R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T ro p ic a l 2 9 (2 ) :1 5 3 -1 6 3 , m a r -a b r , 1 996.
T H E F M L (F U C O S E M A N N O S E L IG A N D ) O F L E 1S H M A N IA
D O N O V A N I . A N E W T O O L IN D IA G N O S IS , P R O G N O SIS ,
T R A N S F U S IO N A L C O N T R O L A N D V A C C IN A T IO N A G A IN S T
H U M A N K A L A -A Z A R
Claris» B. Palatnik de Sousa, Elza M . G om es, E dilm a Paraguai de Souza, W ania R. dos Santos, Sirley R. de M acedo, Linnete V. de M edeiros and
Kleber Luz
T h e F u c o se -M a n n o se L ig a n d (FM L) o f L cishm ania donovani is a c o m p le x g ly c o p r o te ic fr a c tio n . Its p o te n tia l u se a s a to o l f o r d ia g n o s is o f h u m a n v is c e ra l le ish m a n ia sis w a s te s te d w ith h u m a n s e r a fr o m N a ta l, R io G ra n d e d o N o rte , B razil. T he FM L-EL1SA test, s h o w e d 1 00% s e n s itiv ity a n d 9 6 % sp e c ific ity , id en tifyin g p a tie n ts w ith o v e rt k a la -a za r (p < 0 .0 0 1 , w h e n c o m p a r e d to n o r m a l s e r a ), a n d su b je c ts w ith su b c lin ic a l infection. M o r e th a n 2 0 % a p p a re n tly h e a lth y s u b je c ts w ith p o s itiv e rea c tio n to F M L d e v e lo p e d o v e rt k a la -a z a r d u rin g th e fo llo w in g 1 0 m o n th s. In th e s c r e e n in g o f h u m a n b lo o d d o n n o rs , a p r e v a le n c e o f 5 % o f s o r o r e a c tiv e su b je c ts w a s d e te c te d , a tta in in g 7 7 % in a sin g le d a y. T he G P 3 6 g ly c o p r o te in o f F H L is sp e c ific a lly re c o n iz e d b y h u m a n k a la -a za r se ra . T h e im m u n o p ro te c tiv e effe c t o f F M L on e x p e rim e n ta l L . do n o v an i in fectio n w a s te ste d in sw is s a lb in o m ic e . T he p r o te c tio n s c h e e m e s in c lu d e d th r e e w e e k ly d o s e s o f F M L , su p p le m e n te d o r n o t w ith sa p o n in b y th e s u b c u ta n e o u s o r in tr a p e r ito n e a l ro u te s a n d c h a lle n g e w ith 2 x 1 0 7 a m a stig o te s o f L eishm ania donovani. A n e n h a n c e m e n t o f 8 0 .0 % in a n tib o d y re s p o n se ( p < 0 . 0 0 1 ) a n d re d u c tio n o f 8 5 .5 % p a r a s ite liv e r b u rd e n ( p < 0 . 0 0 1 ) w a s d e te c te d in a n im a ls im m u n ize d w ith F M L sa p o n in , u n re s p e c tiv e ty o f the im m u n iza tio n route.
K e y -w o rd s: G lyco c o n ju g a te . L eishm ania donovani. D ia g n o sis. P ro g n o sis. K a la -a za r. V isc e ra l le ish m a n ia sis. B lo o d tra n sfu sio n . L e is h m a n ia l a ntigens.
V isceral leishm aniasis or kala-azar is a chronic and frequently lethal disease, caused by parasites o f the L e ish m a n ia d o n o v a n i com plex. T he disease is characterized by fever, m alaise, loss o f w eight, hepatom egaly, splenom egaly, anem ia, leukopenia, h y p e r g a m m a g lu b u lin e m ia a n d p r o g r e s s iv e suppression o f the cellular im m une response. Kala- azar is often fatal if untreated after the onset o f sy m p to m s. F u rth e rm o re , c h e m o te ra p y show s several un d esirab le colateral effects. T he total num ber o f L e ish m a n ia infected people all over the w o rld is being estim ated in tw elve m illions. Five h u n d re d th o u san d o f th ese cases, each year, correspond to visceral leishm aniasis25. This num ber
In stitu to d e M ic r o b io lo g ia . U n iv e r s id a d e F e d e r a l d o R io d e J a n e ir o . C e n tr o d e H e m a to lo g ia e H e m o te r a p ia -H E M O N O R T E . H o sp ita l d e D o e n ç a s I n fe c c io s a s G is e ld a T r ig u e ir o . D ep artam en to
d e I n f e c t o lo g ia . U n iv e r s id a d e F e d e r a l d o R io G ra n d e d o N o r te .
A d d r e s s l o : D r a . C la r isa B . P a la tn ik d e S o u s a . In stitu to d e M ic r o b io lo g ia /C C S /U F R J . C . U n iv e r sitá r ia . I. d o F u n d ã o . C P : 6 8 0 4 0 , 2 1 9 4 1 - 5 9 0 R io d e J a n e ir o , R J.
R e c e b id o p ara p u b lic a ç ã o e m 0 8 / 0 1 / 9 6 .
is probably underestim ate since an accurate and uniform early diagnose o f disease has n o t y et been established. V isceral leishm aniasis is spreading out in the W orld due to the increased resistance o f parasites to chem oterapy and o f insect vectors to insecticides41. InB razil, the disease is dissem inating from the N orth and N o rth -E ast regions o f the country and has recently been described in the State o f Rio de Janeiro, p reviously considered as not endem ic20.
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R, M a c e d o S R , M e d e ir o s L V , L u z K. T h e F M L (F u c o se M a n n o s e L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w to o l in d ia g n o s is , p r o g n o s is , tr a n s fu s io n a l c o n tr o l a n d v a c c in a tio n a g a in s t h u m a n k a la - a z a r . R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 9 :1 5 3 -1 6 3 , 1 9 9 6 .
d isap p ear a fte r its treatm ent. T he sorologycal diagnosis o f kala-azar has been proposed as an altern ativ e by several w ork s, based on antigens that sh o w d if f e r e n t d e g re e s o f p u r ific a tio n , and developping assays w ith diverse sensitivity and specifficity ranges5 32 35 38. T h e stability o f these antigens has also been discussed32. N evertheless, a non invassive m ethod o f m onitorization o f the disease w as n o t yet n ot available in Brazilian Public H ealth Services.
I n f e c t e d m o n o c y te s , a s w e ll a s , p o l y m o r p h o n u c l e a r n e u t r o p h i l e s 7 19 a n d eosin o p h iles9 w ere described in the blood, as potential carriers o f L. d o n o v a n i parasites from the skin to viscera and bone m arrow . In older stu d ies19 1, and in som e recent reports on hum an kala-azar22, th e presence o f infective parasites in th e secreta o f the u pper respiratory and digestive tracts, as w ell as in u rin e and faeces w ere cited. O nly o ccasio n ally , inoculation o f leishm aniasis by the transfusional via w ere cited10. Recent indirect evidences su p p o rt the need o f the study o f the blood m ediated contam ination w ith kala-azar in hum ans. L e is h m a n ia tro p ic a w as identified in bone m arrow o f am erican soldiers returning from Operation Desert Storm w ith viscerotropic leishm aniasis, and for this reason all individuals that traveled to that region w ere recom m ended to b e deferred as blood donors12. N o tw ith stan d in g , the existence o f a blood-m ediated contam ination is up to now not adm itted as a potential d anger fo r leishm anial infections, and no co n tro l o f blood is perform ed for the detection o f leishm aniasis in b lood banks, even in regions reported to b e endem ic for kala-azar, and expected to h arb o r a large n um ber o f subjects w ith subclinical infection.
