- Email: [email protected]
Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen
JOURNAL OF IMMUNOlOGICAL METHODS ELSEVIER
Journal of Immunological
Methods
i 93 (I 996) 9- 15
Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen G. Senaldi a,*, H. Xiao-su ‘, D.C. Hoessli ‘, C. Bordier b a WHO-Immunology
Research and Training b Bordier
’
Depnrtrnenf
NfParasitolo~q.
Cer~tre, Department Afjinig
Products.
of PathalaR?: Crissier.
West China Unil~ersi~
Received 30 June 1995; revised 20 November
Unic,ersi@
of GeneL,a.
Geneva, Sukerland
Switxrland
NfMedical
Sciences. Chengdu,
1995: accepted 3 January
China
1996
Abstract Field medicine in tropical areas needs laboratory assays which are inexpensive and easy to perform. To meet this need a semi-quantitative dot-enzyme immunoassay (EIA) was developed for the detection of an L. donornni-related circulating antigen and tested for clinical relevance in the diagnosis of visceral leishmaniasis (VL). The dot-EIA probes serum spotted on nitrocellulose for the presence of the antigen using a monoclonal antibody raised against L. n’orro~~~i promastigotes, a peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum and chloronaphthol as peroxidase substrate. The intensity of dot staining by chloronaphthol is read by eye and scored. The dot-EIA was used to test the following groups: 69 patients with VL from Brazil, Kenya, China and France, nine patients with cutaneous leishmaniasis, 38 patients with tropical diseases other than VL, five patients with rheumatoid arthritis and 40 health blood donors. The specificity of the assay was 96.7% (3/92 false positive) and the sensitivity 98.5% (I /69 false negative). A quantitative EIA for the detection of serum antibodies which makes use of a crude, soluble L. in@zrum promastigote extract as capture antigen and which was used as the reference method. proved to be more specific (98.9%) but similarly sensitive (98.57~). It should be possible to produce a kit, suitable for large scale application at low cost in order to facilitate routine use of the dot-EIA in the diagnosis of VL. Keyords:
Visceral
leishmaniasis:
Dot-enzyme
immunoassay;
Circulating
antigen
1. Introduction
Abbreviations: munoassay; MAb. niasis: OD, optical I Corresponding 15-2-A-250. 1810 1789. USA. Tel.:
VL, visceral leishmaniasis: EIA, enzyme immonoclonal antibody; CL. cutaneous leishmadensity; PBS, phosphate buffered saline. author. At: Amgen Inc., Amgen Center M/S Dehavilland Drive, Thousand Oaks, CA 91320I J305.4474061: Fax: 1.805.447 1939.
0022- 1759/96/$15.00 0 I996 Elsevier Science B.V. All rights reserved PI/ SOO12- 1759(96)00037-3
Visceral leishmaniasis (VL) is a disease caused by protozoa belonging to the genus Leishmania. in particular L. donoLlani. L. kfantum and L. chagasi. VL is widespread in tropical areas where it represents a major health problem. It is a severe disease which is usually lethal if untreated (Zuckerman and Lainson, 1977).
The diagnosis of VL is based on the demonstration of the causative parasites in bone marrow aspirates or spleen or liver biopsies by microscopy or culture (Muigai and Bryceson, 1982). Collecting and processing these specimens for microbiological analysis, however, requires extensive facilities and experience. The demonstration of specific serum antibodies also permits clinically conclusive, albeit indirect, diagnosis of VL (Bray, 1976). Several serological methods have been developed to detect circulating anti-l. dmmwni antibodies, based on immunoprecip itation. complement fixation, haemagglutination, immunofluorescence, and radio- and enzyme immunoadsorption. but their diagnostic value is limited by false negative and false positive results (Bray, 1976. 1980). However, modem methods which incorporate in a quantitative enzyme-immunoassay (EIA) the use of specific monoclonal antibodies and purified preparations of parasite antigens (Jaffe et al.. 1984; Jaffe and McMahon Pratt, 1987: Jaffe and Zalis. l988a, b; Reed et al.. 1990) and a method based on immunoblotting test serum (Bogdan et al., 1990) give sensitive and specific results of satisfactory reliability in the clinical context. Unfortunately these methods are too expensive to introduce in tropical areas. In particular, parasite preparations. especially if purified, are expensive, and both quantitative EIA and immunoblotting require special equipment and expertise. It is known that cultured Leishnarzia promastigotes and amastigotes release structural components of their cell surface (Schnur, 1982>, and circulating antigens of parasitic origin have been demonstrated in patients with VL (Evans and Pearson. 1988; Xiaosu et al.. 1988; Sinha et al., 1988; Mary et al., 1993; Azazi et al., 1994). Bearing in mind the needs of field medicine in tropical areas we have developed methods for the detection of an L. dr>llorsani-related circulating antigen. which are sufficiently sensitive and specific to be clinically reliable and which are at the same time inexpensive and easy to perform. Semi-quantitative dot-EIA and paddle-EIA were set up using a monoclonal antibody (MAb) raised against L. donormi promastigotes and both satisfied specific practical requirements in terms of cost and performance. Their clinical relevance was tested and compared to those of two quantitative microplate-EIA. one developed
for the detection of the same antigen using the above MAb, and another aimed at detecting serum antibodies to L. kfnrltlfm.
