FOLLOW-UP OF NEUROCYSTICERCOSIS PATIENTS AFTER TREATMENT USING AN ANTIGEN DETECTION ELISA
NGUEKAM*, ZOLI A.P.*. ONGOLO-ZOGO P.**, DORNY P.***. BRANDT J.*** & GEERTS S.***
Summary:
Seven patients with active neurocysticercosis (NCC) received an eight days treatment with albendazole and were followed up using computed tomography (CT-scan) and a monoclonal antibody based ELISA for the detection of circulating antigen (Ag-ELISA).
Only three patients were cured as was shown by CT-scan and by the disappearance of circulating antigens one month after treatment. After a second course of albendazole therapy, two other patients became seronegative. CT-scan showed the disappearance of viable cysts in all persons who became seronegative whereas patients who were not cured remained seropositive. These preliminary results show that this Ag-ELISA is a promising technique for monitoring the success of treatment of NCC patients because of the excellent correlation between the presence of circulating antigens and of viable brain cysts.
KEY WORDS : Taenia solium, CT-scan, ELISA, circulating antigen, neuro- cysticercosis, albendazole.
N
eurocysticercosis (NCC) is an infection o f the central n e r v o u s system by the larval stage o f Taenia solium. It is r e c o g n i z e d as a c o m m o n c a u s e o f neurological d i s e a s e in d e v e l o p i n g countries (Schantz et al., 1 9 9 8 ) . T h e p r e s e n c e o f T. solium meta- c e s t o d e s in the n e r v o u s system leads to a variety o f clinical manifestations d e p e n d i n g u p o n the n u m b e r , size, viability and location within the brain and the host inflammatory reaction (White, 2 0 0 0 ) . Albendazole and praziquantel are the drugs o f c h o i c e for the treat- ment o f NCC. F o l l o w - u p after treatment is usually d o n e using c o m p u t e d t o m o g r a p h y ( C T - s c a n ) , w h i c h is an e x p e n s i v e t e c h n i q u e for the p e o p l e o f d e v e l o - ping countries ( T s a n g ¿4 Wilson, 1 9 9 5 ) . Considering the i m p o r t a n c e o f NCC and the increasing poverty in t h e s e countries, it is n e c e s s a r y to investigate alterna- tive t e c h n i q u e s , w h i c h c o u l d r e d u c e at least the cost o f evaluation o f the efficacy o f the treatment. S i n c e an antigen detection ELISA (Ag-ELISA) has b e e n deve-* U n i v e r s i t y of D s c h a n g . P . O . B o x 9 6 D s c h a n g . C a m e r o o n .
** C e n t r a l H o s p i t a l of Y a o u n d é . D e p a r t m e n t o f R a d i o l o g y . Y a o u n d e . C a m e r o o n .
*** Institute o f T r o p i c a l M e d i c i n e , N a t i o n a l e s t r a a t , A n t w e r p , B e l g i u m . C o r r e s p o n d e n c e : S t a n n y G e e r t s , Institute o f T r o p i c a l M e d i c i n e , Natio- n a l e s t r a a t 1 5 5 , B - 2 0 0 0 A n t w e r p . E-mail: s g e e r t s @ i t g . b e
Résumé : SUIVI DE PATIENTS ATTEINTS D E NEUROCYSTICERCOSE APRÈS TRAITEMENT EN UTILISANT UN T E S T ELISA POUR LA DÉTECTION D'ANTIGÈNES
Sept patients atteints de neurocysticercose active (NCC) ont été traités à l'albendazole pendant huit jours et ont été suivis en utilisant la tomodensitométrie et un ELISA pour la détection d'antigènes circulants à base d'anticorps monoclonaux (Ag-ELISA).
Seulement trois patients étaient guéris comme indiqué par le CT- scan et par la disparition des antigènes circulants un mois après traitement. Après un deuxième traitement à Talbendazole, deux outres patients sont devenus séronégatifs. Le CT-scan montrait la disparition de tous les cysticerques vivants chez les personnes qui étaient devenus séronégatifs, tandis que les patients qui n'étaient pas guéris restaient séropositifs. Ces résultats préliminaires montrent que l'Ag-ELISA est une technique prometteuse pour évaluer le succès d'un traitement de la neurocysticercose en raison de la corrélation excellente entre l'Ag-ELISA et la tomodensitométrie.
