Improvement of blood and fly gut processing for PCR diagnosis of

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1996034387
IMPROVEMENT OF BLOOD AND FLY GUT PROCESSING
FOR PCR DIAGNOSIS OF TRYPANOSOMOSIS
PENCHENIER L.*, DUMAS V.**, GREBAUT P.*, REIFENBERG J.M***& CUNY G.****
Summary :
W e have adapted a simple and efficient technique to detect
trypanosomes in human blood, without DNA purification, and
increased the sensitivity threshold to 1 parasite in 1 ml. W e have
then applied it for detection of parasites in midguts of tsetse flies,
negative by microscopy. This technique has been developed for
field conditions and could greatly facilitate epidemiological
studies.
KEY WORDS : PCR, diagnosis, Trypanosoma.
Résumé : AMÉLIORATION DU TRAITEMENT DU SANG ET DES INTESTINS
DE MOUCHE POUR LE DIAGNOSTIC DE LA TRYPANOSOMOSE par PCR
Nous avons adapté une technique simple et efficace pour la
détection par PCR des trypanosomes dans le sang humain, sans
purification d'ADN, et augmenté le seuil de sensibilité à un
parasite par ml. Nous l'avons ensuite appliquée à la détection de
parasites dans des intestins de glossines infectées, négatives à
l'observation microscopique. Cette technique a été développée
pour une utilisation dans des conditions de terrain et pourrait
faciliter les études épidémiologiques.
MOTS CLES : PCR, diagnostic, Trypanosoma.
INTRODUCTION
D
iagnosis o f Trypanosoma
sp. in blood is dif­
ficult, either b e c a u s e o f low parasitemias or
due to the limits o f parasitological techniques:
centrifugation on mini-anion e x c h a n g e columns
(mAEC) n e e d s fresh b l o o d (Lanham and Godfrey,
1970), is delicate to perform and is almost forsaken for
mass detection; haematocrit centrifugation technique
(HTC) is much easier to achieve ( W o o , 1971), but lacks
in sensitivity (detection o f o n e parasite for 75 ul o f
b l o o d theoritically). PCR t e c h n o l o g y can bring the
required sensitivity and specificity, but direct amplifi­
cation from b l o o d s a m p l e s is very often difficult,
b e c a u s e o f h a e m o g l o b i n derivatives which cause inhi­
bition o f the reaction. T o overcome these problems and
to increase the sensitivity treeshold, w e have adapted
a simple and efficient method to detect o n e parasite
in 1 ml o f b l o o d , without DNA purification, and tested
it on samples stored in field conditions.
In tsetse flies, detection o f a single parasite by PCR
amplification has b e e n achieved in probóscides and in
salivary glands, but failed for midguts without DNA
* Laboratoire de recherche sur les trypanosomes OCEAC, BP 288,
Yaoundé, Cameroun.
** Laboratoire d'Épidémiologie des Maladies à Vecteurs, ORSTOM,
*** Laboratoire de pathologie tropicale, CIRAD/EMVT and **** Labo­
ratoire des Rétrovirus, ORSTOM, 911 Avenue Agropolis, BP 5045,
34032 Montpellier Cedex 1, France.
Correspondance : G. Cuny. Tél. : 04 67 61 75 98 - Fax : 04 67 54 78 00
- e-mail: [email protected].
Parasite, 1996, 4, 387-389
purification ( G i b s o n et al, 1 9 8 8 ; Masiga et al, 1992).
W e have then applied the same procedure to amplify
trypanosomes in midguts o f experimentally infected
flies.
MATERIALS AND METHODS
B
lood was taken from an healthy volunteer, on
dry or EDTA tubes, aliquoted by o n e ml frac­
tions, and o n e to five parasites
(Trypanosoma
brucei gambiense,
ITMAP 1 8 4 1 ) were added by serial
dilutions or by using a micromanipulator apparatus
(Leica, Wetzlar, Germany).
Samples w e r e treated with the Ready AMP™ g e n o m i c
purification kit (Promega, Madison, WI, USA) with
slight modifications: briefly, 1ml o f sterilized-nuclease
free water were added to each sample, and tubes were
incubated at room temperature for 10 minutes, with
vortex shaking every 1-2 minutes; after centrifugation
(two minutes at 13,000 g) in a microcentrifuge, supernatants w e r e discarded and 100 ul o f resin added. Pel­
lets were resuspended and incubated for 20 minutes
at 56 °C, vortexed vigorously and put at 100 °C for
10 m i n u t e s . After 2 m i n u t e s o f centrifugation at
13,000 g, supernatants containing single sranded DNA
w e r e stored at 4 °C or used directly.