2. Materials
and methods
2.1. Seru Sera were obtained from 69 patients with VL acquired in different parts of the world (40 from Brazil (provided by R. Badaro. Laboratory of Immunology, Edgard Santos Hospital, Federal University of Bahia, Salvador, Brazil), 16 from Kenya (provided by J. Jephthah-Ochola. Kenya Medical Research Institute. Nairobi. Kenya), eight from China and five from France (provided by C. Mary, Laboratory of Parasitology. Faculty of Medicine, University of Marseilles, Marseilles. France)]; from nine patients with cutaneous leishmaniasis (CL) (provided by C.L. Jaffe, MacArthur Centre for Molecular Biology of Tropical Diseases. Department of Biophysics, Weizmann Institute of Science. Rehovot. Israel); from 38 patients with various infectious tropical diseases other than VL of whom 12 presented with a history of multiple infections (provided by N. Weiss, Swiss Tropical Institute, Basel, Switzerland. and by J. Jephthah-Ochola); from five patients with rheumatoid arthritis attending a clinic for rheumatic diseases in Geneva and from 40 healthy subjects recruited among Geneva blood donors. Among the tropical infectious diseases other than VL there were ten cases of strongyloidiasis, nine of filariasis, seven of amebiasis, seven of schistosomiasis, five cases of Echinocotc~4s graizfilosiu infestation, five of malaria. five of toxocariasis, two of African trypanosomiasis. two of tuberculosis and one of Fnsciolrr hepticcr infestation. The patients with tropical infectious diseases other than VL and those with RA were considered together in this study and referred to as patients with diseases other than leishmaniasis. The five sera which gave the highest optical density CODI values in the quantative EIA established for the detection of the circulating antigen (see below) were pooled to obtain a positive control serum: five blood donor sera were also pooled to obtain a negative control serum.
G. Senaldi et al. /Journal
2.2. Monoclonal
of Immunological Methods 193 f 19961 9- 15
antibody
The mouse anti-L. donoL>ani suchuan promastigote I B 1-C2-B3 MAb was used in this study (Xiao-su et al.. 1988). It is an IgM produced by a hybridoma obtained by fusion of SP2/0 myeloma cells and spleen cells from BALB/c mice previously immunised with cultured promastigotes fixed in glutaraldehyde. This MAb reacts with three major immunoblot bands in an L. donovani suchuan lysate and it is able to detect antigen in the circulation of patients with VL (Xiao-su et al., 1988). Ascitic fluid containing the 1B l-C2-B3 MAb was obtained by growing the hybridoma in the peritoneum of BALB/c mice. 2.3. Detection qf L. donor!ani-related antigen by dot-EfA
circulating
1 ~1 of the sera to be tested, diluted l/25 in phosphate buffered saline (PBS), pH 7.4, were spotted on 4 X 7 mm nitrocellulose rectangles (Schleicher and Schuell, Dassel, Germany), mounted on a plastic strip as a support (obtained by M. Deres, Grands Places Medical Centre, Freiburg, Switzerland). Negative and positive control sera were also spotted on each strip. After allowing the serum to dry for 30 min, the strip was incubated for 30 min in PBS containing 0.05% Tween 20 and 5% milk powder, in order to block non-specific binding sites. The strip was then incubated for 2 h with lBl-C2-B3 MAb ascites diluted 1/ 1000 in PBS containing 0.05% Tween 20 and 2.5% milk powder (PBS-T-M), washed in five changes of 5 min each of PBS containing 0.05% Tween 20 (PBS-T), further incubated for 2 h in peroxidase-conjugated rabbit anti-mouse IgG, IgA and IgM antiserum (Zymed Laboratories, San Francisco, CA, USA) diluted l/500 in PBS-T-M, washed again as above and finally incubated for 20 min in PBS containing 0.2% chloronaphthol and 0.01% H,O,. The intensity of the blue/violet staining was evaluated visually by three independent observers unaware of sample identities and scored as 0, 1, 2 or 3. with 0 representing a colour intensity weaker than or equal to the one generated by the negative control and 3 the intensity equal to or stronger than the one generated by the positive control. Final scores were
I1
the means of the scores attributed by the single observers. A sample scored 1 or more was considered positive. 2.4. Detection of L. donorani-related antigen by paddle-EIA or microplate-EIA
circulating
Plastic paddles (Nunc, Glostrup, Denmark) or EIA microplate wells (Nunc) were coated overnight at 4°C with 500 and 100 ~1, respectively, of test serum diluted l/500 in PBS (in duplicate for the microplate-EIA), blocked with PBS containing 0.05% Tween 20 and 5% milk powder for 30 min at 37°C and incubated for 1 h with lBl-2C-B3 MAb ascites diluted l/1000 in PBS-T-M. After washing in five changes of PBS-T, paddles or microplate wells were further incubated for 1 h in peroxidase-conjugated rabbit anti-mouse IgG. IgA and IgM antiserum (Zymed Laboratories) diluted I/ 1000 in PBS-T-M, washed again as above and eventually incubated for 20 min in phosphate/citric acid buffer containing 0.1% ABTS (Sigma, St. Louis, MO, USA) and 0.01% H?O?. The intensity of the ensuing green staining obtained by paddle-EIA was evaluated and scored as described for the dot-EIA. Staining intensity obtained by microplate-EIA was measured in a microplate reader at 405 nm and expressed in OD units. A sample was considered positive when it generated an OD greater than 1.5 times the OD value generated by the negative control serum. 2.5. Detection
qf serum antibodies to L. in~antum
EIA microplate wells pre-coated with L. infanturn soluble antigens (Bordier Affinity Products, Crissier, Switzerland) were blocked with PBS containing 0.3% Tween 20 and 0.1% bovine serum albumin (dilution buffer) for 15 min and incubated with 100 ul of test serum (in duplicate) diluted l/500 in dilution buffer. After washing with PBS containing 0.3% Tween 20 microplate wells were incubated with 100 ~1 of peroxidase-conjugated goat anti-human IgG antiserum (Sigma) diluted l/1000 in dilution buffer, washed again and incubated with the ABTS containing buffer. Staining intensity was measured in a microplate reader, expressed in OD units and judged positive or negative as above.
12
3
1
+ ..
.
LC
PC
.. 01
9
.
,.