MOTS CLES : Taenia solium, tomodensitométrie, ELISA, antigène circulant, neurocysticercose, albendazole.
loped, w h i c h has a high sensitivity for the detection o f h u m a n cysticercosis (Erhart et al., 2 0 0 2 ) and which allows to distinguish cattle and pigs carrying living cysts from t h o s e harbouring only dead cysts (Brandt et al, 1 9 9 2 ; N g u e k a m et al., in p r e s s ) , it was decided to evaluate its u s e in NCC patients. T h e p u r p o s e o f this study was to c o m p a r e the Ag-ELISA test with CT- s c a n to evaluate the efficacy o f an a l b e n d a z o l e treat- ment o f NCC.
MATERIALS AND METHODS
PATIENTS
S
even patients with active lesions o f NCC from the West province o f Cameroon identified by serology (Ag-ELISA) and confirmed by brain CT-scan (in Y a o u n d e Central Hospital) w e r e included in this study.NCC was asymptomatic in all but two o f them, w h o were epileptics (Batoula 94 and K E ) . Five o f them w e r e w o m e n and two men. Their age ranged between seven and 7 3 years (with a mean o f 39-9 ± 24.9 years). T h e patients harboured an average o f five viable cysts (range:
1 to 12) and o f 7.6 calcified cysts (range: 0 to 25) in the
P a r a s i t e , 2 0 0 3 , 10, 6 5 - 6 8
Note de recherche 65
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2003101p65
NGUEKAM, ZOLI A.P., ONGOLO-ZOGO P. ETAL.
brain. T h e localisation o f the cysticerci was parenchymal a n d / o r subarachnoidal. Informed consent was obtained from e a c h adult and from the parents o f the two young girls (seven and 15 years old) included in the study.
TREATMENT PROTOCOL
T h e patients w e r e treated with a l b e n d a z o l e (Alben®
4 0 0 mg, Smithkline B e e c h a m ) at a d o s a g e o f 15 m g / k g b o d y w e i g h t / d a y for eight days as d e s c r i b e d by Del Brutto et al. ( 1 9 9 9 ) . T o prevent adverse reactions, p r e d n i s o l o n e (Solupred® 5 mg, Laboratoire H o u d é ) at 10 mg p e r p e r s o n thrice a day w a s given from o n e day prior to the a l b e n d a z o l e treatment until at least four days after the e n d o f it (Groll, 1 9 8 2 ) . T h e two epi
leptic patients c o n t i n u e d to receive anti-epileptic treat
ment (Gardenal®).
Six to n i n e m o n t h s after the e n d o f the first treatment, the patients w h o r e m a i n e d seropositive w e r e treated again with the s a m e d o s e o f a l b e n d a z o l e for a period o f o n e month. In o n e patient ( K E ) the first treatment w a s not a c o u r s e o f eight days, but o f o n e month.
ANTIGEN DETECTION E L I S A FOR CYSTICERCOSIS ( A G - E L I S A )
T h e patients w e r e sampled before the beginning o f the treatment, o n e and three m o n t h s after the first treat
m e n t and in those patients w h o r e c e i v e d a s e c o n d treatment, o n e m o n t h later. T h e serum samples w e r e tested using a m o n o c l o n a l antibody b a s e d antigen detection ELISA as d e s c r i b e d b y Brandt et al. ( 1 9 9 2 ) but slightly modified according to Pouedet et al. ( 2 0 0 2 ) . T h e sera w e r e pre-treated using trichloroacetic acid and used in ELISA at a final dilution o f 1/4. T w o m o n o clonal antibodies ( M o A b ) w e r e used in a sandwich ELISA. MoAb B 1 5 8 C 1 1 A 1 0 w a s used for coating and a biotinylated MoAb B 6 0 H 8 A 4 w a s included as detector antibody. O r t h o p h e n y l e n e diamine a n d H202 w e r e used as c h r o m o g e n - s u b s t r a t e solution. After arresting the reaction with 4 N H2S O4 the plates w e r e read using an ELISA reader (Labsystem Multiskan RC) at 4 9 2 nm.
