/ Ü L U M E 51
July
1 9 9 4
The A m e r i c a n Journal of
TROP1CAL MEDICINE
AND
HYGIENE
OFFICIAL O R G A N OF
THE A M E R I C A N SOCIETY O F TROPICAL MEDICINE A N D HYGIENE
NUMf3ER 1
THE A M E R I C A N J O U R N A L OF
TROPICAL MEDICINE AND HYGIENE
(ISSN 0002-9637)
Editor,
MCWU.SON
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A s s o c i a t e E d i t o r M A R K L . EBERHARD
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RONALU
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BENENSON
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BURKE
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F R A N K L I N A . NF.VA
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METTE
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BARNETT L. CLINE
Term
1994
El A I N E J O N G
1994
MICIIELE
1995
P r e s i d e n t - E l ect
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W I L L I A M A. PETRI
m
S e c r e t a r y - Ti e asm -e i •
PETER
WELLER
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© This paper meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper).
VOLUME
51
AUGUST
1994
NUMBER
2
CONTENTS
Amoebiasis—180
Filariasis—244
M a l a r i a — 1 2 3 . 170, 183, 198,
204, 214, 224. 233
Schistosomiasis— 162, 190
Scrub typhus—149
Shigellosis/S al mone 11 os i s—219
Spotted fever—138
Slrongyloidiasis—175
Viral encephalitis—154
EPIDEMIOLOGY
The D i e l m o project: a longitudinal study of natural malaria infection and the mechanisms of protective
i m m u n i t y in a Community Iiving in a holoendemic area of S e n e g a l — J e a n - F r a n c o i s
Trape,
C h r i s t o p h e R o g i e r , L a s s a n a K o n a t e , Nafissatou D i a g n e , H i l a i r e B o u g a n a l i , B r u n o C a n q u e ,
F a b r i c e L e g r o s , Assane B a d j i , G o r a Ndiaye, Papa Ndiaye, K a r i m a B r a h i m i , Ousmane Faye,
Pierre D r u i l h e , and L u i z Pereira da Silva
123
Identification o f spotted fever group rickettsiae isolated from D e r m a c e n t o r m a r g i n a t u s and Ixodes
ricinus ticks collected in S w i t z e r l a n d — L o r e n z a B e a t i , P i e r r e - F r a n c o i s H u m a i r , A n d r e A e s c h l i mann, and Didier Raoult
.
138
Prevalence o f antibodies to rickettsiae in the human population o f suburban B a n g k o k — D a n i e l S t r i c k m a n , P a n i t a T a n s k u l , C h i r a p a E a m s i l a , and D a r y l J . K e l l y
149
Viruses isolated from mosquitoes collected in Sri L a n k a — J . S. M . P e i r i s , P r i y a n i e H . A m e r a s i n g h e ,
F e l i x P. A m e r a s i n g h e , C h a r l e s H . C a i i s h e r , L . P a r a k r a m a P e r e r a , C h i n n i a h K . A r u n a g i r i ,
N a v a r a t n a B . M u n a s i n g h a , a n d S. H . P. P a r a k r a m a K a r u n a r a t n e
154
PREVF:NTION A N D C O N T R O L
lmpact of annual Screening and chemotherapy with praziquantel on Schistosomiasis japonica on Jishan Island, P e o p l e s R e p u b l i c of C h i n a — P e t e r M . W i e s t , G u a n l i n g VVu, S h a o j i Z h o n g , S t e p h e n T . M c G a r v e y , J i a n h u a Y u a n , R e m i g i o M . O l v e d a , P i e r r e A . S. Peters, a n d G . R i c h a r d O l d s
162
CLINICAL STUDIES
R i s k factors for anemia in youna children in rural M a l a w i — S t e p h e n C . R e d d , J a c k J . W i r i m a , a n d
R i c h a r d W . Steketee
7.
-
170
DIAGNOSTIC STUDIES
Prospective evaluation of enzyme-Iinked immunosorbent assay and immunoblot methods for the diagnosis o f endemic S t r o n g y l o i d e s s i e r c o r a l i s infection—John F. L i n d o , D a v i d J . C o n w a y , N e i l S.