LV
NC
LC
LV
PC
NC
Fig. 1. Results (scores) of the dot-EIA for the detection of an L. &nol,arGelated circulating antigen obtained in patients with visceral leishmaniasis (VL) or cutaneous leishmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
Fig. 2. Results (scores) of the paddle-EIA for the detection of an L. ck,rlol,clni-rrlated circulating antigen obtained in patients with visceral leishmaniasis (VL) or cutaneous lekhmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
2.6. Statistical
whether considered as a whole or divided into healthy subjects, patients with CL and patients with diseases other than leishmaniasis. Values were not significantly different between the various groups without VL (Table 1) (Figs. l-4). However. patients with CL had OD values for serum antibodies to L. infallturn which were higher than in patients with diseases other than leishmaniasis (Table 1). The specificity and sensitivity for VL of the different methods are given in Table 2. The detection of serum antibodies to L. infarztum by microplate-EIA proved to be the most specific (98.9%) and sensitive (98.5%) method. The detection of the circulating
analyis.
Scores and OD values were compared Wilcoxon’s rank sum test and correlated Spearman’s rank correlation analysis.
by the by the
3. Results Dot- and paddle-EIA obtained when detecting serum antibodies to L. patients with VL than
scores and EIA OD values the circulating antigen or infantum were higher in in subjects without VL,
Table 1 Median (range) of EIA scores (dot-EIA and paddle-EIA for the detection of an L. donoc,ckrelated circulating antigen) and optical density values (microplate-EIA (M/P-EIA) for the detection of the circulating antigen and of anti-l. infnntutn serum antibodies (Abs-EIA) in patients and healthy subjects Dot-EIA VL Non-VL NC CL PC
1.7 (O-2.7) O(O-1.7) 0 (O-0.3) 0 (0-O) 0 (O-1.7)
:’
Paddle-EIA
M/P-EIA
2 0 0 0 0
-307 (73-783) _ 66 (O-212) 51(29-1951 63 G-123) 71 (O-212)
(O-3) J (O-2) (0-O) (O-2) (O-2)
Abs-EIA il
1307 (110-3783) 39 (O-336) 8 (O-336) 70 (O-175) h 38 (O- 184)
a
VL = visceral leishmaniasis: non-VL = all the subjects studied except those with VL; NC = healthy subjects: CL = patients with cutaneous leishmaniasis: PC = patients with diseases other than leishmaniasis: M/P = microplate. ’ = p < 0.001 as compared to non-VL, NC. CL and PC. h = JJ < 0.05 as compared to PC.
G. Senaidi et al. /Journal
600 0 z x
of Immunological
Methods 193
400
. ..
2 t.. *
8
200
13
Table 2 Specificity and sensitivity of the EIA for the detection of an L. donor,&-related circulating antigen (dot-EIA, paddle-EIA. M/PEIA) and for the detection of anti-l. irzfantumserum antibodies (Abs-EIA)
..
P
C19%) 9- 15
5.O: .‘<‘. .=: . + .
.
.
. . . :-
m3
.
.
w.
x. --
V’
LV
NC
Lb
PC
Fig. 3. Results (OD values) of the microplate-EIA for the detection of an L. donollarii-related circulating antigen obtained in patients with visceral leishmaniasis (VLl or cutaneous leishmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
antigen by dot-EIA was (96.7%) and as sensitive Interestingly, there was was negative for serum tive in dot-EIA for the
second best for specificity as microplate-EIA (98.5%). one patient with VL who antibodies but scored posicirculating antigen. Of the
.
2
NC
LC
PC
Fig. 4. Fig. 4. Results (OD values) of the EIA for the detection of anti-l. infanturn serum antibodies obtained in patients with visceral leishmaniasis (VLI or cutaneous leishmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
False positives Specificity t%) False negatives Sensitivity (%I
Dot-EIA
Paddle-EIA
M/P-EIA
Abs-EIA
3/92 96.7 l/69 98.5
3/92 96.7 14/69 79.7
9/92 90.2 6/69 91.3
I /92 98.9 I /69 98.5
A b
a 92 were the subjects not affected by visceral leishmaniasis (i.e., healthy subjects. patients with cutaneous leishmaniasis or diseases other than leishmaniasis) considered as a whole group. ’ 69 were the patients with visceral leishmaniasis. M/P = microplate.
three non-VL patients who were positive in dot-EIA, but negative for serum antibodies, two had tuberculosis.
4. Discussion This study illustrates the possibility of detecting an f.. donorlalzi-related circulating antigen in the serum of patients with VL by semiquantitative dotEIA and paddle-EIA. both of which are inexpensive and easy to perform, and by quantitative microplateEIA. It also shows that the dot-EIA is able to provide reliable identification of patients with VL which is comparable in specificity and sensitivity to quantitative EIA for the detection of anti-l. infanturn serum antibodies. Antigens of parasitic origin have been demonstrated in the circulation of patients with L. &nopani infection, although their biochemical characterisation still awaits definition (Evans and Pearson, 1988; Xiao-su et al., 1988; Sinha et al., 1988; Mary et al., 1993; Azazi et al., 1994). EIA methods aimed at detecting L. donor& circulating antigens have been developed for use in the diagnostic laboratory. thus providing the possibility for direct diagnosis of VL (Xiao-su et al., 1988; Sinha et al., 1988; Azazi et al., 19941. The novelty of the present study is the establishment of (1) an upgraded version of the dot-EIA method published by Xiao-su et al. (1988). which is improved in sensitivity and is capable of
14
G. Srrmldi et al. / Jortrnnl
of Immur~ological
large scale application; and (2) a novel microplateEIA, which differs from the EIA proposed by Sinha et al. (1988) and Azazi et al. (1994) in that it makes use of a MAb instead of antisera against the circulating antigen and it directly detects antigen instead of relying on competition with peroxidase-labelled promastigote soluble antigen (Sinha et al., 1988) or on preliminary precipitation and dissociation of antigen containing circulating immune complexes (Azazi et al., 19941. The fact that the dot-EIA showed an elevated degree of sensitivity when tested on a series of geographically heterogeneous VL subjects suggests that it might be suitable for the diagnosis of VL caused by different strains of Leishrnania protozoa. The dot-EIA also showed high specificity. Of the three false positive cases that were found two were cases of tuberculosis and one of amebiasis. Thus. both tuberculosis cases included in our series of pathological controls were false positive. supporting the possibility that antigenic cross-reactivity exists between Leishmania and Mycobacteria. We only studied two cases of African trypanosomiasis, but neither was responsible for a false positive result, which is interesting since serological tests for the diagnosis of VL often show weak specificity when challenged with sera from patients with 7’ppanosoma infections. The dot-EIA described in this paper is suitable for the screening of clinical cases where there is suspicion of VL and the practice of invasive biopsies followed by demanding microbiological investigations could be confined to individual screened as positive. To provide health care workers with the opportunity of using the dot-EIA for screening. an inexpensive kit which is appropriate for large scale production is currently under development. This would permit applications of the dot-EIA in the less well-equipped laboratories. We calculate that the production cost of such a kit for 120 tests is US$ 60 (US$ 0.5 per test). The quantitative microplate-EIA for antigen detection established in this study may prove useful for monitoring the course of L. domx~arzi infection. However, further studies are required to evaluate the the reproducibility of the assay and its relevance in predicting relapses and remissions in association with the differing clinical presentations of VL.
