Research/Recherche Variant-specific trypanolytic antibodies in sera from patients infected with Trypanosoma brucei rhodesiense W.K. Isharaza1 & N. Van Meirvenne2 Infections with salivarian trypanosomes are characterized by the successive development of parasite populations of distinct variable antigen types (VATs), the corresponding antibodies accumulating in the blood of the host. Sixty VATs had been cloned during previous studies on variable antigen repertoires of Trypanosoma brucei rhodesiense; 12 of these were selected for immunolysis tests against 85 sera from T.b. rhodesiense patients in Busoga (Uganda). One variant, ETat 1/1, reacted with 59 out of 65 sera that contained detectable lytic antibodies. ETat 1/1 in combination with two other variants ETat 1/14 and Utat 1/1, covered all the seropositive sera; other VA Ts showed varying degrees of reactivity. The results suggest that sera from T.b. rhodesiense patients contain easily detectable VAT-specific antibodies and that their corresponding antigens might be used in the preparation of serodiagnostic reagents for the disease.
The cell membrane of salivarian trypanosomes is covered by a monolayer of glycoproteins. These surface glycoproteins are highly immunogenic and elicit trypanocidal antibodies. However, the organisms are able to change their surface coats and form new antigenic variants before the antibodies eliminate the majority of parasites in the blood. As a result, infection is maintained until the host succumbs to the disease. The continuous appearance of new variants results in fluctuating parasitaemia which makes parasitological diagnosis difficult. Currently, there are several serological techniques for the diagnosis of human trypanosomiasis. Up to now, the Trypanosoma brucei rhodesiense serological tests have been based on common antigen/antibody reactions whereas in routine diagnosis of T.b. gambiense, which has less antigenic variation compared with T.b. rhodesiense (1-3),a variable antigen/antibody reactions have been used.
The immunofluorescent antibody test (IFAT) (4,5) and enzyme-linked immunosorbent assay (ELISA) are based on both common and variable antigen/ antibody reactions 6-9). Specific tests for only variable antigen/antibody reactions are trypanolysis (10), indirect haemagglutination (11), and direct agglutination (1) which has recently been developed further into the card agglutination test of stained trypanosomes (CATT), in which a well-defined variable antigen type (VAT) was identified for diagnosis of T.b. gambiense (12). The large number of different VATs belonging to different serodemes that are circulating in the field make it difficult to identify suitable VATs for use in serodiagnosis, especially of T.b. rhodesiense. When rodent models were used in comparative studies of variable antigen repertoires of T.b. rhodesiense isolates from Uganda, several predominant VATs were detected (3).a The objective of the present study was to screen these VATs for specific antibodies in patients' sera and to assess their potential use in serodiagnosis.
' Principal Research Officer, Uganda Trypanosomiasis Research Organisation, (U.T.R.O.) P.O. Box 96, Tororo, Uganda. Requests for reprints should be sent to this author. 2 Chief, Serological Laboratory, Prince Leopold Institute for Tropical Medicine, Antwerp, Belgium. Reprint No. 5042
Isharaza, W.K. Trypanosoma brucei rhodesiense: antigenic diversity and host protective immunity. Ph.D. thesis, Free University, Brussels, 1984.
Bulletin of the World Health Organization, 68 (1): 33-37 (1990)
@© World Health Organization 1990
Introduction
'
33
W.K. Isharaza et al.
Materials and methods VA Ts and sera Clones of the following VATs circulating in Uganda
used: ETat 1/1, 1/2, 1/3, 1/10, 1/14, 1/18 and 1/19 (13); and UTat 3/1, 3/7, 1/1 and 4/1 (4). AnTat 25/1 from an isolate of an unrelated serodeme from Rwanda (15) was also included. All these VATs had been cloned in mice as described by Van Meirvenne et al. (10). The sera were obtained from T.b. rhodesiense patients in Busoga (Uganda) and grouped according to parasitological diagnosis: Group A- trypanosomes seen either in blood, cervical gland juice or cerebrospinal fluid of patients admitted to the Uganda Trypanosomiasis Research Organisation hospital (69 sera); Group B- trypanosomes seen in patients' blood during field surveys but gland juice and cerebrospinal fluid not examined (16 sera); Group C- negative control sera from Caucasians with no history of trypanosomiasis (5 sera); were
Group
D-
negative control
no
sera
from Africans with
history of trypanosomiasis (5 sera).
