Acta Tropica, 50(1992)87, 88
87
© 1991 ElsevierSciencePublishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00160
Short Communication
Fluorescent antibody titres after recovery from visceral leishmaniasis Debasis Basu 1, K.K. Mallik 1, Amal Bhattacharya z a n d Anil K u m a r G h o s h 3 1Department of Tropical Medicine, School of Tropical Medicine, Calcutta. India, 2Department of Zoology, University of Calcutta, India and 3Department of Genetic Engineering, Indian Institute of Chemical Biology, Calcutta, India
(Received 14 January 1991;accepted 24 April 1991) Key words: Human serum; Antileishmanialantibodies; Post kala-azar dermal leishmaniasis; Immunology
Following recovery from visceral leishmaniasis (VL), a proportion of cases in India develop post kala-azar dermal leishmaniasis (PKDL). During VL, the levels of specific antibody and of polyclonal IgG are high (WHO, 1984). It has been believed that failure of the antibody titre to fall may be an early indicator of relapse (MansonBahr and Bell, 1987). On the other hand, indirect immunofluorescent titres may persist for years after resolution of disease (Locksley, 1991). P K D L sera have been shown to contain specific antibodies but the titres are lower (Haldar et al., 1981). A study is in progress to find out whether persistence of specific antibody or the lack of it, following recovery from VL, has any relation to the development of PKDL. The titres of antibodies of IgG class directed against intact promastigotes were evaluated using the indirect fluorescent-antibody test. Live promastigotes were harvested from log-phase culture in tissue-culture medium (M-199) and then diluted in phosphate-buffered saline (pH 7.2) so as to obtain 100 promastigotes in 50 ~tl of suspension. Fixation was done using cold methanol. Promastigotes employed were of an established pathogenic strain MHOM/IN/1983/AG83, originally obtained from a case of VL. Serially diluted sera were employed from three different groups of patients - cases of active VL, cases of P K D L and cases who even 3 to 4 years after recovery from VL had not developed P K D L . Ten cases from each group were studied and the titres compared. Sera from normal individuals and patients with other common diseases of the reticulo-endothelial system were also studied. Fluorescence was developed using FITC-tagged goat-antihuman IgG (Sigma, St. Louis, MO). Titres below 20 were considered negative. The results are presented in Table 1. It was observed that sera from VL patients had high titres exceeding 640 and in 6 cases exceeding 1280. Titres in P K D L patients were less than 80. For those in the 'follow-up n o n - P K D L ' group, the titre was Correspondence address." A.K. Ghosh, Department of Genetic Engineering, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India.
88 TABLE 1 Number of cases with antibody levels exceeding particular titres IFA Titres
VL PKD L Non-PKDL
l0
20
-
3
-
-
Total 40 7 -
80
160
320
640
1280
5
4
6
3
2
-
10 10 10
v a r i a b l e b u t was consistently a b o v e 160 a n d the highest value exceeded 640. Sera o f n o r m a l p e r s o n s a n d f r o m o t h e r p a t i e n t s d i d n o t show d e t e c t a b l e titres. It can be inferred t h a t in V L there is a high titre o f specific a n t i - l e i s h m a n i a l I g G directed a g a i n s t antigenic d e t e r m i n a n t s on the surface o f the p r o m a s t i g o t e s . Cases w h o d e v e l o p e d P K D L h a d low titres, b u t cases o f c o m p a r a b l e d u r a t i o n w h o h a d n o t d e v e l o p e d P K D L h a d higher titres, i n d i c a t i n g t h a t P K D L m a y be a s s o c i a t e d with lack o f persistence o f such specific a n t i - p r o m a s t i g o t e I g G . T h e r e is s o m e evidence t h a t P K D L m a y be related to a fresh i n o c u l a t i o n o f p r o m a s t i g o t e s , since it occurs c o m m o n l y in areas where n a t u r a l t r a n s m i s s i o n o f VL is c o n t i n u i n g ( S a n y a l et al., 1979). T h e low a n t i b o d y titres f o u n d in the present series o f P K D L cases c o u l d s u p p o r t a possible i m m u n o l o g i c a l e x p l a n a t i o n for this observation. F u r t h e r studies are being d o n e to find o u t the titre c h a n g e in the intervening p e r i o d , especially at 6 m o n t h s after recovery from VL, when the high globulin level has generally subsided.
Acknowledgements
This w o r k was s u p p o r t e d by the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in T r o p i c a l Disease ( T D R ) . We are grateful to Dr. S a m i t A d h y a o f the I n d i a n I n s t i t u t e o f C h e m i c a l Biology, C a l c u t t a , for his gift o f the L. donovani strain a n d for o t h e r l a b o r a t o r y facilities.
References
Haldar, J.P., Saha, K.C. and Ghose, A.C. (1981) Serological profiles in Indian post kala-azar dermal leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 75, 514-517. Locksley, R.M. (1991) Leishmaniasis. In: Wilson et al. (Eds.), Harrison's Principles of Internal Medicine, International Edition (12th Edn.), Vol. I, McGraw Hill, New York, NY, pp. 789-791. Manson-Bahr, P.E.C. and Bell, D.R. (1987) In: Manson's Tropical Diseases, 19th Edition, ELBS/Bailliere Tindall, London, p. 104. Sanyal, R.K., Alam, S.N., Kaul, S.M. and Wattal, B.L. (1979) Some observations on epidemiology of current outbreak of kala-azar in Bihar. J. Commun. Dis 11, 170-182. WHO (1984) The Leishmaniases, WHO Tech Report Series No. 701, Geneva, p. 20.
