THE FML (FUCOSE MANNOSE LIGAND) OF LE1SHMANIA DONOVANI. A NEW TOOL IN DIAGNOSIS, PROGNOSIS, TRANSFUSIONAL CONTROL AND VACCINATION AGAINST HUMAN ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Revista da Sociedade B rasileira de M edicina Tropical
29(2):153-163, m ar-abr, 1996.
THE FML (FUCOSE MANNOSE LIGAND) OF LE1SHMANIA
DONOVANI. A NEW TOOL IN DIAGNOSIS, PROGNOSIS,
TRANSFUSIONAL CONTROL AND VACCINATION AGAINST
HUMAN KALA-AZAR
Claris» B. Palatnik de Sousa, Elza M. Gomes, Edilma Paraguai de Souza,
Wania R. dos Santos, Sirley R. de Macedo, Linnete V. de Medeiros and
Kleber Luz
The Fucose-M annose Ligand (FML) o f Lcishmania donovani is a complex glycoproteic
fraction. Its potential use as a tool fo r diagnosis o f human visceral leishmaniasis was tested with
human sera fro m Natal, Rio Grande do Norte, Brazil. The FML-EL1SA test, show ed 100%
sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when
com pared to norm al sera), and subjects with subclinical infection. M ore than 20% apparently
healthy subjects with positive reaction to FML developed overt kala-azar during the follow ing
10 months. In the screening o f human blood donnors, a prevalence o f 5% o f sororeactive
subjects was detected, attaining 7 7 % in a single day. The GP36 glycoprotein o f FHL is
specifically reconized by human kala-azar sera. The immunoprotective effect o f FML on
experimental L. donovani infection was tested in swiss albino mice. The protection scheem es
included three weekly doses o f FML, supplemented or not with saponin by the subcutaneous or
intraperitoneal routes and challenge with 2 x 107 amastigotes o f Leishm ania donovani. An
enhancem ent o f 8 0 .0 % in antibody response (p < 0 .001) and reduction o f 85.5 % parasite liver
burden (p < 0 .001) was detected in animals immunized with FML saponin, unrespectivety o f the
immunization route.
Key-words: Glycoconjugate. Leishmania donovani. Diagnosis. Prognosis. Kala-azar.
Visceral leishmaniasis. Blood transfusion. Leishmanial antigens.
Visceral leishmaniasis or kala-azar is a chronic is probably underestimate since an accurate and
and frequently lethal disease, caused by parasites of uniform early diagnose o f disease has not yet been
the Leishmania donovani complex. The disease is established. Visceral leishmaniasis is spreading out
characterized by fever, malaise, loss of weight, in the W orld due to the increased resistance of
hepatomegaly, splenomegaly, anemia, leukopenia, parasites to chemoterapy and o f insect vectors to
h y p e rg a m m a g lu b u lin e m ia and p ro g re s s iv e insecticides41. InBrazil, the disease is disseminating
suppression o f the cellular immune response. Kala- from the North and North-East regions o f the
azar is often fatal if untreated after the onset of country and has recently been described in the State
sym ptom s. F urtherm ore, chem oterapy shows o f Rio de Janeiro, previously considered as not
several undesirable colateral effects. The total endemic20.
number o f Leishmania infected people all over the The current diagnostic in endemic and epidemic
world is being estimated in twelve millions. Five areas is still based on clinical analysis and
hundred thousand o f these cases, each year, identification o f parasites in bone-marrow aspirates.
correspond to visceral leishmaniasis25. This number This very invasive method can give positive results
in cases of advanced kala-azar. The cure o f the
disease cannot be monitored, and preclinical patients
Instituto de M icro b io lo g ia . U niversidade Federal do R io de
are not detected, their clinical evolution is not
Janeiro. Centro de H em atologia e Hem oterapia-H EM O N O R TE.
Hospital de D oenças InfecciosasG iseldaTrigueiro. Departamento
followed-up, and they are a potential reservoir of
de In fectologia. U niversidade Federal do R io Grande do Norte. the disease in endemic areas. Anti -Leishmania
A d d re ss lo: D ra. C larisa B . Palatnik de Sou sa. Instituto de circulating antibodies can be detected in human
M icrob iolog ia /C C S /U F R J . C . U niversitária. I. do Fundão.
serum soon after infection: they attain high titers
CP: 6 8 0 4 0 , 2 1 9 4 1 -5 9 0 R io de Janeiro, RJ.
R eceb id o para p u blicação em 0 8 /0 1 /9 6 . with the evolution o f the disease and progressively
153Sousa CBP, Gomes EM , Souza EP, Santos WR, M acedo SR, M edeiros LV , L u z K. The FML (Fucose M annose
L igand) o/L eish m an ia donovani. A new tool in diagnosis, pro g n o sis, transfusional control and vaccination against
hum an kala-azar. R evista da Sociedade Brasileira de M edicina Tropical 29:153-163, 1996.
disappear after its treatm ent. The sorologycal human visceral leishmaniasis, no vaccine is up to
diagnosis o f kala-azar has been proposed as an now available. Only few studies on immunization
alternative by several works, based on antigens that against Leishmania donovani have been undertaken
show d ifferen t degrees o f p u rificatio n , and in the murine model, using total parasites26 18 14 or
developping assays with diverse sensitivity and purified antigens17.