A t k i n s , A l b e r t E . B i a n c o , R a l p h D . R o b i n s o n , a n d D o n a l d A . P. B u n d y
175
PATHOLOGY
B r a i n abscess due to infection with Entamoeba
histolytica—Kenji
Ohnishi, Misako Murata, H i rooki K o j i m a , N o b u h i k o Takemura, Tomio Tsuchida, and H i r o s h i Tachibana
180
Immune complexes and nephropathies associated with P l a s m o d i u m i n u i infection in the rhesus monk e y — L a i l a F. N i m r i and Norbert I L Lanners
183
L a c k o f ultrasonographic evidcnce for severe hepatosplenic morbidity in Schistosomiasis mansoni in
M a l i — R u d i g e r K a r d o r f f , M a m a d o u T r a o r e , A b d o u l a y e D i a r r a , IVIoussa S a c k o , M o u s s a M a i g a ,
D o r i s F r a n k e , U d o Vester, U l r i k e H a n s e n , H a m m a r A . T r a o r e , S a h a r i F o n g o r o , H e l m u t G o r gen, R o l f K ö r t e , B r u n o G r y s e e l s , E k k e h a r d D o e h r i n g - S c h w e r d t f e g e r , and J o c h e n H . H . E h r i c h
190
DRUG STUDIES
Sulfated glycoconjugates as disrupters of P l a s m o d i u m f a l c i p a r u m erythrocyle rosettes—Stephen J .
R o g e r s o n , J o h n C . Reeder, F a d w a A I - Y a m a n , a n d G r a h a m V. B r o w n
198
Clinical. cfficacy and pharmacokinetics of micronized halofantrine for the treatment of acute u n c o m p l i cated falciparum malaria in nonimmune patients—O. B o u c h a u d , L . K . B a s c o , C . G i l l o t i n , F.
G i m e n e z , O . R a m i l i a r i s o a , B . G e n i s s e l , E . B o u v e t , R . F a r i n o t t i , J . L e B r a s , a n d J . P. C o u l a u d ...
204
Determination o f (ifty percent inhibitory concentrations (1C ) of antimalarial drugs against P l a s m o d i u m
f a l c i p a r u m parasites in a serum-free m e d i u m — A y u b O . O f u l l a , A l o y s S. O r a g o , J o h n I.
G i t h u r e , J a m e s P. B u r a n s , G l a d y s M . A l e m a n , A n t h o n y J . J o h n s o n , a n d S a m u e l K . M a r t i n
214
Short-course norfloxacin and trimethoprim-sulfamethoxazole treatment of shigellosis and salmonellosis
in E g y p t — S a m i r B a s s i l y , K e n n e t h C . H y a m s , N a b i l A . E I - M a s r y , Z o h e i r F a r i d , E l e a n o r C r o s s ,
A u g u s t L . B o u r g e o i s , E z z a t A y a d , and R i c h a r d G . H i b b s
219
50
Contents
c o n t i n u e d on i n s i d e back
cover
Contents continued from outside back cover
VACCINE STUDIES
Seleclion o f differcnt strains of P l a s m o d i u m f a l c i p a r u m for testing blood-stage vaccines in A o l u s
nancy-
m a i m o n k e y s — W i l l i a m E . C o l l i n s , G . G a l e G a l l a n d , J o A n n S. S u l l i v a n , a n d C a r l a L . M o r r i s
224
BASIC/MOLECULAR BIOLOG Y
Sporogonic development o f cullured P l a s m o d i u m f a l c i p a r u m in six species of laboratory-reared
les mosquitoes—Jefferson A . V a u g h a n , B r u c e H . N o d e n , a n d J o h n C . B e i e r
..
Rates o f acquisition and Ioss of W u c h e r e r i a b a n c r o f t i infection in C u l e x c/uincptefasciatus—S.
m a n i a n , A . M a n o h a r a n , K . D . R a m a i a h , a n d P. K . D a s . .
233
Subra244
• üassified Advertisement
Hrrata
Anophe-
250
— -
--
- -
-..
