Specific and sensitive immunological diagnosis of

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A m J. Trop. A4ed. Hvg.,35(1), 1986, pp. 86-93

Copyright CI 1986 by The American Society ofTropical Medicine and Hygiene

SPECIFIC AND SENSITIVE IMMUNOLOGICAL DIAGNOSIS . OF CHAGAS’ DISEASE BY COMPETITIVE ANTIBODY ENZYME IMMUNOASSAY USING A TRYPANOSOMA CR UZI-SPECIFIC MONOCLONAL ANTIBODY J. L. LEMESRE,* D. AFCHAIN,* O. OROZCO,* M. LOYENS,* F. S. BRENIERE,** P. DESJEUX,** Y. CARLIER,+ U. MARTIN,$ J. A. NOGUEIRA-QUEIROZ,§ D. LE RAY,ll AND A. CAPRON* *Centre d’Iw”ologie et de Biologie Parasitaire, Institut Pasteur, 15 rue Cainille Guérin, 59019 Lille Cédex, France, +*Instituto Boliviano de Biologia de Altura, L a Paz, Bolivia,

+Laboratoire de Parasitologie, Faculté de Médecine, ULB, Bruxelles, Belghin, $Centro de Investigaciones Sobre Endemias Nacionales, Santa Fe, Argentina, §Nucleo de Medicina Tropical, Fortaleza, Brazil, and IlInstitut de Médecine Tropicale, Anvers, Belgium

Abstract. Coexistence of Chagas’ disease with leishmaniasis and T. rangeli infectionin endemic areas and cross-reactivity between corresponding etiological agents can confuse the immunodiagnosis of Chagas’ disease. A discriminative serological test could therefore represent a major advance in specific immunodiagnosis. A competitive antibody enzyme immunoassay against a component 5-enriched preparation, using a T. cruzi species-specific monoclonal antibody has allowed development of a specific serodiagnosis of Chagas’ disease with high sensitivity (96.6% in undetermined and chronic phases of infection). This test can differentiate Chagas’ disease from other cross-reacting parasitic diseases in areas where concomitant infections are unknown or suspected. The parasitic protozoan Trypanosoma cruzi is the causative agent of Chagas’ disease (American trypanosomiasis). At least 24 million people in Central and South America are estimated to be infected with T. cruzi. In the initial acute phase of Chagas’ disease, lethal in about 10% of cases, trypanosomes can usually be detected in the circulating blood. In the undeterminate and chronic phases of infection, parasitemia is very low and imniunoserological diagnosis is required. In vast areas of Central and South America, cutaneous, mucocutaneous2. or visceral leishmaniasis‘ and T. rangeli infection^^-^ are associated with T. cruzi infection. Due to a variable degree of cross-reactivity between the corresponding etiological agents8-I0 the precise diagnosis of Chagas’ disease using classical serological tests is not effective in areas where these diseases are coendemic. Attempts have been made to overcome such cross-reactivity using aniastigote or trypomas-

Accepted 1 July 1985.

86

tigote forms of T. cruzi,ll-l 2 “live” T. cruzi antigen,I3 human sera absorption,14 a monospecific serum anti-T. cruzi component 5 to identify specific antibodies,I5 and defined T. cruzi-specific antigens.16.l 7 The World Health Organization has emphasized the need for a discriminative Serological test ensuring the specific diagnosis of South American trypanosomiasis. In previous studies, a T. cruzi-specific component with regard to other Trypanosomatidae (T. rangeli, L.donovani, L.mexicana, L.braziliensis)8-l 5 so called “5,” was identified. Moreover, anti-component 5 precipitating antibodies frequently were found in sera from patients chronically infected with T. cruzi.15- l9 In recent papers,20-22we reported the production of murine monoclonal antibodies directed against component 5 of T, cruzi and the characterization of target antigens corresponding to the 72 Kd glycoprotein and its maturation products (5 1 Kd, 43 Kd and 24 Kd). In the present work, we describe a simple, highly specific and sensitive serological test for Chagas’ disease by competitive (antibody) enzyme immunoassay (CEIA). It uses a 27 Kdenriched fraction as antigen and a species-spe-

cific anti-T, cruzi I the detection of an! the sera of chagasic Chagas’ disease fro

MATE RI.