specifficity ranges5 32 35 38. The stability o f these In recent studies, we described the isolation of
antigens has also been discussed32. Nevertheless, a the FM L (Fucose M annose Ligand) of L. donovani
non invassive method o f monitorization o f the promastigotes, a complex glycoproteic fraction that
disease was not yet not available in Brazilian Public strongly inhibits promastigote27 and amastigote
Health Services. infection o f murine macrophages29. Its proteic
In fe c te d m o n o c y te s , as w e ll as, moiety is mainly composed o f acidic and apolar
p o ly m o rp h o n u c le a r n e u tr o p h ile s 7 19 and aminoacid residues. Among its neutral sugar
eosinophiles9 w ere described in the blood, as com ponents, fucose (10% ), m annose (47% ),
potential carriers o f L. donovani parasites from the g a la c to se (1 2 % ) and g lu c o se (3 0 % ) w ere
skin to viscera and bone marrow. In older described27. FM L developped a species-speciffic
studies19 1, and in some recent reports on human effect on the inhibition o f promastigote uptake by
kala-azar22, the presence o f infective parasites in macrophages in vitro21. This is a potent antigen for
the secreta o f the upper respiratory and digestive rabbits, hamsters and mice283130. Its main antigenic
tracts, as well as in urine and faeces were cited. fraction is a 36kD glycoprotein, recognized by
Only occasionally, inoculation o f leishmaniasis by most mouse IgG anti-FM L monoclonal antibodies,
the transfusional via were cited10. Recent indirect and present at the surface o f both the promastigote
evidences support the need o f the study o f the blood and amastigote forms o f L. donovani parasites29.
mediated contamination with kala-azar in humans. The protective potential o f FM L on visceral
Leishmania tropica was identified in bone marrow leishmaniasis was analyzed in the isogenic CB-
of american soldiers returning from Operation Desert hamster model31. W e studied the effect o f three
Storm with viscerotropic leishmaniasis, and for this intraperitoneal weekly doses o f FM L ( 100/xg) in
reason all individuals that traveled to that region saponin (100/xg), followed by an intracardiac
were recommended to be deferred as blood donors12. injection of 107amastigotes. Protection was speciffic
Notwithstanding, the existence o f a blood-mediated and highly significant (87.7% , p < 0 .0 1 ) in the
contamination is up to now not admitted as a enhancement of anti-FM L antibodies titers, o f the
potential danger for leishmanial infections, and no sp len o c y te p ro life ra tiv e re sp o n se , and the
control o f blood is performed for the detection o f intradermal delayed hypersensitivity reaction to
leishmaniasis in blood banks, even in regions antigen, as well as in the decrease o f the parasite
reported to be endemic for kala-azar, and expected burden in spleen and o f splenom egaly31. An
to harbor a large num ber of subjects with subclinical analogous study in the inbred balb/c mice model
infection. showed an average o f 84.2% (pCO .O O l) o f
The analysis o f the protective potential of protection in antibody titers, splenocyte in vitro
Leishmanial antigens to cutaneous leishmaniasis in proliferation and liver parasitic load30.
murine models has been the focus o f detailed In the present study we report the use o f the
studies. The W orld Health Organization encouraged FM L antigen in diagnosis, p ro g n o sis32 and
the development o f vaccines for leishmaniasis, tra n s fu s io n a l c o n tro l o f h u m a n v is c e ra l
using total or partially identified parasite lysates leishmaniasis, monitoring the epidemic and endemic
and crude antigens, that could induce a clear disease in Public Health Services o f Natal, Rio
prophylactic immune response24. This kind of Grande do Norte, Brazil. This work shows the
vaccine has shown to be effective against cutaneous results obtained with the implementation o f the
leishmaniasis1121. The vaccines derived from whole FMLELISA assay in the routine o f the laboratory
killed Leishm ania are in study in Brazil (L . sorologycal screening o f the Hospital de Doenças
amazonensis), Iran (L. major) and Venezuela (L. Infecciosas Giselda Trigueiro and the Centro de
mexicana and L. braziliensis)25. However, for Hematologia e Hemoterapia-HEM ONORTE, with
154Sousa CBP, Gomes EM, Souza E P, Santos WR, M acedo SR, M edeiros LV , L uz K. The FML (Fucose M annose
L igand) o /L eish m an ia donovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
hum an kala-azar. R evista da Sociedade B rasileira de M edicina Tropical 29:153-163, 1996.
the support o f the Fundação Nacional de Saúde, lymphoma (2), typhus (1) and hemophagocytic
FN S. Furtherm ore, prelim inary results o f the disease (1) were obtained from the Departamento de
analysis o f the FM Lvaccine in the outbred swiss Doenças Infecciosas, U FRJ, and Departamento de
albino mouse model, are described. Pediatria UERJ, Rio de Janeiro, RJ.
A series o f 171 sera o f volunteer blood donors
M ATERIAL AND METHODS was obtained from the blood bank Centro de
Hematologia e Hemoterapia-HEM ONORTE, and
H u m a n sera tested for their reactivity w ith FM L. Informed
consent for this study was obtained from all the
A total o f 462 sera was analyzed in this study. subjects included in this study.
A series o f 149 sera was collected by the Fundação
Nacional de Saúde do Rio Grande do Norte, in the F M L-ELISA qu alitativ e assay
peri-urban area o f Natal, RN (North East Brazil), a
focus o f a recent outbreak o f kala-azar. Ten o f the T he F M L com plex w as o b ta in e d from
sera in this series came from subjects that had a Leishm ania donovani (LD -IS/M H O M /SD /O O -
patent kala-azar at the time o f serum collection. A strain IS) promastigotes as previously described29.