N u m b e r 1 (July) o f Volume 51 was mailed to subscribers on 23 August 1994
250
Am. J. Trop.
Med. Hy^.. 51(1). 1994. pp. I 15-1 18
Copyright <D 1994 by The American Society of Tropica! Medicine and Hygiene
DIRECT AMPLIFICATION A N D DIFFERENTIATION OF
PATHOGENIC A N D NONPATHOGENIC E N T A M O E B A
H I S T O L
DNA FROM STOOL SPECIMENS
Y T I C A
S Y L V I A K A T Z W I N K E L - W L A D A R S C H , T H O M A S LOSCHER, AND HEINZ RINDER
A b t e i l u n g für I n f e k t i o n s -
u n d Tropenmedizin
der Universität,
München,
Germany
Abstract.
D i s c r i m i n a t i o n o f p a t h o g e n i c a n d n o n p a t h o g e n i c Entamoeba
h i s t o l y t i c a is o f
great c l i n i c a l i m p o r t a n c e . A s i m p l e a n d r a p i d D N A e x t r a c t i o n m e t h o d that c a n be u s e d
d i r e c t l y w i t h s t o o l s p e e i m e n s w a s d e v e l o p e d w i t h o u t the n e e d f o r p r i o r c u l t i v a t i o n o f the
parasites. T h e entire p r o t o e o l c a n be p e r f o r m e d at r o o m t e m p e r a t u r e i n a 1.5-ml m i c r o c e n t r i f u g e tube f o r m a t . T h e r e is no D N A p r e e i p i t a t i o n step. T h e s u b s e q u e n t nested P o l y m e r a s e c h a i n r e a c t i o n c o n s i s t s o f a n i n i t i a l E. histolytica-speciüc
amplification, followed
by t w o separate a m p l i f i c a t i o n s u s i n g t w o p r i m e r p a i r s s p e c i f i c f o r p a t h o g e n i c a n d n o n p a t h o g e n i c E. h i s t o l y t i c a , r e s p e c t i v e l y . A m p l i f i c a t i o n p r o d u e t s c a n be v e r i f i e d b y r e s t r i c t i o n
e n d o n u c l e a s e d i g e s t s . T h e r e is n o n e e d for h y b r i d i z a t i o n s o r the use o f r a d i o n u c l e o t i d e s .
O n e t r o p h o z o i t e p e r m i l l i g r a m o f s t o o l s a m p l e c o u l d be d e t e c t e d a n d d i f f e r e n t i a t e d i n a
0.1-g speeimen.
A m e b i a s i s is an i n f e c t i o n o f the l a r g e i n t e s t i n e
caused
b y the
histolytica.
parasitic protozoon
Generally, a nonpathogenic
Stool speeimens. S t o o l s a m p l e s (0.1 g) f r o m
Entamoeba
an a s y m p t o m a t i c p e r s o n w i t h o u t m i c r o s c o p i c a l -
f o r m is
ly d e t e c t a b l e p a r a s i t e s w e r e m i x e d w i t h d e f i n e d
d i s c r i m i n a t e d f r o m a p a t h o g e n i c o n e that c a u s e s
numbers o f H K - 9 trophozoites. Speeimens
i n v a s i v e a m e b i a s i s . B o t h f o r m s w e r e first d i f f e r -
a l s o o b t a i n e d f r o m three patients p o s i t i v e f o r E.
e n t i a t e d b y c h a r a c t e r i s t i c i s o e n z y m e patterns.
has been s u g g e s t e d
separate s p e c i e s .
genomic
2
that these f o r m s
this report, those d i f f e r e n c e s
sequences o f a small subunit
on
3
described. ™
i n the
It
constitute
R e c e n t l y , differences
l e v e l h a v e a l s o been
1
In
nucleotide
ribosomal R N A
Polymerase chain reaction ( P C R ) technique
to
detect a n d differentiate b o t h f o r m s at the s a m e
time.
a first attempt to a v o i d
methods,
E.
time-consuming
histolytica
D N A from
stool samples was amplified using freezing with
ice. an ultrasonic generator, and a 24-hr i n 6
e u b a t i o n s t e p . In an effort to find a m e t h o d that
is
on
microscopic examination.