T he analysis o f the protective potential o f L e is h m a n ia l antigens to cutaneous leishm aniasis in m urine m odels has been the focus o f detailed studies. T he W orld H ealth O rganization encouraged th e developm ent o f vaccines for leishm aniasis, using total o r p artially identified parasite lysates and cru d e an tig en s, that could induce a clear pro p h y lactic im m une response24. T his kind o f vaccine has show n to be effective against cutaneous leishm aniasis1121. T h e vaccines derived from w hole k ille d L e is h m a n ia a re in study in B razil (L . a m a zo n e n sis), Iran (L. m a jo r) and V enezuela (L. m e x ic a n a a n d L . b ra zilie n sis)25. H ow ever, for
hum an visceral leishm aniasis, no vaccine is up to now available. O nly few studies on im m unization against L e ish m a n ia d o n o v a n i have been undertaken in the m urine m odel, using total parasites26 18 14 or purified an tig en s17.
In recent studies, w e described the isolation o f the F M L (F ucose M annose L igand) o f L. d o n o v a n i prom astigotes, a com plex glycoproteic fraction that strongly inhibits p rom astigote27 and am astigote infection o f m urine m acrophages29. Its proteic m oiety is m ainly com posed o f acidic and apolar am inoacid resid u es. A m o n g its n e u tra l su g a r c o m p o n e n ts, fu c o se (1 0 % ), m a n n o se (4 7 % ), g a la c to s e (1 2 % ) a n d g lu c o s e (3 0 % ) w e r e described27. F M L developped a species-speciffic effect on the inhibition o f p ro m astig o te uptake by m acrophages in vitro21. T h is is a po ten t antigen for rabbits, ham sters and m ice283130. Its m ain antigenic fraction is a 36kD g lycoprotein, recognized by m ost m ouse IgG a n ti-F M L m onoclonal antibodies, and present at the surface o f both the prom astigote and am astigote form s o f L . d o n o v a n i parasites29. T h e p ro te c tiv e po ten tial o f F M L on v isceral leishm aniasis w as analyzed in the isogenic CB- ham ster m odel31. W e studied the effect o f three intraperitoneal w eekly doses o f F M L ( 100/xg) in saponin (100/xg), follow ed by an in tracard iac injection o f 107am astigotes. Protection w as speciffic and highly significant (8 7 .7 % , p < 0 .0 1 ) in the enhancem ent o f a n ti-F M L antibodies titers, o f the s p le n o c y te p r o lif e r a tiv e r e s p o n s e , a n d th e intraderm al delayed hypersensitivity reaction to antigen, as w ell as in the decrease o f th e parasite b u rd en in spleen and o f sp le n o m e g a ly 31. A n analogous study in the inbred balb /c m ice m odel show ed an a v e ra g e o f 8 4 .2 % (p C O .O O l) o f protection in antibody titers, splenocyte in vitro proliferation and liv er parasitic load30.
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e ir o s L V , L u z K. T h e F M L (F u c o se M a n n o s e L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w to o l in d ia g n o s is , p r o g n o s is , tr a n s fu s io n a l c o n tr o l a n d v a c c in a tio n a g a in s t h u m a n k a la - a z a r . R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 9 :1 5 3 - 1 6 3 , 1 9 9 6 .
the su p p o rt o f the Fundação N acional d e Saúde, F N S . F u rth e rm o re , p relim in ary results o f the analysis o f the F M L vaccine in the outbred swiss albino m ouse m odel, are described.
M A T E R IA L A N D M ET H O D S
H u m a n s e ra
A total o f 462 sera w as analyzed in this study. A series o f 149 sera w as collected by the Fundação N acional de Saúde do R io G rande do N orte, in the p eri-urban area o f N atal, R N (N orth E ast Brazil), a focus o f a recent outbreak o f kala-azar. Ten o f the sera in this series cam e from subjects that had a patent kala-azar at the tim e o f serum collection. A series o f 26 sera o f kala-azar patients, befo re and d u rin g th e ir treatm ent, w as obtained at the H ospital de D oenças Infecciosas G iselda T rigueiro, N atal. A ll the 36 patients w ith clinical sym ptom s o f acute k a la -a z a r (lo ss o f w e ig h t, ly m p h a d e n o p a th y , hepatom egaly a n d /o r splenom egaly, and fever) w ere subm itted to b one m arro w aspiration. P arasitologic analysis confirm ed the presence o f L e ish m a n ia am astigotes in the G iem sa-stained sm ears. A fter the serum collection, patients w ere subm itted to anti- L e ish m a n ia treatm ent. T h irty five sera o f patients that have had kala-azar w ere collected at least three m onths after the end o f the treatm ent w ith antim onials o r a m p h o te ric in -B , d u rin g th e ir clin ical and serological follow -up32.
T w enty one persons w ith no patent signs o f disease, neighbors and relatives o f kala-azar patients, o r ow ners o f dogs w ith L. d o n o v a n i infection, w ere also included in the study. W e also analyzed the reactivity w ith F M L o f 21 sera o f patients w ith cutaneous o r m ucocutaneous leishm aniasis from R io de Janeiro and M inas G erais states, obtained from D r. S .M . C outinho, F undação O sw aldo Cruz, (R io d e Jan eiro ), 22 sera o f fully characterized patients w ith chronic Chagas disease from Bam bui, M inas G erais, and 18 sera from healthy adult blood d onors from the H ospital U niversitário Clem entino F ra g a F ilh o , U niversidade Federal do R io d e Janeiro, that w ere screened and considered negative for C hagas d isease, H IV I and II, h ep atitis, and syphilis32. F o r differential control diagnosis, sera o f patients w ith fever a n d /o r hepatosplenom egaly due to P a ra c o c c id io id e s bra silie n sis infections (1),
lym phom a (2), typhus (1) and hem ophagocytic disease (1) w ere obtained from the D epartam ento de D oenças Infecciosas, U F R J, and D epartam ento de P ediatria U E R J, R io de Jan eiro , RJ.
A series o f 171 sera o f v o lu n teer blood donors w as obtained from the b lo o d bank C entro de H em atologia e H em oterapia-H E M O N O R T E , and tested fo r th eir reactivity w ith F M L . Inform ed consent fo r this study w as obtained from all the subjects included in this study.