series o f 26 sera o f kala-azar patients, before and Titration o f normal human, kala-azar, Chagas’
during their treatment, was obtained at the Hospital disease and cutaneous leishmaniasis patient’s sera
de Doenças Infecciosas Giselda Trigueiro, Natal. pools against FM L antigen was performed in order
All the 36 patients with clinical symptoms o f acute to establish the optimal antigen concentration. The
kala-azar (loss o f w eight, lym phadenopathy, micro-ELISA method was done as described32,
hepatomegaly and/or splenomegaly, and fever) were using FM L diluted in carbonate buffer (pH 9.6), to
submitted to bone m arrow aspiration. Parasitologic sensitize flat-bottom 96-well plates (Haemobag,
analysis confirmed the presence o f Leishmania Ribeirão Preto, SP, Brazil). A ntibodies w ere
amastigotes in the Giemsa-stained smears. After the detected by peroxidase-labeled protein-A (Sigma,
serum collection, patients were submitted to anti- St Louis, MO). Reaction was developed with O-
Leishmania treatment. Thirty five sera o f patients phenyldiamine (Sigma), interrupted with IN sulfuric
that have had kala-azar w ere collected at least three acid, and monitored at 492nm. Sera were analyzed
months after the end o f the treatment with antimonials by double blind tests, in triplicates, and results
o r am photericin-B , during their clinical and expressed as mean values. Positive and negative
serological follow-up32. control sera were included in each test. The
Twenty one persons with no patent signs of absorbance values o f sera were compared at 1:100.
disease, neighbors and relatives of kala-azar patients, The cut o ff limit between the normal sera from a
or owners of dogs with L. donovani infection, were non-endem ic area and sera o f patients w ith
also included in the study. We also analyzed the parasitologically confirmed kala-azar, was identified
reactivity with FM L o f 21 sera of patients with using the Youden’s J index43. S ignificance o f the
cutaneous or mucocutaneous leishmaniasis from differences between groups o f sera was established
Rio de Janeiro and Minas Gerais states, obtained by a standard t test.
from Dr. S.M . Coutinho, Fundação Oswaldo Cruz,
(Rio de Janeiro), 22 sera o f fully characterized W estern Blot analysis
patients with chronic Chagas disease from Bambui,
M inas Gerais, and 18 sera from healthy adult blood FM L was submitted to SDS-PAGE under
donors from the Hospital Universitário Clementino reducing conditions, in 10% slab “baby” gels and
FragaFilho, Universidade Federal do Riode Janeiro, transferred to nitrocellulose. Strips were incubated
that were screened and considered negative for with human sera diluted in blocking buffer, overnight
Chagas disease, HIV I and II, hepatitis, and at 4°C , washed and developed with peroxidase-
syphilis32. For differential control diagnosis, sera labeled protein-A followed by diaminobenzidine
o f patients with fever and/or hepatosplenomegaly (Sigma).
due to Paracoccidioides brasiliensis infections (1),
155Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) o /L eish m a n ia donovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista da Sociedade Brasileira de M edicina Tropical 29:153-163, 1996.
Vaccination assays absorbances at 492nm, up to the end-point titer)32.
Statistical comparison o f the groups was done by
2,5 month old swiss albino female mice were the Student t test.
obtained from the Bioterio Central, Instituto de
M icrobiologia da U FR J. Groups of 6-8 females RESULTS
were immunized w ith three either subcutaneous or
intraperitoneal doses o f 150/xg FM L and 100 /xg Standardization o f ELISA experiments was
saponin (Riedel-de Haen, Seelze, Germany) in done using FM L in dilutions from 0.03 to 2p.g per
0.2m l sterile 0.95% saline, with weekly interval. well, reacted with pools o f normal human sera from
Saline and saponin treated controls were included. a non-endemic area, sera o f patients w ith kala-azar,
Seven days after immunization, sera were collected tegumentar leishmaniasis and Chagas disease, in
and animals were challenged by intravenous inj ection dilution 1:2000. The results presented in the Figure
o f 2 x 107 L. donovani amastigotes, obtained from 1, indicated the antigen concentration o f 0 . 125/xg /
infected ham ster’s spleens6. Animals were sacrificed well as the highest that gave no unspecific reaction.
by etherization, 15 days after infection, and their All the studied samples were titered and scored in
liver and spleen parasite-load was monitored in this condition.
Leishman-Donovan Units o f Stauber of Giemsa The individual absorbance values in FM L-
stained imprintings (L D U =num ber of amastigotes/ ELISA assay for 1:100 diluted sera are represented
1000 cell nuclei x mg organ weight). Sera o f in Figure 2A. The mean value o f Abs 1:100 (X +
animals before and after infection were analyzed by SE = 0.452 ± 0.038) o f patients w ith acute kala-
ELISA using 2/ig FM L/w ell and reacted with a azar was significantly different from the one of
Peroxidase labeled anti-mouse IgG (Sigma, Co.). the normal subjects o f a nonendemic area
ELISA results were expressed as scores (sum o f all (p < < 0.001). Eight standard error values separated
Figure 1 - Determination o f FML optimal concentration fo r ELISA lest.
ELISA test o f FML reactivity with a normal serum, and sera from patients with tegumenttir leishmaniasis, kala-azar
or Chagas disease. The arrow indicates the concentration used in ELISA assay standardization.
156Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) fj/ L eishm ania donovani. A new tool in diagnosis, prognosis, transfusionul control and vaccination against
human kala-azar. Revista da Sociedade Brasileira de Medicina Tropical 29:153-163, 1996.
the two means. The use o f mean value + 2SD in the (RE) (n = 59). We interpret these subjects as a
FML-ELISA assay attain the maximal Youden subclinical but active Leishmania infection, in view
v a lu e = l, corresponding to the absence o f fa lse o f their clinical evolution. During the ten months
p o sitive or fa ls e negative results (cut off value = following the first study of 41 sera from the kala-
0.204). Sera o f patients with chronic Chagas disease azar outbreak region, nine o f the inhabitants o f the
sh o w ed , as ex p ected from sta n d a rd iz a tio n studied endemic area spontaneously searched
experiments, a very reduced reactivity (Abs 1:100 medical treatment for the severe disease (NKA).
mean value 0.149). In the endemic area, (Figure Kala-azar was diagnosed by identification o f
2B). the FM L-ELISA assay functioned as a highly amastigotes in the bone-marrow aspirate. Among
discriminating tool. It allowed to distinguish the these, six had had high titers of anti-FM L antibodies
non-reactive sera from the endemic area (NE) (n and the highest titers had been observed in two
= 86), from the group of subjects without any sign patients that eventually died of severe kala-azar.
of leishmanial infection but reactive with FML During the same period, at our knowledge, no
Figure 2 - The FML-ELISA assay in an endemic and non-endemic region fo r human visceral leishmaniasis.
