Bailey, J.W.

Doua, F.

Godfrey, D.G.

Laveissière, Claude

Truc, Philippe

Horizon / Pleins textes

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1994) 88, 419-421 fi” 9 I t 
A comparison of parasitological methods for the diagnosis of gambian 
trypanosomiasis in an area of low endemicity in Côte d‘Ivoire 
419 
P. T r ~ c ~ ~ ~ ,  J.W. Bailey3, F. Doua4, C. Laveissière2 and D.G. Godfrey‘ ’Department of Veterinaty Medicine, 
Universi&! of Bristol, Langford House, Langford, Bristol, BS18 7DU, UK; 21nstitut Pierre RichetlOCCGE, B.P. 1500, OI 
Bouaké, Côte d‘Ivoire; 3Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 SQA, UK; 4Projet de 
Recherches Cliniques sur la Trypanosomiase, B.P. 1425, Daloa, Côte d’Ivoire 
Abstract 
The card agglutination test for trypanosomiasis (CATT) was used to examine 8974 inhabitants in 14 village 
areas south-west of Daloa, Côte d’Ivoire; 114 (1.3%) were UTT+ or k ,  and were further examined by one 
or more of 6 methods for the direct detection of trypanosomes: lymphatic gland puncture, stained thick 
blood film (TBF), haematocrit centrifugation technique (HCT), mini-anion exchange column (MAEC), 
quantitative buffy coat method (QBC)@, and kit for in vitro isolation of trypanosomes (KIVI). Trypanosomes 
were seen by at least one method in 16 (14.0%) of the CATT+ group. Blood from 356 of the 8860 CATT- 
group was inoculated into RIVI; trypanosomes grew from the blood of 1 person. Eleven of the 17 patients 
with detectable trypanosomes were screened by all 6 methods: 6 were HCT+; 7 were gland+; 10 were 
MAEC+; 10 were KIVIS;  11 were both TBF+ and QBC@+. One CATT+ patient was KIVI+ but otherwise 
negative, although TBF was not done. The overall prevalence of trypanosomes was 0.2% rising to 0.8% in 
one village area. The results support previous evidence that a reappraisal of procedures is required in the 
customary system of surveillance for gambian sleeping sickness. 
Introduction 
Recently the quantitative buffy coat ( Q B P )  and the 
kit for in vitro isolation (KIVI) techniques have been pro- 
posed for the parasitological diagnosis of gambian try- 
panosomiasis (BAILEY & SMITH, 1992; TRUC et al., 
1992); low numbers of trypanosomes in the blood are 
easily detected. QBCB gives a quick result whereas KIVI, 
although perhaps more sensitive, is limited in value be- 
cause of the long wait before the organisms multiply ade- 
quately in culture. 
The initial field trial with the QBC@ was in Uganda 
(BAILEY ¿? SMITH, 1992), where it performed well 
against several other techniques for detecting trypano- 
somes in putative patients; these had positive responses 
to the standard initial screening of whole blood with the 
card agglutination test for trypanosomiasis (CATT; 
MAGNUS et al., 1978), and trypanosomes were found by 
either lymph gland juice aspiration or the haematocrit 
centrifugation technique (HCT; WOO, 1970). However, 
the largely neglected examination of stained thick blood 
films (TBF) also appeared to be efficient; the technique 
confirmed infection in 95% of the patients parasitaemic 
by QBCm, 
By contrast, according to LUMSDEN et al. (1981) TBF 
missed 37% of the infections found in Cóte d’Ivoire by 
another sensitive detection method, mini-anion-exchange 
column and centrifugation (MAEC). This may indicate 
that parasitaemias are lower in Côte d‘Ivoire than in 
Uganda, where the circulating organisms are now known 
to be genetically different (ENYARU et al., 1993). 
Thus, the opportunity was taken to evaluate the 
methods together during a routine Côte d’Ivoire govern- 
ment survey when CATT was used for the initial screen- 
ing. At the same time, surveys were carried out on the 
sympatric animal and tsetse fly populations; the results 
of these will be described elsewhere. 