F M L -E L IS A q u a lita tiv e a ssa y
T h e F M L c o m p le x w a s o b ta in e d fro m L e is h m a n ia d o n o v a n i (L D -IS /M H O M /S D /O O - strain IS) prom astigotes as prev io u sly described29. T itration o f norm al hum an, kala-azar, C hagas’ disease and cutaneous leishm aniasis p a tie n t’s sera pools against F M L antigen w as perform ed in o rd er to establish the optim al antigen concentration. T he m icro-E L ISA m ethod w as d one as described32, using F M L diluted in carbonate bu ffer (pH 9 .6 ), to sensitize flat-bottom 96-w ell plates (H aem obag, R ibeirão P reto , SP, B razil). A n tib o d ie s w ere detected by peroxidase-labeled pro tein -A (Sigm a, St Louis, M O ). R eaction w as developed w ith O- phenyldiam ine (Sigma), interrupted w ith IN sulfuric acid, and m onitored at 492nm . Sera w ere analyzed by double blind tests, in triplicates, and results expressed as mean values. P ositive and negative control sera w ere included in each test. T he absorbance values o f sera w ere com pared at 1:100. T h e cut o f f lim it betw een the norm al sera from a n o n -e n d e m ic a re a a n d se ra o f p a tie n ts w ith parasitologically confirm ed kala-azar, was identified using the Y ou d en ’s J index43. S ig n ific a n c e o f the differences betw een groups o f sera w as established by a standard t test.
W e ste rn B lo t an aly sis
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w t o o l in d i a g n o s i s , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t io n a g a i n s t h u m a n k a l a - a z a r . R e v i s t a d a S o c ie d a d e B r a s i l e i r a d e M e d ic in a T r o p i c a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
V accination assays
2 ,5 m onth old sw iss albino fem ale m ice w ere obtained from th e B ioterio C entral, Instituto de M icro b io lo g ia d a U F R J. G roups o f 6-8 females w ere im m unized w ith three either subcutaneous or intraperitoneal doses o f 150/xg F M L and 100 /xg saponin (R iedel-de H aen, Seelze, G erm any) in 0 .2 m l sterile 0 .9 5 % saline, w ith w eekly interval. Saline and saponin treated controls w ere included. Seven days after im m unization, sera w ere collected and anim als w ere challenged by intravenous inj ection o f 2 x 107 L . d o n o v a n i am astigotes, obtained from infected h am ster’s spleens6. A nim als w ere sacrificed by etherization, 15 days after infection, and their liv er and spleen parasite-load w as m onitored in L eishm an-D onovan U nits o f Stauber o f G iem sa stained im printings (L D U = n u m b e r o f am astigotes/ 1000 cell nuclei x m g organ w eight). Sera o f anim als before and after infection w ere analyzed by EL ISA using 2/ig F M L /w ell and reacted w ith a P ero x id ase labeled anti-m ouse IgG (Sigm a, C o.). EL ISA results w ere expressed as scores (sum o f all
absorbances at 492nm , up to the en d -p o in t titer)32. Statistical com parison o f th e g roups w as done by the Student t test.
R E SU L T S
S tandardization o f E L ISA experim ents w as done using F M L in dilutions from 0 .03 to 2 p .g per w ell, reacted w ith pools o f norm al hum an sera from a non-endem ic area, sera o f patients w ith kala-azar, tegum entar leishm aniasis and Chagas disease, in dilution 1:2000. T he results presented in the F ig u re
1, indicated the antigen concentration o f 0 . 125/xg / w ell as the highest that gave no unspecific reaction. All the studied sam ples w ere titered and scored in this condition.
T he individual absorbance values in F M L - E L ISA assay for 1:100 diluted sera are represented in F ig u re 2A . T h e m ean value o f A bs 1:100 (X + SE = 0 .4 5 2 ± 0 .0 3 8 ) o f patients w ith acute kala- azar w as significantly d ifferent from the o ne o f the norm al subjects o f a nonendem ic area (p < < 0.001). Eight standard error values separated
F ig u r e 1 - D e te r m in a tio n o f F M L o p ti m a l c o n c e n tr a tio n f o r E LIS A le s t.
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e
L ig a n d ) fj/ L e is h m a n ia d o n o v a n i. A n e w t o o l in d i a g n o s i s , p r o g n o s i s , tr a n s f u s io n u l c o n t r o l a n d v a c c i n a t io n a g a i n s t h u m a n k a la - a z a r . R e v is t a d a S o c ie d a d e B r a s i le i r a d e M e d i c in a T r o p ic a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
the tw o m eans. T he use o f mean value + 2SD in the F M L -E L IS A assay attain the m aximal Youden v a l u e = l , corresponding to the absence o f f a l s e p o s i t i v e o r f a l s e n e g a t i v e results (cut o ff value = 0 .2 0 4 ). Sera o f patients w ith chronic Chagas disease s h o w e d , as e x p e c te d fro m s ta n d a r d iz a tio n experim ents, a very reduced reactivity (A bs 1:100 m ean value 0 .1 4 9 ). In the endem ic area, (Figure 2B). the F M L -E L IS A assay functioned as a highly discrim inating tool. It allow ed to distinguish the non-reactive sera from the endem ic area (N E) (n = 86), from the group o f subjects w ithout any sign o f leishm anial infection but reactive w ith F M L
(RE) (n = 59). W e interpret these subjects as a subclinical but a c tiv eL e i s h m a n ia infection, in view o f their clinical evolution. D uring the ten m onths follow ing the first study o f 41 sera from the kala- azar outbreak region, nine o f the inhabitants o f the stu d ie d endem ic area sp o n ta n e o u sly search ed m edical treatm ent for the severe disease (N K A ). K ala-azar w as diag n o sed by id e n tific a tio n o f am astigotes in the bone-m arrow aspirate. A m ong these, six had had high titers o f anti-F M L antibodies and the highest titers had been observed in two patients that eventually died o f severe kala-azar. D uring the sam e period, at our know ledge, no
F ig u r e 2 - T h e F M L -E L IS A a s s a y in a n e n d e m ic a n d n o n -e n d e m ic r e g io n f o r h u m a n v i s c e r a l le is h m a n ia s is . In A : F M L -E L IS A r e a c tiv i ty w ith s e r a o f n o r m a l s u b je c ts f r o m a n o n -e n d e m ic a r e a (N N ), p a ti e n ts w ith k a la - a z a r
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L (F u c o s e M a n n o s e L i g a n d ) o /L e is h m a n ia d o n o v a n i. A n e w t o o l in d i a g n o s i s , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t i o n a g a in s t h u m a n k a l a - a z a r . R e v is t a d a S o c i e d a d e B r a s i l e i r a d e M e d ic in a T r o p ic a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
subjects w ith negative reaction to F M L developed kala-azar. C onsequently, am ong 41 sera that had show n h igh o r low reactivity w ith F M L but had not clinical signs o f kala-azar, m ore than 20% developed the o v ert disease in the ten m onth period. T his nu m b er is p robably underestim ated, since a full clinical and serological survey o f the sam e area has n ot been com pleted32. Sera o f patients w ith fever and hepatosplenom egaly due to P a r a c o c c i d i o i d e s b r a s i l i e n s i s in fe c tio n (1 ), ly m p h o m a (2) and hem ophagocytic disease (1) gave also negative results in the F M L -E L IS A assay. T hese results determ ined 100% sensitivty and 96 % specificity.
A random m onitoring o f an ti-F M L reactivity am ong 171 v olunteer blood donors at the blood b a n k C en tro d e H e m a to lo g ia e H em o terap ia- H E M O N O R T E , N atal, w as perform ed, testing an average o f 2 4 .4 diary do n o r sam ples, fo r a period o f seven consecutive days. T he results obtained are sum m arized in T able 1. T he FM L -E L IS A test show ed the clearly positive reaction for 8 donors. T h e o th e r 163 sam ples show ed no reactiv ity (X + S E = 0 .1 3 8 + 0 .0 0 2 ). T his values represent a total o f 5% o f sam ples and w ere distributed attaining even 17% in a single day, no one o f w hich had a p o sitiv e reaction for C hagas’s disease, hepatites, H IV n o r syphilis o r a decreased hem atocrite value. C onversely, am ong the nonreactive donors, two cases o f hepatites B w ere detected indicating that also in this assay the FM L -E L ISA show ed to be hig h ly specific.