Glossina
morsitans
morsitans
from CIRAD/ORSTOM
insectarium was used in the experimental infection: five
teneral flies were fed on a mouse infected with Trypa­
nosoma congolense (savannah type, Satiri/87/CRTA/235)
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387
P E N C H E N I E R L., D U M A S V . , G R E B A U T P., R E I F E N B E R G J . M . & C U N Y G .
or six on a healthy rabbit, as a control for experiment.
Seven days after flies were dissected. From e a c h fly,
the midgut was carefully removed and examined under
the m i c r o s c o p e . T h e dissecting instruments w e r e
cleaned by immersion in bleach and briefly rinsed in
water, before dissection o f a n e w fly, to avoid conta­
mination. Each midgut w a s then put in 5 0 pi o f phy­
siological serum, 100 pi o f resin w e r e added and each
sample w a s processed as above.
PCR reactions were carried out in 5 0 ul final volume
containing 1x Cetus reaction buffer, 2 0 pmoles o f each
primer, 200 pM o f dNTPs, 10 pi o f sample and o n e unit
o f Ampli T a q DNA polymerase (Perkin-Elmer, Norwalk,
CT, USA). Amplification conditions were: 4 0 cycles with
denaturation step at 9 4 °C for 3 0 s., annealing step at
60 °C (77 brucei) or 67 °C (T. congolense)
for 3 0 s.,
and polymerisation step at 72 °C for o n e minute, on
a T e c h n e PHC-3 ( T e c h n e , Cambridge, UK). After a final
elongation at 72 °C for 5 minutes, 10 pi o f reactions
were run in 1.5 % agarose gel containing 0.5 mg/ml
of ethidium bromide a n d photographed under UV
light.
Primers for amplification o f T. brucei and T. congolense
savannah
are those described b y Masiga et al., 1992.
Fig. 1. — Amplification o f T. brucei in b l o o d s t o r e d at 4 °C :
Marker M., ox 1 7 4 D N A / H a e l l l ; 1 ) four t r y p a n o s o m e s in E D T A t u b e
c o n t a i n i n g b l o o d ; 2 ) o n e t r y p a n o s o m e in E D T A t u b e c o n t a i n i n g
b l o o d ; 3) n e g a t i v e control o f PCR, b l o o d without parasite; 4 ) four try­
p a n o s o m e s in dry t u b e c o n t a i n i n g b l o o d ; 5 ) o n e t r y p a n o s o m e in
dry t u b e c o n t a i n i n g b l o o d ; 6) 1 0 p g o f T. brucei DNA.
RESULTS
S
ince the aim o f our studies w a s to perform this
technique for field diagnosis, w e decided to test
the lower limit o f detection in 1 ml b l o o d and
storage conditions o f the blood for efficient detection:
blood taking was done on EDTA or dry tubes, trypanosomes added by micromanipulation and samples were
kept at 4 °C for o n e week. Those stored in EDTA tubes
were then processed directly. For the others, clots were
first disrupted mechanically (by pipetting in and out)
before treatment. As shown in figure 1, specific ampli­
fication (as evidenced by control experiments, lanes 3
and 6 ) o f 164 b p repeated monomer fragments occurred
whatever the storage conditions. For dry tubes, however,
bands are less intense and particularly oligomer frag­
ments (lanes 4 and 5 ) probably due to loss o f material
in the clots, which cannot b e completely disrupted.
From the five teneral flies fed on a mouse infected with
T. congolense
savannah,
only two flies appeared to be
parasited at high rate, by microscopic observation, the
three others being negative. All midguts were treated as
described in Materials and Methods section and subjected
to PCR amplification. Results are presented in figure 2.
As e x p e c t e d , the 3 1 6 b p repeated bands (specific o f
T. congolense
savannah)
are amplified in the positive
flies (lanes 1 and 2 ) as well as from the midgut o f a
n o n infected fly w h e r e about 100 trypanosomes w e r e
added (lane 3 ) . Interestingly, from the three negatives
by microscopy, o n e shows amplification o f T. congo-
388
Fig. 2. — Amplification o f T. congolense in midguts o f tsetse flies.