E x t r a c t i o n s of D N A . F o r the c o m p a r i s o n o f
DNA
extraction methods,
5000 H K - 9 tropho-
z o i t e s w e r e a d d e d to 0.1 g o f s t o o l . I n a m o d i fication
o f an Echinococcus
extraction method
multilocularis D N A
used w i t h fox feces,
0 . 1 - g s t o o l s a m p l e s w e r e transferred
to
7
these
1.5-ml
m i c r o c e n tri fuge tubes, 3 3 . 3 |xl o f 1 M K O H a n d
9.3 uJ o f 1 M D T T w e r e a d d e d a n d m i x e d thor-
cultivation
dry
cysts
C y s t c o n c e n t r a t i o n s w e r e not d e t e r m i n e d .
the
5
g e n e w e r e u s e d to d e v e l o p a s i m p l e yet s e n s i t i v e
In
histolytica
were
fast a n d s i m p l e e n o u g h
to be p e r f o r m e d
at
r o o m t e m p e r a t u r e w i t h o u t the n e e d for s p e c i a l
e q u i p m e n t , a p r o t o e o l w a s d e v e l o p e d to detect
o n e t r o p h o z o i t e per m i l l i g r a m i n a 0 . 1 - g s t o o l
o u g h l y b y s t i r r i n g w i t h a pipette t i p , f o l l o w e d b y
brief shaking.
min,
of
8
A f t e r i n e u b a t i o n at 6 5 ° C f o r 15
the s a m p l e s w e r e n e u t r a l i z e d w i t h 4.3 |xl
2 5 % H C l , buffered w i t h 80 |xl o f 2 M T r i s -
HC1
( p H 8.3), a n d the S u s p e n s i o n w a s m i x e d
again. T h e D N A was extracted by shaking with
250
(PCI,
JJLI o f
phenol:chloroform:isoamyl alcohol
2 5 : 2 4 : 1 ) saturated w i t h 10 m M T r i s ( p H
8.0), 1 m M E D T A ( S i g m a , St. L o u i s , M O ) . T h e
sample.
phases w e r e s e p a r a t e d b y a 4 - m i n s p i n i n a m i crocentrifuge.
MATERIALS A N D METHODS
Entamoeba
h i s t o l y t i c a trophozoites. T r o p h o -
was
trans-
ferred to a n e w tube a n d the D N A w a s
The
aqueous
phase
further
p u r i f i e d b y a d s o r p t i o n to 5 jxl o f a s i l i c a g e l Sus-
z o i t e s o f the p a t h o g e n i c strain H K - 9 w e r e g r o w n
pension ( G e n e c l e a n ; B i o 101, L a J o l l a , C A ) .
in
DNA
a x e n i c c u l t u r e u s i n g T P S - 1 m e d i u m as
de-
scribed by D i a m o n d and cryopreserved in liquid
nitrogen.
7
For
The
was eluted in 38.2 |xl o f water.
comparison, some
0.1-g
stool
samples
w e r e first w a s h e d b y s u s p e n d i n g t h e m i n 7 0 0 u,l
115
116
KATZWINKEL-WLADARSCH AND OTHERS
o f 0 . 9 % N a C l . A f t e r c e n t r i f u g a t i o n , the
super-
p r o d u e t s c o u l d be d e t e c t e d . T o generate a r a p i d
natant w a s d i s c a r d e d . S o m e s a m p l e s w e r e P C I -
p r o t o e o l , l o n g e r i n e u b a t i o n t i m e s w e r e not c o n -
e x t r a c t e d three t i m e s a n d others w e r e not e x -
s i d e r e d . In contrast, the a l k a l i n e l y s i s
tracted. T h e P r o t e i n a s e K d i g e s t i o n p r o c e s s u s e d
y i e l d e d a 0 . 9 - k b b a n d . In an attempt to s i m p l i f y
w a s a d o p t e d f r o m a Standard p r o t o e o l .
