r o l s was 0.033 ± 0.012, the u p p e r l i m i t (cut-off v a l u e ) of the a b s o r b a n c e of n e g a t i v e c o n t r o l s was c a l c u lated to b e 0.1 b y m u l t i p l y i n g the m e a n absorb a n c e v a l u e b y 3. T h e a b s o r b a n c e of p o s i t i v e c o n t r o l s e r a i s o l a t e d f r o m 17 J a p a n e s e p a t i e n t s w i t h a m o e b i c l i v e r a b s c e s s and c o l i t i s w a s far a b o v e t h i s v a l u e (0.280 to 1.500). H e n c e , the absorb a n c e of 0.1 w a s c o n s i d e r e d t o b e r e a s o n a b l e as
C a b o C i t y area. Absorbance at 405 nm ( crude extract antigen of amoeba )
TABLE 5 R e s u l t of s e r o l o g i c a l e x a m i n a t i o n for a m o e b i a s i s b y G D P a n d ELISA
on the o u t p a t i e n t s of I M I P h o s p i t a l a n d the i n h a b i
Healthy control I (Japanese) 1 Amebiasis (Japanese)
tants of C a b o C i t y area ELISA
GDP Cyst
+
—
Total
Trichuriasis Hookworm disease
+
0
12
2
10
12
-
0
144
15
129
144
Total
0
156
17
139
156
+
0
54
7
47
54
0
405
8
397
405
E.nana infection I. bütschlH Infection
Strongyloidiasis
IMIP patients
Cabo inhabitants Total
0
459
15
444
459
+
0
66
9
57
66
0
549
23
526
549
0
615
32
583
615
Schistosomiasis mansoni i-fiflli Infection
* n « Total Total
i.b&m Infection infection
F i g . 2 — R e s u l t of c r o s s r e a c t i v i t y of s e r a i s o l a t e d f r o m t h e
C y s t s of E . h i s t o l y t i c a were f o u n d at 7.7% of t h e I M I P p a t i e n t s and 11.8% of the C a b o i n h a
out
p a t i e n t s of I M I P h o s p i t a l a n d i n h a b i t a n t s of C a b o
City
a r e a , w h o w e r e i n f e c t e d w i t h s i n g l e o r a few s p e c i e s o f p a r a s i t e s , with Entamoeba histolytica antigen on E L I S A .
O K A Z A K I , M.; O K A Z A K I , M.; M I R A N D A , P . ; N E T O , J . ; D I E G U E S , V . ; A L V E S , J . ; C A U A S , M., T A N A H F
M.; K O B A Y A
S H I , S . ; K A N E K O , N . ; N A G A K U R A , K . ; K O B A Y A S H I , M.; M O T T A , S . ; T A T E N O , S . & T A K E U C H I , T . — logical and serological studies on amoebiasis area, Northeast Brazil.
a n d o t h e r i n t e s t i n a l p a r a s i t i c i n f e c t i o n s in R e c i f e a n d its
R e v . I n s t . M e d . t r o p . S á o P a u l o , 30 (4):
Parasito suburban
313 321, 1988.
1
the u p p e r l i m i t of n e g a t i v e s e r a . F i g . 2 a l s o s h o -
U s i n g the s a m e t e c h n i q u e , A S A M I et al.
