Frézil, Jean-Louis

Gouteux, Jean-Paul

Lemesre, J.L.

Louembet, M.T.

Noireau, François

Nzoukoudi, M.Y.

Horizon / Pleins textes

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TRANSACTIONS F THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1988) 82, 237-240 9 0 2  237 
Serodiagnosis of sleeping sickness in the Republic of the Congo: 
com pa rison of indirect immun of lu orescent anti body test 
and card agglutination test 
F. Noireau, J. L. Lemesre, M. Y .  Nzoukoudi, M. T. Louembet, J. P. Gouteux and J. L. 
Frezil Laboratoire d’Entomologie Médicale et de Parasitologie, Centre O R S T O M  de Brazzaville, BP 181, 
Republic of the Congo 
Abstract 
The card agglutination test for trypanosomiasis 
(CATT) was evaluated and compared to the classical 
immunofluorescent antibody test (IFAT) in the im- 
munological diagnosis of Gambian trypanosomiasis. 
Tests were performed on serum and whole blood. 
Cross-reactions were found in the CATT with sera 
from patients suffering from parasitic infections other 
than sleeping sickness, but could be largely overcome 
by selecting 1/10 as the specific threshold dilution. At 
1/40 dilution no false positive result was observed in 
the IFAT. At the specific threshold dilution, the 
sensitivity of IFAT was 94*7%, compared with 91.6% 
for the CATT. On whole blood, a more convenient 
sample in the field, IFAT specificity (100%) was 
greater than that of the CATT (94*3%), as was its 
sensitivity (92% compared with 82.5%). In view of its 
simplicity and rapidity of execution, the CATT is an 
efficient serological test to detect new foci. When 
greater sensitivity is required, IFAT should be 
preferred to CATT. 
Introduction 
At least 45 million people in Africa are estimated to 
be exposed to trypanosomiasis. In the People’s 
Republic of the Congo, more than 600 new cases are 
reported annually. The causative organism, Typano- 
soma brucei gambime, can usually be detected in the 
circulating blood, lymph nodes or cerebrospinal fluid 
by repeated microscopical examination. However, 
because of the relatively low sensitivity of parasitolo- 
gical techniques, serological tests are indispensable 
for large scale screening of the population at risk. 
Only a few comparative evaluations of serological tests 
have been carried out (WHO, 1976; KNOBLOCK et 
al., 1984); and their usefulness is still disputed. 2 
hnunoassays are currently used in the Congo: the 
indirect immunofluorescent antibody test (IFAT), 
considered as the reference method, and more recent- 
ly the card agglutination test for trypanosomiasis 
(CATT), described by MAGNUS et al. (1978). We 
Present here an evaluation and comparison of these 
serological techniques in the diagnosis of Gambian 
sleeping sickness. 
Materiais and Methods 
Serum samples 
Sera were collected from 366 Congolese persons of 
(1) 95 patients with parasitologically confirmed 
trypanosomiasis; their mean age was 28.9k16.1 years 
(range 4-74); the male/female ratio was 1.17. 
(2) 105 healthy Congolese living in Brazzaville, an 
aea free of human trypanosomiasis. 
the following 3 groups. 
(3) 195 patients with one or more of the 3 following 
parasitic infections. (a) Filariasis (Loa loa and/or 
Mansonella perstans); 115 patients belonging to two 
ethnic groups-Bantu (81) and Pygmy (34). The 
diagnosis of filariasis was based upon clinical (Calabar 
swelling, worm migration under the conjunctiva) or 
parasitological data. (b) Schistosomiasis: 52 patients 
with parasitologically confirmed Schistosoma haemato- 
bium infection. (c) Toxoplasmosis: 28 patients with 
positive serological, diagnosis (high titre obtained in 
passive agglutination test). 
Except for sub-group (3b), these patients had not 
been exposed to the risk of Trypanosoma infection. 
The mean age of the subjects of groups (2) and (3) was 
37.7t-16.5 years (range 6-80) and the male/female 
ratio was 1.57. 
Serum samples were stored frozen at -20°C until 
used. 
Dried blood samples 
4 groups. 
gically confirmed trypanosomiasis. 
same origin as group 4. 
exposed to Onchocerca volvulus infection. 
perstans infection. 
Test systems 
I F A T  on serum. Details of this test have been 
described by W É R Y  et al. (1970). The test was carried 
out according to the mofications of FRÉZIL et al. 