Methods
193 (19961 Y-15
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 R. Badaro (Laboratory of Immunology, Edgard Santos Hospital. Federal University of Bahia, Salvador, Brazil), J. Jephthah-Ochola (Kenya Medical Research Institute, Nairobi, Kenya), C.L. Jaffe (MacArthur Centre for Molecular Biology of Tropical Diseases, Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel), C. Mary, (Laboratory of Parasitology, Faculty of Medicine, University of Marseilles, Marseilles. France) and N. Weiss (Swiss Tropical Institute, Base], Switzerland) for providing sera. We thank M. Deres (Grands Places Medical Centre, Freiburg. Switzerland) for the supply of technical material. We also thank C. Tougne for technical assistance and G. Del Giudice for helpful discussions. This study is dedicated to the memory of Vaclav Houba. who conceived it.
References Azazi, A.A., Devaney, E. and Chance. M.L. (19941 A PEG-ELISA for the detection of Lrishmnniu donowmi antigen in circulating immune complexes. Trans. R. Sot. Trop. Med. Hyg. 88. 62-66. Bogdan, C., Stosiek. N.. Fuchs, H.. Rollinghoff. H. and Solbach, W. (1990) Detection of potentially diagnostic leishmanial antigens by western blot analysis of sera from patients with kala-arar or multilesional cutaneous leishmaniasis. J. Infect. Dis. 162. 1417-1418. Bray, R.S. (1976) Immunodiagnosis of leishmaniasib. In: S. Cohen and E. Sadun (Eds.1, Immunology of Parasitic Infections. Blackwell Scientific Publications. Oxford. pp. 65-76. Bray, R.S. (1980) Leishmaniasis. In: V. Houha (Ed.), Immunological Investigation of Tropical Parasitic Diseases. Churchill Livingstone. Edinburgh. pp. 65-7-l. Evans. T.G. and Pearson. R.D. (19881 Identification of leishmanial antigens in the sera of patients with American visceral leishmaniasis. Infect. Immun. S6. 3 139-3 144. Jaffe, C.L. and McMahon Pratt, D. (1987) Serodiagnostic assay for visceral leishmaniasis employing monoclonal antibodies. Trans. R. Sot. Trop. Med. Hyg. 81. 587-S94. Jaffe, CL. and Zalis, M. ( 1988al Purification of two L. domwmi membrane proteins recognised by sertt from patients with visceral leishmaniasis. Mol. Biochem. Parasitol. 77, 53-62. Jaffe. C.L. and Zalis. M. (1988b) Use of purified parasite proteins from L. &noi,(l~ij for the rapid serodiagnosis of visceral leishmaniasis. J. Infect. Dis. 157, 1212- 1220. Jaffe. C.L.. Bennet. E.. Grimaldi, Jr.. G. and McMahon Pratt. D.
G. Senaldi et al. /Journal
of Immunological Methods 193 (1996) Y-15
(1984) Production and characterisation of species-specific monoclonal antibodies against L. donor,ani for immunodiagnosis. J. Immunol. 133, 440-447. Mary, C., Ange, G., Dunan, S., Lamouroux, D.. Quilici, M. (1993) Characterisation of a circulating antigen involved in immune complexes in visceral leishmaniasis patients. Am. J. Trop. Med. Hyg. 49, 492-501. Muigai. R. and Bryceson, A. (1982) The diagnosis and management of leishmaniasis. Postgrad. Doctor (Africa) 4. 280-288. Reed, S.. Shreffler, W.G., Bums, Jr.. J.M.. Scott. J.M., Da Gloria Orge, M.. Ghalib. H.W., Siddig. M. and Badaro. R. (1990) An improved serodiagnostic procedure for visceral leishmaniasis. Am. J. Trop. Med. Hyg. 43. 632-639. Schnur, L.P. (1982) The immunological identification and charac-
15
terisation of leishmanial sticks and strains, with special reference to excreted factor serotyping. In: M.L. Chance and B.C. Walton (Eds.1. Biochemical Characterisation of kishmnnia. World Health Organisation/Tropical Disease Research, Geneva. pp. 25-47. Sinha. R.. Arora, S.K. and Sehgal. S. (19881 A sensitive ELISA for detection of circulating antigens in kala-azar patients. Ind. J. Med. Res. 88, 138-140. Xiao-su, H.. Qing. L., Fang-Zing. L.. Tao-lin. Y.. Ya-Jin, W.. Zhi, Q., Ping, L. and Ling. W. (1988) Kala-azar infected serum circulating antigens and their characteristic detected by monoclonal antibody. Chin. Med. J. 101. l-6. Zuckerman, A. and Lainson, R. (1977) Leishmania. In: J.P. Kreier (Ed.), Protozoa. Academic Press, New York, pp. 37-39.