9
the m e t h o d , it w a s f o u n d that the i n i t i a l w a s h i n g
Nested P C R . P r i m e r c o n s t r u e t i o n w a s b a s e d
on
pathogenic
and
nonpathogenic
step w i t h 0 . 9 % N a C l c o u l d be o m i t t e d w i t h o u t
sequences
a d e t e c t a b l e l o s s o f p r o d u e t . H o w e v e r , the P C I
4
e x t r a c t i o n p r o v e d to be e s s e n t i a l , but n o c h a n g e
from a small subunit ribosomal R N A gene.
For
the first P C R , the p r i m e r p a i r E H - 1 ( T T T G T A
in
T T A G T A C A A A ) and E H - 2 ( G T A ( A , G ) T A
times.
TTG
A T A T A C T ) , w h i c h speeified a 0.9-kb
fragment,
was
u s e d . F o r the
method
second
(nested)
y i e l d resulted from
repeating
it t w o
more
V o l u m e of speeimens a n d detection limit.
Since stool components
are k n o w n
to
inhibit
P C R , t w o d i f f e r e n t p r i m e r p a i r s that w e r e spe-
P C R a m p l i f i c a t i o n s , 0 . 0 3 g , 0 . 0 8 g , 0.2 g , a n d
cific
0.5 g o f s t o o l at a c o n c e n t r a t i o n o f 5 0 H K - 9
GGC
f o r the
pathogenic
form
(EHP-1: A A T
C A A T T C A T T C A A T G and
EHP-2:
trophozoites per m i l l i g r a m were extracted and
T C T A G A A A C A A T G C T T C T C T ) o r the
the D N A w a s a m p l i f i e d as d e s c r i b e d . T h e o p t i -
nonpathogenic form ( E H N - 1 : A G T G G C C A A
m a l a m o u n t o f s t o o l w a s f o u n d to be b e t w e e n
T T T A T G T A A G T and E H N - 2 :
TTT A G A
0.08 g a n d 0 . 2 g . U s i n g a s a m p l e s i z e o f 0.1 g ,
A A C A A T G T T T C T T C ) were used. A l l p r i m -
the d e t e c t i o n l i m i t w a s f o u n d to be o n e t r o p h o -
ers w e r e o b t a i n e d f r o m G e n z e n t r u m ( M a r t i n s -
z o i t e p e r m i l l i g r a m o f s t o o l w h e n 1,000, 3 0 0 ,
ried,
Germany). The P C R s were done using a
h o t start t e c h n i q u e i n w h i c h the 3 8 . 2 - | x l D N A
extracts o r 2 (xl o f the first P C R i n 3 6 . 2 jxl w a t e r
w e r e d e n a t u r e d at 9 6 ° C f o r 2 m i n after the a d d i t i o n o f 1 (xl e a c h o f 5 0 m M S o l u t i o n s o f the
primers and two drops o f mineral o i l . After c o o l i n g t o 8 0 ° C , 9.8 [xl o f a f r e s h l y p r e p a r e d m i x t u r e
o f 5 [L\ o f b u f f e r ( 1 0 0 m M T r i s - H C l , p H 8.3,
5 0 0 m M K C l ) , 4 pJ o f 5 0 m M M g C l , 0.5 u,l o f
2
dNTP-mix
(25 m M e a c h ; U n i t e d
States B i o -
c h e m i c a l C o r p . , C l e v e l a n d , O H ) , a n d 0.5 jxl (5
U/jxl) o f T a q
Polymerase (Amersham Buchler,
B r a u n s c h w e i g , G e r m a n y ) was added. Fifty c y c l e s w e r e p e r f o r m e d w i t h d e n a t u r a t i o n at 9 2 ° C
for 6 0 sec, a n n e a l i n g at 4 3 ° C f o r 6 0 sec f o r E H 1/-2 o r at 6 2 ° C f o r 6 0 sec f o r b o t h E H P - 1 1 - 2 a n d
EHN-1/-2,
a n d e x t e n s i o n at 7 2 ° c f o r 9 0
sec.
