The serological diagnosis of human hydatid disease by time-resolved fluoroimmunoassay

Journal of Infection (I99i) 2z, I 3 5 - I 4 t

T h e s e r o l o g i c a l d i a g n o s i s o f h u m a n h y d a t i d disease by t i m e resolved fluoroimmunoassay A. Aceti,*~ A. Pennica,* Antonella Teggi,T A. Grilli,* Marta Caferro,* D. Celestino,* Oriana Leri,* A. Sebastiani* and F. De Rosat * Institute of the Clinic of Tropical and Infectious Diseases and t Institute of Infectious Diseases, 2nd Chair, 'La Sapienza' University, Rome, Italy Accepted for publication 15 August 1990

Summary The use of a new immunoassay, time-resolved fluoroimmunoassay (TR-FIA), in the diagnosis of human hydatid disease has been evaluated. This technique, which is based on the labelling of antibodies with europium (Eu), was compared with a wellestablished method, the enzyme linked immunosorbent assay (ELISA). Of Io2 patients with hydatid disease, 97 (95"I %) were positive according to T R - F I A and 83 (8I'4 ~/o) according to ELISA. The rate of non-specificity for other parasitic infections (n = 206) was 8.7 % for T R - F I A and I7"5 % for ELISA. It is concluded that T R - F I A is more sensitive and more specific than E L I S A in the diagnosis of human hydatid disease. Introduction

T h e immunological diagnosis of h u m a n hydatid disease, caused b y the larval stage of Echinococcus granulosus, is generally based on the detection of specific circulating antibodies. F o r this purpose, the sensitivity and specificity of various techniques have been evaluated and several have p r o v e d to be very useful clinically. 1' ~ E v e n so, there are limitations to such diagnostic tests which have varying degrees of b o t h false-positive and false-negative results as well as both low and high degrees of s e n s i t i v i t y ) Recently, a non-isotopic i m m u n o a s s a y has been developed based on the labelling of antibodies with e u r o p i u m (Eu) and conversion of the specifically b o u n d non-fluorescent Eu-label after i m m u n o r e a c t i o n in chelate solution with a long fluorescence life-time. Fluorescence is then m e a s u r e d with a timeresolved fluorometer after a delay selected to eliminate almost completely the b a c k g r o u n d fluorescence which decays rapidly. 4 This assay, time-resolved f l u o r o i m m u n o a s s a y ( T R - F I A ) , has been successfully applied in various f i e l d s ) -~ T h e aim of the present s t u d y was to evaluate the use of T R - F I A in the diagnosis of h u m a n hydafid disease. T h e technique was also c o m p a r e d with a well-established test, the enzyme linked i m m u n o s o r b e n t assay ( E L I S A ) . 1: Address correspondence to: D r Antonio Aceti, Istituto di Clinica delle Malattie Tropicali e Infettive~ Policlinico U m b e r t o I °, o o I 6 i Roma, Italia.

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© I99I T h e British Society for the Study of Infection

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A. ACETI

ET AL.

Materials and m e t h o d s Source o f s p e c i m e n s

Samples of serum were collected from IO2 patients with hydatid disease (7I hepatic, 2I p u l m o n a r y and IO with multiple sites of localisation, confirmed in all cases by surgery) and from 2o6 patients with parasitic diseases other than hydatid disease (82 infected by Schistosoma mansoni a n d / o r 23 by S. haematobium, 42 with filariasis, 4I with invasive amoebiasis, 23 with malaria, I3 with taeniasis, and five with ascaridiasis). Samples from IO6 healthy Italians were also examined. Antigen

Partly purified antigen was prepared from crude sheep hydatid fluid according to the m e t h o d of Oriol et al. 9 T h e fluid was concentrated by means of a M W IOOOO cut-off filtration m e m b r a n e (PM Io, Amicon). Dialysis against o'oo5 M acetate buffer, p H 5, precipitated the euglobulins and parasite proteins which were then collected by centrifuging at 5o0oo g for 6o min. T h e precipitated proteins were redissolved in o'2 M phosphate buffer, p H 8, and the globulin fraction removed b y ' salting o u t ' with 4o % a m m o n i u m sulphate. T h e dialysis and 'salting o u t ' were repeated once. T h e s u p e m a t a n t was dialysed against IOOO x volume of phosphate buffer for 24 h. T h e protein content was then estimated by the Lowry procedure.l° T R - F I A test T h e wells of microtitre plates were coated with hydatid antigen by adding to each well o.2 ml antigen at a protein concentration of 5 #g/m1 in o.2 M borate buffer, p H 9"3. Plates were then left overnight at 4 °C. After coating, the plates were washed five times with physiological saline containing o'o5 % T w e e n 2o. T h e n o'2 ml of each test serum, diluted I in 2oo, was added and allowed to react for 2 h at 4o °C. T h e plates were washed as before and, after swine antihuman I g G labelled with Eu (Iong/we11) (Pharmacia, L K B Wallac) had been added, were incubated for I h at 4o °C. T h e specimens of serum and the conjugate were diluted in o'o5 M Tris-HC1 buffer, p H 7"7, containing o'5 % BSA and o'o5 % bovine globulin, in order to reduce non-specific binding. Any free Eu was b o u n d by adding 1o/~M of diethylene-triamine-penta-acefic acid (Pharmacia, L K B Wallac). At the end of the period of incubation, and after a washing cycle, the b o u n d Eu was dissociated from the solid-phase antibody and transferred into a fluorescent chelate by adding o'2 m1/we11 of an ' e n h a n c e m e n t solution' consisting of O'I M acetone-potassium hydrogen phthalate, p H 3.2, containing I5/~M 2naphthoyltrifluoroacetate, 5o/zM tri(n-octyl)phosphine oxide and o" 1% T r i t o n x-Ioo (Pharmacia, L K B Wallac) (Plate I). After being shaken for xo rain by an automatic device, the fluorescence was measured in a fluorometer with a xenon flash-lamp (I23O Arcus fluorometer, Pharmacia, L K B Wallac). A I #s excitation pulse at 34o n m was given and, after a delay time of o'4 ms, the single p h o t o n emission was counted for o'4 ms at 613 nm. T h e cycle was repeated IOOO times during the total counting time of i s. T h e optimal concentrations of all reagents were estimated by chequer board titrations. All tests were done in duplicate. T h e performance of the assay was m o n i t o r e d by