Antigen preparati0 Trypanosoma cr mastigotes culturel viously described’ tion at 400 x g fo four times with H Six grams (wet we in 100 nil 0.1% Na passages through Bio-LKB) at 18,OO at 26,000 x g f o r was dialyzed again 4”C, lyophilized ax tract of T. cruzi (T resuspended in 4 1 with an equal voli solution (2: 1). The trifuged at 1,000 aqueous phase was in the same way. ( rated and the ren: precipitated by the ano1 for 4 hr at -i precipitate was wa resuspended in 2 IT was centrifuged, d: for 24 hr at 4°C an extraction by chloi precipitation crea1 preparation ((2,-EI trophoresis results this fraction.” Monoclonal anlibc

A murine IgG, our laboratory ha ognize the compor Kd glycoprotein ar Kd, 43 Kd and 2 duced in BALWc were isolated fron tation with 50% : The pellet was dis

SPECIFIC IMMUNOLOGICAL DIAGNOSIS OF CHAGAS' DISEASE

cific anti-T. cru:; monoclonal antibody, allows the detectiorrof anti-component 5 antibodies in the sem of chagasic patients, and can differentiate Chagas' disease from leishmaniasis.

87

alyzed overnight. The solution was then submitted to ion exchange chromatography on a DEAE-Trisacryl column. The purified h5ab was labeled with alkaline phosphatase (grade I from calf intestine, Boehringer) by the one-step glutaraldehyde method previously described.')

MATERIALS AND METHODS

Antigen preparafion Trypanosoma cruzi (Tehuantepec sirain) epimastigotes cultured in GLSH medium as previously described8 were collected by centrifugation at 400 x g for 15 min at 4°C and washed four times with Hank's balanced salt solution. Six grams (wet weight) of epimastigoxs frozen in 100 ml 0.1% NaCl were disintegrated by four passages through an hydraulic press (X press, Bio-LKB) at 18,000 psi. It was then ccntrifuged at 26,000 x g for 1 hr at 4°C. The supernatant was dialyzed against distilled water for 24 hr at 4"C, lyophilized and used as a total soluble extract of T. cruzi (TSE). Twenty mg of TSE were resuspended in 4 ml distilled water and added with an equal volume of ch1oroform;methanol solution ( 2 1 ) . The mixture was shaken and centrifuged at 1,000 x g for 30 min at 4°C. The aqueous phase was collected and extracted twice in the same way. Organic solvents were evaporated and the remaining aqueous fraction was precipitated by the addition of 3 volumes of ethanol for 4 hr at -20°C. After centrifugation, the precipitate was washed with ethanol. dried, and resuspended in 2 ml distilled water. The solution was centrifuged. dialyzed against distilled water for 24 hr at 4°C and lyophilized. This successive extraction by chloroformhnethanol and ethanol precipitation created a Component 5-enriched preparation (C,-EP) as verificd by immunoelectrophoresis results of mouse scra immunized with this fraction.21

Monoclonal antibody /.\lab!

A murine IgG, blab ~11-190.'30~ produced in our laboratory has been dcmonsirated to recognize lhe component 5 corrc:;ponding to the 7 1 Kd glycoprotein and its ma1ur;ltion products ( jI Kd. 4 3 Kd and 24 Kd).2'.Z1Ascitcs were produced in BALBk mice and immunoglobulins were isolated from the ascitic lluid by precipitation u i t h 50% saturated ammonium sult-ate. The pellet was dissolved In O.?o NaCI and di-

SDS-polyacn*fatnide gcl electrophoresis (SDS-

PAGE)

. .