Products were v i s u a l i z e d on a 1 3 % agarose gel
c o n t a i n i n g 0.2 (xg/ml o f e t h i d i u m b r o m i d e ( S i g -
1 0 0 , 3 0 , a n d 10 t r o p h o z o i t e s , r e s p e c t i v e l y , w e r e
tested i n a 0 . 1 - g s t o o l s a m p l e .
Specificity of the P C R p r i m e r s . T h e p r i m e r
p a i r E H - 1 a n d E H - 2 is c o m p l e m e n t a r y to b o t h
pathogenic and nonpathogenic sequences
a n d the n o n p a t h o g e n i c f o r m s d i f f e r at the f o u r t h
base. T h e E H - 2 p r i m e r w a s therefore c o n s t r u e t e d t w o - f o l d d e g e n e r a t e l y , i.e., as a m i x t u r e w i t h
h a l f c o r r e s p o n d i n g to the p a t h o g e n i c
sequence
a n d the o t h e r h a l f c o r r e s p o n d i n g to the n o n p a t h o g e n i c s e q u e n c e . T h e first, the p r e a m p l i f i c a t i o n
P C R w i t h E H - 1 a n d E H - 2 , is f o l l o w e d b y t w o
a d d i t i o n a l P C R s , e a c h o f w h i c h is s p e c i f i c f o r
e i t h e r the p a t h o g e n i c o r the n o n p a t h o g e n i c seq u e n c e . T h e p r i m e r s u s e d f o r these reactions are
located downstream o f E H - 1 and E H - 2 , m a k i n g
this a nested P C R to i n c r e a s e s e n s i t i v i t y . T h e
t w o p r i m e r p a i r s u s e d i n these s e c o n d
ma).
Restriction endonuclease digests. B a n d s e x c i s e d f r o m the agarose g e l w e r e s i l i c a g e l - p u r i f i e d as d e s c r i b e d a b o v e , e l u t e d i n 9.6 juul o f w a ter, a n d after the a d d i t i o n o f 1.2 jxl o f b u f f e r ( N r .
4; N e w E n g l a n d B i o l a b s , B e v e r l y , M A ) , digeste d w i t h 0.8 \x\ ( 1 0 U / u J ) o f D r a
I (Appligene,
I i i k i r c h , F r a n c e ) f o r 6 0 m i n at 3 7 ° C , f o l l o w e d b y
the a d d i t i o n o f 0.4 fxl ( 1 0 U / f i l ) o f Sau
96 I
with
the e x c e p t i o n o f E H - 2 , i n w h i c h the p a t h o g e n i c
PCRs
w e r e c o n s t r u e t e d i n s u c h a w a y that m u t a t i o n s
specifying pathogenic or nonpathogenic
forms
are p l a c e d near the 3 ' e n d to i n c r e a s e s p e c i f i c i t y
at the s a m e t i m e . T h i s is i n a g r e e m e n t w i t h an
e a r l i e r O b s e r v a t i o n that a n a b s o l u t e r e q u i r e m e n t
f o r P C R p r i m e r s to f u n e t i o n is a perfect m a t c h
o f at least t w o bases at the 3 ' e n d .
1 0
The primer
p a i r E H P - 1 a n d E H P - 2 , as w e l l as the p r i m e r
( N e w E n g l a n d B i o l a b s ) a n d further i n e u b a t i o n
p a i r E H N - 1 a n d E H N - 2 , w e r e f o u n d to selec-
at the s a m e t e m p e r a t u r e f o r a n o t h e r 9 0 m i n .
tively amplify pathogenic or nonpathogenic E .
h i s t o l y t i c a D N A , r e s p e c t i v e l y ( F i g u r e 1).