S ince the F M L com ponents can b e separated by electro p h o resis, w e have analyzed by W estern Blot the reactivity o f hum an sera w ith F M L (F igure 3).
T a b le 1 - B lo o d d o n o r s a m p le s r e a c t i v e in th e F M L -E L IS A a s s a y .
Donor number FML-ELISA
reactivy at 492nm
Date
462. .506 30.8.94
463. .291 31.8.94
464. .231 1.9.94
465. .271 1.9.94
466. .250 1.9.94
467. .232 1.1.94
468. .220 1.9.94
469. .232 5.9.94
All the samples were tested at 1/100 sera dilution. Results represent the absorbency values at 490nm. Normal sera gave values below 0.204.
T he 55kD a com ponent o f F M L w as recognized by norm al sera from kala-azar endem ic and n o n endem ic area, and sera from patien ts w ith tegum entar leishm aniasis, k ala-azar o r C hagas disease. This antigen is thus non-specific. Conversely the G P36 glycoprotein band w as only detected in W estem B Iotsbykala-azarpatients’ sera. No labeling w as detected w ith sera o f patients w ith tegum entar leishm aniasis, Chagas disease, o r norm al sera. T he G P36 antigen o f F M L show s to be a m arker o f hum an kala-azar.
F inally the analysis o f the p rotective potential o f F M L in experim ental vaccination against visceral leishm aniasis w as perform ed in the outbred swiss albino m odel. W e have tested th e potential activity
F ig u r e 3 - S D S -P A G E a n d W e s te r n B lo t a n a ly s is o f F M L a n tig e n o fL . d o n o v a n i u s in g h u m a n s e r a . M o le c u la r w e ig h t s t a n d a r d s a n d F M L (1 5 0 ^ g ) a n a ly z e d b y S D S
-P A G E a n d s t a in e d w ith C o o m a s s ie B r il lia n t B lu e R 2 5 0 , a r e in d ic a te d o n th e le ft s id e . B lo tts c o n ta in ig 1 - 3 u g F M L
w e r e tr e a te d w ith : a : s e r u m o f a p a t i e n t w ith c o n fir m e d k a la - a z a r ( 1 1 / 1 1 ) , b : s e r u m o f a n o r m a l d o n o r f r o m e n d e m ic a r e a , c : s e r u m o f a p a t i e n t w ith te g u m e n ta r le is h m a n ia s is (5 /5 ) , d : s e r u m o f a p a t i e n t w ith C h a g a s ’
d is e a s e (5 /5 ). e : s e r u m o f n o r m a l h e a th y d o n o r f r o m n o n e n d e m ic a r e a (2 /8 ). T h e a r r o w in d ic a te s th e G P 3 6 b a n d . T h e n u m b e r in b r a c k e t s r e p r e s e n t th e s a m p le s
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e
L i g a n d ) o / L e is h m a n ia d o n o v a n i. A n e w t o o l in d i a g n o s i s , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t i o n a g a in s t h u m a n k a l a - a z a r . R e v i s t a d a S o c ie d a d e B r a s i l e i r a d e M e d i c in a T r o p ic a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
o f F M L in p rotection o f outbred sw iss albino m ice against the infection w ith L . d o n o v a n i. W ecom pared the adm inistration o f F M L w ith Saponin through the intraperitoneal o r subcutaneous route. R esults o f a n ti-F M L antibodies are sum m arized in F igure 4A . Before the infection a pronounced speciffic response to F M L (p < 0.001 ) w as detected in animals im m unized w ith F M L and saponin, h igher by the intraperitoneal than the subcutaneous route. A fter the infection, both group o f mice showed an increased response w hereas no response w as detected in any control g roup. Taken together, these results show the slow establishm ent o f im m une response in m ice, even w hen infected by this relatively high d o se o f L . d o n o v a n i am astigotes. Indeed, no
im m unoglobulin secretion to F M L w as detected in untreated infected anim als, at 15 days o f infection. N otew orthy, the specific p re-stim ulation w ith F M L and saponin trig g ered the a n ti-F M L an tib o d y synthesis. T he p rotective effect o f this response is show n by the dim inished parasite b urden in h o s t’s tissues. Indeed, the parasite counts in liv er w ere significantly decreased ( p < 0 .0 0 1 ), in the sam e order o f m agnitude as the im m unoglobulins to F M L ex p an d ed F ig u re 4B . A t th is sta g e o f experim ental m urine infection, parasites are found only in liv er, and they infect the spleen only 30 days later (data not show n). N o significant differences w ere detected betw een th e saline and th e saponin treated anim als, in any param eter ( p > 0 .3 ) . Taken
F ig u r e 4 - E ffe c t o f F M L o n L e is h m a n ia d o n o v a n i in fe c tio n in s w is s a lb in o m ic e . In A : a n tib o d y r e s p o n s e .o p e n c o lu m n s : s e v e n d a y s a fte r th r e e in tr a p e r ito n e a l (IP) o r s u b c u ta n e o u s (S C ) in je c tio n s o f c o n tr o l s a lin e s o lu tio n (C ), lO O fig s a p o n in (S ), o r 1 5 0 jig F M L c o m b in e d to lO O fig s a p o n in (SF M L). S h a d e d c o lu m n s : id e m , 1 5 d a y s a f te r in fe c tio n w ith
L . donovani. R e s u lts r e p r e s e n t s c o r e m e a n v a lu e s o f t h e p o o l o f s e r a o f t w o in d e p e n d e n t e x p e r im e n ts d o n e in tr ip l ic a te s ,
u s in g 4 - 5 m i c e in e a c h g r o u p . In B : liv e r p a r a s i t i c b u r d e n ., o f th e s a m e g r o u p s in A , a f te r 1 5 d a y s in fe c tio n w ith L. donovani. R e s u lt s a r e e x p r e s s e d a s p e r c e n t s o f th e a v e r a g e o f L D U v a lu e s (c o u n ts in 1 0 0 0 c e ll s ) o f l i v e r im p r in ts o f
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w t o o l in d i a g n o s i s , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t io n a g a i n s t h u m a n k a l a - a z a r . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d ic in a T r o p ic a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
to g u eth er these results represent an average o f 80.0 % increase in speciffic antibody response and a 8 5.5% protectio n in reduction o f parasite liver burden. A lthough the hum oral response w as higher in the case o f intraperitoneal adm inistration, no signifficant d ifference betw een the vias was shown in w hat concern to protective effect.
D ISC U SSIO N
E L IS A tests using crude native antigens are usually d one in dilu tio n s o f 2 to 5/ig /w ell15 l6. In an im proved E L ISA assay, the recom binant GP63 antigen o f L . c h a g a s i w as used in concentration o f 1 /xg/well35 39. T h e F M L is thus a very sensitive antigen, and standard ELISA tests can be done in dilu tio n s increased by an o rd er o f m agnitude. This sensitivity can b e probably advantageously used in developm ent o f rapid and autom ated tests for kala- azar. This property o f F M L can be possibly attributed to its g lycoproteic nature. Previous studies have attributed the species-specific characteristics o f
L e is h m a n ia g ly c o c o n ju g a te s to th e ir g ly c id ic
m oiety28.