Journal of Immunological
Methods
i 93 (I 996) 9- 15
Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen G. Senaldi a,*, H. Xiao-su ‘, D.C. Hoessli ‘, C. Bordier b a WHO-Immunology
Research and Training b Bordier
’
Depnrtrnenf
NfParasitolo~q.
Cer~tre, Department Afjinig
Products.
of PathalaR?: Crissier.
West China Unil~ersi~
Received 30 June 1995; revised 20 November
Unic,ersi@
of GeneL,a.
Geneva, Sukerland
Switxrland
NfMedical
Sciences. Chengdu,
1995: accepted 3 January
China
1996
Abstract Field medicine in tropical areas needs laboratory assays which are inexpensive and easy to perform. To meet this need a semi-quantitative dot-enzyme immunoassay (EIA) was developed for the detection of an L. donornni-related circulating antigen and tested for clinical relevance in the diagnosis of visceral leishmaniasis (VL). The dot-EIA probes serum spotted on nitrocellulose for the presence of the antigen using a monoclonal antibody raised against L. n’orro~~~i promastigotes, a peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum and chloronaphthol as peroxidase substrate. The intensity of dot staining by chloronaphthol is read by eye and scored. The dot-EIA was used to test the following groups: 69 patients with VL from Brazil, Kenya, China and France, nine patients with cutaneous leishmaniasis, 38 patients with tropical diseases other than VL, five patients with rheumatoid arthritis and 40 health blood donors. The specificity of the assay was 96.7% (3/92 false positive) and the sensitivity 98.5% (I /69 false negative). A quantitative EIA for the detection of serum antibodies which makes use of a crude, soluble L. in@zrum promastigote extract as capture antigen and which was used as the reference method. proved to be more specific (98.9%) but similarly sensitive (98.57~). It should be possible to produce a kit, suitable for large scale application at low cost in order to facilitate routine use of the dot-EIA in the diagnosis of VL. Keyords:
Visceral
leishmaniasis:
Dot-enzyme
immunoassay;
Circulating
antigen
1. Introduction
Abbreviations: munoassay; MAb. niasis: OD, optical I Corresponding 15-2-A-250. 1810 1789. USA. Tel.:
VL, visceral leishmaniasis: EIA, enzyme immonoclonal antibody; CL. cutaneous leishmadensity; PBS, phosphate buffered saline. author. At: Amgen Inc., Amgen Center M/S Dehavilland Drive, Thousand Oaks, CA 91320I J305.4474061: Fax: 1.805.447 1939.
0022- 1759/96/$15.00 0 I996 Elsevier Science B.V. All rights reserved PI/ SOO12- 1759(96)00037-3
Visceral leishmaniasis (VL) is a disease caused by protozoa belonging to the genus Leishmania. in particular L. donoLlani. L. kfantum and L. chagasi. VL is widespread in tropical areas where it represents a major health problem. It is a severe disease which is usually lethal if untreated (Zuckerman and Lainson, 1977).
The diagnosis of VL is based on the demonstration of the causative parasites in bone marrow aspirates or spleen or liver biopsies by microscopy or culture (Muigai and Bryceson, 1982). Collecting and processing these specimens for microbiological analysis, however, requires extensive facilities and experience. The demonstration of specific serum antibodies also permits clinically conclusive, albeit indirect, diagnosis of VL (Bray, 1976). Several serological methods have been developed to detect circulating anti-l. dmmwni antibodies, based on immunoprecip itation. complement fixation, haemagglutination, immunofluorescence, and radio- and enzyme immunoadsorption. but their diagnostic value is limited by false negative and false positive results (Bray, 1976. 1980). However, modem methods which incorporate in a quantitative enzyme-immunoassay (EIA) the use of specific monoclonal antibodies and purified preparations of parasite antigens (Jaffe et al.. 1984; Jaffe and McMahon Pratt, 1987: Jaffe and Zalis. l988a, b; Reed et al.. 1990) and a method based on immunoblotting test serum (Bogdan et al., 1990) give sensitive and specific results of satisfactory reliability in the clinical context. Unfortunately these methods are too expensive to introduce in tropical areas. In particular, parasite preparations. especially if purified, are expensive, and both quantitative EIA and immunoblotting require special equipment and expertise. It is known that cultured Leishnarzia promastigotes and amastigotes release structural components of their cell surface (Schnur, 1982>, and circulating antigens of parasitic origin have been demonstrated in patients with VL (Evans and Pearson. 1988; Xiaosu et al.. 1988; Sinha et al., 1988; Mary et al., 1993; Azazi et al., 1994). Bearing in mind the needs of field medicine in tropical areas we have developed methods for the detection of an L. dr>llorsani-related circulating antigen. which are sufficiently sensitive and specific to be clinically reliable and which are at the same time inexpensive and easy to perform. Semi-quantitative dot-EIA and paddle-EIA were set up using a monoclonal antibody (MAb) raised against L. donormi promastigotes and both satisfied specific practical requirements in terms of cost and performance. Their clinical relevance was tested and compared to those of two quantitative microplate-EIA. one developed
for the detection of the same antigen using the above MAb, and another aimed at detecting serum antibodies to L. kfnrltlfm.
