I
MPROVEMENT 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 s p . in b l o o d is difficult, either b e c a u s e o f l o w parasitemias or due to the limits o f parasitological techniques: c e n t r i f u g a t i o n o n m i n i - a n i o n e x c h a n g e c o l u m n s ( m A E C ) n e e d s fresh b l o o d ( L a n h a m and Godfrey, 1 9 7 0 ) , is delicate to perform and is almost forsaken for mass detection; haematocrit centrifugation t e c h n i q u e (HTC) is much easier to achieve ( W o o , 1 9 7 1 ) , but lacks in sensitivity (detection o f o n e parasite for 7 5 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 c a t i o n 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 w h i c h c a u s e inhi bition o f the reaction. T o o v e r c o m e these problems and to increase the sensitivity treeshold, w e have adapted a simple and efficient m e t h o d to detect o n e parasite in 1 ml o f b l o o d , without DNA purification, and tested it o n s a m p l e s stored in field conditions.
In tsetse flies, detection o f a single parasite by PCR amplification has b e e n a c h i e v e d in p r o b ó s c i d e s 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].
purification ( G i b s o n et al, 1 9 8 8 ; Masiga et al, 1 9 9 2 ) . W e have then applied the s a m e procedure to amplify t r y p a n o s o m e s in midguts o f experimentally infected flies.
MATERIALS AND METHODS
B
l o o d was taken from an healthy volunteer, o ndry 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 ) w e r e added by serial dilutions or by using a micromanipulator apparatus (Leica, Wetzlar, G e r m a n y ) .
Samples w e r e treated with the Ready A M P ™ g e n o m i c purification kit ( P r o m e g a , Madison, WI, USA) with slight modifications: briefly, 1ml o f sterilized-nuclease free water w e r e added to e a c h sample, and tubes were incubated at r o o m temperature for 10 minutes, with vortex shaking every 1-2 minutes; after centrifugation ( t w o minutes at 13,000 g ) in a microcentrifuge, super-natants w e r e discarded and 100 ul o f resin added. Pel lets w e r e r e s u s p e n d e d and incubated for 2 0 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 c e n t r i f u g a t i o n 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)
Parasite, 1996, 4, 387-389
Note de recherche 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 w e r e dissected. From e a c h fly, the midgut w a s carefully removed and e x a m i n e d under t h e m i c r o s c o p e . T h e d i s s e c t i n g i n s t r u m e n t s w e r e c l e a n e d b y immersion in b l e a c h a n d briefly rinsed in water, before dissection o f a n e w fly, to avoid conta mination. E a c h midgut w a s then put in 5 0 pi o f phy siological serum, 100 pi o f resin w e r e a d d e d a n d e a c h s a m p l e w a s p r o c e s s e d as a b o v e .
PCR reactions w e r e carried out in 5 0 ul final v o l u m e containing 1x Cetus reaction buffer, 2 0 p m o l e s o f each primer, 2 0 0 pM o f dNTPs, 10 pi o f sample a n d 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 6 0 °C (77 brucei) or 67 °C (T. congolense) for 3 0 s., and polymerisation step at 7 2 °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 7 2 °C for 5 minutes, 10 pi o f reactions w e r e run in 1.5 % agarose gel containing 0.5 m g / m l o f ethidium b r o m i d e a n d p h o t o g r a p h e d under UV light.
Primers for amplification o f T. brucei a n d T. congolense savannah are those d e s c r i b e d b y Masiga et al., 1 9 9 2 .