T h e presented results indicate that assays using F M L can be highly sensitive and specific in diagnosis o f k ala-azar. T his antigen can clearly separate visceral leishm aniasis from all the other closely related tegum entar infections caused by parasite o f th e g e n u s L e i s h m a n i a , w ith th e s e n s itiv ity co m p arab le to those rep o rted for recom binant antigens35 39. M o reo v er, this antigen can identify in h a b ita n ts o f endem ic areas w ith a potential subclinical infection, and p o in t out those w ith a h igh risk o f ev o lu tio n tow ards the overt severe disease. It can be also used to follow -up o f the p a ra sito lo g ic cu re. D iffe re n t fo rm p rev io u sly rep o rted data2, the F M L antigen isolated from an A frican strain o f L . d o n o v a n i w as accurate in d ia g n o s is o f S o u th A m e r ic a n v is c e r a l leishm aniasis, representing apparently a tool that can b e used thro u g h o u t the diagnostics o f diseases caused by the L . d o n o v a n i com plex.
T w o cases o f a positive reaction o f F M L w ith sera o f a p atient w ithout o vert kala-azar w ere observed. T h e first w as a patient w ith chronic dissem inated m uco-cutaneous leishm aniasis. This serum h ad h igh titers o f IgG and IgM antibodies against L . b r a z ilie n s is , b ut the parasite could n ot be
isolated n o r characterized. T h e second w as from a patient w ith C hagas’s disease. In both exam ples the p o ssibility o f a concom itant infection w ith L .
d o n o v a n i could not be ruled o u t since visceral
leishm aniasis w as also endem ic in these regions. T hese tw o p o sitiv e reactions are at p resen t, o f difficult interpretation. T heir presence has decreased the specificity o f the assay to 9 6 % . T h e F M L antigen developed equal levels o f sensitivity and specificity than the recom binant antigen used by B um s et al (1993)8. H ow ever, d ifferent from the recom binant antigen, F M L is a a glycoproteic com plex and its long lasting stability m akes o f it a good candidate fo r w ork in field assays. W e w ere able to use the sam e batch for perio d over tw o years w ith no signifficant changes in its perfo rm an ce.T h e F M L -E L IS A test is now being used as a new tool fo r diagnosis, p rognosis, cure survey o f visceral leishm aniasis and it is used as an alternative to bone m arrow punction in several hum an cases w ith no false n egative o r p o sitiv e resu lts. T h e test is perform ed routinely in the cited H ospital and Blood bank services. Furtherm ore a clinical and epidem ical survey o f perypherical areas o f N atal, using the F M L -E L IS A test is underw ay.
In this study w e described the presence o f L.
d o n o v a n i s o r o r e a c t i v e i n d i v i d u a l s a m o n g
spontaneous blood donors in the kala-azar endem ic area. T his prevalence attained up to 17% o f diary donors. T hese values are h ig h er than those usually detected for chagasic d onors in R io de Janeiro. Recent results from o u r laboratory dem ostrated that b lood transfusion is an efficient via o f transm ission o f experim ental kala-azar. N o o ne o f the 8 FM L - ELISA positive sera w as reactive in the tests for C hagas’ disease o r o ther diseases, perform ed in the blood bank and therefore th e use o f these b lood package units w ould be considered p ossible, since the presence o f antibodies ag ain st L e is h m a n ia
d o n o v a n i w ould n ot b e detected by the present used
control tools. Since these specific an tibody titers could b e a signal o f active infection and o f a p o t e n t i a l s o u r c e o f b lo o d b o r n e v i s c e r a l leishm aniasis, the extension o f the F M L -E L IS A screening in blood banks and the detailed study o f each sororeactive case is extrem ely necessary.
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R,. M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w t o o l in d ia g n o s is , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t io n a g a i n s t
h u m a n k a l a - a z a r . R e v is t a d a S o c ie d a d e B r a s i l e i r a d e M e d ic in a T r o p i c a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
p a tie n ts’ sera. N o labeling w as detected w ith sera o f patients w ith tegum entar leishm aniasis, Chagas disease, o r norm al sera. T he GP36 has show n to be the m ost specific fraction o f the F M L antigen. The G P 36 com ponent o f F M L could correspond in liv in g p ara site s to a lo w e r m olecu lar w eight com ponent since its extraction procedure, favours the glycidic enrichm ent that could retard ist migration in SDS P A G E . T he specificity o f a 32-35 kD a band fo r kala-azar w as suggested by Reed et a l. ( 1987)34, w ho used a total soluble extract o f L c h a g a s i w ith sera o f patients from Brazil. C irculating antigens o f 30-35 k D a w ere detected in sera o f hum an patients by m onoclonal specific antibodies42 and w ere recovered from kidneys o f L . d o n o v a n i infected h am sters35. T h e G P36 m ay be thus shed in vivo by parasites, representing a circulating antigen. The isolation o f G P36 antigen o f F M L is in progress.
F in ally , the reported results indicate that F M L m ay be a potential candidate for vaccination in visceral leishm aniasis. B oth in trap erito n eal or subcutaneous im m unizations w ere effective. T hese results are p articularly signifficant in view o f the o u tbred natu re o f the experim ental m odel. Indeed a h ig h in tra g ro u p v a ria tio n w as expected. T his protective effect o f F M L and saponin is in agree to previous evidences o f the protective potential against experim ental visceral leishm aniasis in CB ham sters and B alb/c m ice30 31. In o u r conditions, saponin show ed n o r toxic neith er unspeciffic effects. Its use as adjuvant w as com pared to C o r y n e b a c te r iu m
p a r v u m , F reu n d A djuvant, A lhydrogel, B o r d e te lla
p e r tu s s is and M uram yl dipeptide and considered to
be advantageous4. In agreem ent w ith o u r results, saponin has been previously show n to be a good p o t e n t i a t o r o f a n ti- p r o to z o a n im m u n ity in experim ental vaccines against T c r u z p 1. H ow ever, saponins are discrim inated as adjuvant because o f th eir hem olytic pro p erties3. F o r this reason, w e started the com parative analysis o f the hem olytic and ajuvant potential o f different kind o f saponins. U sing the pu rified antigen D P 72 for im m unization o f balb/c m ice against L. d o n o v a n i infection, a protective effect o f the sam e o rd er o f m agnitude was re p o rte d 16 33. H ow ever, in those w orks, for pro te c tio n o r ly m p h o cy te p ro liferatio n assays, respectively, after sensitization w ith the antigen, theanim als received different dosesofprom astigotes. It is consequently not clear if the described effects
w ere d ue only to D P 7 2 a d m in istra tio n o r to prom astigote injection o r to both o f them . T w o other antigens o f L e is h m a n ia , extensively studied w ere candidates fo r vaccination against cutaneous leishm aniasis in m ice, how ever, it w as recently show n that G P63 is unable to induce lym phocyte proliferation in hum an leishm aniasis23 and that the ability o f LPG to induce a protective response in mice is due to contam inant proteins o r glycoproteins, c a lle d L PG a s so c ia te d p ro te in s ( L P G A P ) 17. F urtherm ore subcutaneous LPG vaccination against m u r in e te g u m e n ta r l e i s h m a n i a s i s b y th e subcutaneous route show ed to induce an increase o f lesion size, instead o f p ro te c tio n 13. R ecently, the im m unization w ith one o f the L P G A P flagellar proteins, present in a w ide range o f L e is h m a n ia species and in A frican try p a n o so m e s, in the recom binant form , stim ulates prim ed lym phocytes to p roliferate in v itr o 40. T h e use o f F M L o f
L e is h m a n ia d o n o v a n i and o f its com ponents in
sorologycal screening for visceral leishm aniasis show s high sensitivity, specificity, stability and com paratively easier perform ance. Its use in Blood banks w ould im prove the q uality control to prevent blood borne infections. F u rth erm o re it show ed a signifficant potential on experim ental vaccination against kala-azar. The study o f the m olecular aspects related to the described F M L potentials is under progress.