The prevalence of human sleeping sickness was found 
to be extremely low, so, unfortunately, decisive conclu- 
sions on the diagnostic tests used cot&I.pot be drawn; 
however, the indications reported here’may be of interest 
for future trials. 
Methods 
Study area 
The survey area stretched from about 10 to 30 km 
south-west of the town of Daloa in Côte d’Ivoire. The 
population of several ethnic groups was, as in many areas 
of the region (HERVOUËT & LAVEISSIERE, 1987), in- 
Address for correspondence, Dr P. Truc, Institut Pierre 
RichedOCCGE, B.P. 1500,Ol Bouaké, Côte d’Ivoire. 
a 
.. 
.I ... . -  
i ,_ . .  . i?- 
>. . ” 
volved mostly in coffee and cocoa production, with sub- 
sistence agriculture and limited livestock husbandry. 
The inhabitants, many of whom dwelt in outlying campe- 
ments, were asked to attend a series of screening centres 
arranged, over a period of 10 d, in the nearest of 14 large 
villages (28 October-6 November 1992). 
Techniques 
The Ivorian surveillance team used CATT (MAGNUS et 
al., 1978) with finger-prick blood to screen the 8974 
people who attended the centres. From most of those 
who were CATT+ or CATTI, 8-0 mL of venous blood 
were taken into a syringe containing 0.5 m L  of 5% 
Liquoïde (Roche); 5.0 mL of the mixture was inoculated 
into a KIVI vial (AERTS et al., 1992; TRUC et al., 1992). 
The remainder was used for the HCT (WOO, 1970), the 
MAEC (LUMSDEN et al., 1981), and to provide plasma 
for a further CATT. Finger-prick blood was taken for one 
Q B P  tube (BAILEY & SMITH, 1992). If a subclavicular 
lymph node of a patient was palpable, the fluid extruded 
after puncture was examined for trypanosomes. A thick 
film of 5 pL of blood was also made from a finger-prick 
and stained later in the laboratory by Field’s method 
(FIELD et al., 1963). The whole of the film was examined 
under a ~ 4 0  microscope objective, which took about 30 
min. 
In addition, blood samples for KIVI inoculation were 
taken from 356 of those attending the survey who were 
CATT-; during each of the first 9 days of screening, 32- 
49 individuals were selected without any known bias on 
our part. All were examined by HCT as well, but field 
logistics severely limited the number that could be addi- 
tionally examined by QBCm. 
In Cóte d’Ivoire, the KIVIS were kept in the shade at 
the ambient temperature (2C-2S°C) until arrival in the 
UK where they were maintained at 27°C; weekly examin- 
ations were continued up to a maximum of 8 weeks after 
inocula tion. 
Results 
Altogether 469 KIVIS were inoculated; 6 (1.3%) were 
subsequently discarded when found to be contaminated 
with bacteria or fungi, which inhibit multiplication of 
trypanosomes in vitro. 
