Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2003101p65
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.***
Résumé
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, neurocysticercosis, albendazole.
:
SUIVI DE PATIENTS ATTEINTS DE NEUROCYSTICERCOSE APRÈS
TRAITEMENT EN UTILISANT UN TEST 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 CTscan 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.
N
eurocysticercosis (NCC) is an infection o f the
central nervous system by the larval stage o f
Taenia solium. It is recognized as a c o m m o n
c a u s e o f neurological disease in developing countries
(Schantz et al., 1998). T h e p r e s e n c e o f T. solium metac e s t o d e s in the nervous system leads to a variety o f
clinical manifestations depending upon the number,
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 treatment o f NCC. Follow-up after treatment is usually
d o n e using c o m p u t e d tomography (CT-scan), which
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 developing countries (Tsang ¿4 Wilson, 1 9 9 5 ) . Considering
the importance o f NCC and the increasing poverty in
these countries, it is necessary to investigate alternative techniques, which could reduce at least the cost
o f evaluation o f the efficacy o f the treatment. Since
an antigen detection ELISA (Ag-ELISA) has b e e n deve-
* University 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 .
** Central 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 .
Cameroon.
*** Institute o f T r o p i c a l M e d i c i n e , Nationalestraat, 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 , Nationalestraat 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
Parasite, 2 0 0 3 , 10, 6 5 - 6 8
loped, which has a high sensitivity for the detection
of human cysticercosis (Erhart et al., 2 0 0 2 ) and which
allows to distinguish cattle and pigs carrying living
cysts from those harbouring only dead cysts (Brandt
et al, 1992; Nguekam et al., in press), it was decided
to evaluate its use in NCC patients. T h e purpose o f
this study was to c o m p a r e the Ag-ELISA test with CTscan to evaluate the efficacy o f an albendazole treatment 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
Yaounde Central Hospital) were included in this study.
NCC was asymptomatic in all but two of them, who were
epileptics (Batoula 94 and K E ) . Five o f them were
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 of five viable cysts (range:
1 to 12) and o f 7.6 calcified cysts (range: 0 to 25) in the
Note de recherche
65
NGUEKAM, ZOLI A.P., ONGOLO-ZOGO P. ETAL.
brain. T h e localisation o f the cysticerci was parenchymal
and/or subarachnoidal. Informed consent was obtained
from each 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 albendazole (Alben®
4 0 0 mg, Smithkline B e e c h a m ) at a dosage o f 15 m g / k g
b o d y weight/day for eight days as described by Del
Brutto et al. ( 1 9 9 9 ) . T o prevent adverse reactions,
prednisolone (Solupred® 5 mg, Laboratoire H o u d é ) at
10 mg per person thrice a day w a s given from o n e
day prior to the albendazole treatment until at least
four days after the end o f it (Groll, 1982). T h e two epi­
leptic patients continued to receive anti-epileptic treat­
ment (Gardenal®).
Six to nine months after the end o f the first treatment,
the patients w h o remained seropositive w e r e treated
again with the s a m e d o s e o f albendazole for a period
o f o n e month. In o n e patient ( K E ) the first treatment
w a s not a course o f eight days, but o f o n e month.
ANTIGEN DETECTION E L I S A FOR CYSTICERCOSIS
(AG-ELISA)
T h e patients w e r e sampled before the beginning o f the
treatment, o n e and three months after the first treat­
ment and in those patients w h o received a s e c o n d
treatment, o n e month 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 described b y Brandt et al. ( 1 9 9 2 )
but slightly modified according to Pouedet et al. (2002).
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 (MoAb) w e r e used in a sandwich
ELISA. MoAb B 1 5 8 C 1 1 A 1 0 was used for coating and a
biotinylated MoAb B 6 0 H 8 A 4 was included as detector
antibody. O r t h o p h e n y l e n e diamine and H 0 w e r e
used as chromogen-substrate solution. After arresting
the reaction with 4 N H S O the plates w e r e read using
an ELISA reader (Labsystem Multiskan RC) at 4 9 2 nm.
2
2
2
4
Patients
Before
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 taeniasis or cysticercosis in the family) and o n e reference
positive s e r u m from a C a m e r o o n i a n patient with
confirmed cysticercosis (by 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 was c o m p a r e d with the mean 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, 1981). 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 was considered as positive.