R E SU M O
O F M L (L ig a m e d e F u c o s e - M a n o s e ) d e L eishm ania donovani é u m a f r a ç ã o g li c o p r o l e i c a c o m p le x a . O s e u
p o te n c ia l n o d ia g n ó s tic o d a le is h m a n io s e v i s c e r a l h u m a n a f o i te s ta d o co m s o r o s p r o v e n i e n te s d e N a ta l, R io G r a n d e d o N o r te , B ra sil. O te s te d e F M L -E L IS A m o s tr o u 1 0 0 % d e s e n s ib il id a d e e 9 6 % d e e s p e c i f i c i d a d e , k le n tif ic a n d o p a c ie n t e s c o m c a la z a r d e c la r a d o ( p < 0 . 0 0 1 , c o m p a r a d o s c o m s o r o s n o r m a is ) e in d iv íd u o s c o m in f e c ç ã o s u b c lín ic a . M a i s d e 2 0 % d o s s o r o r r e a t i v o s a s s i m p t o m á t i c o s d e s e n v o l v e r a m a d o e n ç a n o p r a z o d e 1 0 m e s e s . N a a n á lis e d e d o a d o r e s d e s a n g u e , 5 % d e s o r o r e a ti v o s , a tin g in d o a té 17%> n u m ú n ic o d i a f o r a m d e te c ta d o s . A g lic o p r o te ín a G P 3 6 d o F H L é r e c o n h e c id a es p e c ific a m e n te
p o r s o r o s d e p a c i e n t e s c o m c a la z a r . O p o t e n c i a l im u n o p r o te to r d o F M L n o c a la z a r e x p e r i m e n t a l f o i te s ta d o n o m o d e l o sw is.s a lb in o em c o m b in a ç ã o c o m
s a p o n in a p e l a s v ia s s u b c u tâ n e a s e /o u in t r a p e r i to n e a l s e g u id o d e d e s a f io c o m 2x 1 (P a m a s tig o la s d e Leishm ania donovani. U m a u m e n to d e 8 0 .0 % n a r e s p o s t a d e
a n tic o r p o s e s p e c íf ic o s ( p < 0 . 0 0 1 ) e a r e d u ç ã o d e 8 5 . 5 %
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e
L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w t o o l in d ia g n o s is , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t io n a g a in s t h u m a n k a l a - a z a r . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d ic in a T r o p ic a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
d a c a r g a p a r a s i t á r i a n o f í g a d o ( p < 0 . 0 0 1 ) f o i d e te c ta d o n o s a n i m a i s v a c i n a d o s c o m F M L e s a p o n i n a , in d e p e n d e n te m e n te d a v ia d e a d m in is tr a ç ã o .
P a la v r a s - c h a v e s : G lic o c o n j u g a d o . L eishm ania d o n o v an i. C a la z a r . T r a n s f u s ã o d e s a n g u e . D ia g n ó s tic o .
A C K N O W L E D G M E N T S
T his research has been supported by FN S, F IN E P , C N P Q , R H A E -C N P Q and C E PG -U F R J. D r. T elm a C. B arros F re ire from H E M O N O R T E , is acknow ledgeded for sam ple collection facilities.
R E FE R E N C E S
1. A sh fo rd D A , B a d aró R , E u la lio C , F re ire M , M iran d a C , Z a lis M , D a v id , JR . S tu d ies o n th e c o n tro l o f visceral leish m a n ia sisrv a lid a tio n o f th e F a lc o n assay screen in g test-en zy m e lin k ed im m u n o so rb en t assay (Fast-E lisa^ ) fo r field d ia g n o sis o f can in e v isc eral leish m an iasis. T h e A m e ric a n Jo u rn a l o f T ro p ic a l M e d icin e an d H y g ien e 4 8 :1 -8 , 1993.
2 . B ad aró R , R e ed S G , B arrai A , O rg e G , Jo n e s T C . E v a lu a tio n o f th e m icro en zy m e lin k ed im m u n o so rb en t a s s a y f o r a n t i b o d i e s in a m e r i c a n v i s c e r a l Ieish m an iasis:an tig en se lectio n fo r d etectio n o f infection- sp e cific re sp o n se s. T h e A m erican Jo u rn a l o f T ro p ical M e d icin e a n d H y g ie n e 3 5 :7 2 -7 8 , 1986.
3 . B o m fo rd R . S a p o n in an d o th e r h aem o ly sin s (V itam in A , alip h atic am in e s, p o ly e n e an tib io tics) a s ad ju v an ts for SR B C in th e m o u se . In tern atio n al A rch iv es o f A llerg y and A p p lied Im m u n o lo g y 6 3 :1 7 0 -1 7 7 , 1980.
4 . B o m fo rd R . T h e c o m p a ra tiv e selectiv ity o f ad ju v an ts for h u m o ra l an d cell m ed iated im m unity II. E ffect o n delay ed ty p e h y p e rse n sitiv ity in th e m o u se , g u in e a p ig , and cell m ed iated im m u n ity to tu m o r an tig en s in th e m ouse o f F re u n d ’s in co m p lete and co m p lete ad ju v an t, alh y d ro g el,
C orynebacterium p a r v u n i, Bordetella p ertu ssis, m u ram y l d ip e p tid e a n d s a p o n in . C lin ic a l a n d E x p e rim e n ta l Im m u n o lo g y 3 9 :4 3 5 -4 4 1 , 1984.
5 . B o ro w y N K , S ch ell D , S c h aeffer C , O v erath P . D iagnostic o f h u m a n a f r ic a n tr y p a n o s o m ia s is a n d v is c e ra l leish m an iasis b a se d o n d etectio n o f an tip arasite-en zy m e an tib o d ie s. J o u rn a l o f In fec tio u s D ise ases 1 6 4 :4 2 2 -4 2 5 , 1991.
6 . B radley D J, K irkley J . R egulation o f L eish m an iap o p u latio n w ith in th e h o st. I. V a ria b le c o u rse o f Leishm ania donovani
in fectio n in m ice. C lin ical and E x p erim en tal Im m u n o lo g y 3 0 , 1 19-1 2 9 , 1977.
7 . B ru m p t E . L e K a la-azar. In: M a sso n e C ie (ed) P re cis de Parasitologie, 6 em e edition, C o llectio n d e Precis M édicaux, P aris p . 2 5 6 -2 7 7 , 1949.