(1974). A Congolese T. b. gambiense stock (MHOM/ 
CG/ORBZV/113) was maintained in rats for antigen 
production. Sera were tested at twofold dilutions 
ranging from 1/20 to 1/160. 
IFAT on dried blood. Dried blood samples were 
collected on Whatman No. 1 filter paper as described 
by BAILEY et al. (1967). Discs of 5 mm diameter were 
punched out of each sample. The resulting dilution of 
blood in phosphate-buffered saline (pH 7.2) was 
approximately equal to a 1/10 serum dilution (WÉRY 
et al., 1970). 
CATT on whole blood. This qualitative screening 
test, and the other CATT procedures, were carried 
out according to the method of the manufacturer 
(Smith Kline-RIT, lot number 47/7). Agglutination 
patterns of 1+  or more were considered as positive. 
CATT on s e ”  (qualitative method). This screening 
system used 5 pl of serum. 
CATT on serum (quantitative method). Sera with 
These were collected from persons in the following 
(4) 87 patients from Bouenza focus with parasitolo- 
(5) 154 other trypanosomiasis patients from the 
(6) 255 individuals free of trypanosomiasis but 
(7) 141 patients exposed to Loa loa and Mansonella 
ORSTOM Fonds Documentaire 
238 
Table 1-Tests used for each group of samples of various geographical o n a s  
Number of Geographical 
Group patients ongm 
1 95 Bouenza & 
Parasitology' Sample Tests used 
T serum IFAT & CATT 
Plateaux 
2 105 Pool O serum IFAT & CATT 
3 195 Lekoumou, Pool O serum IFAT & CATT 
& Bouenza 
4 87 Bouenza T blood IFAT 
5 154 Bouenza T blood CATT 
6 255 Pool O blood IFAT 
7 141 Lekoumou O blood CATT 
IT = trypanosomiasis patients; o = patients free of trypanosomiasis. 
NR 
- 
B 
B 
8 
8 
8 
a 
a 
a 
a 
* 
a 
- 
- 
t 
t 
t 
t 
t 
t 
t 
t 
t 
t 
t 
t 
t 
t 
4- 
t 
+ 
t 
t 
-t 
t 
i - 
- 
. 
m . 
m . 
m 
m 
m . 
m 
m . 
IFAT 
II I : I  I 
CATT 
Titres Titres 
Figure. Distribution of end-titres in sera from: 95 trypanosomiasis cases ., 105 healthy patients (O), 115 patients with filariasis (FI), 52 patients 
with schistosomiasis (O) and 28 patients with toxoplasmosis (w. 
NR = no reaction. 
positive qualitative tests were titrated at twofold 
dilutions from 1/5 to 1/80. 
Sample origins and ranges of tests applied to each 
group of samples are summarized in Table 1. 
Results 
IFAT results 
The Figure shlows the distribution of the IFAT 
titres obtained with sera from the different groups. 
Analyzing individuals free of trypanosomiasis (groups 
2 and 3), no false positive reaction was observed at a 
serum dilution of 1/40, so this titre was selected as the 
specific threshold dilution. Using samples collected 
from T .  b .  gambiense patients, IFAT sensitivity was 
94*7%, and its specificity was 100%. Only 5 patients 
reacted negatively. Among them, 2 did not present 
any cerebrospinal fluid alteration. Whatever the type 
of sample used (serum or dried blood), there was no 
significant difference in the IFAT sensitivities 
obtained using serum and dried blood (94.7% and 
92% respectively). The specificity of IFAT performed 
on dried blood samples was 100% (group 6). 
CATT results 
Titres given by CATT on sera collected from 
patients free of trypanosomiasis are shown in the 
Figure; they formed a unimodal distribution around 
1/20. The 1/10 dilution was considered as the specific 
--- 
Table 2-S, 
Parasitic iní 
Filariasis 
Schistosox 
Toxoplasr 
Healthy 1 
Table %-Sei 
samples frai 
results 
No. 
("/.I 
threshold d! 
dilutions, f 
observed in 
diseases (gr 
(group 2). 
The qua 
reactions w 
(Table 2). 
microfilarial 
33.5% of p: 
the qualitar 
without mil 
more specif 
(75.3% con 
As showr 
91.6% whei 
used. Althc 
the 115 dilu 
preferable 1 
samples no 
when teste( 
performed 
that observ 
its lack 01 
method. 1 
whole bloo 
type of SaIr 
(94.3%), a: 
serum dilu 
Discussior 
The dial 
' field condit 
sensitivity 
which are : 
large-scale 
Tests used 
IFAT & CATT 
IFAT & CATT 
IFAT & CATT 
IFAT 
CATT 
IFAT 
CATT 
0, 52 patients 
5 patients 
It present 
r the type 
re was no 
isitivities 
1.*7% and 
erformed 
6). 
ed from 
1 in the 
1 around 
: specific 
239 
Table 2-SpecScity of the CATT on serum: numbers of samples giving negative results 
Qualitative 
screening Serum dilution 
test 1/5 1/10 
Parasitic infection 