Immunolysls test Test sera were dispensed in aliquots of 5 41 into Cooke's microtitration plates. Parasitized blood from mice infected with a given VAT two or three days previously was suspended in guinea pig serum to give a concentration of 12 trypanosomes per microscope field at a magnification of x 400. Aliquots of 25 pl of this suspension were added to the serum samples. The resulting mixtures contained patients' sera at a dilution of 1/6 and 10 trypanosomes per microscope field. These plates were sealed and shaken for a few minutes on a plate shaker before incubation at room temperature for two hours. The plates were shaken again and samples of wet films were taken for phasecontrast microscopic examination. At least 10 fields per sample were examined and the percentage of lysed trypanosomes was recorded. Among the 85 sera there were 8 which could not be examined against all the antigens; these incomplete series have nevertheless given results on a range of cross reactions.
Results Immunospeclficlty of the lysas test Three ranges of immunolysis were scored as 1-10% (0), 10-90% (±) and 90-100% (+). None of 34
the ten negative control sera in groups C and D reacted with any of the VATs tested, thus demonstrating absence of non-specific trypanolysis. Therefore, any lysis observed in the test sera of groups A and B (Table 1) was attributed to trypanosome-induced lytic antibodies in the patients. In view of the unequivocal results among the negative controls, those sera scored as + or + were considered as seropositive. The latter group may have contained specific antibodies whose titres were slightly too low for the respective VATs, especially since all sera in this category (with the exception of No. 19, 157, S157 and S213) fully lysed one or more VATs. Reactivity of VATs In groups A and B, 18 sera out of the 77 that were completely tested showed no trypanolytic activity on any of the 12 VATs tested although trypanosomes had been observed in these patients. Of these seronegative patients, 13 belonged to group A in which the cerebrospinal fluid had been examined and only two patients (No. 84 and 166) had positive CSF. It is possible the infections were still at a very early stage and/or the antibody titres, if any, were too low to be detected. Alternatively, these patients may have been infected with serodeme(s) unrelated to those of the VATs tested. Out of the 85 test sera in groups A and B, 65 reacted with one or more of these VATs; 46 of the positive sera reacted with six or more VATs out of the eleven from Busoga. When assessing the relative VAT reactivities as defined in Table 2, only those sera that lysed one or more VATs were considered. The highest number of positive sera and a relative reactivity of 90.8% was recorded for ETat 1/1. Etat 1/14, 1/18, 1/19 and Utat 1/1 also showed high relative reactivities of 75% and above. Slightly lower values were obtained from UTat 3/1 and 4/1 as well as Etat 1/2 and 1/10. ETat 1/3 and UTat 3/7 gave significantly low relative reactivities of 33.9%; these two VATs are relatively less predominant in their respective repertoires.b AnTat 25/1, which originates from Rwanda and belongs to a serodeme entirely different from those of Uganda, showed the lowest relative reactivity (18%).
Discussion All the VATs showed positive reactions with sera but in varying proportions. Sixty-five sera contained detectable VAT-specific lytic antibodies. Among the VAT antigens for use in serodiagnosis, ETat 1/1 appeared to be the best candidate; of the 65 seropositives, only 6 were negative to this VAT. WHO Bulletin OMS. Vol 68 1990.