Total soluble extract and C,-EP were dissolved in 4 0 pl of sodium dodecyl sulfate (SDS)-containing slab gel buffer with 5% 8-mercaptoethano1 and boiled for 3 min. Insoluble material was removed by centrifugation at 2,000 x g for 10 min before samples were applied to the gel. SDS-PAGE was camed out on vertical slab gels according to the method of Laemmli'4 using 1G% polyacrylamide gel. After electrophoresis the gel was stained with Coomassie blue, destained and then dried under vacuum.

Competitive atitilodj. etizj.me i m t ~ ~ i i t ~ o m s a y (CEI.4)

Polypropylene beads (6.5 mm) were incubated overnight at room temperalure by gentle agitation in 0.015 hl carbonate-0.035 M bicarbonate buffer (pH 9.6) containing the antigen (see Results for concentrations). 'After washing three times in PBS-Tween (0.01 hi phosphate buffer, pH 7.2, 0.1'30Tween 70). the beads were incubated with PBS +- 0.19'0bovine serum albumin for 2 hr and washed twice in PBS-Ttveen. then once in phosphate buffcrcii s ~ ! i n c(PBS). CEIA was performed in disposable polystyrene tubes. Coated bcads wwc incubated for 3 hr at 37°C in a misturc of 100 pl diluted hlab labclcd wijh alkalinc phosphatase. 100 pl dilutcd human serum sample and 159 p1 PBS-Tween. Tubes were empticd by saction and hcads were tvashcd three times in PtYj-Ttvccn and transferred to another tube: the amount of enzyme fised to the bcads W S dctcrmiricd using 300 pl of enzyme substrate ( 1 mg ml 4-nitrophcnylphosphate in 0.5 h4 Na,CO,. 0.001 lí LlgCl, bufIèr. pH 10.4). After I hr incubation at 37°C. further reaction was stoppcd b> addition of 300 pl 2 N NaOH and thc rcss!!:r+ >ello~v color \vas measurrd inaspC.StrOI)ti:!:.)I:?"ir.r;;t 4~15nm. Thc CEIA test involved t h c inIiih!;:on o f binding of

88

LEMESRE ET AL.

**

.

eBy*

-94

. . . .

-:>.-y.

24 -.-

H u m a n sera

B

A-

,

. .

- ..!

.f

1

The control group was comprised of50 healthy patients with a negative serology for T. cruti, including 20 Europeans, 20 Bolivians and 10 Argentinians. The T. cruzi-inkcted group included 15 Argentinian children ( 1-10 years) with evident acute phase Chagas' disease (Romaña sign, generai edema, presence of blood parasites) and 89 Bolivian patients with chronic phase Chagas' disease and a positive serology for T. cmzi (immunofluorescence. complement fixation test, immunoelectrophoresis and ELISA). They lived in lowland areas of Bolivia where leishmaniasis has never been found. The third group was infected by other Trypanosomatidae. including LPishniania braziliensis ( 1 5 Bolivian patients with evident lesions of mucocutaneous leishmaniasis and a negative serology for T. c r u 5 who lived in areas where Chagas' disease had never been found), L. Iropica (6 African patients with evident cutaneous leishmaniasis), L. donovani ( I 4 mediterranean patients with visceral leishmaniasis), and T. gambiense( 1 1 African patients with sleeping sickness). A founh group was infected by other protozoa including Toxoplasma gondii (1 4 European patients), Plasnrodiirn? (IO African patients), and Entanzoeba hisio[wica (3 African patients). The fifth group was comprised of patients living in areas with possible mixed infection: 40 Brazilians clinically and serologically confirmed as being infected with Schis:osonta niansoni and 47 Bolivian patients with clinical and serological confirmation of L. braziliessis infection, living in the Yungas \‘alley. RESULTS

Cotnposirwti ~ f : I i CI-EP c ori:igqn FIGURE1. Protein patterns after Coomassie blue staining of 500 p g of total soluble extract of T.rnizi (B) and 50 pg of the component Senrichcd preparation (A) by SDS-P.4GE. The positions of migra~ionof the marker proteins (hi, x IO-?) are indicated.

l

alkalin phosphatasc-labcled blab by sera from T. crrrzi-infectcd patients: low extinction values indicate the presence of anti-component 5 antibodies in human serum. A total of 1.750 scra can be tested usine 2.5 mg of C,-EP and 3.5 mg of purified Mab.