Filariasis No. 81/115 104/115 113/115 
(%I (70.4) (90-4) (98.3) 
Schistosomiasis No. 46/52 48/52 51/52 
(%) (88.5) (92.3) (98.1) 
Toxoplasmosis No. 18/28 26/28 28/28 
Healthy patients No. 105/105 1051105 105/105 
(%I (64-3) (92.8) ( 100) 
(%> ( 100) (100) (100) 
Table 3-Sensitivity of the CATT on serum: numbers of 
samples from trypanosomiasis patients giving positive 
results 
Qualitauve 
screerung Serum dilution 
test 115 1/10 
No. 92/95 92/95 87/95 
(%I (96.8) (96.8) (91.6) 
threshold dilution for the CATT (Table 2). At lower 
dilutions, false positive reactions were frequently 
observed in patients suffering from other parasitic 
diseases (group 3), but never in healthy patients 
(group 2). 
The qualitative CATT gave many non-specific 
reactions when samples of group 3 were analysed 
(Table 2). In filariasis patients, the presence of 
microfilariae seemed to have no a u e n c e  on the test. 
33.5% of patients with microfilariae were positive by 
the qualitative test, compared with 27.7% of those 
without microfilaraemia. The CATT appeared to be 
more specific in the Bantu group than in the Pygmies 
(75.3% compared with 58.8% P ~ 0 . 1 ) .  
As shown in Table 3, the CATT had a sensitivity of 
91.6% when the specific threshold dilution (1110) was 
used. Although we observed greater sensitivity with 
the 1/5 dilution, we considered that 1/10 dilution was 
preferable because it was more specific. Among the 3 
samples non-reactive to CATT, 2 were also negative 
when tested by IFAT. When the qualitative test was 
performed on sera, the sensitivity was comparable to 
that observed when 1/5 dilution was used. However, 
its lack of specificity precludes adoption of this 
method. The sensitivity of CATT performed on 
whole blood was lower (127/154; 82.5%). With t h i s  
type of sample (group 7) , the test specificity was high 
(94*3%), and was similar to that obtained with 1/5 
serum dilution. 
Discussion 
The diagnosis of Gambian trypanosomiasis under 
field conditions presents some difficulties. The lack of 
sensitivity of the classical parasitological methods, 
which are moreover time-consuming, precludes their 
large-scale application. An initial serological screening 
test is therefore required. The easiest type of sample 
to use for mass surveys in the field is blood collected 
by fhger prick. For testing whole blood, IFAT, 
because of its great specificity and sensitivity, is 
preferable to CATT. But, due to its simplicity and 
rapidity of execution, CATT must be preferred to 
IFAT for detecting new foci. The high specificity of 
the CATT on whole blood confirms the results of 
BAFORT et al. (1986), but better specificity was 
obtained by testing serum at the 1/10 dilution. 
In our experience the h e  agglutination observed 
with some sera tested at 115 or lower dilutions is a 
non-specific reaction, possibly caused by other parasi- 
tic infections. This particular phenomenon may be 
masked by red cells when the test is performed on 
whole blood. 
Our results confirm the statements of FREZIL et al. 
(1977) and DWALLET et al. (1979) that a positive 
IFAT is fully reliable evidence for the diagnosis of. 
Gambian sleeping sickness. In the Congo, the very 
high specificity of this test might be the consequence 
of the absence of trypanosomes of the subgenus 
Typanozoota other: than T. b. gambiense (see NOIREAU 
et al., 1986). In conclusion, even if the value of 
serological tests for individual diagnosis of trypanoso- 
miasis remains controversial, we are convinced that 
their value for mass surveys is beyond doubt. 
Acknowledgements 
This investigation received hancial support from the 
UNDPNorld BanldWHO Special Programme for Research 
and Training in Tropical Diseases. We are indebted to Smith  
Kline- RIT (Biological Division), who generously provided 
the CATT kits, and we are also grateful to Drs N. Van 
Meirvenne and M. Tibayrenc for invaluable help in correc- 
tion of this manuscript. 
240 
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C. J. Leake 
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Serodiagnosis of sleeping sickness in the Republic of the Congo : comparison of indirect immunofluorescent antibody test and card agglutination test

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