RESULTS
S
ince the aim o f our studies w a s to perform thist e c h n i q u e for field diagnosis, w e d e c i d e d to test the lower limit o f detection in 1 ml b l o o d a n d storage conditions o f the blood for efficient detection: blood taking was done on EDTA or dry tubes, trypa-nosomes added by micromanipulation and samples were kept at 4 °C for o n e week. T h o s e stored in EDTA tubes were then processed directly. For the others, clots were first disrupted mechanically ( b y 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 b e 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 a n d 2 ) as well as from the midgut o f a n o n infected fly w h e r e about 100 t r y p a n o s o m e s w e r e added (lane 3 ) . Interestingly, from the three negatives by microscopy, o n e s h o w s amplification o f T.
congo-Fig. 1. — A m p l i f i c a t i o n o f T. brucei in b l o o d s t o r e d at 4 °C : M a r k e r 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 c o n t r o l o f PCR, b l o o d w i t h o u t 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.
Fig. 2 . — A m p l i f i c a t i o n o f T. congolense in m i d g u t s o f t s e t s e flies. L a n e s a r e : 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 w i t h 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 P C R ; 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 t h e t w o o t h e r s remain negative (lanes 4 and 6 ) as control flies (data not s h o w n ) .
IMPROVEMENT OF BLOOD AND FLY GUT PROCESSING FOR P C R DIAGNOSIS OF TRYPANOSOMOSIS
DISCUSSION
M
ass detection o f african h u m a n trypanoso-mosis in d o n e in t w o steps: first serological tests (Card Agglutination Test for T r y p a n o -s o m o -s i -s ) b y b l o o d taking (Magnu-s et al., 1 9 7 8 ) , a n d then search o f parasites in seropositive patients by lym-p h a t i c g l a n d lym-puncture, HTC o r mAEC t e c h n i q u e s . W h e n the p r e s e n c e o f the parasite is confirmed, a treat-ment c a n b e a c h i e v e d ( P e n c h e n i e r a n d J a n i n , 1 9 9 D ; but, in many cases, parasites are not evidenced in sero-positive patients, d u e to limitations o f parasitological t e c h n i q u e s ( P e n c h e n i e r et al., 1 9 9 1 ) . Since mass d e t e c -tion is d o n e in distant villages, o u r m e t h o d offers the advantage that blood samples c a n b e kept until the e n d o f field work, b e f o r e p r o c e s s i n g in the laboratory. Although any m e t h o d o f b l o o d c o n s e r v a t i o n s e e m s to w o r k well, b l o o d taking o n anticoagulant c o a t e d tubes would b e m o r e suitable for PCR diagnosis.Moreover, c o m p a r e d to b l o o d dotting (a drop o f b l o o d is about 6 0 pi), the sensitivity threshold is n o t i c e a b l y i n c r e a s e d s i n c e w e detect o n e parasite in 1 ml o f b l o o d , allowing then a m o r e accurate diagnosis. Epidemiological studies are also c o n c e r n e d , particularly t h o s e c o n c e r n i n g the animal reservoir: the pig is a potential reservoir for human trypanosomosis (Mehlitz, 1986) a n d lives in c l o s e contact with h u m a n popula-tions. 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 t e c h n i q u e . PCR contaminations can b e a major p r o b l e m in a large epidemiological survey: these first experiments allowed us to assess o u r conditions o f s a m p l e collection in the field, a n d laboratory structures a n d manipulations in the OCEAC center.
Direct PCR on midguts o f tsetse flies underlines t w o important features: 1 ) the opportunity to detect para-sitemia in negative flies by c o m m o n parasitological 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 c a n then provide information on the sites o f contamination by identifying recently infected y o u n g flies. Even if the p r e s e n c e o f t r y p a n o s o m e s d o e s not imply that the fly b e c o m e s infective, this t e c h n i q u e is o f great interest for t h e d e t e c t i o n o f immature parasites.
Finally, PCR diagnosis will b e very useful for veteri-nary surveys, particularly in detection o f t r y p a n o s o m e s in animals with l o w parasitemia in the b l o o d , for e x a m p l e trypanotolerant cattle a n d wild fauna.
ACKNOWLEDGEMENTS
W
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Reçu le 13 février 1996 Accepté le 26 septembre 1996
Note de recherche 389