2. Materials
and methods
2.1. Seru Sera were obtained from 69 patients with VL acquired in different parts of the world (40 from Brazil (provided by R. Badaro. Laboratory of Immunology, Edgard Santos Hospital, Federal University of Bahia, Salvador, Brazil), 16 from Kenya (provided by J. Jephthah-Ochola. Kenya Medical Research Institute. Nairobi. Kenya), eight from China and five from France (provided by C. Mary, Laboratory of Parasitology. Faculty of Medicine, University of Marseilles, Marseilles. France)]; from nine patients with cutaneous leishmaniasis (CL) (provided by C.L. Jaffe, MacArthur Centre for Molecular Biology of Tropical Diseases. Department of Biophysics, Weizmann Institute of Science. Rehovot. Israel); from 38 patients with various infectious tropical diseases other than VL of whom 12 presented with a history of multiple infections (provided by N. Weiss, Swiss Tropical Institute, Basel, Switzerland. and by J. Jephthah-Ochola); from five patients with rheumatoid arthritis attending a clinic for rheumatic diseases in Geneva and from 40 healthy subjects recruited among Geneva blood donors. Among the tropical infectious diseases other than VL there were ten cases of strongyloidiasis, nine of filariasis, seven of amebiasis, seven of schistosomiasis, five cases of Echinocotc~4s graizfilosiu infestation, five of malaria. five of toxocariasis, two of African trypanosomiasis. two of tuberculosis and one of Fnsciolrr hepticcr infestation. The patients with tropical infectious diseases other than VL and those with RA were considered together in this study and referred to as patients with diseases other than leishmaniasis. The five sera which gave the highest optical density CODI values in the quantative EIA established for the detection of the circulating antigen (see below) were pooled to obtain a positive control serum: five blood donor sera were also pooled to obtain a negative control serum.
G. Senaldi et al. /Journal
2.2. Monoclonal
of Immunological Methods 193 f 19961 9- 15
antibody
The mouse anti-L. donoL>ani suchuan promastigote I B 1-C2-B3 MAb was used in this study (Xiao-su et al.. 1988). It is an IgM produced by a hybridoma obtained by fusion of SP2/0 myeloma cells and spleen cells from BALB/c mice previously immunised with cultured promastigotes fixed in glutaraldehyde. This MAb reacts with three major immunoblot bands in an L. donovani suchuan lysate and it is able to detect antigen in the circulation of patients with VL (Xiao-su et al., 1988). Ascitic fluid containing the 1B l-C2-B3 MAb was obtained by growing the hybridoma in the peritoneum of BALB/c mice. 2.3. Detection qf L. donor!ani-related antigen by dot-EfA
circulating
1 ~1 of the sera to be tested, diluted l/25 in phosphate buffered saline (PBS), pH 7.4, were spotted on 4 X 7 mm nitrocellulose rectangles (Schleicher and Schuell, Dassel, Germany), mounted on a plastic strip as a support (obtained by M. Deres, Grands Places Medical Centre, Freiburg, Switzerland). Negative and positive control sera were also spotted on each strip. After allowing the serum to dry for 30 min, the strip was incubated for 30 min in PBS containing 0.05% Tween 20 and 5% milk powder, in order to block non-specific binding sites. The strip was then incubated for 2 h with lBl-C2-B3 MAb ascites diluted 1/ 1000 in PBS containing 0.05% Tween 20 and 2.5% milk powder (PBS-T-M), washed in five changes of 5 min each of PBS containing 0.05% Tween 20 (PBS-T), further incubated for 2 h in peroxidase-conjugated rabbit anti-mouse IgG, IgA and IgM antiserum (Zymed Laboratories, San Francisco, CA, USA) diluted l/500 in PBS-T-M, washed again as above and finally incubated for 20 min in PBS containing 0.2% chloronaphthol and 0.01% H,O,. The intensity of the blue/violet staining was evaluated visually by three independent observers unaware of sample identities and scored as 0, 1, 2 or 3. with 0 representing a colour intensity weaker than or equal to the one generated by the negative control and 3 the intensity equal to or stronger than the one generated by the positive control. Final scores were
I1
the means of the scores attributed by the single observers. A sample scored 1 or more was considered positive. 2.4. Detection of L. donorani-related antigen by paddle-EIA or microplate-EIA
circulating
Plastic paddles (Nunc, Glostrup, Denmark) or EIA microplate wells (Nunc) were coated overnight at 4°C with 500 and 100 ~1, respectively, of test serum diluted l/500 in PBS (in duplicate for the microplate-EIA), blocked with PBS containing 0.05% Tween 20 and 5% milk powder for 30 min at 37°C and incubated for 1 h with lBl-2C-B3 MAb ascites diluted l/1000 in PBS-T-M. After washing in five changes of PBS-T, paddles or microplate wells were further incubated for 1 h in peroxidase-conjugated rabbit anti-mouse IgG. IgA and IgM antiserum (Zymed Laboratories) diluted I/ 1000 in PBS-T-M, washed again as above and eventually incubated for 20 min in phosphate/citric acid buffer containing 0.1% ABTS (Sigma, St. Louis, MO, USA) and 0.01% H?O?. The intensity of the ensuing green staining obtained by paddle-EIA was evaluated and scored as described for the dot-EIA. Staining intensity obtained by microplate-EIA was measured in a microplate reader at 405 nm and expressed in OD units. A sample was considered positive when it generated an OD greater than 1.5 times the OD value generated by the negative control serum. 2.5. Detection
qf serum antibodies to L. in~antum
EIA microplate wells pre-coated with L. infanturn soluble antigens (Bordier Affinity Products, Crissier, Switzerland) were blocked with PBS containing 0.3% Tween 20 and 0.1% bovine serum albumin (dilution buffer) for 15 min and incubated with 100 ul of test serum (in duplicate) diluted l/500 in dilution buffer. After washing with PBS containing 0.3% Tween 20 microplate wells were incubated with 100 ~1 of peroxidase-conjugated goat anti-human IgG antiserum (Sigma) diluted l/1000 in dilution buffer, washed again and incubated with the ABTS containing buffer. Staining intensity was measured in a microplate reader, expressed in OD units and judged positive or negative as above.
12
3
1
+ ..
.
LC
PC
.. 01
9
.
,.