Journal of Infection

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Plate I. Principle for the release of europium (EU) after the immunoassay has been completed. As a final step before fluorescence is measured, an enhancement solution is added. This consists of o'i M acetone-potassium hydrogen phthalate, pH 3"2, containing IS#M 2naphthoyltrifluoroacetate, 5o/gM tri(n-octyl)phosphine oxide and o'I % Triton X-ioo. The europium ion dissociates from the labelled antibody and forms a new fluorescent chelate in solution.

A. ACETI ET AL.

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4O 55 5O 25 I/J

o. 20 15 I0 5 0

I

I

I

I

I

I

I

I

I

I

I

I

2

:3

4

5

6

7

8

9

I0

II

FU

Fig. I. Standard calibration curve for the control used in the T R - F I A test (cps x io-3). Coefficient of correlation between cps and F U : o'99.

comparing the mean signal values of the positive and negative controls. In addition, each plate contained a 'calibration' control consisting of a pool of IO positive samples o f s e r u m and a high titre of antibody. T h i s was diluted from in 200 in doubling dilutions to I in 204 800, so allowing the construction of a standard curve as described below u n d e r analysis of data. ELISA test T h i s assay was p e r f o r m e d b y a microtitration p r o c e d u r e n according to the m e t h o d described earlier. 12 In the present study, the optimal antigen concentration was 5 # g / m l , the optimal dilutions for the serum samples and for the conjugate (goat antihuman I g G labelled with peroxidase, Nordic) were I in 2oo and I in 2ooo respectively, as determined b y a c h e q u e r - b o a r d titration. All tests were p e r f o r m e d in duplicate. T h e cut-off value was calculated b y adding twice standard deviations (S.D.) to the mean of absorbance of negative controls. Calculation of TR-FIA

T h e readings in counts per second (cps) obtained for each dilution of the calibration control were used to construct a standard curve in the following manner. T h e highest dilution of the control with a cps value over the negative m e a n plus 3 S.D.s (I in I o 2 4 o o dilution) was arbitrarily designated as being equal to one T R - F I A unit ( F U ) . T h e results were plotted as cps values vs. dilutions of the calibration control (Fig. I). T h e results, which usually covered a range from 4500 to 35000 cps, were entered into a p r o g r a m m e d calculator. T h e plate was accepted if the correlation coefficient of this standard curve was > 0"98. F o r each test serum, the result was calculated from the standard curve and expressed in F U x sample dilution (200). Specimens with a F U value > 200 were considered as positive. Results

T h e distribution of T R - F I A F U values observed in persons belonging to various categories s h o w e d a clear discrimination b e t w e e n those with hydatid

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Table I Distribution of T R - F I A values, expressed as arbitrary units (FU), obtained with serum samples from various groups Origin of serum samples Other Hydatid disease parasitic diseases Healthy persons (102 samples) (206 samples) (lO6 samples)

(%)

T R - F I A values (FU) < 200 200--500 5o1-1ooo 1OO1-2OOO 2OOl-4OOO 4OOl-8OOO > 8000

5 15 20 22 16 I5 9

(%)

(49) (14'7) (19'6) (21"6) (15"7) (14"7) (8"8)

188 (91-3) 9 (4"4) 7 (3"4) 2 (ro) o o O

(%) lO6 (IOO) o o o o o O

Table II Sensitivity of T R - F I A and E L I S A in relation to the location of cysts

Location (number of patients) Liver

TR-FIApositive (number)

TR-FIA ELISAsensitivity positive (%) (number)

ELISA sensitivity (%)