The C,-EP was analyzed by SDS-PAGE foilowed by Coomassic blue swizing. Figure l shotvs a comparison between protein patterns ol'thc T. criizi total soluhlr extract (TSEt (SOO pg, lanr 13) and thc C,-EP (50 p g , lane A). More than 2 0 polypcptidc chzins could be clcarly distinguislicd in TSE pattern 2nd a major band of 74.003 daltons with some minor contsminating proteins were idcntilicd in the C,-EP profilc. Approsimatcly. 2.5 n i 5 a ! ' C ,-EP n z < cibt:iincd ii,rrii 21) mg of TSE.

89

SPECIFIC IM hl 11NOLOCiICAL DI A--.

IL-.

.../-*

3A and B), which obtained a 2.3 higher difference in OD values bet\veen positive and negative sera with C,-EP than wirh TSE. This increase in the sensitivity of rhe test was used in all subsequent studies. using the 5 pg ml C,-EP coating.

Sensirisil!: and specificicy08CEM Figure 4 shows the CEIA OD values in the control group (0.92 = (2.17) significantly higher than those of the acute (0.50 2 0.30, P < 0.00 1) and those with the chronic phase (O. 19 t O. 14, P c 0.001). There nas a clear cut-off point be-

LEMESRE ET.AL.

OD 405om I

1

..

pl t-

..

iH

... ..

...

.....

Ii

..

1

CHRONIC PHASE

ACUTE CONTROL PHASE

TOXO.

AMOEE.

MALARIA

T. CAMB.

LOONO.

L.ERAZ.

L.TROP.

FIGLIRE 4. Sensitivity and specificity studies of CEIA using c o m r . r x n t 5-enriched prtr?zrr;::. . . ,..; . i - i component 5 Mab and sera from different patient groups. Chronic p t b s e= patients with ;-:.:-I;. . ,. .....'.,'. Acute phase = patients with acute phase of the Chagas' disease; Control = healthy subjec:; Is.-: i.-: :- :?...:il i' ...: ....: .> and Argentina: Toxo = Toxoplasmosis; Amoeb. = Amoebiasis: ma!:i:ia: T.gunth. = ,+ï::.:in ir: F.: L. dono. = visceral leishmaniasis: L. bru--.= mucocutaneous leishmmiasis: L.trop. = CE:;;.^. ,L\ i t r ~ . ; : : ~ ! ~ , : , ; ~ The Student's r-test shows a significant difference bctween Chagas' disease group and 0th:: ;rt12:3. - - - ctpt~ca! density cut-off point.

.

1.;

i i

tween chagasic a n d non-chagasic paticnts using the OD 0.5s [calculated a s m (control group)-' SD: i.e., 0.93 - 2 x (0.17) = 0.581. Thc sensitivity of CEIA positive detection in relation to the cut-off value was 66.7% in acutc infection a n d attained 96.6% in the cli:unic phase of Chagas' disease. As sho\vn in Figure 4. CELA OD valucs in scm from patients with o t h e r 7 F p a n o s o m a t i d x or protozoal infections were betwecn 0.6 and 1.3 and showed a significant ditrcrrncc from those o f chronic Chqgas' diseasc.

I

I5 scra samples. respectivei! !Fig. 5). gi\.inc c\.id e n c r of Chagas' infection ir: 7.j0;>01' scnisii!soniiasis cascs and in 31.4"; of 'I: + m a n i a s i s cases.

P

I .