In A: FML-ELISA reactivity with sera o f normal subjects from a non-endemic area (NN), patients with kala-azar
diagnosed by the parasitological analysis o f bone marrow aspirates (KA), patients with cutaneous or muco-cutaneous
leishmaniasis (CML), patients fo r differential diagnosis (DD) (Paracoccidioides brasiliensis, lymphoma, typhous and
hemophagocytic disease), patients with chronic Chagas ’ disease (CH). In B: normal subjects from an area endemic
fo r kala-azar (NE), subject from the endemic area without overt disease, but reactive with FML (RE), subjects from
the endemic area, that had been reactive fo r FML in the first screening o f sera, but did not presen t at the time overt
disease, which developed severe kala-azar in the subsequent ten months period (NKA), and kala-azar patients that have
been submitted to anti-parasite treatment and have been cured from the leishmanial infection (CKA). Dots represent
absorbance values at sera dilution o f 1:100; the horizontal line represents the cut off value (0.204).
157Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
L igand) o /L eish m a n ia donovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista da Sociedade Brasileira de M edicina Tropical 29:153-163, 1996.
subjects with negative reaction to FM L developed The 55kDa component o f FM L was recognized by
kala-azar. Consequently, among 41 sera that had normal sera from kala-azar endemic and non
shown high or low reactivity with FM L but had not endemic area, and sera from patients with
clinical signs o f kala-azar, more than 20% developed tegumentar leishmaniasis, kala-azar or Chagas
the overt disease in the ten month period. This disease. This antigen is thus non-specific. Conversely
num ber is probably underestimated, since a full the GP36 glycoprotein band was only detected in
clinical and serological survey of the same area has W estemBIotsbykala-azarpatients’ sera. No labeling
not been completed32. Sera o f patients with fever was detected with sera o f patients w ith tegumentar
and hepatosplenomegaly due to P aracoccidioides leishmaniasis, Chagas disease, or normal sera. The
b ra silien sis infection (1), lym phom a (2) and GP36 antigen o f FM L shows to be a marker of
hemophagocytic disease (1) gave also negative human kala-azar.
results in the FM L-ELISA assay. These results Finally the analysis o f the protective potential
determined 100% sensitivty and 96 % specificity. of FM L in experimental vaccination against visceral
A random monitoring o f anti-FM L reactivity leishmaniasis was performed in the outbred swiss
among 171 volunteer blood donors at the blood albino model. We have tested the potential activity
bank Centro de H em atologia e Hem oterapia-
HEM O N O RTE, Natal, was performed, testing an
average o f 24.4 diary donor samples, for a period
o f seven consecutive days. The results obtained are
summarized in Table 1. The FM L-ELISA test
showed the clearly positive reaction for 8 donors.
The other 163 samples showed no reactivity
(X + S E = 0 .1 3 8 + 0.002). This values represent a
total o f 5% o f samples and were distributed attaining
even 17% in a single day, no one o f which had a
positive reaction for Chagas’s disease, hepatites,
HIV nor syphilis or a decreased hematocrite value.
Conversely, among the nonreactive donors, two
cases o f hepatites B were detected indicating that
also in this assay the FM L-ELISA showed to be
highly specific.
Since the FM L components can be separated by
electrophoresis, we have analyzed by Western Blot
the reactivity o f human sera with FM L (Figure 3).
Table 1 - Blood donor sam ples reactive in the FML-
ELISA assay.
Donor number FML-ELISA Date
reactivy at 492nm Figure3 - SDS-PAGE and Western Blot analysis o f
FMLantigen ofL. donovani using humansera. Molecular
462. .506 30.8.94 weight standards and FML (150^g) analyzed by SDS-
463. .291 31.8.94 PAGE and stained with Coomassie Brilliant Blue R250,
464. .231 1.9.94 are indicated on the left side. Blotts containig 1-3ug FML
465. .271 1.9.94 were treated with: a: serum o f a patient with confirmed
466. .250 1.9.94 kala-azar (11/11), b: serum o f a normal donor from
467. .232 1.1.94 endemic area, c: serum o f a patient with tegumentar
468. .220 1.9.94 leishmaniasis (5/5), d: serum o f a patient with Chagas ’
469. .232 5.9.94 disease (5/5). e: serum o f normal heathy donor from
nonendemic area (2/8). The arrow indicates the GP36
All the samples were tested at 1/100 sera dilution. Results
represent the absorbency values at 490nm. Normal sera band. The number in brackets represent the samples
gave values below 0.204. assayed that show ed the same profile o f reactivity.
158Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) o /L eish m a n ia donovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista da Sociedade Brasileira d e M edicina Tropical 29:153-163, 1996.
o f FM L in protection o f outbred swiss albino mice immunoglobulin secretion to FM L was detected in
against the infection with L. donovani. Wecompared untreated infected animals, at 15 days o f infection.