Variant-specific trypanolytic antibodies In
sera from
patients Infected with T.b. rhodeslensee
Table 1: VAT-specific immunolysis of seiected VATs by sera from patients Infected with T.b. rhodesiensee Variable antigen type (VAT)"
0 v o
In
0
o
Group A 0 0 0 0 0 + 0 0 + + + + 0 +
20 22 152 27 30 31 32 33 34 35 40 166 191
+ + + + + + 0 0 0 + 0 + + + 0 + + + + 0 + +
44
54 55 57 59 64 65 66 67 68 69 71 72 73 74 75 76 81 82
0
+ + +
+ + +
0 0 0 + 0 0 0 + 0 0 0 + 0 + 0
+
0 0
0
+ + 0
0 0 + 0 + 0 0 0 + + + 0 + 0 0 0 0 +
841 92 149
+
153 154
0 +
155
+
156 157
0
a1 +
0
+
83 84
0 0 0 0 0 + + 0 + 0 0 0 + + + 0 + 0 + 0 0 0 + + 0 + 0 0 + ++ + + + ++ + + 00 0 0 + 0 + + 0 + + + + + 0 +0 00 0 0 + 0 + + + 0 + 00 0 + ++ 0 + + + + + + 0 + 0 + 0 0 0 0 0 + + + + 0 0 + 0 0 0 + + + 0 + 0 0 + 0 + 0 + + + 0 + + 00 0 0 0 0 0 0 0 0 0 0 + 0 + 0 + 0 + +0 0 + 0 + + + + 0 0 0 0 0 0 0 + 0 + 0 0 + + 0 0 +0 0 0 0 0 0 0 0 0 ++ 0 0 0 0 0 0
0 0
+ =
D
o
o
D
+ 0 0 0
+ 0 0 0 0 0 0
+ + + + + + + +
+
0
0 0 0
0
+
+ + +
0
+ + +
+ + +
0
+ +
0 0
+
+ +
0
+
+ 0
+ + 0
0 +
0 0 +
163
0
164
+
165
185 187 188
+ 0 + + + + + + + 0 + + +
190 192
+ +
169
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
159 160
172 176 177 178 179 180 181 183
193 194 195 197 S132 S167
0
+ 0 0
+
0 + 0 +
0 + 0 +
0 0 0 0 00 + 0 0 0 0 00 + + 0 +0 + + + + 0 + ++ + 0 + + 0 + + + + + + + 0 00 + 00 + + 0 0 0 0 +0 + + + 0 00 + + 0 0 0 0 0 0 ++ +0 + + 0 +0 + +0 + 0 00 + + + 0 0 0 0 + +0 +0 + 0 + + + 0 +0 + 0 + 0 + 0 0 0 0 0 + 0 ++ ++ 0 0 + 0 0 +0 +0 + 00 00 + + 0 0
0 0
0 0
0
+ + + + + +
0 0 +
0
+ +0 0 0 0 0
+ 0
0 0 +
0
Group B 8140 8146 S157 S201 8203 8205 8206 S207 8208 8211 8213 S214 8215 8218 8220 S223
0 0
+ +
00 0 00 00
0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0
0 0 0 0
+
+ + + + + + 0 0 0 0 0 0 0 0 0 0
+0 0 00 0 00 0 0 +0 0 00 + 0 +0
0 0
0
0 0 0 0
0 0 0
0
0
0
0
0 + 00 0 00 0 0 0 0 + 0 0 + + 0 0 0 00 0 0 0 0 0
+ + + + + + + + + + + + + + + +
0 0
complete lysis; 0 = no lysis; ±
bBS=blood seen
Serum w w w w w w w
Group A
+
19
Parasitological
s0tu0 stauSb0
0
Seu
17
Variable antigen type (VAT)"
aaiooia
= partial lysis; blank = not tested. film or smear; GJ=cervical gland juice; CSF=cerebrospinal fluid. Parasitological status: trypanosomes seen (+) or not
(0).
However, of these six tera, two lysed both ETat 1/14 and Utat 1/1 while, of the remaining four sera, one lysed ETat 1/14 alone and three lysed UTat 1/1. Hence when these three VATs are considered as a group, they cover the whole range of sera that WHO Bulletin OMS. Vol 68 1990.
contained detectable VAT-specific antibodies. This suggests that a limited number of serodemes are circulating in the T.b. rhodesiense epidemic area and that some variable antigens expressed by all these serodemes may exist.
W.K. Isharaza et al. Table 2. Relative reactivities of VATs determined by specific immunolysis In sera from patients infected with T.b. rhodeslense VATs tested ETat 1/1 ETAT 1/18 UTat 1/1 ETat 1/14 ETat 1/19 ETat 1/2 UTat 4/1 UTat 3/1 ETat 1/10 ETat 1/3 UTat 3/7 AnTat 25/1
No. of sera tested (T)
Negative to all VATs (N)
Positive to one or more VATs (P)
No. of positives per VAT (0)
Relative reactivity (1000/P)
85 80 85 85 80 85 84 77 85 80 77 79
20 19 20 20 19 20 20 18 20 18 18 19
65 61 65 65 61 65 65 59 65 62 59 60
59 48 50 49 46 48 47 41 44 21 20 11
90.8 78.7 76.9 75.4 75.4 73.8 73.4 69.5 67.7 33.9 33.9 18.3
' T=total number of T.b. rhodesiense patients' sera which were tested against a particular VAT; N=sera in which no lytic activity was detected for any of the 12 VATs tested; P = number of sera which had shown lytic activity to one or more of the 12 VATs tested; 0 = total sera showing lytic activity to a particular VAT; 100Q/P = relative reactivity of a given VAT, i.e., the percentage of sera with detectable VAT-specific antibodies which lysed that VAT.