LV
NC
LC
LV
PC
NC
Fig. 1. Results (scores) of the dot-EIA for the detection of an L. &nol,arGelated circulating antigen obtained in patients with visceral leishmaniasis (VL) or cutaneous leishmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
Fig. 2. Results (scores) of the paddle-EIA for the detection of an L. ck,rlol,clni-rrlated circulating antigen obtained in patients with visceral leishmaniasis (VL) or cutaneous lekhmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
2.6. Statistical
whether considered as a whole or divided into healthy subjects, patients with CL and patients with diseases other than leishmaniasis. Values were not significantly different between the various groups without VL (Table 1) (Figs. l-4). However. patients with CL had OD values for serum antibodies to L. infallturn which were higher than in patients with diseases other than leishmaniasis (Table 1). The specificity and sensitivity for VL of the different methods are given in Table 2. The detection of serum antibodies to L. infarztum by microplate-EIA proved to be the most specific (98.9%) and sensitive (98.5%) method. The detection of the circulating
analyis.
Scores and OD values were compared Wilcoxon’s rank sum test and correlated Spearman’s rank correlation analysis.
by the by the
3. Results Dot- and paddle-EIA obtained when detecting serum antibodies to L. patients with VL than
scores and EIA OD values the circulating antigen or infantum were higher in in subjects without VL,
Table 1 Median (range) of EIA scores (dot-EIA and paddle-EIA for the detection of an L. donoc,ckrelated circulating antigen) and optical density values (microplate-EIA (M/P-EIA) for the detection of the circulating antigen and of anti-l. infnntutn serum antibodies (Abs-EIA) in patients and healthy subjects Dot-EIA VL Non-VL NC CL PC
1.7 (O-2.7) O(O-1.7) 0 (O-0.3) 0 (0-O) 0 (O-1.7)
:’
Paddle-EIA
M/P-EIA
2 0 0 0 0
-307 (73-783) _ 66 (O-212) 51(29-1951 63 G-123) 71 (O-212)
(O-3) J (O-2) (0-O) (O-2) (O-2)
Abs-EIA il
1307 (110-3783) 39 (O-336) 8 (O-336) 70 (O-175) h 38 (O- 184)
a
VL = visceral leishmaniasis: non-VL = all the subjects studied except those with VL; NC = healthy subjects: CL = patients with cutaneous leishmaniasis: PC = patients with diseases other than leishmaniasis: M/P = microplate. ’ = p < 0.001 as compared to non-VL, NC. CL and PC. h = JJ < 0.05 as compared to PC.
G. Senaidi et al. /Journal
600 0 z x
of Immunological
Methods 193
400
. ..
2 t.. *
8
200
13
Table 2 Specificity and sensitivity of the EIA for the detection of an L. donor,&-related circulating antigen (dot-EIA, paddle-EIA. M/PEIA) and for the detection of anti-l. irzfantumserum antibodies (Abs-EIA)
..
P
C19%) 9- 15
5.O: .‘<‘. .=: . + .
.
.
. . . :-
m3
.
.
w.
x. --
V’
LV
NC
Lb
PC
Fig. 3. Results (OD values) of the microplate-EIA for the detection of an L. donollarii-related circulating antigen obtained in patients with visceral leishmaniasis (VLl or cutaneous leishmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
antigen by dot-EIA was (96.7%) and as sensitive Interestingly, there was was negative for serum tive in dot-EIA for the
second best for specificity as microplate-EIA (98.5%). one patient with VL who antibodies but scored posicirculating antigen. Of the
.
2
NC
LC
PC
Fig. 4. Fig. 4. Results (OD values) of the EIA for the detection of anti-l. infanturn serum antibodies obtained in patients with visceral leishmaniasis (VLI or cutaneous leishmaniasis (CL) or diseases other than leishmaniasis (PC) and in healthy subjects (NC). The horizontal line marks the threshold between positive and negative results.
False positives Specificity t%) False negatives Sensitivity (%I
Dot-EIA
Paddle-EIA
M/P-EIA
Abs-EIA
3/92 96.7 l/69 98.5
3/92 96.7 14/69 79.7
9/92 90.2 6/69 91.3
I /92 98.9 I /69 98.5
A b
a 92 were the subjects not affected by visceral leishmaniasis (i.e., healthy subjects. patients with cutaneous leishmaniasis or diseases other than leishmaniasis) considered as a whole group. ’ 69 were the patients with visceral leishmaniasis. M/P = microplate.
three non-VL patients who were positive in dot-EIA, but negative for serum antibodies, two had tuberculosis.
4. Discussion This study illustrates the possibility of detecting an f.. donorlalzi-related circulating antigen in the serum of patients with VL by semiquantitative dotEIA and paddle-EIA. both of which are inexpensive and easy to perform, and by quantitative microplateEIA. It also shows that the dot-EIA is able to provide reliable identification of patients with VL which is comparable in specificity and sensitivity to quantitative EIA for the detection of anti-l. infanturn serum antibodies. Antigens of parasitic origin have been demonstrated in the circulation of patients with L. &nopani infection, although their biochemical characterisation still awaits definition (Evans and Pearson, 1988; Xiao-su et al., 1988; Sinha et al., 1988; Mary et al., 1993; Azazi et al., 1994). EIA methods aimed at detecting L. donor& circulating antigens have been developed for use in the diagnostic laboratory. thus providing the possibility for direct diagnosis of VL (Xiao-su et al., 1988; Sinha et al., 1988; Azazi et al., 19941. The novelty of the present study is the establishment of (1) an upgraded version of the dot-EIA method published by Xiao-su et al. (1988). which is improved in sensitivity and is capable of
14
G. Srrmldi et al. / Jortrnnl
of Immur~ological
large scale application; and (2) a novel microplateEIA, which differs from the EIA proposed by Sinha et al. (1988) and Azazi et al. (1994) in that it makes use of a MAb instead of antisera against the circulating antigen and it directly detects antigen instead of relying on competition with peroxidase-labelled promastigote soluble antigen (Sinha et al., 1988) or on preliminary precipitation and dissociation of antigen containing circulating immune complexes (Azazi et al., 19941. The fact that the dot-EIA showed an elevated degree of sensitivity when tested on a series of geographically heterogeneous VL subjects suggests that it might be suitable for the diagnosis of VL caused by different strains of Leishrnania protozoa. The dot-EIA also showed high specificity. Of the three false positive cases that were found two were cases of tuberculosis and one of amebiasis. Thus. both tuberculosis cases included in our series of pathological controls were false positive. supporting the possibility that antigenic cross-reactivity exists between Leishmania and Mycobacteria. We only studied two cases of African trypanosomiasis, but neither was responsible for a false positive result, which is interesting since serological tests for the diagnosis of VL often show weak specificity when challenged with sera from patients with 7’ppanosoma infections. The dot-EIA described in this paper is suitable for the screening of clinical cases where there is suspicion of VL and the practice of invasive biopsies followed by demanding microbiological investigations could be confined to individual screened as positive. To provide health care workers with the opportunity of using the dot-EIA for screening. an inexpensive kit which is appropriate for large scale production is currently under development. This would permit applications of the dot-EIA in the less well-equipped laboratories. We calculate that the production cost of such a kit for 120 tests is US$ 60 (US$ 0.5 per test). The quantitative microplate-EIA for antigen detection established in this study may prove useful for monitoring the course of L. domx~arzi infection. However, further studies are required to evaluate the the reproducibility of the assay and its relevance in predicting relapses and remissions in association with the differing clinical presentations of VL.