the administration o f FM L with Saponin through Noteworthy, the specific pre-stimulation with FM L
the intraperitoneal or subcutaneous route. Results and saponin triggered the anti-FM L antibody
o f anti-FM L antibodies are summarized in Figure synthesis. The protective effect o f this response is
4A. Before the infection a pronounced speciffic shown by the diminished parasite burden in host’s
response to FM L (p < 0.001 ) was detected in animals tissues. Indeed, the parasite counts in liver were
immunized w ith FM L and saponin, higher by the significantly decreased (p < 0.001), in the same
intraperitoneal than the subcutaneous route. After order o f magnitude as the immunoglobulins to
the infection, both group of mice showed an increased FM L expanded Figure 4B. At this stage o f
response whereas no response was detected in any experimental murine infection, parasites are found
control group. Taken together, these results show only in liver, and they infect the spleen only 30 days
the slow establishment of immune response in later (data not shown). No significant differences
mice, even when infected by this relatively high were detected between the saline and the saponin
dose o f L. don ovan i amastigotes. Indeed, no treated animals, in any parameter (p > 0 .3 ). Taken
Figure 4 - Effect o f FML on Leishmania donovani infection in swiss albino mice. In A: antibody response.open
columns: seven days after three intraperitoneal (IP) or subcutaneous (SC) injections o f control saline solution (C), lOOfig
saponin (S), or 150jig FML combined tolOOfig saponin (SFML). Shaded columns: idem, 15 days after infection with
L . donovani. Results represent score mean values ofthe pool o f sera oftw o independent experiments done in triplicates,
using 4 -5 mice in each group. In B: liver parasitic burden., o f the same groups in A, after 15 days infection with L.
donovani. Results are expressed as percents o f the average o f LDU values (counts in 1000 cells) o f liver imprints o f
infected mice, o f two independent experiments, using 4 to 5 mice in each group. Horizontal bars represent standard
errors. FML saponin treated mice, after infection, gave significantly different results (p < 0.001) when com pared to
all other groups.
159Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) o /L eish m a n ia don ovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista d a Sociedade Brasileira de M edicina Tropical 29:153-163, 1996.
toguether these results represent an average o f 80.0 isolated nor characterized. The second was from a
% increase in speciffic antibody response and a patient with Chagas’s disease. In both examples the
85.5% protection in reduction of parasite liver possibility o f a concomitant infection with L.
burden. Although the humoral response was higher donovani could not be ruled out since visceral
in the case o f intraperitoneal administration, no leishmaniasis was also endemic in these regions.
signifficant difference between the vias was shown These two positive reactions are at present, of
in what concern to protective effect. difficult interpretation. Their presence has decreased
the specificity o f the assay to 96% . The FM L
DISCUSSION antigen developed equal levels o f sensitivity and
specificity than the recombinant antigen used by
ELISA tests using crude native antigens are Bums et al (1993)8. However, different from the
usually done in dilutions o f 2 to 5/ig/w ell15 l6. In an recombinant antigen, FM L is a a glycoproteic
improved ELISA assay, the recombinant GP63 complex and its long lasting stability makes o f it a
antigen of L. chagasi was used in concentration of good candidate for work in field assays. W e were
1 /xg/well35 39. The FM L is thus a very sensitive able to use the same batch for period over two years
antigen, and standard ELISA tests can be done in with no signifficant changes in its performance.The
dilutions increased by an order of magnitude. This FML-ELISA test is now being used as a new tool
sensitivity can be probably advantageously used in for diagnosis, prognosis, cure survey o f visceral
development o f rapid and automated tests for kala- leishmaniasis and it is used as an alternative to bone
azar. This property o f FM Lcan be possibly attributed marrow punction in several human cases with no
to its glycoproteic nature. Previous studies have false negative or positive results. The test is
attributed the species-specific characteristics of performed routinely in the cited Hospital and Blood
Leishmania glycoconjugates to their glycidic bank services. Furthermore a clinical and epidemical
moiety28. survey o f perypherical areas o f Natal, using the
The presented results indicate that assays using FML-ELISA test is underway.
FM L can be highly sensitive and specific in diagnosis In this study we described the presence o f L.
of kala-azar. This antigen can clearly separate do n o va n i s o ro re a c tiv e in d iv id u a ls am o n g
visceral leishmaniasis from all the other closely spontaneous blood donors in the kala-azar endemic
related tegumentar infections caused by parasite of area. This prevalence attained up to 17% o f diary
the gen u s L eishm ania, w ith the sen sitiv ity donors. These values are higher than those usually
comparable to those reported for recombinant detected for chagasic donors in Rio de Janeiro.
antigens35 39. M oreover, this antigen can identify Recent results from our laboratory demostrated that
inhabitants o f endem ic areas with a potential blood transfusion is an efficient via o f transmission
subclinical infection, and point out those with a o f experimental kala-azar. No one o f the 8 FM L-
high risk o f evolution towards the overt severe ELISA positive sera was reactive in the tests for
disease. It can be also used to follow-up of the Chagas’ disease or other diseases, performed in the
parasitologic cure. D ifferent form previously blood bank and therefore the use o f these blood
reported data2, the FM L antigen isolated from an package units would be considered possible, since
African strain o f L. donovani was accurate in the presence o f antibodies against Leishmania
d ia g n o s is o f S o u th A m erican v isc e ra l donovani would not be detected by the present used
leishmaniasis, representing apparently a tool that control tools. Since these specific antibody titers
can be used throughout the diagnostics o f diseases could be a signal o f active infection and o f a
caused by the L. donovani complex. p o te n tia l s o u rc e o f b lo o d b o rn e v is c e ra l
Two cases o f a positive reaction o f FM L with leishmaniasis, the extension o f the FM L-ELISA
sera o f a patient without overt kala-azar were screening in blood banks and the detailed study o f
observed. The first was a patient with chronic each sororeactive case is extremely necessary.
disseminated muco-cutaneous leishmaniasis. This W e further analyzed by W estern Blot the
serum had high titers o f IgG and IgM antibodies reactivity o f human sera with FM L. Isolated GP36
against L. braziliensis, but the parasite could not be was only detected in W estern Blots by kala-azar
160Sousa CBP, Gomes EM, Souza EP, Santos WR,. M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) o /L eish m a n ia d on ovani. A new to o l in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista da Sociedade Brasileira d e Medicina Tropical 29:153-163, 1996.