SPECIFIC IMMUNOLOGICAL DIAGNOSIS OF CHAGAS' DISEASE

serodiagnosis for Chagas' disease, particularly in areas with concomitant infections. Previous studies. using immunoelectrophoresis'8 and a microdouble diffusion test,15 have demonstrated the high frequency of anti-comChagas' 5disease. ponent antibodies Moreinrecefi'.!y, sera of murine patients hlabs with

,

(II- 190/30. III- 160118 and 1-35/67) have been produced against component 520.2 1 which recognized a glycoprotein of 72 Kd (GP72) and its maturation products (5 1, 43 and 24 Kd). These molecules are exclusively expressed in epimastigote and amastigote forms of T. cruzizi and not on other trypanosomatid parasites of humans (Leishmania and T. rangeli).a Preliminary results2' using Mab II-190/30 in a competitive radioimmunoassay suggest its potential value for specific serodiagnosis of Chagas' disease. Thus, the use ofa species-specific Mab in a competitive enzyme immunoassay allows precise immunodiagnosis.2' It possesses all the advantages of constant specificity and reproducibility of a monoclonal reagent. Finally, this enzyme immu-

Recently, two sensitive and specific tests using .purified antigens of T. cruzi (25 Kd and 90 Kd) have been developed.Ib.l i However, they are very expensive and require large quantities of purified antigens, precluding a large field application. The present simple chemical extraction procedure from a total hydrosoluble extract of T. cru5 can isolate a measurable quantity of 14 Kd-enriched fraction. The 24 Kd antigen. one of the maturation products of the GP72,21.?2seems to br produced by a proteolytic degradation occumng during extraction. No false positive reactions wwc observed with rhe CEIA test using sera from healthy individuals as neIl as from patients with othcr parasitic infections. This was due to the specificity of thc component 5 of T. crici lvith regard to othcr Tq-panosomatidae as previously demonstrated." However, further evaluations of the usefulness of this test are required in arcas where T. rutigc~!; infections are associated Lvith Chagas' disease. >lore than 95% of thc pawnis wirh undctcrminate and chronic phases d Chagas' discasc presented specilic high lt'vcls of anti-component 5 antibodies. This high scnsiiiviiy was not only di.pLyxicnt on the nion:*clo:::!! P2:ig~'lit but alsu on [he use of a componcnt 5-cnrlchcd fraction

91

O D [ ~ S ~ ~ \

CH

'.O.

.. .. e..

... ..

... .. ... ..

_____ ---- - -----

---c---

- - ---------- --

05.

0.1

.

H

..

FIGURE5. Detection of mixec infections in two groups of sera samples from South American patients (S. mam. = Brazilian patients with schistosomiasis;t. bru:. = Bolivian patients with mxocutaneous leishmaniasis). --- Optical density cut-off point.

In fact, when a crude extraci of T. criizi was used, the sensitivity of the CEIA tes! was lower: On the other hand. only 66.?0 of the patients with acute Chagas' disease stowed anti-component 5 antibodies. Several h:;.otheses can account for these results: a) the low level of anticomponent 5 antibodies: b) ths eventual weak affinity of corresponding IgG. o: cl the presence of IghI antibodies and 'or imnicze complexes in sera of acute cascs. The level of associated Chtys' disease dctectcd in cocndcniic 3rcas !sct:j!osomiasis and leishnianiasis endemic arms. r:iFicctively) dcmonstraies that the C'EI.4 te51 Trovidcs an cxrrcmcly precise diagnosis of C'h2;as' disease. Indeed. i t is very important :a obtain such con!irniation o!' T. t w z i infectto:. since the therapcutic management 01' leishn?;lr.iasis and Chagas' disea5e arc very different. I n victv cif its simplictty. sp:.:iiity and scnsitivity. the CEIA test I S rccornr.:-xlcJ fora spc-

92

LESlESKE ET AL.

cific diagnosis a n d for large screening of undcm i n a t e and chronic phases of Chagas' disease. especially in areas where several parasitic diseases are coendemic.

1I.

ACKNOWLEDGhl ESTS

T h i s investigation received financial support from the trypanosomiasis c o m p o n c n t o f the U N D P i W o r l d BanWWHO Special Programme for Research a n d Training in Tropical Diseases. The authors wish to thank D. H e n v a n d J. L. Neyrinck for their excellent technical assistance and C. Colson and M. F. Massard for thcir sccretarial help. W e are also grateful to Drs. F. Darcy a n d F. Santoro for invaluable help in correcrion o f t h i s manuscript.

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