RESULTS
Verification
D N A extraction methods. A f t e r 9 0 m i n o f
digestion
with Proteinase
K , no
amplification
o f the
P C R produets by
re-
striction endonuclease digest. T h e results o f
the P C R a m p l i f i c a t i o n s w e r e c o n f i r m e d by a re-
A M P L I F I C A T I O N O F /;. H I S T O L Y T I C A
n
H
p
A
p
n
1 17
DNA
B
n
p
C
p n
kb
2.01.0
-
0.5
-
F I G U R E 1. Polymerase chain reaction ( P C R ) amplification produets o f cultured trophozoites from the pathogenic strain H K - 9 of E n t a m o e b a h i s t o l y t i c a (H) and three nonpathogenic patient speeimens ( A , B , and C ) . The
second, nested P C R shown was performed with the primer pair E H P - 1 and E H P - 2 (p) or with the primer pair
E H N - 1 and E H N - 2 (n), respectively. kb = kilobases.
striction
Dral
endonuclease
a n d Sau
double
digestion
with
9 6 I. T h e D r a \ r e s t r i c t i o n site is
c o m m o n to b o t h f o r m s a n d c o n f i r m s the
pres-
tains a Sau
3'
9 6 I r e s t r i c t i o n site 0.2 k b f r o m the
e n d o f the n o n p a t h o g e n i c
sequence w i t h
a
base m u t a t i o n i n the p a t h o g e n i c f o r m . T h e r e f o r e ,
ence o f E . h i s t o l y t i c a D N A b y p r o d u c i n g a 0 . 5 5 -
the
kb band. T h e r e m a i n i n g 0.35-kb fragment
b a n d s o f 0.55 k b a n d 0.35 k b , u s u a l l y w i t h s o m e
con-
pathogenic
D N A exhibits
characteristic
o f the u n d i g e s t e d 0 . 9 - k b b a n d r e m a i n i n g . T h e
nonpathogenic
D N A y i e l d s a band o f 0.55
kb
a n d a c o n f l u e n t d o u b l e b a n d o f 0 . 1 5 - 0 . 2 k b , often w i t h a p a r t i a l d i g e s t i o n p r o d u e t o f 0.7
kb
(Figure 2).
DISCUSSION
T h e p r o t o e o l d e s c r i b e d p r o v i d e s a m e t h o d to
s e n s i t i v e l y a n d s e l e c t i v e l y detect a n d d i f f e r e n t i ate b e t w e e n
histolytica
without
pathogenic
and nonpathogenic E .
D N A directly from stool
the
need
for
speeimens
prior cultivation. Apart
f r o m the often u n s u c c e s s f u l a n d t i m e - c o n s u m i n g
c u l t i v a t i o n attempts, p o s s i b l e m i s d i a g n o s e s
by
o n e strain d i s p l a c i n g another i n m i x e d i n f e c t i o n s
are b e i n g a v o i d e d . T h e m e t h o d is fast a n d s i m ple
because
all extraction
steps c a n
be
per-
f o r m e d at r o o m t e m p e r a t u r e i n a 1.5-ml m i c r o centrifuge
tube
format.
Neither
ice
nor
r e f r i g e r a t i o n is r e q u i r e d . T h e r e is n o p r e e i p i t a tion
step,
eliminating problems
with
small
amounts o f D N A . N o laborious hybridizations
F I G U R E 2. Restriction endonuclease digestions of
the produets from polymerase chain reactions reactions
as described in Figure 1. a, nonpathogenic E n t a m o e b a
h i s t o l y t i c a D N A . b, pathogenic H K - 9 D N A . U = undigested; D = digested with D r a I and Sau 96 I. kb =
kilobases.
or the use o f r a d i o n u c l e o t i d e s are r e q u i r e d . T h e
w h o l e p r o c e d u r e c a n be p e r f o r m e d i n o n e d a y .
Financial support: This work was supported by the
Friedrich-Baur-Stiftung (Nr. 13/93) and by the A k a demie für Tiergesundheit e.V.
118
KATZW1NKFL-WLADARSCH A N D OTHERS
Authors' address: Sylvia Katzwinkel-Wladarsch, Thomas Loscher, and Heinz Rinder, Department of Tropical M e d i c i n e and Infectious Diseases, University o f
M u n i c h , Germany.
5.
Reprint requests: Heinz Rinder, Department of T r o p i cal Medicine and Infectious Diseases, University o f
M u n i c h , Leopoldstrasse 5, 80802 M u n i c h , Germany.
6.
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