Negative results obtained with sera of parasitologically positive cases may be explained by a number of factors. T.b. rhodensiense infection is characterized by high parasitaemias with acute and severe clinical symptoms during the early stages. Case detection should therefore be possible parasitologically before antibody titres to the respective VATs have risen to levels that are high enough to give serologically positive results. In rodent models inoculated with small numbers of trypanosomes, antibodies to some of the VATs reach detectable levels after three or more weeks.b A similar situation could arise in human infections, especially if the metacyclic inoculum of the infective tsetse's bite was devoid of the VATs used in these tests. Another explanation is that serologically negative patients could have been infected with different serodeme(s). The occasional seropositives observed for AnTat 25/1 may be explained by the isotype phenomenon among trypanosomes whereby totally unrelated serodemes occasionally express one or two similar VATs. In conclusion, there appear to be VAT antigens that could be used for the serodiagnosis of Tb. rhodesiense infections. However, careful screening of a number of fairly ubiquitous VATs is essential and these could be used either singly or in combinations. Furthermore, given the extensive antigenic diversity among Tb. rhodesiense serodemes from different areas, such serodiagnostic reagents may have to be based on serodemes circulating in the respective foci. I
See footnote a
(p. 33).
Acknowledgements This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. We are grateful to M. Akol and M. Magnus for technical assistance.
R6sum6 Anticorps trypanolytiques sp6cifiques de variant dans le s6rum de sujets Infest4s par Trypanosoma brucel rhodeslense Les infestations par des trypanosomes salivaires sont caracterisees par le developpement de populations successives de parasites appartenant a des types antigeniques variables (VAT) distincts, les anticorps correspondants s'accumulant dans le sang de l'hote. Lors d'etudes ante rieures sur les repertoires d'antigenes variables de Trypanosoma brucei rhodesiense, 60 VAT ont ete clones; douze d'entre eux ont et retenus pour des epreuves d'immunolyse sur 85 serums de malades atteints de trypanosomiase a T. b. rhodesiense a Busoga (Ouganda). L'un des variants, ETat 1/1, reagissait avec 59 des 65 s6rums contenant des anticorps trypanolytiques decelables. Associ6 a deux autres variants, ETat 1/14 et Utat 1/1, ce variant couvrait l'ensemble des serums positifs; les autres VAT r6agissaient a des degres divers. WHO Bulletin OMS. Vol 681990.
Variant-specific trypanolytic antibodies In sera from patients Infected with T.b. rhodesaenue
Ces resultats semblent indiquer que les serums de malades infest6s par T. b. rhodesiensecontiennent des anticorps specifiques de VAT facilement decelables et que les antig6nes correspondants pourraient dtre utilises pour preparer des reactifs de serodiagnostic de la maladie.
References 1. Gary, A.R. Variable agglutinogenic antigens of Trypanosoma gambiense and their distribution among isolates of trypanosomes collected from different places in Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66: 263284 (1972). 2. Kazyumba, G.L. et al. Antigenic-relationships of Trypanosoma gambiense populations isolated from sleeping sickness patients in Zaire. In: Proceedings of the 17th Meeting of the International Scientific Council for Trypanosomiasis Research and Control, Arusha, 19-24 October 1981. (O.A.U./I.S.C.T.R.C. Publication No. 112), 1981, pp. 131-136. 3. Isharaza, W.K. & Van Melrvenne, N. Comparative studies of variable antigen repertoires of Trypanosoma brucei rhodesiense from Busoga (Uganda) and Kagera (Rwanda). In: Proceedings of the 17th International Scientific Council for Trypanosomiasis Research and Control, Arusha, 19-24 October 1981. (O.A.U./I.S.C.T.R.C. Publication No. 112), 1981, pp. 125-130. 4. W6ry, M. et al. The diagnosis of human African trypanosomiasis (T.b. gambiense) by the use of fluorescent antibody test. Standardisation of an easy technique to be used in mass surveys. Annales de la Soci6t6 belge de M6dicine tropicale, 50: 613-634 5.