wed t h a t s e r u m s a m p l e s i s o l a t e d f r o m the I M I P
d e m o n s t r a t e d t h a t p r e v a l e n c e of N. a m e r i c a n u s
already
patients infected with various parasites did not
i n the p a t i e n t s of U F P E h o s p i t a l w a s m u c h hi-
s h o w a p o s i t i v e r e a c t i o n e x c e p t for one c a s e w i t h
gher t h a n t h a t of A . d u o d e n a l e . S i n c e M A R Z O 1 4
t r i c h u r i a s i s . F r o m t h i s o b s e r v a t i o n , it is l i k e l y
CHI & CHIEFFI
t h a t t h e r e are s c a r c e l y c r o s s r e a c t i o n s b e t w e e n
v a l e n c e rate of N. a m e r i c a n u s i n the i n h a b i t a n t s
E . h i s t o l y t i c a a n t i g e n a n d sera i s o l a t e d f r o m va-
of P a r a n á S t a t e , s o u t h e r n B r a z i l , it s e e m s l i k e l y
r i o u s p a r a s i t i c i n f e c t i o n s , and t h a t our
also d e m o n s t r a t e d h i g h e r pre-
ELISA
t h a t N. a m e r i c a n u s is the m a i n h o o k w o r m s p e -
s e e m s to be s p e c i f i c a n d h a v e a s u f f i c i e n t s e n s i -
c i e s i n B r a z i l . T h i s t e c h n i q u e also d e m o n s t r a t e d
t i v i t y o n e p i d e m i o l o g i c a l s u r v e y of a m o e b i a s i s .
the h i g h p r e v a l e n c e rate of S . s t e r c o r a l i s i n b o t h areas. I n C a b o area, a p p r o x i m a t e l y
10% of the
A s s h o w n i n T a b l e 5, the p o s i t i v e r e a c t i o n
i n h a b i t a n t s were infected w i t h this parasite. S i n -
of E L I S A w a s o b s e r v e d i n 32 o u t of 615 s e r a e x a -
ce test t u b e c u l t i v a t i o n m e t h o d a l r e a d y s h o w e d
m i n e d . N i n e (13.6%) o u t of 66 s e r a i s o l a t e d f r o m
5.1% of p a t i e n t s i n the U F P E h o s p i t a l were infec-
the I M I P p a t i e n t s and the C a b o
ted w i t h S . s t e r c o r a l i s , S . s t e r c o r a l i s i n f e c t i o n
inhabitants,
who discharged E. h i s t o l y t i c a cystis in stools, exhibited positive reaction on
their
1
a p p e a r s still c o m m o n in R e c i f e area.
ELISA,
w h i l e t w e n t y - t h r e e s e r a (4.1 %) o u t of 549 c y s t - n e -
S . m a n s o n i egg w a s d e t e c t e d i n o n l y 2 out-
gative s u b j e c t s were also j u d g e d positive on E L I
p a t i e n t s of I M I P h o s p i t a l , w h e r e a s
SA.
tely 10% of the i n h a b i t a n t s i n C a b o C i t y area
approxima-
w e r e f o u n d p o s i t i v e for t h e e g g s b y stool e x a m i Ninety-eight stool s p e c i m e n s were i n o c u l a -
nation. A n epidemiological study on schistoso-
ted i n t o B a l a m u t h m e d i u m a n d t h e n t r o p h o z o i -
miasis in S ã o L o u r e n ç o village in P e r n a m b u c o ,
te g r o w t h w a s e x a m i n e d . N o t r o p h o z o i t e g r o w t h
i n 1975 a n d 1979, A S A M I et al. d e t e c t e d s c h i s t o -
w a s o b s e r v e d i n all s t o o l s e x a m i n e d
s o m e e g g s f r o m o v e r 40% of p r i m a r y s c h o o l p u -
although
2
16 o u t of 98 s t o o l s w e r e p o s i t i v e for E . h i s t o l y t i c a
pils. B A R B O S A
cysts.
l e n c e r a t e of S . m a n s o n i i n f e c t i o n i n the p r i m a r y
4
also d e m o n s t r a t e d h i g h p r e v a -
s c h o o l p u p i l s in P e r n a m b u c o S t a t e . S c h i s t o s o m i a s i s i s , t h e r e f o r e , still c o n s i d e r e d to be o n e
DISCUSSION
of the m o s t i m p o r t a n t p a r a s i t i c d i s e a s e s i n the O u r p r e s e n t i n v e s t i g a t i o n i n d i c a t e s t h a t the o u t - p a t i e n t s of I M I P h o s p i t a l a n d t h e
r u r a l s e c t o r of P e r n a m b u c o S t a t e .