CT-SCAN OF THE BRAIN
The CT-scans were performed in Y a o u n d e Central Hos­
pital using a S o m a t o m AR STAR scanner ( S i e m e n s
Medical Systems, Erlangen, Germany) before and after
contrast fluid injection (Telebrix®35, Guerbet, France).
Brain image slices o f five m m thickness were transferred
on negatives and scan reports were produced, indica­
ting the number and types o f lesions compatible to NCC.
T w o CT-scans were 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
was performed six to ten months later, whereas the
patients, w h o r e c e i v e d a s e c o n d treatment, w e r e
scanned 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 Table I and Figures 1 and 2.
A G - E L I S A RESULTS
O n e month after the end o f the first course o f alben­
dazole three patients b e c a m e negative in the Ag-ELISA
treatment
CT-scan
S e x / a g e / e p i l e p t i c status
n o . l i v / n o . calc
After treatment
CT-scan
ELISA ratio
n o . l i v / n o . calc
ELISA ratio
- B L 21
M/73/N
1/1
1.8
0/1
0.7*
- B a l e o 01
F/07/N
2/0
1.9
0/0
0.8
1.3
11.5
0/0
0.6
0/3
0.8*
- B F K P 137
M/60/N
3/0
- BMKP 194
F/60/N
1/6
- Batoula 94
F/32/E
2/25
F/15/N
6°/ 25
1276
48.7
- Metchou 378
12.5
0/12
0.7
- KE
F/32/E
10/15
11.0
4°/15
18.9
20.6*
*: 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 cysts; 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. — Ag-Elisa 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
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F O L L O W - U P O F NEUROCYSTICERCOSIS USING
ELISA
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
Fig. 1. - Antigen m o n i t o r i n g results in a c t i v e NCC patients (initial
Flisa ratio > 1 0 ) .
Fig. 2. - Antigen m o n i t o r i n g results in a c t i v e NCC patients (initial
Flisa ratio < 1 0 ) .
(Table I ) . Patient ( K E ) received only o n e treatment
course during o n e month and remained seropositive
o n e month after therapy. A s e c o n d Ag-ELISA per­
formed on serum samples 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 (BL 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 seven ( 7 1 . 4 % ) patients.
In the two others, either a reduction o f the number o f
viable cysts ( B a t o u l a 9 4 ) or the p r e s e n c e o f ring
e n h a n c e d cysts indicating a process o f degeneration
(KE) was observed after treatment (Table I). In all
patients where the viable cysts disappeared after treat­
ment a negative ELLSA result (ratio < 1) was obtained
whereas a persistence o f even 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 now. CT-scan in Cameroon 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 each patient
involved in the present study to approximately 152.2 €
( 1 0 0 , 0 0 0 CFA) whereas the cost o f a test o f an AgParasite. 2 0 0 3 . 10. 6 5 - 6 8
C
omputed tomography is an useful imaging tech­
nique 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 compared this technique with an Ag-ELISA as an
alternative method for the follow-up o f neurocysticer­
cosis patients after cysticidal treatment. T h e Ag-ELISA
has b e e n shown to detect the excretory-secretory pro­
ducts o f viable cysticerci in cattle (Brandt et al., 1992),
pigs (Nguekam et al, in press) and man (Erhart et al,
2002). These latter authors reported a sensitivity of 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 same for
the two techniques, 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 albendazole courses, s h o w e d a complete
disappearance o f the viable cysts in the brain whereas
the two remaining seropositive patients (Batoula 94 and
KE) showed 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 antibody-based Elisa used 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 one 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 antibody-based Ag-ELISA is a promising tech­
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 developing countries where
poverty is an increasing reality. Currently, studies on
a larger number o f NCC patients are going on in order
to validate the Ag-ELISA as an alternative for CTscan.
67
N G U E K A M , Z O L I A.P.. O N G O L O - Z O G O
ETAL.
P.
ACKNOWLEDGEMENTS
T
his study was carried out with the financial sup­
port o f the Belgian Directorate General for Inter­
national Cooperation (DGIC, Brussels) and o f
the University o f Dschang. T h e research w a s d o n e
within t h e framework agreement b e t w e e n DGIC a n d
the Institute o f Tropical Medicine, Antwerp.
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