(1970). W6ry, M.
et al. The diagnosis of human African trypanosomiasis (T.b. gambiense) by the use of fluorescent antibody test. 2. First results of field
WHO Bulletin OMS. Vol 68 1990.
application. Annales de la Soci6t6 be/ge de Medicine tropicale, 50: 711-730 (1970). 6. Voller, A. et al. A serological study on human T. rhodesiense infections using a microscale ELISA. Tropical medicine and parasitology, 26: 247-251
(1975).
7. Van Knapen, et al. Enzyme-linked immunosorbent assay (ELISA) and its application to serodiagnosis of African trypanosomiasis. Annales de la Societ6 belge de M6dicine tropicale, 57: 282-292 (1977). 8. Vervoort, T. et al. Enzyme-linked immunosorbent assay (ELISA) with variable antigen for serodiagnosis of T.b. gambiense trypanosomiasis. Annales de la Societ6 be/ge de M6dicine tropicale, 56: 177182 (1978). 9. Roffl, J. et al. Diagnostic immunoenzymatique (ELISA) de la trypanosomiase humaine africaine; etude compar6e de plusieurs antigenes. M6dicine tropicale, 39: 637-641 (1979). 10. Van Melrvenne, N. et al. Antigenic variation in syringe passaged populations of Trypanosoma (Trypanozoon) brucei. 1. Rationalisation of the experimental approach. Annales de la Societ6 belge de Medicine tropicale, 55: 1-23 (1975). 11. Bone, G.J. & Charlier, J. l'hemagglutination indirecte en capillaire: une methode de diagnostic de la trypanosomiase applicable sur le terrain. Annales de la Soci6t6 belge de M6dicine tropicale, 55: 559-569
(1975).
12. Magnus, E. et al. A card agglutination test with stained trypanosomes (C.A.T.T.) for the serological diagnosis of T.b. gambiense trypanosomiasis. Annales de la Soci6t6 belge de M6dicine tropicale, 58: 169-176 (1978). 13. McNelllage, G. J. C. et al. Antigenic type of first relapse variants arising from a strain of Trypanosome (Trypanozoon) brucei. Experimental parasitology, 25: 1-7 (1969). 14. Van Melrvenne, N. et al. Comparisons of variable antigen types produced by trypanosome strains of subgenus Trypanozoon. Annales de la Soci6t6 belge de M6dicine tropicale, 57: 409-423 (1977).
37
The cell membrane of salivarian trypanosomes is covered by a monolayer of glycoproteins. These surface glycoproteins are highly immunogenic and elicit trypanocidal antibodies. However, the organisms are able to change their surface coats and form new antigenic variants before the antibodies eliminate the majority of parasites in the blood. As a result, infection is maintained until the host succumbs to the disease. The continuous appearance of new variants results in fluctuating parasitaemia which makes parasitological diagnosis difficult. Currently, there are several serological techniques for the diagnosis of human trypanosomiasis. Up to now, the Trypanosoma brucei rhodesiense serological tests have been based on common antigen/antibody reactions whereas in routine diagnosis of T.b. gambiense, which has less antigenic variation compared with T.b. rhodesiense (1-3),a variable antigen/antibody reactions have been used.
The immunofluorescent antibody test (IFAT) (4,5) and enzyme-linked immunosorbent assay (ELISA) are based on both common and variable antigen/ antibody reactions 6-9). Specific tests for only variable antigen/antibody reactions are trypanolysis (10), indirect haemagglutination (11), and direct agglutination (1) which has recently been developed further into the card agglutination test of stained trypanosomes (CATT), in which a well-defined variable antigen type (VAT) was identified for diagnosis of T.b. gambiense (12). The large number of different VATs belonging to different serodemes that are circulating in the field make it difficult to identify suitable VATs for use in serodiagnosis, especially of T.b. rhodesiense. When rodent models were used in comparative studies of variable antigen repertoires of T.b. rhodesiense isolates from Uganda, several predominant VATs were detected (3).a The objective of the present study was to screen these VATs for specific antibodies in patients' sera and to assess their potential use in serodiagnosis.
' Principal Research Officer, Uganda Trypanosomiasis Research Organisation, (U.T.R.O.) P.O. Box 96, Tororo, Uganda. Requests for reprints should be sent to this author. 2 Chief, Serological Laboratory, Prince Leopold Institute for Tropical Medicine, Antwerp, Belgium. Reprint No. 5042
Isharaza, W.K. Trypanosoma brucei rhodesiense: antigenic diversity and host protective immunity. Ph.D. thesis, Free University, Brussels, 1984.