patients’ sera. N o labeling was detected with sera of were due only to D P72 adm inistration or to
patients with tegumentar leishmaniasis, Chagas promastigote injection or to both o f them. Two
disease, or normal sera. The GP36 has shown to be other antigens of Leishmania, extensively studied
the most specific fraction o f the FM L antigen. The were candidates for vaccination against cutaneous
GP36 component o f FM L could correspond in leishmaniasis in mice, however, it was recently
living parasites to a low er m olecular weight shown that GP63 is unable to induce lymphocyte
component since its extraction procedure, favours proliferation in human leishmaniasis23 and that the
the glycidic enrichment that could retard ist migration ability o f LPG to induce a protective response in
in SDS PAGE. The specificity o f a 32-35 kDa band mice is due to contaminant proteins or glycoproteins,
for kala-azar was suggested by Reed et al. ( 1987)34, called LPG associated p ro tein s (L P G A P )17.
who used a total soluble extract o f L chagasi with Furthermore subcutaneous LPG vaccination against
sera o f patients from Brazil. Circulating antigens of m u rin e te g u m e n ta r le is h m a n ia s is by th e
30-35 kDa were detected in sera of human patients subcutaneous route showed to induce an increase of
by monoclonal specific antibodies42 and were lesion size, instead o f protection13. Recently, the
recovered from kidneys o f L. donovani infected immunization with one o f the LPGAP flagellar
hamsters35. The GP36 may be thus shed in vivo by proteins, present in a wide range o f Leishmania
parasites, representing a circulating antigen. The species and in African trypanosom es, in the
isolation o f GP36 antigen o f FM L is in progress. recombinant form, stimulates primed lymphocytes
Finally, the reported results indicate that FM L to proliferate in vitro40. The use o f FM L of
may be a potential candidate for vaccination in Leishmania donovani and o f its components in
visceral leishm aniasis. Both intraperitoneal or sorologycal screening for visceral leishmaniasis
subcutaneous immunizations were effective. These shows high sensitivity, specificity, stability and
results are particularly signifficant in view of the comparatively easier performance. Its use in Blood
outbred nature o f the experimental model. Indeed a banks would improve the quality control to prevent
high intragroup variation was expected. This blood borne infections. Furtherm ore it showed a
protective effect o f FM L and saponin is in agree to signifficant potential on experimental vaccination
previous evidences o f the protective potential against against kala-azar. The study o f the molecular aspects
experimental visceral leishmaniasis in CB hamsters related to the described FM L potentials is under
and Balb/c mice30 31. In our conditions, saponin progress.
showed nor toxic neither unspeciffic effects. Its use RESUMO
as adjuvant was compared to Corynebacterium
parvum, Freund Adjuvant, Alhydrogel, Bordetella O FML (Ligame de Fucose-Manose) de Leishm ania
pertussis and M uramyl dipeptide and considered to donovani é uma fração glicoproleica complexa. O seu
be advantageous4. In agreement with our results, potencial no diagnóstico da leishmaniose visceral humana
fo i testado com soros provenientes de Natal, Rio Grande
saponin has been previously shown to be a good
do Norte, Brasil. O teste de FML-ELISA mostrou 100%
p o te n tia to r o f a n ti-p ro to z o a n im m unity in
de sensibilidade e 96% de especificidade, klentificando
experimental vaccines against T cruzp1. However, pacientes com calazar declarado ( p < 0 .001, comparados
saponins are discriminated as adjuvant because of com soros normais) e indivíduos com infecção subclínica.
their hemolytic properties3. For this reason, we M ais de 20% dos so ro rrea tivo s assim ptom áticos
started the comparative analysis o f the hemolytic desenvolveram a doença no p ra zo de 10 meses. Na
and ajuvant potential o f different kind of saponins. análise de doadores de sangue, 5% de sororeativos,
Using the purified antigen DP72 for immunization atingindo até 17%> num único dia foram detectados. A
of balb/c mice against L. donovani infection, a glicoproteínaGP36doFHL éreconhecida especificamente
p o r soros d e pacien tes com calazar. O potencial
protective effect o f the same order o f magnitude
imunoprotetor do FML no calazar experimental fo i
was reported16 33. However, in those works, for
testado no modelo swis.s albino em combinação com
protection or lym phocyte proliferation assays, saponina pelas vias subcutâneas e/ou intraperitoneal
respectively, after sensitization with the antigen, seguido de desafio com 2x 1(P amastigolas de Leishmania
theanimals received different dosesofpromastigotes. donovani. Um aumento de 80.0% na resposta de
It is consequently not clear if the described effects anticorpos específicos ( p < 0 .001) e a redução de 85.5 %
161Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) o /L eish m a n ia donovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista d a Sociedade Brasileira d e M edicina Tropical 29:153-163, 1996.
da carga parasitária no fíg a do ( p < 0 .001 )foi detectado Science USA 90:775-779, 1993.
nos an im a is va c in a d o s com FML e sa p o n in a , 9. Castellani A Chalm ers AJ. V isceral L eishm aniasis In:
independentemente da via de administração. Baillière, T indall, C ox (eds) M anual ofT ropicalM edicine,
3 rt* edition, U niversity series, L ondon p . 1289-1307,
1919.
Palavras-chaves: Glicoconjugado. Leishm ania
10. Cohen C , Corazza F, D e Mol P, Brasseur D . Leishmaniasis
donovani. Calazar. Transfusão de sangue. Diagnóstico. acquired in Belgium. L ancet 3 3 8:128, 1991.
11. Convit J, Castellano PL, R ondon A, Penardi M E , Ulrich
ACKNOW LEDGM ENTS M , Castes M , Bloom B, G arcia L. Im m unotherapy versus
chem oterapy in localized C utaneous Leishm aniasis. The
Lancet 1:401-404, 1987.