8 . B urns J r JM , S h re ffle r W G , B en so n D R , G halib H W , B ad aró R , R eed S G . M o le c u la r ch ara c te riz a tio n o f a k in e sin -re la te d an tig e n o f L eish m an ia ch ag asi th at detects sp e cific an tib o d y in A frica n and A m e ric a n V isc era l L e ish m a n ia sis. P ro c eed in g s o f th e N atio n al A cad em y o f
S cien ce U S A 9 0 :7 7 5 -7 7 9 , 1993.
9 . C a stellan i A C h a lm e rs A J. V isc era l L eish m an iasis In:
B aillière, T in d a ll, C o x (eds) M a n u al o fT ro p ic a lM e d ic in e , 3 rt* ed itio n , U n iv ersity se ries, L o n d o n p . 1 2 8 9 -1 3 0 7 , 1919.
10. C o h en C , C o razza F , D e M ol P, B rasseu r D . L eish m an iasis acq u ired in B elg iu m . L a n c e t 3 3 8 :1 2 8 , 1991.
11. C o n v it J , C a stellan o P L , R o n d o n A , P en ard i M E , U lrich M , C astes M , B loom B, G arcia L . Im m u n o th e ra p y v e rsu s c h e m o terap y in lo calized C u ta n e o u s L e ish m a n ia sis. T h e L an cet 1:40 1 -4 0 4 , 1987.
12. G ro g l M , D au g ird a JL , H o o v e r D L , M ag ill A J, B erm an J . S u rvaivability and infectivity o fv isc e ro tro p ic Leishm ania tropica fro m O p eratio n D esert S to rm p artic ip a te s in h u m a n b lo o d p r o d u c ts m a in ta in a n c e u n d e r b lo o d b a n k in g co n d itio n s. T h e A m erican Jo u rn a l o f T ro p ic a l M e d icin e a n d H y g ien e 4 9 :3 0 8 -3 1 5 , 1993.
13. H an d m an E , M itch ell G F . T h e g ly c o c o n ju g a te d eriv ed fro m a Leishm ania m a jo r re c e p to r fo r m a c ro p h a g e s is a su p p re s so g e n ic d ise ase p ro m o tin g a n tig e n in m u rin e C u tan e o u s L eish m an iasis. P arasite Im m u n o lo g y 8 :2 5 5 -2 6 3 , 1986.
14. H o lb ro o k T W , C o o k JA , P a rk e r B W . Im m u n iza tio n ag ain st Leishm ania d onovani: g lu c a n a s a n a d ju v a n t w ith killed p ro m astig o tes. T h e A m erican Jo u rn a l o f T ro p ic a l M e d icin e an d H y g ien e 3 0 :7 6 2 -7 6 8 , 1981.
15. Jaffe C L Z alis M . U se o f p u rifie d p a ra site p ro te in s fro m
Leishm ania donovani for the rapid se ro d iag n o sis o f v isceral leish m an iasis. Jo u rn a l o f In fectio u s D ise ases 157, 1212-
1220, 1988.
16. Ja ffe C L , R a ch am im N , S a rfste in R . C h a ra c te riz a tio n o f tw o p ro tein s fro m Leishm ania d o n o v a n i an d th e ir u se fo r v acc in a tio n a g ain st v isc e ra l le ish m a n ia sis. J o u rn a l o f Im m u n o lo g y 1 4 4 :6 9 9 -7 0 6 , 1990.
17. Ja rd im A , T o lso n D L , T u rc o S J, P e a rso n T W , O lafso n R W.L e is h m a n ia d o n o v a n i l i p o p h o s p h o g l y c a n T ly m p h o cy te reactiv e co m p o n e n t is a tig h tly asso ciated p ro te in co m p lex . Jo u rn a l o flm m u n o lo g y 1 4 7 :3 5 3 8 -3 5 4 4 , 1991.
18. Ja rec k i-B lac k JC , Ja m es E R , K irsh tein JW , K irsh tein JD , G lassm an A B. Leishm aniadonovani: im m u n izatio n ag ain st in fectio n a s a fu n ctio n o f p a ra site g ro w th p h ase. T h e A m erican Jo u rn a l o f T ro p ic a l M e d icin e an d H y g ien e 3 5 :1 1 1 7 -1 1 2 1 , 1986.
19. K udo R R . F am ily 5 T ry p an o so m atid ae D oflein. In: T h o m a s C C (ed) P ro to z o o lo g y , 4 th ed itio n , T h o m a s B o o k s, U SA p . 3 5 5 -3 5 6 , 1960.
2 0 . M a rz o c h i, M C A , M a rzo c h i K B F , C a rv a lh o R W . V isc era l L eish m an iasis in R io d e Ja n e iro . P arasito lo g y T o d a y 1 0 :3 7 -4 0 , 1994.
2 1 . M a y rin k W , W illiam P , C o sta C A , M a g a lh ã e s P A , M e lo H N , D ia s M , L im a A O , M ic h alic k M S M , C a rv a lh o E F , B arros G C , S essa P A , A le n c a r JE . A n e x p erim en tal vaccin e ag ain st A m erican derm al L eish m an iasis ex p erien ce in th e S tate o f E sp irito S an to B razil. A n n als o f T ro p ic a l M e d icin e an d P arasito lo g y 7 9 :2 5 9 -2 6 9 , 1985.
2 2 . M e b rah tu Y B , H en d rick s L D , O ste r C N , L a w y e r PG , P erk in s P V , P am b a H , K o ech D , R o b e rts C R . L eishm ania d o n o v a n i p a r a s i t e s in t h e n a s a l s e c r e t i o n s , to n sillo p h ary n g eal m u co sa, and u rin e c e n trifu g a te s o f v isc eral leish m an iasis p atien ts in K e n y a . A m erican Jo u rn al
S o u s a C B P , G o m e s E M , S o u z a E P , S a n to s W R , M a c e d o S R , M e d e i r o s L V , L u z K . T h e F M L ( F u c o s e M a n n o s e L ig a n d ) o /L e is h m a n ia d o n o v a n i. A n e w t o o l in d i a g n o s i s , p r o g n o s i s , tr a n s f u s io n a l c o n t r o l a n d v a c c i n a t i o n a g a i n s t
h u m a n k a l a - a z a r . R e v i s t a d a S o c i e d a d e B r a s i le i r a d e M e d i c i n a T r o p ic a l 2 9 : 1 5 3 - 1 6 3 , 1 9 9 6 .
o f T ro p ic a l M e d icin e and H y g ien e 4 8 :5 3 0 -5 3 5 , 1993. 2 3 . M e n d o n ç a S C F , R u ssell D G , C o u tin h o S G . A n aly sis o f
th e h u m a n T cell re sp o n s iv e n e ss to p u rified an tig en s o f
L eishm ania: L y p o p h o sp h o g ly c a n (L P G ) and g ly co p ro tein 63 (G P 6 3 ). C lin ical an d E x p erim en tal Im m unology 83:4 7 2 - 4 7 8 , 1991.
2 4 . M o d a b b e rF . E x p e rie n c e s w ith v a c c in e s a g ain st cu tan eo u s leish m an iasis o f m e n an d m ice. P arasito lo g y 98 (S ):4 9 -6 0 , 1989.