Bulletin of the World Health Organization, 68 (1): 33-37 (1990)
@© World Health Organization 1990
Introduction
'
33
W.K. Isharaza et al.
Materials and methods VA Ts and sera Clones of the following VATs circulating in Uganda
used: ETat 1/1, 1/2, 1/3, 1/10, 1/14, 1/18 and 1/19 (13); and UTat 3/1, 3/7, 1/1 and 4/1 (4). AnTat 25/1 from an isolate of an unrelated serodeme from Rwanda (15) was also included. All these VATs had been cloned in mice as described by Van Meirvenne et al. (10). The sera were obtained from T.b. rhodesiense patients in Busoga (Uganda) and grouped according to parasitological diagnosis: Group A- trypanosomes seen either in blood, cervical gland juice or cerebrospinal fluid of patients admitted to the Uganda Trypanosomiasis Research Organisation hospital (69 sera); Group B- trypanosomes seen in patients' blood during field surveys but gland juice and cerebrospinal fluid not examined (16 sera); Group C- negative control sera from Caucasians with no history of trypanosomiasis (5 sera); were
Group
D-
negative control
no
sera
from Africans with
history of trypanosomiasis (5 sera).
Immunolysls test Test sera were dispensed in aliquots of 5 41 into Cooke's microtitration plates. Parasitized blood from mice infected with a given VAT two or three days previously was suspended in guinea pig serum to give a concentration of 12 trypanosomes per microscope field at a magnification of x 400. Aliquots of 25 pl of this suspension were added to the serum samples. The resulting mixtures contained patients' sera at a dilution of 1/6 and 10 trypanosomes per microscope field. These plates were sealed and shaken for a few minutes on a plate shaker before incubation at room temperature for two hours. The plates were shaken again and samples of wet films were taken for phasecontrast microscopic examination. At least 10 fields per sample were examined and the percentage of lysed trypanosomes was recorded. Among the 85 sera there were 8 which could not be examined against all the antigens; these incomplete series have nevertheless given results on a range of cross reactions.
Results Immunospeclficlty of the lysas test Three ranges of immunolysis were scored as 1-10% (0), 10-90% (±) and 90-100% (+). None of 34
the ten negative control sera in groups C and D reacted with any of the VATs tested, thus demonstrating absence of non-specific trypanolysis. Therefore, any lysis observed in the test sera of groups A and B (Table 1) was attributed to trypanosome-induced lytic antibodies in the patients. In view of the unequivocal results among the negative controls, those sera scored as + or + were considered as seropositive. The latter group may have contained specific antibodies whose titres were slightly too low for the respective VATs, especially since all sera in this category (with the exception of No. 19, 157, S157 and S213) fully lysed one or more VATs. Reactivity of VATs In groups A and B, 18 sera out of the 77 that were completely tested showed no trypanolytic activity on any of the 12 VATs tested although trypanosomes had been observed in these patients. Of these seronegative patients, 13 belonged to group A in which the cerebrospinal fluid had been examined and only two patients (No. 84 and 166) had positive CSF. It is possible the infections were still at a very early stage and/or the antibody titres, if any, were too low to be detected. Alternatively, these patients may have been infected with serodeme(s) unrelated to those of the VATs tested. Out of the 85 test sera in groups A and B, 65 reacted with one or more of these VATs; 46 of the positive sera reacted with six or more VATs out of the eleven from Busoga. When assessing the relative VAT reactivities as defined in Table 2, only those sera that lysed one or more VATs were considered. The highest number of positive sera and a relative reactivity of 90.8% was recorded for ETat 1/1. Etat 1/14, 1/18, 1/19 and Utat 1/1 also showed high relative reactivities of 75% and above. Slightly lower values were obtained from UTat 3/1 and 4/1 as well as Etat 1/2 and 1/10. ETat 1/3 and UTat 3/7 gave significantly low relative reactivities of 33.9%; these two VATs are relatively less predominant in their respective repertoires.b AnTat 25/1, which originates from Rwanda and belongs to a serodeme entirely different from those of Uganda, showed the lowest relative reactivity (18%).