Blood from one person, who was both CATT- and 
HCT-, infected a KIVI (Table); since the culture did not 
become positive until 18 d after inoculation, in the UK, 
the original blood sample was not tested by any addi- 
tional method. None of the remaining CATT- people 
whose blood was also inoculated into KIVI gave rise to a 
positive culture; all were HCT-, and no trypanosome 
O. R.S.T.Q,MVI. Fonds Documentaire 
420 
Table. Detection of trypanosomes in patients in an endemic area of Côte d'Ivoirea 
c 
Survey 
centre 
CATT KIVI 
Blood Plasma 3E$: HCT MAEC TBF QBC@ Result Day positive . ..._~ - 
+ + - + + + + + 16 
- + ND + N D  N D  + 13 rt 
- ND N D  ND + 18 
- + + + + 30 - N D  Ziguedia-Goza + + + + + + 12 
- + + 16 + + 12 + b  N D  
Ziguedia-Goza + + + + 
Ziguedia-Goza * + 
Ziguedia-Goza + + + + + + + + - + + + ..*& + + 12 
- + + 4- 12 Ziguedia-Goza 
14 + + + 
+ + + Ziguedia-Goza + + Guédékiprea 
Guédékiprea + + + + + 14 
- + 30 Gbieguhé + Gbieguhé ct: + 
Guediboua + + + -k + + + + 9 
Kékegoza * + + + + + + + 13 
Kékegoza * + + - + + + + 18 
Kiboua + + + + + N D  N D  + 10 
"Abbreviations: CATT, card agglutination test for trypanosomes; HCT , haematocrit centrifuge technique; MAEC, 
mini-anion exchange column; TBF, thick blood film; QBC@, quantitative buffy coat technique; KIVI, kit for in vitro 
isolation of trypanosomes; ND, not done. 
bInitial result; repeat MAEC was positive. 
'Fungal contamination may have prevented trypanosome growth. 
Gboghué 
Gokra 
Zoboua - 
- - 
C - 
- 
- + b  + - - + + - - + + - ND 
N D  - - - 
was seen in any of the 35 who were also examined by 
QBC?. 
One hundred and fourteen persons were CATTS or 
CATT+ (using whole blood or plasma), and 16 of them 
had trypanosomes demonstrated by at least one other 
method (Table). Because of organizational problems in a 
large, fast moving field survey, only 11 of these candidate 
patients were examined by all 6 parasitological proce- 
dures. Six of them were HCT+; 7 had infected glands, 
of whom 3 were HCT- . Ten were initially MAEC+, but 
after the QBC@ was reported positive, 1 patient who was 
MAEC- at first was found to be positive by repeat tests. 
Discounting this repeat MAEC result, 4 MAEC+ pa- 
tients were initially HCT-. All l l of the comprehens- 
ively examined patients were QBC@+ and TBF+,  while 
10 gave rise to positive KIVI cultures; fungal contamina- 
tion may have inhibited trypanosome proliferation in the 
twelfth. 
One of the 6 incompletely examined patients with de- 
monstrable trypanosomes was HCT- but KIVI+ , 
QB@+ and TBF+; MAEC was not done. Another was 
KIVI+, but QBC@-, HCT- and MAEC-; TBF was not 
done. A third was KIVI+ and QBC@+ but was MAEC+ 
only when re-tested. 
Unexpectedly, the prevalence of infection in the whole 
area, as demonstrated by the presence of parasites, was 
only 0.2%. However, this figure was about 4 times 
higher in one survey centre, Ziguedia-Goza; trypano- 
somes occurred in 61782 (04%) of those examined at the 
primary screenmg. 
Discussion 
The few infections found meant that an esrimate of 
comparative diagnostic efficiency could not be reliably 
made. Nevertheless , the study supported previous find- 
ings that QBCB, KIVI and TBF are efficient diagnostic 
techniques. The many apparently uninfected CATT f 
people in the population (97/8974) remain of some con- 
cern; although, unfortunately, the blood was not exam- 
ined by TBF, trypanosomes were found only by KIVI in 
one CATT+ person. It may be that trypanosomes were so 
sparse in the blood that they were undetectable by any 
other blood examination method. They may have also 
been lurking solely in sites away from the bloodstream, 
such as the cerebrospinal fluid. Since positive lymph 
nodes were always confirmed by another technique, it 
seems unlikely that lymph is, at any stage, an exclusive 
environment for trypanosomes, although again the ques- 
tion of concentration must be considered. Nonetheless, 
the discovery by KIVI, as before (TRUC et al. , 1992), of an 
otherwise undiagnosed patient poses the question of how 
many other such people occur in this part of Côte 
d'Ivoire. 