Eight negative reference control sera from local p e o p l e o f the region o f D s c h a n g (without any history o f tae- niasis or cysticercosis in the family) and o n e reference p o s i t i v e s e r u m from a C a m e r o o n i a n p a t i e n t with confirmed cysticercosis ( b y CT-scan) w e r e included in e a c h ELISA run. T h e optical density ( O D ) o f e a c h serum sample w a s c o m p a r e d with the m e a n o f the eight negative reference sera at a probability level o f P= 0.001 to determine the result using a modified Stu
dent test (Sokal & Rohlf, 1 9 8 1 ) . T h e ELISA values w e r e e x p r e s s e d as a ratio b y dividing the O D o f the test sample b y the O D o f the cut-off value. An ELISA ratio > 1 w a s c o n s i d e r e d as positive.
CT-SCAN OF THE BRAIN
T h e CT-scans w e r e performed in Y a o u n d e Central Hos
pital using a S o m a t o m AR STAR s c a n n e r ( S i e m e n s Medical Systems, Erlangen, G e r m a n y ) before and after contrast fluid injection (Telebrix®35, Guerbet, France).
Brain image slices o f five m m thickness w e r e transferred o n negatives and scan reports w e r e produced, indica
ting the number and types o f lesions compatible to NCC.
T w o CT-scans w e r e made for e a c h patient: o n e before and o n e after treatment. For the group o f patients w h o b e c a m e seronegative after the first treatment, CT-scan w a s performed six to ten months later, w h e r e a s the p a t i e n t s , w h o r e c e i v e d a s e c o n d t r e a t m e n t , w e r e s c a n n e d three to five months later. T h e radiologist was not aware o f the serological results o f the patients.
RESULTS
T
he serological results and brain CT-scan status o f each patient before and after albendazole therapy are presented in T a b l e I and Figures 1 and 2.A G - E L I S A RESULTS
O n e m o n t h after the e n d o f the first c o u r s e o f alben
dazole three patients b e c a m e negative in the Ag-ELISA
Patients B e f o r e treatment After treatment
S e x / a g e / e p i l e p t i c s t a t u s
C T - s c a n
n o . l i v / n o . calc ELISA ratio
C T - s c a n
n o . l i v / n o . calc ELISA ratio
- B L 2 1 M / 7 3 / N 1/1 1.8 0 / 1 0.7*
- B a l e o 01 F / 0 7 / N 2 / 0 1.9 0 / 0 0 . 8
- B F K P 1 3 7 M/60/N 3 / 0 1.3 0/0 0.6
- B M K P 1 9 4 F / 6 0 / N 1/6 1 1 . 5 0 / 3 0 . 8 *
- B a t o u l a 9 4 F / 3 2 / E 6°/ 2 5 4 8 . 7 2 / 2 5 20.6*
- M e t c h o u 3 7 8 F / 1 5 / N 1276 12.5 0 / 1 2 0 . 7
- K E F / 3 2 / E 1 0 / 1 5 1 1 . 0 4 ° / 1 5 18.9
*: after a s e c o n d t r e a t m e n t ; °: i n c l u d i n g ring e n h a n c e d c y s t s ; M: m a l e ; F: f e m a l e ; E: e p i l e p s y ; N: n o e p i l e p s y .
T a b l e I. — A g - E l i s a a n d C T - s c a n results b e f o r e a n d after t r e a t m e n t w i t h a l b e n d a z o l e o f p a t i e n t s w i t h n e u r o c y s t i c e r c o s i s .
66 Note de recherche P a r a s i t e , 2 0 0 3 , 10, 6 5 - 6 8
F O L L O W - U P O F N E U R O C Y S T I C E R C O S I S U S I N G E L I S A
Fig. 1. - A n t i g e n m o n i t o r i n g results in a c t i v e NCC p a t i e n t s (initial Flisa ratio > 1 0 ) .
Fig. 2. - A n t i g e n m o n i t o r i n g results in a c t i v e N C C p a t i e n t s (initial Flisa ratio < 1 0 ) .
( T a b l e I ) . Patient ( K E ) received only o n e treatment course during o n e month and remained seropositive o n e m o n t h after therapy. A s e c o n d Ag-ELISA per
formed o n serum s a m p l e s taken three months post treatment did not s h o w any additional seronegative patients (Figs 1, 2 ) . O n e month after a s e c o n d treat
ment o f the three patients, w h o w e r e not cured, two o f them ( B L 21 and BMKP 1 9 4 ) b e c a m e seronegative.
C T - S C A N RESULTS
CT-scan o f the brain after o n e or two courses o f alben
dazole therapy s h o w e d that viable cysts had c o m p l e tely disappeared in five out o f s e v e n ( 7 1 . 4 % ) patients.