70

98'6

60

84"5

17

8I.O

13

61.9

(71) Lung

(2i) Multiple sites

IO

IOO

IO

IOO

(io)

disease and those in control groups (Table I). W h e n a threshold limit of 2oo F U was taken in order to distinguish between reactive and unreactive persons according to T R - F I A , 97/IO2 (95 %) patients with hydatid disease were found to be positive. A lower degree of sensitivity was shown by E L I S A which indicated as positive only 83 ( 8 I ' 4 % ) of the samples from patients with hydatid disease (X2 7"98; P : 4 x io-3). W h e n the sensitivity of the two tests was evaluated in relation to the site of cysts, T R - F I A revealed a higher reliability than E L I S A in locating both hepatic and p u l m o n a r y cysts (Table II). In addition, IO samples from patients with surgically proven E. granulosus infection, but with a borderline E L I S A result (optical density value ranging from the mean of negative controls plus 2 S.D.s to the mean of negative controls plus 4 S.D.S), were also tested by T R - F I A . F r o m the results obtained (Table III), it is evident that this test offers better discrimination between reactive and unreactive specimens than E L I S A . T h e rate of non-specificity for other parasitic infections was 8"7 % for T R F I A (nine patients with schistosomiasis, five with filariasis, three with taeniasis

Diagnosis of hydatid disease by TR-FIA

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Table III TR-FIA and E L I S A values in arbitrary units (FU) in respect of

ro selected serum samples from patients with hydatid disease (TR-FIA cutoff: 2oo; E L I S A cut-off: 237) Serum code

TR-FIA value (FU)

E L I S A value (OD49~ X IO3)

335 88I 439 641 946 479 814 2II 350 308

237 258 249 250 26I 247 260 216 245 225

E23 E27 E34 E41 E45 E67 E68 E79 E8I E96

Table IV Results obtained by means of TR-FIA and E L I S A in respect of

serum samples from various groups Number tested Hydatid disease Other parasitic diseases Healthy persons

ioz 206 IO6

TR-FIA positive (%)

ELISA positive (%)

97 (95"I) 83 (8I'4) 18 (8"7) 36 (I7'5) o o

and one with ascaridiasis) and I7-5~/o for E L I S A (I 9 patients with schistosomiasis, Io with filariasis, four with taeniasis and three with ascaridiasis). Statistical analysis of the data showed a significantly higher specificity for T R - F I A as compared to E L I S A (X ~ 6"I; P : I ' 3 X IO-2). No false-positive reactions were obtained with either technique in tests of samples from healthy persons (Table IV). Discussion

In the present study we have shown that T R - F I A may be of considerable value in the diagnosis of h u m a n hydatid disease. Successful application of this technique has also been reported in respect of other parasitic infections. 18,14 A serological test may be made more reliable by the use of standardised conditions. We used a calibration control on each plate and, for the plate to be accepted, a correlation coefficient > o'98 was required. This control allowed us to record results in a standardised manner rather than directly as cps values. Our results demonstrate that T R - F I A is more sensitive and more specific than E L I S A . T h e results we obtained with the latter technique accord with most of the findings reported by others. 15-19 In the E L I S A test, cross-reactions

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A. ACETI E T AL.

in respect o f parasitic or non-parasitic diseases have required the use of diagnostic titres in order to obtain an acceptable degree of specificity. As a result, the high sensitivity of E L I S A , one of its m a i n advantages, has been decreased to a degree similar to that of other traditional serological methods. T h e greater sensitivity o f T R - F I A m a y d e p e n d on the use of a readily detectable ' tracer '. Since the limit o f detection o f lanthanides by fluorescence is as low as IO-13 M, the use of e u r o p i u m makes it possible to achieve a high degree o f sensitivity. In addition, the use of the low molecular weight Euchelate disturbs the binding of low-affinity antibodies to the solid surface antigen less t h a n the use of an enzyme label. T h i s m a y also explain the better sensitivity of T R - F I A . O f special note, is the high rate of positivity (80"9 %) obtained by use of T R - F I A for samples f r o m patients with cysts in the lung. I n d e e d , the sensitivity of existing serological tests in the diagnosis of p u l m o n a r y echinococcosis is generally not satisfactory. 1' ~ It m a y be that it is the sensitivity of T R - F I A that allows detection o f the low concentration of antibodies p r o d u c e d in p u l m o n a r y h y d a t i d disease. T h e higher specificity shown by T R - F I A seems to be related to the lack of ' b a c k g r o u n d ' which, in E L I S A , m a y p r o d u c e false-positive results. Because o f the long fluorescence decay time o f lanthanides (over IO4 times longer than that of the average b a c k g r o u n d fluorescence), it is possible, by selecting a suitable delay time, to reduce non-specific ' n o i s e ' . I n addition, the Eu-labels are stable, and d e t e r m i n a t i o n o f emitted fluorescence is easy. By contrast, in E L I S A the e n z y m e - s u b s t r a t e reaction is sensitive to t e m p e r a t u r e and results are in the f o r m of unstable coloured products. On the basis of the present study, we conclude that T R - F I A is a highly reliable technique in the diagnosis of h u m a n h y d a t i d disease. References

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