This research has been supported by FNS,
12. Grogl M , D augirda JL , H oover D L , M agill AJ, Berman
FIN EP, CNPQ, RHAE-CNPQ and CEPG-UFRJ. J . Survaivability and infectivity ofviscerotropic Leishmania
Dr. Telm a C. Barros Freire from HEM ONORTE, tropica from Operation Desert Storm participates inhum an
is acknowledgeded for sample collection facilities. b lood p roducts m aintainance u n d e r b lood b anking
conditions. T he A m erican Journal o f T ropical M edicine
and H ygiene 49:308-315, 1993.
REFERENCES 13. H andm an E, Mitchell G F. T he glycoconjugate derived
from a Leishmania major receptor for m acrophages is a
1. A shford DA , Badaró R , Eulalio C, Freire M , M iranda C, suppressogenic disease prom oting antigen in m urine
Zalis M , D avid, JR . Studies on the control o f visceral Cutaneous Leishm aniasis. Parasite Im m unology 8:255-
leishm aniasisrvalidation o f the Falcon assay screening 263, 1986.
test-enzym e linked im m unosorbent assay (Fast-Elisa^ ) 14. Holbrook T W , Cook JA , P arker BW . Im m unization
for field diagnosis o f canine visceral leishmaniasis. The against Leishmania donovani: glucan as an adjuvant with
A m erican Journal o f T ropical M edicine and H ygiene killed prom astigotes. T he A m erican Journal o f Tropical
48:1-8, 1993. M edicine and H ygiene 30:762-768, 1981.
2. Badaró R, Reed SG , Barrai A, O rge G, Jones T C . 15. Jaffe C L Zalis M . Use o f purified parasite proteins from
Evaluation o f the m icroenzym e linked im m unosorbent Leishmania donovani for the rapid serodiagnosis o f visceral
assay f o r a n tib o d ie s in a m e r ic a n v is c e r a l leishm aniasis. Journal o f Infectious D iseases 157, 1212-
Ieishm aniasis:antigen selection for detection o f infection- 1220, 1988.
specific responses. T he A m erican Journal o f Tropical 16. Jaffe C L , Racham im N , Sarfstein R. C haracterization o f
M edicine and H ygiene 35:72-78, 1986. two proteins from Leishmania donovani and their use for
3. Bom ford R . Saponin and other haem olysins (Vitamin A, vaccination against visceral leishm aniasis. Journal o f
aliphatic am ines, polyene antibiotics) as adjuvants for Im m unology 144:699-706, 1990.
SRBC in the m ouse. International Archives o f Allergy and 17. Jardim A, T olson D L , T urco S J, Pearson T W , Olafson
Applied Im m unology 63:170-177, 1980. R W .L eishm ania d o n o v a n i lip o p h o s p h o g ly c a n T
4. B om ford R . T h e com parative selectivity o f adjuvants for lym phocyte reactive com ponent is a tightly associated
hum oral and cell mediated immunity II. Effect on delayed protein com plex. Journal oflm m unology 147:3538-3544,
type hypersensitivity in the m ouse, guinea pig, and cell 1991.
m ediated im m unity to tum or antigens in the mouse o f 18. Jarecki-Black JC , Jam es ER, Kirshtein JW , K irshtein JD ,
F reu n d ’s incom plete and com plete adjuvant, alhydrogel, Glassman AB. Leishmaniadonovani: im m unization against
Corynebacterium parvuni, Bordetella pertussis, m uram yl infection as a function o f parasite grow th phase. The
d ip ep tid e and sap o n in . C linical and E xperim ental Am erican Journal o f Tropical M edicine and Hygiene
Im m unology 39:435-441, 1984. 35:1117-1121, 1986.
5. Borowy NK, Schell D, Schaeffer C , Overath P. Diagnostic 19. Kudo R R . Family 5 Trypanosom atidae Doflein. In: Thomas
o f h u m an afric a n try p an o so m iasis and visceral CC (ed) Protozoology, 4th edition, T hom as Books, USA
leishm aniasis based on detection o f antiparasite-enzym e p. 355-356, 1960.
antibodies. Journal o f Infectious Diseases 164:422-425, 20. M arzochi, M C A , M arzochi K B F, C arvalho RW . Visceral
1991. Leishm aniasis in Rio de Janeiro. Parasitology T oday
6. Bradley D J, Kirkley J . Regulation o f Leishm aniapopulation 10:37-40, 1994.
w ithin the host. I. V ariable course o f Leishmania donovani 2 1. M ayrink W , W illiam P , Costa C A , M agalhães PA, M elo
infection in m ice. Clinical and Experim ental Immunology H N , D ia sM , Lima AO, M ichalick M SM , C arvalho EF,
30, 119-129, 1977. Barros G C, Sessa PA , A lencar JE . An experim ental
7. Brum pt E. Le K ala-azar. In: M asson e Cie (ed) Precis de vaccine against American dermal Leishmaniasis experience
Parasitologie, 6 eme edition, Collectionde Precis Médicaux, in the State o f Espirito Santo Brazil. Annals o f Tropical
Paris p. 256-277, 1949. M edicine and Parasitology 79:259-269, 1985.
8. Burns Jr JM , Shreffler W G, Benson DR, Ghalib HW, 22. M ebrahtu YB, H endricks L D , O ster C N , L aw yer PG,
Badaró R, Reed SG . M olecular characterization o f a Perkins PV, Pam ba H, Koech D, R oberts CR . Leishmania
kinesin-related antigen o f Leishmania chagasi that detects d o n o v a n i p a r a s ite s in th e n a s a l s e c r e ti o n s ,
specific antibody in African and A m erican Visceral tonsillopharyngeal mucosa, and urine centrifugates o f
Leishm aniasis. Proceedings o f the National Academy o f visceral leishmaniasis patients inK enya. Am erican Journal
162Sousa CBP, Gomes EM, Souza EP, Santos WR, M acedo SR, M edeiros LV, Luz K. The FML (Fucose Mannose
Ligand) o /L eish m a n ia d on ovani. A new tool in diagnosis, prognosis, transfusional control and vaccination against
human kala-azar. Revista da Sociedade Brasileira de M edicina Tropical 29:153-163, 1996.
o f T ropical M edicine and H ygiene 48:530-535, 1993. 33. Racham im N , Jaffe C L . Pure protein from Leishmania
23. M endonça S C F , Russell D G , Coutinho SG. Analysis o f donovani protects mice against both Cutaneous and Viceral
the hum an T cell responsiveness to purified antigens o f Leishm aniasis. Journal o f Im m unology 150:2322-2331,
Leishmania: Lypophosphoglycan (LPG) and glycoprotein 1993.