inhabi infected
O u r p r e s e n t e x a m i n a t i o n also d e m o n s t r a t e d
with intestinal parasites. T h e overall p r e v a l e n c e
a h i g h p r e v a l e n c e r a t e of i n t e s t i n a l p r o t o z o a . I n
t a n t s of C a b o C i t y a r e a w e r e h e a v i l y
r a t e of the I M I P p a t i e n t s a n d C a b o i n h a b i t a n t s
the r u r a l area of P e r n a m b u c o , t h e p r e v a l e n c e
w e r e 71.2 a n d 9 1 . 9 % , r e s p e c t i v e l y , w h i c h
r a t e of all i n t e s t i n a l p r o t o z o a e x c e p t for E . h i s t o -
are
m u c h h i g h e r t h a n t h o s e of c e n t r a l a n d s o u t h e r n Brazil
7
18
2 7
28
. T h e r e was significant difference
i n t h e p r e v a l e n c e r a t e of h o o k w o r m s , A . l u m b r i -
l y t i c a reported here were m u c h higher than tho se p u b l i s h e d b e f o r e
2
7
18
. T h e high prevalence
r a t e of i n t e s t i n a l p r o t o z o a , i n c l u d i n g p a t h o g e n i c
c o i d e s a n d S . m a n s o n i , b u t n o t of T . t r i c h i u r a ,
and n o n - p a t h o g e n i c , is c o n s i d e r e d to be d e p e n
b e t w e e n t h e I M I P p a t i e n t s ( u r b a n area) a n d the
d e n t o n the c o n t a m i n a t i o n of d r i n k i n g w a t e r a n d
C a b o i n h a b i t a n t s ( r u r a l area). T h e h i g h e r p r e v a -
food w i t h p r o t o z o a n c y s t in a c c o r d a n c e w i t h
l e n c e of t h e s e p a r a s i t e s i n s u c h r u r a l s e c t o r s s u g -
i n c o m p l e t e s t o o l d i s p o s a l b y p o o r s a n i t a r y facili
g e s t s t h a t t h e s e i n f e c t i o n s are a p a r t i c u l a r p r o
ties. A m o n g t h e s e i n t e s t i n a l p r o t o z o a , G . l a m -
b l e m in agricultural areas w i t h a b u n d a n t water
b l i a and E. h i s t o l y t i c a were c o n s i d e r e d impor-
r e s o u r c e , w h i c h p l a y a n i m p o r t a n t role i n m a i n
t a n t i n t h i s area i n r e s p e c t to t h e i r p a t h o g e n i c i t y .
taining these parasitic infections.
T h e d i f f e r e n c e i n p r e v a l e n c e r a t e of G . l a m b l i a b e t w e e n the I M I P p a t i e n t s a n d the C a b o i n h a b i -
T e s t tube cultivation method revealed that approximately
87 to 9 3 % of t h e I M I P
patients
t a n t s is i n t e r e s t i n g . A l t h o u g h t h i s m a y be at l e a s t p a r t i a l l y d e p e n d e n t o n the d i f f e r e n c e i n age
and C a b o i n h a b i t a n t s , w h o d i s c h a r g e d h o o k -
d i s t r i b u t i o n b e t w e e n these two g r o u p s , there
w o r m e g g s , w e r e i n f e c t e d w i t h N. a m e r i c a n u s .
w a s still a d i f f e r e n c e i n the p r e v a l e n c e r a t e of
O K A Z A K I , M ; O K A Z A K I , M.; M I R A N D A , P . ; N E T O , J . ; D I E G U E S , V . ; A L V E S , J . ; C A U A S , M.; T A N A B E , M..
K O B A Y A
S H I , S . ; K A N E K O , N . ; N A G A K U R A , K . ; K O B A Y A S H I , M.; M O T T A , S . ; T A T E N O , S . & T A K E U C H I , T . — P a r a s i t o logical and serological studies on amoebiasis and other intestinal area. Northeast Brazil.
parasitic infections
in R e c i f e a n d its
suburban
R e v . I n s t . M e d . t r o p . S ã o P a u l o , 30 (4): 313 321, 1988.