2 5 . M o d a b b e r , F . L e i s h m a n i a s is . I n : W o r ld H e a lth O rg a n iz a tio n (ed) T ro p ic a l D ise ase R e se a rc h . P ro g ress. 1 9 9 1 -1 9 9 2 , G en ev a p .7 7 -8 7 , 1993.
2 6 . O ’D aly C JA , C a b re ra Z . Im m u n iza tio n o f h a m ste rs w ith T L C K - k ille d p a r a s ite s in d u c e d p r o te c tio n a g a in s t
L eishm ania in fectio n . A cta T ro p ic a 4 3 :2 2 5 -2 3 2 , 1986. 2 7 . P a la tn ik C B ,B o r o je v ic R ,P re v ia to JO ,M e n d o n ç a -P re v ia to
L . I n h ib itio n o f L e ish m a n ia d o n o v a n i p ro m a stig o te in te rn a liz a tio n in to m u rin e m a c ro p h a g e s b y chem ically d efin ed p a ra site g ly c o c o n ju g a te lig an d s. In fec tio n and Im m u n ity 5 7 :7 5 4 -7 6 3 , 1989.
2 8 . P alatn ik C B , P re v iato JO , M e n d o n ça-P rev iato L , Borojevic R . A n ew a p p ro a c h to p h y lo g e n y o f Leishm ania: species- sp e cificity o f g ly c o c o n ju g a te lig an d s fo r p ro m astig o te in te rn a liz a tio n in to m u rin e m acro p h ag es. P arasito lo g y R e se a rc h 7 6 :2 8 9 -2 9 3 , 1990.
2 9 . P alatn ik -d e -S o u sa C B , D u tra H S , Borojevic R . Lrishftum id do n o va n i su rfa c e g ly c o c o n ju g a te G P 3 6 is th e m ajo r im m u n o g e n co m p o n e n t o f th e F u co se -M an n o se L igand (F M L ). A cta T ro p ic a 5 J .5 9 - 7 2 , 1993.
3 0 . P alatn ik -d e -S o u sa C B , P arag u ai de S ou za E , G o m es E M , B o ro jev ic R . E x p erim en tal m u rin e Leishm ania donovani
in fectio n : im m u n o p ro te c tio n b y th e F u co se M a n n o se L ig a n d (F M L ). B razilian Jo u rn a l o fM e d ic a l a n B iologycal R e se a rc h 2 7 :5 4 7 -5 5 1 , 1994.
3 1 . P alatn ik -d e -S o u sa C B , B u n n M o re n o M M , P arag u ai de S o u za E , B orojevic R . T h e F M L v accin e (F u co se M annose L ig a n d ) p ro te c ts h a m ste rs fro m ex p erim en tal k ala-azar. Jo u n a l o f th e B razilian S o ciety fo r th e A d v an cem en t o f S c ie n c e . C iên c ia e C u ltu ra . 4 6 :2 9 0 -2 9 6 , 1994.
3 2 . P alatn ik d e S o u sa C B , G o m es E M , P arag u ai d e S ou za E, P alatn ik M , L u z K G , B orojevic R . T h e F u co se M an n o se L ig a n d o i Leishm ania d onovani in d ia g n o sisa n d pro g n o sis o f v isc e ra l leish m an iasis (k ala-a zar). T ra n s a c tio n s o f the R o y a l S o ciety o f T ro p ic a l M e d icin e an d H y g ien e 89:(In p re s s ), 1995.
3 3 . R a ch am im N , Ja ffe C L . P u re p ro te in fro m L eishm ania d onovani p ro tects m ice ag ain st b o th C u tan e o u s an d V iceral L eish m an iasis. J o u rn a l o f Im m u n o lo g y 1 5 0 :2 3 2 2 -2 3 3 1 , 1993.
3 4 . R eed S G , B a d aro R , C h e rry L lo y d R M Id en tificatio n o f sp ecific c ro ss re a c tiv e a n tig e n o f L e ish m a n ia d o n o v an i ch ag asi b y h u m a n in fectio n s e ra . J o u rn a l o f Im m u n o lo g y 1 3 8 :1 5 9 6 -1 6 0 1 , 1987.
3 5 . R eed S G , S c h re ffle r W G , B u rn s JM J , S co tt JM , O rg e M G , G h alib M S , B a d aro R . A n im p ro v e d sé ro d ia g n o stic p ro ced u re fo r visceral leish m an iasis. T h e A m erican Jo u rn al o f T ro p ic a l M e d icin e an d H y g ie n e 4 3 :6 3 2 -6 3 9 , 1990. 3 6 . S arto ri A , V iana d e O liv eira A , R o q u e B a rre ira M C , R o ssi
M A , C am pos-N eto A . Im m une co m p lex g lo m eru lo n ep h ritis in ex p rim en tal k a la -a z a r. P arasite Im m u n o lo g y 9 :9 3 -1 0 3 ,
1987.
3 7 . S co tt M T , B a h r G , M o d a b b e r F , A fch ain D , C h e d id L . A d ju v an t req u irem en t fo r p ro te c tiv e im m u n iz a tio n o f m ice u sin g Trypanosom a cruzi 9 0 K cell su rface g ly c o p ro te in . In ternational A rch iv es o f A llerg y and A p p lied Im m u n o lo g y 7 4 :3 7 3 -3 7 7 , 1984.
3 8 . Scott J E , S c h re ffle r W G , G h alib H W , El A sad A , S id d ig M , B ad aro R , R e ed S G . A rap id a n d sim p le d iag n o stic test f o r activ e v isc eral le ish m an iasis. T h e A m e ric a n J o u rn a l o f T ro p ic a l M e d icin e and H y g ie n e 4 4 :2 7 2 -2 7 7 , 1991. 3 9 . S c h re ffle r W G , B u rn s JM J, B a d aro R , G h alib H W , B utton
L L , M c M a ste r R , R e ed S G . A n tib o d y re sp o n s e s o f v isc eral leish m n iasis p atien ts to G P 6 3 , a m a jo r su rface g ly c o p ro te in o f Leishm ania sp e c ie s. J o u rn a l o f In fectio u s D ise ases 1 6 7 :4 2 6 -4 3 0 , 1993.
4 0 . T o iso n D L , Ja rd im A J, S c h n u r L F , S te b e c k C , T u c k e y C , B eecro ft R P , T e h H S , O la fso n R W , P e a rso n T W . T h e k in eto p lastid m em b ran e p ro te in 11 o f L eish m an i d o n o v an i a n d A frican try p a n o so m e s is a p o ten t stim u la to r o f T - ly m p h o cy tep ro liferatio n . Infection and Im m unity 6 2 :4 8 9 3 - 4 8 9 9 , 1994.
4 1 . W o rld H ealth O rg a n iz a tio n . K a la -a z a r su rg e s o n tw o fro n t. T D R N ew s 3 7 :1 -2 , 1991.
4 2 . X iao-S u H , Q in g L , F an g -K in g L , T a o -L in Y , Y a-Jin W , Z h iQ , P in g Q , L ing L .K a la -a z a r in fected se ru m circu latin g an tig en s and th e ir ch a ra c te ristic s d etected b y m o n o clo n al a n tib o d ies. C h in ese M e d ical Jo u rn a l 1 0 1 :1 -6 , 1988. 4 3 . Y o u d en J. In d ex fo r ratin g d iag n o stic te sts. C a n c e r, 3 :3 2 -