L a n e s are: M a r k e r ( M ) a s in figure 1; 1 ) a n d 2 ) m i d g u t s o f i n f e c t e d
flies; 3) m i d g u t o f n o n i n f e c t e d fly with 1 0 0 t r y p a n o s o m e s ; 5 ) , 6)
a n d 7), m i d g u t s o f i n f e c t e d flies n e g a t i v e b y m i c r o s c o p i c o b s e r v a ­
tion; 6). n e g a t i v e c o n t r o l o f PCR; 7), 10 p g o f T congolense DNA.
lense s e q u e n c e s ( l a n e 5 ) w h e r e a s the t w o others
remain negative (lanes 4 and 6 ) as control flies (data
not s h o w n ) .
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Parasite, 1996, 4, 387-389
IMPROVEMENT OF BLOOD AND FLY GUT PROCESSING FOR P C R DIAGNOSIS OF TRYPANOSOMOSIS
DISCUSSION
REFERENCES
M
GIBSON W.C.. DIKES P. & GASHUMBA J . K . Species-specific
DNA probes for identification of African tiypanosomes in
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ass detection o f african human trypanosomosis in d o n e in t w o steps: first serological
tests (Card Agglutination Test for Trypano-
s o m o s i s ) by b l o o d taking (Magnus et al., 1978), and
then search o f parasites in seropositive patients by lymphatic gland
puncture,
HTC or mAEC t e c h n i q u e s .
W h e n the presence o f the parasite is confirmed, a treatment can b e achieved ( P e n c h e n i e r and Janin, 1 9 9 D ;
but, in many cases, parasites are not evidenced in seropositive patients, d u e to limitations o f parasitological
techniques ( P e n c h e n i e r et al., 1991). Since mass detection is d o n e in distant villages, our method offers the
advantage that blood samples can b e kept until the end
o f field work, before processing in the laboratory.
Although any m e t h o d o f b l o o d conservation s e e m s to
w o r k well, b l o o d taking on anticoagulant coated tubes
would b e more suitable for PCR diagnosis.
Moreover, compared to blood dotting (a drop o f blood
is about 6 0 pi), the sensitivity threshold is noticeably
increased since w e detect o n e parasite in 1 ml o f
b l o o d , allowing then a more accurate diagnosis.
Epidemiological studies are also concerned, particularly
those concerning the animal reservoir: the pig is a
potential reservoir for human trypanosomosis (Mehlitz,
1986) and lives in c l o s e contact with human populations. Preliminary investigations in the Y e m e s s o a region
( C a m e r o u n ) strengthens the validity o f our technique.
PCR contaminations can b e a major problem in a large
epidemiological survey: these first experiments allowed
us to assess our conditions o f sample collection in the
field, and laboratory structures and manipulations in
the OCEAC center.
Direct PCR on midguts o f tsetse flies underlines two
important features: 1) the opportunity to detect parasitemia in negative flies by c o m m o n
parasitological
LANIIAM S.M. & GODFREY D . G Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology, 1970, 28, 521-534.
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BAUME F., FADAT G , CHANFREAU B . & EOZENOU P. Le problème de l'interprétation du GATT dans le dépistage delà trypanosomiase humaine à Trypanosoma brucei gambiense. Annales de la Société Belge de Médecine Tropicale,
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t e c h n i q u e s ; 2 ) the advantage o f minimizing loss o f
material that c a n o c c u r with DNA purification. It can
then provide information on the sites o f contamination
Reçu le 13 février 1996
Accepté le 26 septembre 1996
by identifying recently infected young flies. Even if the
p r e s e n c e o f trypanosomes d o e s not imply that the fly
b e c o m e s infective, this technique is o f great interest
for the detection o f immature parasites.
Finally, PCR diagnosis will b e very useful for veterinary surveys, particularly in detection o f trypanosomes
in animals with
low parasitemia
in the b l o o d , for
e x a m p l e trypanotolerant cattle and wild fauna.
ACKNOWLEDGEMENTS
W
e thank D o m i n i q u e Cuisance and J e a n Louis Frézil for critical reading
o f the
manuscript.
Parasite, 1996, 4, 387-389
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