G . l a m b l i a i n c o m p a r i n g the s a m e a g e g r o u p s from two areas. Therefore, the direct t r a n s m i s 5
s i o n ( p e r s o n to p e r s o n ) , i n l a r g e
communities
s u c h as s c h o o l and/or p l a y g r o u n d , m a y be w o r t h
zing serological and parasitological techniques a s w e l l a s a n e s t a b l i s h m e n t of a x e n i c or m o n o x e n i c s t r a i n of E . h i s t o l y t i c a i s o l a t e d f r o m t h i s r e g i o n s e e m s to b e a b s o l u t e l y n e c e s s a r y .
c o n s i d e r a t i o n for p r e v a l e n c e of t h i s p a r a s i t e . RESUMO S i x t y - s i x o u t of 615 p e r s o n s w e r e d e m o n t r a t e d to b e p o s i t i v e for E . h i s t o l y t i c a c y s t b y s t o o l
E s t u d o s s o r o l ó g i c o s e p a r a s i t o l o g i e s na A m e ¬
e x a m i n a t i o n . I t i s , therefore, a s s u m e d t h a t E .
bíase e em outras infecções parasitárias intesti-
histolytica infection still distributed widely
nais em R e c i f e e áreas c i r c u n v i z i n h a s , nordeste
in
R e c i f e area. T o estimate the reliable m o r b i d i t y
do B r a s i l
of i n v a s i v e a m o e b i a s i s , s e r o l o g i c a l t e s t s w e r e e m p l o y e d . A s s h o w n i n T a b l e 5, n o n e of t h e s e r a
E x a m e s p a r a s i t o l ó g i e s foram realizados
was positive on G D P a l t h o u g h these specimens
e m 187 p a c i e n t e s d o H o s p i t a l d o I M I P e 464 h a b i -
i n c l u d e d s e r a f r o m the s u b j e c t s w i t h E . h i s t o -
tantes de v á r i o s vilarejos no m u n i c í p i o do C a b o ,
lytica c y s t in their stools and/or with various intestinal s y m p t o m s . I n the previous s t u d i e s i9. 26 Gop
50 K m à s u d e s t e d e R e c i f e , d u r a n t e o s m e s e s
1 3
de a b r i l a a g o s t o . A p r o x i m a d a m e n t e 71% d o s
c o n s i d e r e d to b e o n e of t h e m o s t
p a c i e n t e s e x a m i n a d o s do I M I P e 92% dos e x a m i -
r e l i a b l e s e r o l o g i c a l t e s t s for d i a g n o s i s of a m o e -
nados do C a b o apresentavam-se infectados c o m ,
b i a s i s , s i n c e it is w e l l correlated w i t h the c l i n i c a l
no m í n i m o , u m a espécie de p a r a s i t a i n t e s t i n a l .
w
a
s
c o u r s e of s y m p t o m a t i c a m o e b i a s i s . O u r o b s e r v a t i o n , t h e r e f o r e , m a y b e e x p l a i n e d a s f o l l o w s ; the-
H o u v e u m a diferença m í n i m a n a t a x a de
re w a s n o c a s e w i t h i n v a s i v e a m o e b i a s i s a m o n g
p r e v a l ê n c i a de T r i c h u r i s t r i c h i u r a entre as d u a s
the s u b j e c t s e x a m i n e d i n this s t u d y ; rather, all
áreas, entretanto a p r e v a l ê n c i a de A s c a r i s l u m -
of t h e m w i t h c y s t i n s t o o l m a y b e a s y m p t o m a t i c
bricoides, família
c y s t c a r r i e r a c c o r d i n g to t h e c o n c e p t e s t a b l i s h e d
loides stercoralis, Schistosoma mansoni e Enta-
by S A R G E A U N T and his colleagues
2 2
2 3
2 4
. In
Ancylostomidae, Strongy¬
m o e b a h i s t o l y t i c a foi m a i o r e n t r e o s h a b i t a n t e s
o t h e r w o r d s , a v i r u l e n t s t r a i n s of E . h i s t o l y t i c a
do C a b o . S o m e n t e a G i a r d i a l a m b l i a apresentou
p r i m a r i l y d i s t r i b u t e a r o u n d R e c i f e a n d its subur-
u m a t a x a de p r e v a l ê n c i a m a i o r nos p a c i e n t e s
b a n area. T h i s speculation is supported by pre
do I M I P .