Any estimate must remain largely a matter of conjec- 
ture. Extrapolation from the one positive KIVI result 
among the 356 CATT- people sampled by that method 
may imply that 25 others among the whole CATT- popu- 
lation of 8860 were also infected. A more extensive inves- 
tigation is obviously required. 
The simplest and cheapest diagnostic technique is 
TBF; the waiting period is merely the time taken for 
staining and examining the film in a simple laboratory. 
KIVI is confirmed as valuable in diagnosis (TRUC et al., 
1992), although it was originally designed to overcome 
the difficulties of isolating Trypanosoma brucei gambiense. 
Its prime disadvantages as a routine tool are high cost 
and the long period of waiting for positive results , in this 
survey up to 30 d; moreover, infections may be missed 
because of microbial contamination inhibiting trypano- 
some multiplication. However, the sensitivity of KIVI is 
illustrated by the discovery of infection in, not only a 
CATT- patient, but also a CATT+ patient in whom try- 
panosomes were not detected by other sensitive direct 
techniques. 
QBC@ has a distinct advantage in giving a clear result 
in less than 10 min in the field. The initial capital outlay 
is high, but the equipment should last a considerab!e 
time. The running cost for the special capillaries approxi- 
mates that of the MAEC, which is not an easy method to 
use in the field. QBC@ appears to be at least as efficient as 
MAEC, which works well in the hands of an expert 
team, such as the Ivorian unit that conducted the routine 
part of this survey. A comparable, well organized team 
could use the QBC@ to considerable advantage and in- 
crease the numbers of people surveyed. 
The success of the TBF is perhaps the most surprising 
feature of this survey, confirming the earlier observations 
in Uganda (BAILEY & SMITH, 1992); it appeared better 
than another widely used technique, HCT. The un- 
fashionable but cheap TBF must be further re-evaluated 
in the field. 
Acknowledgements 
We thank the technical staff and many others in Côte 
D'Ivoire for their assistance in both the field and laboratory, and 
our colleagues in the UK for their technical help and advice. 
42 1 
Financial support was provided by the UNDPNorld 
BanWHO Special Programme for Research and Training in 
Tropical Diseases and by the UK Medical Research Council. 
References 
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scopical Diagnosis of Human Malana. Kuala Lumpur: In- 
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ment and sleeping sickness in the forest belt of West Africa. 
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Lumsden, W. H. R., Kimber, C. D., Dukes, P., Haller, L., 
Stanghellini, A. & Duvallet, G. (1981). Field diagnosis of 
sleeping sickness in the Ivory Coast. 1. Comparison of the 
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Magnus, E., Vervoort, T. & Van Meirvenne, N. (1978). A card 
agglutination test with stained trypanosomes (an) for the 
serological diagnosis of Typatiosoma brucei gambiense. Annales 
de la Société Belge de Médecine Tropicale, 58, 169-176. 
Truc, P., Aerts, D., McNamara, J. J., Claes, Y. ,  Allingham, R. 
Le Ray, D. & Godfrey, D. G. (1992). Direct isolation in vitro 
of Typanosoma brucei from man and other animals, and its 
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Received 10 Augus t  1993; revised 27 September 1993; 
accepted for publication 29 September I993 
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL hlEDlClNE AND HYGIENE (1991) 88, 421-422 
1 Short Report] 
Serodiagnosis of cysticercosis: 
specificity of different antigens and 
enzyme-linked immunosorbent 
assays 
T. F. Ng and R. C .  KO* Department of Zoologl, T h e  
University of Hong Koitg, H o n g  Kong 
Cysticercosis is endemic in Hong Kong and various 
parts of China (KO, 1991). At present, serodiagnosis of 
this disease remains difficult, mainly due to the fact. that 
the commonly used somatic antigens of larval Taenia so- 
liuin cross-react with a wide range of heterologous anti- 
bodies (FLISSER et al., 1982; CHENG & KO, 1991). The 
lack of animal models also hampers the development of 
more reliable assays to identify acute or chronic infec- 
tions. 