87
© 1991 ElsevierSciencePublishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00160
Short Communication
Fluorescent antibody titres after recovery from visceral leishmaniasis Debasis Basu 1, K.K. Mallik 1, Amal Bhattacharya z a n d Anil K u m a r G h o s h 3 1Department of Tropical Medicine, School of Tropical Medicine, Calcutta. India, 2Department of Zoology, University of Calcutta, India and 3Department of Genetic Engineering, Indian Institute of Chemical Biology, Calcutta, India
(Received 14 January 1991;accepted 24 April 1991) Key words: Human serum; Antileishmanialantibodies; Post kala-azar dermal leishmaniasis; Immunology
Following recovery from visceral leishmaniasis (VL), a proportion of cases in India develop post kala-azar dermal leishmaniasis (PKDL). During VL, the levels of specific antibody and of polyclonal IgG are high (WHO, 1984). It has been believed that failure of the antibody titre to fall may be an early indicator of relapse (MansonBahr and Bell, 1987). On the other hand, indirect immunofluorescent titres may persist for years after resolution of disease (Locksley, 1991). P K D L sera have been shown to contain specific antibodies but the titres are lower (Haldar et al., 1981). A study is in progress to find out whether persistence of specific antibody or the lack of it, following recovery from VL, has any relation to the development of PKDL. The titres of antibodies of IgG class directed against intact promastigotes were evaluated using the indirect fluorescent-antibody test. Live promastigotes were harvested from log-phase culture in tissue-culture medium (M-199) and then diluted in phosphate-buffered saline (pH 7.2) so as to obtain 100 promastigotes in 50 ~tl of suspension. Fixation was done using cold methanol. Promastigotes employed were of an established pathogenic strain MHOM/IN/1983/AG83, originally obtained from a case of VL. Serially diluted sera were employed from three different groups of patients - cases of active VL, cases of P K D L and cases who even 3 to 4 years after recovery from VL had not developed P K D L . Ten cases from each group were studied and the titres compared. Sera from normal individuals and patients with other common diseases of the reticulo-endothelial system were also studied. Fluorescence was developed using FITC-tagged goat-antihuman IgG (Sigma, St. Louis, MO). Titres below 20 were considered negative. The results are presented in Table 1. It was observed that sera from VL patients had high titres exceeding 640 and in 6 cases exceeding 1280. Titres in P K D L patients were less than 80. For those in the 'follow-up n o n - P K D L ' group, the titre was Correspondence address." A.K. Ghosh, Department of Genetic Engineering, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India.
88 TABLE 1 Number of cases with antibody levels exceeding particular titres IFA Titres
VL PKD L Non-PKDL
l0
20
-
3
-
-
Total 40 7 -
80
160
320
640
1280
5
4
6
3
2
-
10 10 10
v a r i a b l e b u t was consistently a b o v e 160 a n d the highest value exceeded 640. Sera o f n o r m a l p e r s o n s a n d f r o m o t h e r p a t i e n t s d i d n o t show d e t e c t a b l e titres. It can be inferred t h a t in V L there is a high titre o f specific a n t i - l e i s h m a n i a l I g G directed a g a i n s t antigenic d e t e r m i n a n t s on the surface o f the p r o m a s t i g o t e s . Cases w h o d e v e l o p e d P K D L h a d low titres, b u t cases o f c o m p a r a b l e d u r a t i o n w h o h a d n o t d e v e l o p e d P K D L h a d higher titres, i n d i c a t i n g t h a t P K D L m a y be a s s o c i a t e d with lack o f persistence o f such specific a n t i - p r o m a s t i g o t e I g G . T h e r e is s o m e evidence t h a t P K D L m a y be related to a fresh i n o c u l a t i o n o f p r o m a s t i g o t e s , since it occurs c o m m o n l y in areas where n a t u r a l t r a n s m i s s i o n o f VL is c o n t i n u i n g ( S a n y a l et al., 1979). T h e low a n t i b o d y titres f o u n d in the present series o f P K D L cases c o u l d s u p p o r t a possible i m m u n o l o g i c a l e x p l a n a t i o n for this observation. F u r t h e r studies are being d o n e to find o u t the titre c h a n g e in the intervening p e r i o d , especially at 6 m o n t h s after recovery from VL, when the high globulin level has generally subsided.
Acknowledgements
This w o r k was s u p p o r t e d by the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in T r o p i c a l Disease ( T D R ) . We are grateful to Dr. S a m i t A d h y a o f the I n d i a n I n s t i t u t e o f C h e m i c a l Biology, C a l c u t t a , for his gift o f the L. donovani strain a n d for o t h e r l a b o r a t o r y facilities.
References
Haldar, J.P., Saha, K.C. and Ghose, A.C. (1981) Serological profiles in Indian post kala-azar dermal leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 75, 514-517. Locksley, R.M. (1991) Leishmaniasis. In: Wilson et al. (Eds.), Harrison's Principles of Internal Medicine, International Edition (12th Edn.), Vol. I, McGraw Hill, New York, NY, pp. 789-791. Manson-Bahr, P.E.C. and Bell, D.R. (1987) In: Manson's Tropical Diseases, 19th Edition, ELBS/Bailliere Tindall, London, p. 104. Sanyal, R.K., Alam, S.N., Kaul, S.M. and Wattal, B.L. (1979) Some observations on epidemiology of current outbreak of kala-azar in Bihar. J. Commun. Dis 11, 170-182. WHO (1984) The Leishmaniases, WHO Tech Report Series No. 701, Geneva, p. 20.