v i o u s o b s e r v a t i o n t h a t t h e i n c i d e n c e of a m o e b i c
O cultivo e m t u b o de e n s a i o revelou que a
20
liver abscess at autopsy was extremely low . A n d
prevalência do Necator a m e r i c a n u s em relação
by our preliminary finding that neither G D P nor
a do A n c y l o s t o m a d u o d e n a l e era m u i t o m a i o r
E L I S A detected anti-amoebic antibody in serum
e m a m b a s as áreas e que a do S . stercoralis entre
s a m p l e s i s o l a t e d f r o m 20 or m o r e
hospitalized
p a t i e n t s w h o w e r e c l i n i c a l l y d i a g n o s e d to h a v e
os p a c i e n t e s d o I M I P e d o s h a b i t a n t e s d o C a b o e r a , r e s p e c t i v a m e n t e , 4.5% e 9.6% .
a m o e b i c l i v e r a b s c e s s or c o l i t i s . A a m e b í a s e foi v e r i f i c a d a a t r a v é s d e e x a m e s A s s h o w n i n F i g . 2, e v a l u a t i o n of E L I S A m e -
sorológicos, imunodifusão em gel ( G D P ) e enzi¬
t h o d a s a t o o l for s e r o - d i a g n o s t i c m e t h o d i n d i c a -
m a imunoensaio ( E L I S A ) , usando comoantíge¬
tes t h a t t h i s m e t h o d a p p e a r s s p e c i f i c to E . h i s t o -
n o e x t r a t o b r u t o p r e p a r a d o a p a r t i r d o s trofozoi¬
lytica antigen. However, E L I S A could
detect
t o s de E . h i s t o l y t i c a ( c e p a H M - 1 : I M S S ) , r e a l i z a ¬
a n t i a m o e b i c a n t i b o d y i n 9 o u t of 66 p e r s o n s w i t h
d o s e m 615 s o r o s , o n d e n e n h u m a r e a ç ã o p o s i t i v a
E . h i s t o l y t i c a c y s t i n t h e i r s t o o l s a n d i n 23 of
a p a r e n t e foi o b s e r v a d a a t r a v é s d a i m u n o d i f u s ã o ,
549 p e r s o n s w i t h o u t c y s t . R e c e n t l y O K U S A W A
contudo foram observados resultados positivos
et a l .
1 7
reported that E L I S A could
demonstrate
an I g G class anti-amoebic antibody over 80%
e m 32 d o s 615 c a s o s a t r a v é s d a e n z i m a i m u n o e n saio.
of s e r a i s o l a t e d f r o m t h e a s y m p t o m a t i c c y s t c a r rier. T h e d i f f e r e n c e b e t w e e n t h e s e t w o o b s e r v a -
ACKNOWLEDGEMENT
tions c a n be e x p l a i n a b l e b y the difference in e m p l o y e d E L I S A m e t h o d . T o c h a r a c t e r i z e the epi-
This study was supported by J a p a n Interna-
d e m i o l o g i c a l f e a t u r e of a m o e b i a s i s i n n o r t h e a s t
t i o n a l C o o p e r a t i o n A g e n c y ( J I C A ) , T o k y o , Ja¬
B r a z i l , a n extensive epidemiological survey utili-
pan.
O K A Z A K I , M.; O K A Z A K I , M.; M I R A N D A , P . ; N E T O , J . ; D I E G U E S , V ; A L V E S , J . ; C A U A S , M.; T A N A B E , M.;
K O B A Y A
S H I , S . ; K A N E K O , N . ; N A G A K U R A , K . ; K O B A Y A S H I , M.; M O T T A , S . ; T A T E N O , S . & T A K E U C H I , T . — P a r a s i t o logical and serological studies on amoebiasis and other intestinal parasitic area, Northeast Brazil.
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