In the two others, either a reduction o f the n u m b e r o f viable cysts ( B a t o u l a 9 4 ) o r t h e p r e s e n c e o f ring e n h a n c e d cysts indicating a p r o c e s s o f degeneration ( K E ) was o b s e r v e d after treatment ( T a b l e I ) . In all patients w h e r e the viable cysts disappeared after treat
ment a negative ELLSA result (ratio < 1) was obtained w h e r e a s a persistence o f e v e n few living or d e g e n e rating cysts resulted in positive ELISA values.
COMPARATIVE COSTS O F ELISA
AND CT-SCAN TECHNIQUES IN T H E FOLLOW-UP O F NCC PATIENTS AFTER TREATMENT
Up to n o w . CT-scan in C a m e r o o n can only b e carried out in Y a o u n d e or Douala. B e s i d e s the costs o f scan
ning and the contrast agent, the total cost o f a scan includes therefore also the travel and food e x p e n s e s (for two days). This cost was estimated for e a c h patient involved in the present study to approximately 152.2 € ( 1 0 0 , 0 0 0 CFA) w h e r e a s the cost o f a test o f an Ag-
ELISA (for 4 0 samples or o n e plate) was about 17 € , i.e. 0.425 € per patient. T h e salaries o f the medical doctor or the laboratory technicians are not included in this calculation.
DISCUSSION
C
omputed tomography is an useful imaging technique for the diagnosis o f human neurocysti
cercosis and the assessment o f the efficacy o f anthelminthic drugs in the treatment o f this disease (Padma et al, 1994; Garcia et a l . , 1997; White, 2 0 0 0 ) . Its high cost, however, and the fact that it is often una
vailable in rural regions o f developing countries, where the prevalence o f NCC is high (Tsang & Wilson, 1995), constitute a limitation for its wide use. In this study, w e c o m p a r e d this t e c h n i q u e with an Ag-ELISA as an alternative m e t h o d for the follow-up o f neurocysticer
cosis patients after cysticidal treatment. T h e Ag-ELISA has b e e n s h o w n to detect the excretory-secretory pro
ducts o f viable cysticerci in cattle (Brandt et al., 1 9 9 2 ) , pigs (Nguekam et al, in press) and man (Erhart et al, 2002). These latter authors reported a sensitivity o f 94.4 % and the a b s e n c e o f cross-reactions with sera from human patients infected with Schistosoma, hydatid cysts, Ascaris, Trichuris, filaria, Entamoeba, Plasmo
dium and Trypanosoma.
Although the follow-up period was not the s a m e for the two t e c h n i q u e s , the results obtained in this study w e r e very promising. T h e r e was 100 % agreement bet
w e e n the CT-scan and the Ag-ELISA results. T h e five patients, w h o b e c a m e seronegative o n e month after o n e or two a l b e n d a z o l e courses, s h o w e d a c o m p l e t e disappearance o f the viable cysts in the brain w h e r e a s the two remaining seropositive patients (Batoula 94 and KE) s h o w e d respectively two living cysts and four cysts with ring e n h a n c e m e n t .
T h e sensitivity o f our Ag-ELISA was better than that o f a n o t h e r m o n o c l o n a l a n t i b o d y - b a s e d Elisa u s e d by Garcia et a l . , ( 2 0 0 0 ) in monitoring neurocysticercosis patients after treatment. This latter Ag-ELISA could not detect patients with only o n e viable cyst and/or enhan
cing lesion whereas in this study two patients with o n e single living cysticercus in the brain could b e identi
fied.
T h e s e preliminary results clearly s h o w that this m o n o clonal a n t i b o d y - b a s e d Ag-ELISA is a promising t e c h nique to monitor neurocysticercosis patients after treat
ment. In addition, it is m u c h c h e a p e r than CT-scan in particular for patients o f d e v e l o p i n g countries w h e r e poverty is an increasing reality. Currently, studies o n a larger n u m b e r o f NCC patients are going o n in order to validate the Ag-ELISA as an alternative for CT- scan.
P a r a s i t e . 2 0 0 3 . 10. 6 5 - 6 8
67
N G U E K A M , Z O L I A . P . . O N G O L O - Z O G O P . ETAL.
ACKNOWLEDGEMENTS
T
his study w a s carried out with the financial support o f the Belgian Directorate General for Inter
national Cooperation (DGIC, B r u s s e l s ) and o f the University o f D s c h a n g . T h e research w a s d o n e within t h e framework a g r e e m e n t b e t w e e n DGIC a n d the Institute o f Tropical Medicine, Antwerp.