Discussion All the VATs showed positive reactions with sera but in varying proportions. Sixty-five sera contained detectable VAT-specific lytic antibodies. Among the VAT antigens for use in serodiagnosis, ETat 1/1 appeared to be the best candidate; of the 65 seropositives, only 6 were negative to this VAT. WHO Bulletin OMS. Vol 68 1990.
Variant-specific trypanolytic antibodies In
sera from
patients Infected with T.b. rhodeslensee
Table 1: VAT-specific immunolysis of seiected VATs by sera from patients Infected with T.b. rhodesiensee Variable antigen type (VAT)"
0 v o
In
0
o
Group A 0 0 0 0 0 + 0 0 + + + + 0 +
20 22 152 27 30 31 32 33 34 35 40 166 191
+ + + + + + 0 0 0 + 0 + + + 0 + + + + 0 + +
44
54 55 57 59 64 65 66 67 68 69 71 72 73 74 75 76 81 82
0
+ + +
+ + +
0 0 0 + 0 0 0 + 0 0 0 + 0 + 0
+
0 0
0
+ + 0
0 0 + 0 + 0 0 0 + + + 0 + 0 0 0 0 +
841 92 149
+
153 154
0 +
155
+
156 157
0
a1 +
0
+
83 84
0 0 0 0 0 + + 0 + 0 0 0 + + + 0 + 0 + 0 0 0 + + 0 + 0 0 + ++ + + + ++ + + 00 0 0 + 0 + + 0 + + + + + 0 +0 00 0 0 + 0 + + + 0 + 00 0 + ++ 0 + + + + + + 0 + 0 + 0 0 0 0 0 + + + + 0 0 + 0 0 0 + + + 0 + 0 0 + 0 + 0 + + + 0 + + 00 0 0 0 0 0 0 0 0 0 0 + 0 + 0 + 0 + +0 0 + 0 + + + + 0 0 0 0 0 0 0 + 0 + 0 0 + + 0 0 +0 0 0 0 0 0 0 0 0 ++ 0 0 0 0 0 0
0 0
+ =
D
o
o
D
+ 0 0 0
+ 0 0 0 0 0 0
+ + + + + + + +
+
0
0 0 0
0
+
+ + +
0
+ + +
+ + +
0
+ +
0 0
+
+ +
0
+
+ 0
+ + 0
0 +
0 0 +
163
0
164
+
165
185 187 188
+ 0 + + + + + + + 0 + + +
190 192
+ +
169
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
159 160
172 176 177 178 179 180 181 183
193 194 195 197 S132 S167
0
+ 0 0
+
0 + 0 +
0 + 0 +
0 0 0 0 00 + 0 0 0 0 00 + + 0 +0 + + + + 0 + ++ + 0 + + 0 + + + + + + + 0 00 + 00 + + 0 0 0 0 +0 + + + 0 00 + + 0 0 0 0 0 0 ++ +0 + + 0 +0 + +0 + 0 00 + + + 0 0 0 0 + +0 +0 + 0 + + + 0 +0 + 0 + 0 + 0 0 0 0 0 + 0 ++ ++ 0 0 + 0 0 +0 +0 + 00 00 + + 0 0
0 0
0 0
0
+ + + + + +
0 0 +
0
+ +0 0 0 0 0
+ 0
0 0 +
0
Group B 8140 8146 S157 S201 8203 8205 8206 S207 8208 8211 8213 S214 8215 8218 8220 S223
0 0
+ +
00 0 00 00
0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0
0 0 0 0
+
+ + + + + + 0 0 0 0 0 0 0 0 0 0
+0 0 00 0 00 0 0 +0 0 00 + 0 +0
0 0
0
0 0 0 0
0 0 0
0
0
0
0
0 + 00 0 00 0 0 0 0 + 0 0 + + 0 0 0 00 0 0 0 0 0
+ + + + + + + + + + + + + + + +
0 0
complete lysis; 0 = no lysis; ±
bBS=blood seen
Serum w w w w w w w
Group A
+
19
Parasitological
s0tu0 stauSb0
0
Seu
17
Variable antigen type (VAT)"
aaiooia
= partial lysis; blank = not tested. film or smear; GJ=cervical gland juice; CSF=cerebrospinal fluid. Parasitological status: trypanosomes seen (+) or not
(0).