63 (G P63). Clinical and Experimental Immunology 83:472- 34. Reed SG, Badaro R , C herry L loyd RM Identification o f
478, 1991. specific cross reactive antigen o f Leishm ania donovani
2 4. M o d a b b erF . E xperiences with vaccines against cutaneous chagasi by hum an infection sera. Journal o f Im m unology
leishm aniasis o f m en and m ice. Parasitology 98 (S):49-60, 138:1596-1601, 1987.
1989. 3 5. Reed SG , Schreffler W G , Burns JM J, Scott JM , O rge M G ,
25. M o d a b b e r, F . L e is h m a n ia s is . In: W o rld H ealth G halib M S, Badaro R . A n im proved sérodiagnostic
O rganization (ed) T ropical D isease Research. Progress. procedure for visceral leishm aniasis. T he Am erican Journal
1991-1992, G eneva p .77-87, 1993. o f T ropical M edicine and H ygiene 43:632-639, 1990.
26. O ’D aly C JA , C abrera Z . Im m unization o f ham sters with 36. Sartori A, Viana de Oliveira A, Roque B arreira M C , Rossi
T L C K -k ille d p a ra site s in d u ced p ro te c tio n ag ain st M A , Campos-Neto A. Immune com plex glom erulonephritis
Leishmania infection. Acta T ropica 43:225-232, 1986. in exprim ental kala-azar. Parasite Im m unology 9:93-103,
27. P alatn ik C B ,B o ro je v icR ,PreviatoJO ,M endonça-Previato 1987.
L . In h ibition o f Leishmania donovani prom astigote 3 7 . Scott M T , Bahr G , M odabber F , A fchain D , Chedid L.
internalization into m urine m acrophages by chemically Adjuvant requirem ent for protective im m unization o f mice
defined parasite glycoconjugate ligands. Infection and using Trypanosoma cruzi 90K cell surface glycoprotein.
Im munity 5 7 :754-763, 1989. International Archives o f Allergy and Applied Im m unology
28. Palatnik CB, Previato JO , M endonça-Previato L, Borojevic 74:373-377, 1984.
R . A new approach to phylogeny o f Leishmania: species- 38. Scott JE , Schreffler W G , G halib H W , El A sad A, Siddig
specificity o f glycoconjugate ligands for prom astigote M , Badaro R , Reed SG . A rapid and sim ple diagnostic test
internalization into m urine m acrophages. Parasitology for active visceral leishm aniasis. T h e A m erican Journal o f
R esearch 76:289-293, 1990. Tropical M edicine and H ygiene 44:272-277, 1991.
29. Palatnik-de-Sousa CB , D utraH S , Borojevic R . Lrishftumid 39. Schreffler W G , Burns JM J, Badaro R, G halib H W , Button
donovani surface glycoconjugate GP36 is the m ajor L L, M cM aster R, R eed SG. Antibody responses o f
im m unogen com ponent o f the Fucose-M annose Ligand visceral leishmniasis patients to G P63, a m ajor surface
(FM L). Acta T ropica 5 J.5 9 -7 2 , 1993. glycoprotein o f Leishmania species. Journal o f Infectious
30. Palatnik-de-Sousa CB, Paraguai de Souza E, Gom es EM , Diseases 167:426-430, 1993.
Borojevic R. Experim ental murine Leishmania donovani 40. T oison D L, Jardim AJ, Schnur L F, S teb eck C , T uckey C,
infection: im m unoprotection by the Fucose M annose Beecroft RP, T eh HS, O lafson RW , Pearson T W . T he
L igand (FM L). Brazilian Journal ofM edical an Biologycal kinetoplastid m em brane protein 11 o f Leishm ani donovani
R esearch 27:547-551, 1994. and African trypanosom es is a potent stim ulator o f T-
31. Palatnik-de-Sousa CB , Bunn M oreno M M , Paraguai de lym phocyteproliferation. Infection and Immunity 62:4893-
Souza E, Borojevic R. T he FM L vaccine (Fucose Mannose 4899, 1994.
Ligand) protects ham sters from experim ental kala-azar. 41. W orld Health O rganization. K ala-azar surges on two
Jounal o f the Brazilian Society for the Advancement o f front. T D R N ew s 37:1-2, 1991.
Science. Ciência e C ultura. 46:290-296, 1994. 42. Xiao-Su H, Q ing L, Fang-K ing L, T ao-L in Y, Ya-Jin W ,
32. Palatnik de Sousa CB, Gom es EM , Paraguai de Souza E, Z hiQ , P ingQ , Ling L .K ala-azar infected serum circulating
Palatnik M , Luz KG, Borojevic R. T he Fucose Mannose antigens and their characteristics detected by m onoclonal
L igand o i Leishmania donovani in diagnosisand prognosis antibodies. Chinese M edical Journal 101:1-6, 1988.
o f visceral leishm aniasis (kala-azar). T ransactions o f the 43. Youden J. Index for rating diagnostic tests. C ancer, 3:32-
Royal Society o f T ropical M edicine and H ygiene 89:(In 35, 1950.
p ress), 1995.
163You can also read