REFERENCES
B R A N D T J.R.A., G E F . R T S S., D E D E K E N R., K U M A R V., C E U L E M A N S F., B R I J S L. & F A L L A N . A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysticercosis. International Journal for Parasitology. 1992, 22, 471-477.
D E L B R U T T O O . H . , C A M P O S X . , S A N C H E Z J . & M O S Q U E R A A.
Single-day praziquantel versus 1-week albendazole for neurocysticercosis. Neurology, 1999, 52, 1079-1091.
E R H A R T A., D O R N Y P . , N G U Y E N V A N D E , H A V I E T V I E N , D A N G C A M T H A C H , N G U Y E N DUY T O A N , L E D I N H C O N G , G E E R T S S., S P E Y B R O E C K N., B E R K V E N S D . & B R A N D T J . Taenia solium cys
ticercosis in a village in Northern Viet Nam: Sero-preva- lence study using an ELISA for detecting circulating antigen. Transactions of the Royal Society of Tropical Medi
cine and Hygiene, 2002, 96, 270-272.
G A R C I A H . H . , G I L M A N R . H . , H O R T O N J . , M A R T I N E Z M . , H E R - R E R A G . , A L T A M I R A N O J . , C U B A J . M . , R I O S - S A A V E D R A N., V E R A S - T E G U I M . , B O T E R O J . , G O N Z A L E Z A.E. & tHE C Y S T I C E R C O S I S W O R K I N G G R O U P I N P E R U . Albendazole therapy for neuro
cysticercosis: A prospective double-blind trial comparing 7 versus 14 days of treatment. Neurology, 1997 48, 1421- 1427.
G A R C I A H . H . , P A R K H O U S E R.M.E., G I L M A N R . H . , M O N T E N E G R O T . , B E R N A L T . , M A R T I N E Z S.M., G O N Z A L E Z A.E., T S A N G V.C.W. &
H A R R I S O N L.J.S. Serum antigen detection in the diagnosis, treatment, and follow- up of neurocysticercosis patients.
Transactions of the Royal Society of Tropical Medicine and Hygiene, 2000 94, 673-676.
G R O L L E.W. Chemotherapy of human cysticercosis with pra
ziquantel. In: Flisser. Cysticercosis: present state of know
ledge and perspectives. Academic Press, New York, 1982, 207-218.
N G U E K A M , Z O L I A.P., V O N D O U L., P O U E D E T S . M . R . , A S S A N A E., D O R N Y P., B R A N D T J . & G E E R T S S. Kinetics of circulating anti
gens in pigs experimentally infected with Taenia solium eggs. Veterinary Parasitology, 2003 (in press).
P A D M A M.V., B E H A R I M . , M I S R A N.K. & A H U J A G . K . Albenda
zole in single CT ring lesions in epilepsy. Neurology, 1994, 44, 1344-1346.
P O U E D E T M . S . R . , Z O L I A.P., N G U E K A M , V O N D O U L., A S S A N A E., S P E Y B R O E C K N., B E R K V E N S D . , D O R N Y P., B R A N D T J . & G E E R T S S.
Epidemiological survey of swine cysticercosis in two rural communities of West Cameroon. Veterinary Parasitology.
2002, 106, 45-54.
S C H A N T Z P.M., W I L K I N S P.P. & T S A N G V.C.W. Immigrants, ima
ging, and immunoblots: the emergence of neurocysticer- 68
cosis as a significant public health problem. In: Scheld W.M., Craig W.A. and Hughes J.M. Emerging Infections 2.
ASM Press, Washington, D.C., 1998, 213-242.
S O K A L R.S. & R O H L F J.J. Biometry: the principles and practice of statistics in biological research, 2n d ed. Freeman, New York, 1981, 895 p.
S O T E L O J . Treatment of brain cysticercosis. Surgical Neuro
logy. 1997, 48, 110-112.
T S A N G V.C.W. & W I L S O N M. Taenia solium cysticercosis: an under-recognized but serious public health problem. Para
sitology Today 1995, 11, 124-126.
W H I T E C.A. Jr. Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis, and management. Annual Review of Medicine, 2000, 51, 187-206.
Reçu le 7 septembre 2002 Accepté le 15 novembre 2002
Note de recherche Parasite, 2 0 0 3 , 10, 6 5 - 6 8