Since the excretory-secretory (ES) antigens of some 
helminths are known to be highly specific, the present 
study was undertaken to evaluate the feasibility of using 
ES antigens of C'sticercus cellulosae in serodiagnosis. The 
specificity of ES antigens was compared with that of whole 
cyst (W), membrane (M) and cystic fluid (F) antigens. 
An attempt was also undertaken to determine the eff- 
cacy of 4 recently developed enzyme-linked immuno- 
sorbent assays (ELISAS) in detecting immunoglobulin G 
(IgG) antibodies when different antigens were used. 
ES antigens were obtained by maintaining cysts 
(freshly collected from pork) in mediwm-199 for 3 d in a 
CO2 incubator at 37°C. After ultrafiltration and desalt- 
ing, the concentrated ES sample was centrifuged for 1 h 
at 3000 g to remove tissue debris. W antigens were ob- 
tained by homogenizing intact cysts with a Telflong ho- 
mogenizer. M antigens consisted of the cyst wall and 
protoscolex. F antigens, which were collected by aspira- 
tion using a 27 gauge needle, were centrifuged at 15 O00 
g to remove tissue debris. 
Sera samples were collected from 39 patients admitted 
to various hospitals in Hong Kong who were suspected 
'Author for correspondence. 
a 
to be suffering from cysticercosis because of the presence 
of one or more of the following symptoms: meningitis, 
epileptic fits, presence of cysts in brain or other tissues. 
Heterologous antisera were collected from patients suf- 
fering from other parasitic infections. Negative control 
sera were obtained from members of our laboratory staff. 
The optimal antigenic concentrations for W, M, F and 
ES used to coat microtitration wells were 10, 10, 1 and 5 
pg/mL respectively. Double antibody ELISA was per- 
formed according to VOLLER et al. (1976). Goat anti- 
human IgG conjugated with horse-radish peroxidase 
(HRP) was used as secondary antibody. Triple antibody 
ELISA was performed according to GAMBLE et al. (1983) 
and KO & YEUNG (1989). Goat anti-human IgG and rab- 
bit anti-goat IgG conjugated with HRP were used as sec- 
ondary and tertiary antibodies respectively. o-Phenylene- 
diamine was used as substrate in both double and triple 
antibody ELISAs. The FAST-ELISA (Falcon Assay Screen- 
ing Test ELISA) was modified from the method of HAN- 
COCK & TSANG (1986). For the immunobead ELISA, fer- 
rous metal BionecticB beads (Organon) served as the 
solid support. They were coated with antigens following 
the method of KO & YEUNG (1992). An electromagnetic 
device designed by one of us (R.C.K.) was used to trans- 
fer the beads. Tetramethyl benzidine (TMB) was used as 
the substrate. Samples were considered positive when the 
cut-off value of each ELISA was 3 t h e s  the mean optical 
density (OD) of normal serum samples. 
The triple antibody ELISA was the best. I t  yielded the 
smallest number of false positive reactions with heterolo- 
gous antisera while giving the largest number of positives 
with sera from patients suspected of neurocysticercosis 
(Table). The double antibody ELISA generally yielded 
lower O D  values with positive sera than the triple anti- 
body ELISA. Immunobead ELISA appeared to be the least 
specific assay as it yielded false positive reactions with 
sera from patients with hydatidosis, angiostrongyliasis 
and ascariasis. FAST-ELISA was the least sensitive assay as 
it yielded a false negative reaction even with serum from 
a patient with a positive muscle biopsy. 
Of the 4 antigens tested, ES antigen was the most spe- 
cific and yielded the largest number of cases with positive 
OD. Unlike the other antigens, it did not cross-react 
with the hydatid serum. A separate study (NG, 1991) has 
shown that ES antigen of C. cellulosae consists of 19 poly- 
peptides. The specificity of W and M antigens was fairly 


A comparison of parasitological methods for the diagnosis of gambian trypanosomiasis in an area of low endemicity in Cote d'Ivoire

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