However, of these six tera, two lysed both ETat 1/14 and Utat 1/1 while, of the remaining four sera, one lysed ETat 1/14 alone and three lysed UTat 1/1. Hence when these three VATs are considered as a group, they cover the whole range of sera that WHO Bulletin OMS. Vol 68 1990.
contained detectable VAT-specific antibodies. This suggests that a limited number of serodemes are circulating in the T.b. rhodesiense epidemic area and that some variable antigens expressed by all these serodemes may exist.
W.K. Isharaza et al. Table 2. Relative reactivities of VATs determined by specific immunolysis In sera from patients infected with T.b. rhodeslense VATs tested ETat 1/1 ETAT 1/18 UTat 1/1 ETat 1/14 ETat 1/19 ETat 1/2 UTat 4/1 UTat 3/1 ETat 1/10 ETat 1/3 UTat 3/7 AnTat 25/1
No. of sera tested (T)
Negative to all VATs (N)
Positive to one or more VATs (P)
No. of positives per VAT (0)
Relative reactivity (1000/P)
85 80 85 85 80 85 84 77 85 80 77 79
20 19 20 20 19 20 20 18 20 18 18 19
65 61 65 65 61 65 65 59 65 62 59 60
59 48 50 49 46 48 47 41 44 21 20 11
90.8 78.7 76.9 75.4 75.4 73.8 73.4 69.5 67.7 33.9 33.9 18.3
' T=total number of T.b. rhodesiense patients' sera which were tested against a particular VAT; N=sera in which no lytic activity was detected for any of the 12 VATs tested; P = number of sera which had shown lytic activity to one or more of the 12 VATs tested; 0 = total sera showing lytic activity to a particular VAT; 100Q/P = relative reactivity of a given VAT, i.e., the percentage of sera with detectable VAT-specific antibodies which lysed that VAT.
Negative results obtained with sera of parasitologically positive cases may be explained by a number of factors. T.b. rhodensiense infection is characterized by high parasitaemias with acute and severe clinical symptoms during the early stages. Case detection should therefore be possible parasitologically before antibody titres to the respective VATs have risen to levels that are high enough to give serologically positive results. In rodent models inoculated with small numbers of trypanosomes, antibodies to some of the VATs reach detectable levels after three or more weeks.b A similar situation could arise in human infections, especially if the metacyclic inoculum of the infective tsetse's bite was devoid of the VATs used in these tests. Another explanation is that serologically negative patients could have been infected with different serodeme(s). The occasional seropositives observed for AnTat 25/1 may be explained by the isotype phenomenon among trypanosomes whereby totally unrelated serodemes occasionally express one or two similar VATs. In conclusion, there appear to be VAT antigens that could be used for the serodiagnosis of Tb. rhodesiense infections. However, careful screening of a number of fairly ubiquitous VATs is essential and these could be used either singly or in combinations. Furthermore, given the extensive antigenic diversity among Tb. rhodesiense serodemes from different areas, such serodiagnostic reagents may have to be based on serodemes circulating in the respective foci. I
See footnote a
(p. 33).
Acknowledgements This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. We are grateful to M. Akol and M. Magnus for technical assistance.
R6sum6 Anticorps trypanolytiques sp6cifiques de variant dans le s6rum de sujets Infest4s par Trypanosoma brucel rhodeslense Les infestations par des trypanosomes salivaires sont caracterisees par le developpement de populations successives de parasites appartenant a des types antigeniques variables (VAT) distincts, les anticorps correspondants s'accumulant dans le sang de l'hote. Lors d'etudes ante rieures sur les repertoires d'antigenes variables de Trypanosoma brucei rhodesiense, 60 VAT ont ete clones; douze d'entre eux ont et retenus pour des epreuves d'immunolyse sur 85 serums de malades atteints de trypanosomiase a T. b. rhodesiense a Busoga (Ouganda). L'un des variants, ETat 1/1, reagissait avec 59 des 65 s6rums contenant des anticorps trypanolytiques decelables. Associ6 a deux autres variants, ETat 1/14 et Utat 1/1, ce variant couvrait l'ensemble des serums positifs; les autres VAT r6agissaient a des degres divers. WHO Bulletin OMS. Vol 681990.
Variant-specific trypanolytic antibodies In sera from patients Infected with T.b. rhodesaenue
Ces resultats semblent indiquer que les serums de malades infest6s par T. b. rhodesiensecontiennent des anticorps specifiques de VAT facilement decelables et que les antig6nes correspondants pourraient dtre utilises pour preparer des reactifs de serodiagnostic de la maladie.
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