Improvement of blood and fly gut processing for PCR diagnosis of
Transcription
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) 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 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 ) . Note de recherche 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 tsetse flies. Parasitology, 1988, 97. 63-73. 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. MAGNUS F., VERVOORT T. & VAN MERVENNE N. A card agglutination test with staired trypanosomes (CATT) for serological diagnosis of Trypanosoma b. gambiense trypanosomiasis. Annales de la Société Belge de Médecine Tropicale, 1978, 58, 169-176. MASIGA D.K., SMITH A.J., HAYES P., BROMIDGE T.J. & GIBSON W.C. Sensitive detection of trypanosomes in tsetse flies by DNA amplification. International Journal for Parasitology, 1992, 22, 909-918. MEHLITZ I). Le réservoir animal de la maladie du sommeil à Trypanosoma brucei gambiense. In: Etudes et Synthèses de 1TEMVT, 1986, 18, 156 p. MOSER D.R., COOK G.A., OCHS D.E., BAILEY CP., MCKANI: M.R. & DONELSON J.E. Detection of Trypanosoma congolaise and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction. Parasitology, 1989, 99, 57-66. PENCHENIER L., JANNIN J . , MOULIA-PELAT J.P., ELFASSI DE LA 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, 1991. 71, 221-228. PENCHENIER L. & JANNIN J . Le traitement de la Trypanosomoses Humaine Africaine à Trypanosoma brucei gambiense (in: Some new prospects in epidemiology and fight against Human African Trypanosomiasis by E. Authie et al.). Research and Reviews in Parasitology, 1991, 51, 29-46. Woo P.T.K. Evaluation of the hsematocrite centrifuge and other techniques for the field diagnosis of human trypanosomiasis and filariasis. Acta tropica. 1971, 28, 298-303. 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 Note de recherche 389