Methods
193 (19961 Y-15
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 R. Badaro (Laboratory of Immunology, Edgard Santos Hospital. Federal University of Bahia, Salvador, Brazil), J. Jephthah-Ochola (Kenya Medical Research Institute, Nairobi, Kenya), C.L. Jaffe (MacArthur Centre for Molecular Biology of Tropical Diseases, Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel), C. Mary, (Laboratory of Parasitology, Faculty of Medicine, University of Marseilles, Marseilles. France) and N. Weiss (Swiss Tropical Institute, Base], Switzerland) for providing sera. We thank M. Deres (Grands Places Medical Centre, Freiburg. Switzerland) for the supply of technical material. We also thank C. Tougne for technical assistance and G. Del Giudice for helpful discussions. This study is dedicated to the memory of Vaclav Houba. who conceived it.
References Azazi, A.A., Devaney, E. and Chance. M.L. (19941 A PEG-ELISA for the detection of Lrishmnniu donowmi antigen in circulating immune complexes. Trans. R. Sot. Trop. Med. Hyg. 88. 62-66. Bogdan, C., Stosiek. N.. Fuchs, H.. Rollinghoff. H. and Solbach, W. (1990) Detection of potentially diagnostic leishmanial antigens by western blot analysis of sera from patients with kala-arar or multilesional cutaneous leishmaniasis. J. Infect. Dis. 162. 1417-1418. Bray, R.S. (1976) Immunodiagnosis of leishmaniasib. In: S. Cohen and E. Sadun (Eds.1, Immunology of Parasitic Infections. Blackwell Scientific Publications. Oxford. pp. 65-76. Bray, R.S. (1980) Leishmaniasis. In: V. Houha (Ed.), Immunological Investigation of Tropical Parasitic Diseases. Churchill Livingstone. Edinburgh. pp. 65-7-l. Evans. T.G. and Pearson. R.D. (19881 Identification of leishmanial antigens in the sera of patients with American visceral leishmaniasis. Infect. Immun. S6. 3 139-3 144. Jaffe, C.L. and McMahon Pratt, D. (1987) Serodiagnostic assay for visceral leishmaniasis employing monoclonal antibodies. Trans. R. Sot. Trop. Med. Hyg. 81. 587-S94. Jaffe, CL. and Zalis, M. ( 1988al Purification of two L. domwmi membrane proteins recognised by sertt from patients with visceral leishmaniasis. Mol. Biochem. Parasitol. 77, 53-62. Jaffe. C.L. and Zalis. M. (1988b) Use of purified parasite proteins from L. &noi,(l~ij for the rapid serodiagnosis of visceral leishmaniasis. J. Infect. Dis. 157, 1212- 1220. Jaffe. C.L.. Bennet. E.. Grimaldi, Jr.. G. and McMahon Pratt. D.
G. Senaldi et al. /Journal
of Immunological Methods 193 (1996) Y-15
(1984) Production and characterisation of species-specific monoclonal antibodies against L. donor,ani for immunodiagnosis. J. Immunol. 133, 440-447. Mary, C., Ange, G., Dunan, S., Lamouroux, D.. Quilici, M. (1993) Characterisation of a circulating antigen involved in immune complexes in visceral leishmaniasis patients. Am. J. Trop. Med. Hyg. 49, 492-501. Muigai. R. and Bryceson, A. (1982) The diagnosis and management of leishmaniasis. Postgrad. Doctor (Africa) 4. 280-288. Reed, S.. Shreffler, W.G., Bums, Jr.. J.M.. Scott. J.M., Da Gloria Orge, M.. Ghalib. H.W., Siddig. M. and Badaro. R. (1990) An improved serodiagnostic procedure for visceral leishmaniasis. Am. J. Trop. Med. Hyg. 43. 632-639. Schnur, L.P. (1982) The immunological identification and charac-
15
terisation of leishmanial sticks and strains, with special reference to excreted factor serotyping. In: M.L. Chance and B.C. Walton (Eds.1. Biochemical Characterisation of kishmnnia. World Health Organisation/Tropical Disease Research, Geneva. pp. 25-47. Sinha. R.. Arora, S.K. and Sehgal. S. (19881 A sensitive ELISA for detection of circulating antigens in kala-azar patients. Ind. J. Med. Res. 88, 138-140. Xiao-su, H.. Qing. L., Fang-Zing. L.. Tao-lin. Y.. Ya-Jin, W.. Zhi, Q., Ping, L. and Ling. W. (1988) Kala-azar infected serum circulating antigens and their characteristic detected by monoclonal antibody. Chin. Med. J. 101. l-6. Zuckerman, A. and Lainson, R. (1977) Leishmania. In: J.P. Kreier (Ed.), Protozoa. Academic Press, New York, pp. 37-39.