The use of cotton rat-derived tachyzoite antigens in the development of a sensitive ELISA for the diagnosis of toxoplasmosis

The use of cotton rat-derived tachyzoite antigens in the development of a sensitive ELBA the diagnosis of toxoplasmosis A Fraser, A H Balfour, Department

for

J E Smith

of Pure and Applied

Biology,

University

of Leeds,

Leeds

LS2 9JT, UK

Summary Cotton rats have previously been found to be a reliable source of high quality Toxoplasma gondiitachyzoite antigen. A human IgG enzyme-linked immunosorbent assay (ELlSA) was developed using this antigen, and was evaluated with a panel of 414 human sera to analyse assay performance. Pools of positive and negative sera were included as controls and used to convert readings into enzyme immunoassay units (EIU). Measured against the dye test, sensitivity was calculated as 96.5%, specificity as 98.1%, with a positive predictive value of 99.3% and a negative predictive value of 91.0%. This indicates that cotton rat-derived antigens gave excellent results in this indirect ELISA, and may have uses in other diagnostic tests for T. gondii. Key words:

Toxoplasma

Serodiagn.

Immunother.

gondii, diagnosis, Infect. Disease

human IgG ELISA 1994, Vol. 6, 159-163,

Introduction Toxoplasma gondii is an important

parasite of humans, distributed widely throughout the world. Approximately one third of the world’s population is thought to be infected, though the prevalence in various countries is linked to a number of factors, including climate, hygiene, and diet I. Infection rates vary from 10% in Hong Kong2, to 27% in the UK3 and up to 90% in Guatemala and El Salvador’,5. Infection of immunocompetent patients causes little more than transient ‘flu-like symptoms but can be lifethreatening to the foetus in the first trimester of pregnancyh, and to immunocompromised patients’. Toxoplasmosis has become particularly important in HIV infection, where recrudescence of tissue cysts within the brain leads to fatal toxoplasmic encephalitiss. Rapid accurate diagnosis of infection is therefore of vital importance in the treatment of disease. Definitive diagnosis of the parasite in Britain is by the modified Sabin-Feldman methylene blue dye Received: 17 June 1994 Accepted: 3 July 1994 Correspondence and reprint

requests to: JE Smith, Dept. of Pure and Applied Biology, University of Leeds, Leeds LS2 9JT, UK 0 1994 Butterworth-Heinemarm Ltd 0888-0786/94/030159-05

September

testYJo, which is acknowledged as the gold standard”. However, this assay requires the use of live parasites, restricting its use to a small number of laboratories and presenting a health hazard to workers. Much effort has been put into the development of alternative methods for the detection of toxoplasmosis, including enzymelinked immunosorbent assays (ELISA), latex and haemagglutination tests (ILAT and IHAT) and immunosorbent agglutination assay (ISAGA), though all are less accurate than the dye test12. A number of commercial ELISA assays have been developed to detect anti-toxoplasma antibodies from human sera, the majority of which screen for specific IgM antibodies. The problem with IgM ELISA screening is twofold; a lack of well characterized references exist, preventing true estimates of assay accuracy; also specific IgM presence is primarily an indicator of acute infection and fails to detect most cases of persistent infection”. Screening for the presence of parasitespecific IgG gives a much wider indication of the presence of T. gondii, as IgG is detectable throughout the duration of the infection. Latex agglutination or IHAT assays are commonly used to detect antitoxoplasma IgG and IgM antibodies and specific IgG ELISAs have also been developed14. At present, most ELISA systems rely upon T. gondii antigens derived from tachyzoite extracts. These tachy-

160

Serodiagn. Immunother. Infect. Disease 1994; 6: No 3

zoites are obtained from a number of different sources, including mice 14,tissue culturels and egg chorio-allantoic membranes’h. Recent analysis of T. go&ii tachyzoite antigens from different sources indicated that cotton rat exudates yield prolific numbers of tachyzoites, with very low host cell contamination, producing antigen extracts of high quality. When antigens taken from cotton rat exudates were tested with a murine ELISA system, they were found to give superior diagnostic potential in comparison with the more commonly used mousederived antigens “. To determine whether cotton rat antigens could provide comparable results in a clinical situation, a human IgG ELISA was developed (CRELISA). The assay was tested with a panel of human patient sera supplied by the Public Health Service Toxoplasma Reference Laboratory in Leeds, and the results analysed and compared with commercial assays tested with the same sera. Methods Parasite production T. gondii RH strain tachyzoites

were maintained in adult cotton rats by serial passage. A dose of 5 x 107 tachyzoites were inoculated intraperitoneally and parasites were collected by peritoneal lavage in phosphate-buffered saline (PBS) 3-5 days later. Antigen preparation

Collected parasites were spun down at 25OOg for 15 min and resuspended in 0.83% NH&l solution for 10 min at 37” C to lyse red blood cells. The tachyzoites were then cleaned by two alternative washes with PBS and Hanks balanced salt solution at 3000g for 20 min. The pellets were resuspended in PBS, freeze-thawed three times at -50” C to room temperature (RT) and sonicated on ice for 20 cycles (10 s on, 1 min off). The preparation was spun down at 7500g for 20 min to remove particulate matter, assayed for protein concentration using a BCA protein assay (Pierce, UK), then aliquoted and stored at -50” C until use. Plate coating

Corning 96-well flat-bottom polystyrene ELISA plates (Corning, NY, USA) were used for all assays. The antigen preparation was defrosted and diluted to 4 pg ml-’ with antigen sensitization buffer (200 mM Trisbase buffer, 100 mM KCl, 20 mM ethylene diamine tetra-acetic acid (EDTA), pH 8.6). Each plate was coated with 100 pl of antigen solution per well for 3 h at RT, then washed twice with PBS plus 0.5% Tween 20 (PBST), air-dried and stored in sealed bags at 4” C.

patients referred for routine screening or diagnosis of toxoplasmosis. These sera had previously been tested for anti-toxoplasma antibodies using a modification of the Sabin-Feldman dye test” and in-house IHAT and IgM ELISA assays. Sera were supplied undiluted and heat inactivated at 56” C for 30 min and stored at -50” C until use. Sera were diluted with PBST plus 2% bovine serum albumin (PBST-BSA). The diluent was prepared, filtered through a 0.22 urn membrane and stored frozen in aliquots, to prevent contamination. Pooled positive and negative controls were used with each plate. Sera were diluted with 1 : 1000 with PBSTBSA, the optimum concentration by checkerboard titration. Four replicates of each test serum plus positive, negative and PBST controls were used in each CRELISA plate. Sera were incubated on an orbital shaker at RT for 1 h, washed three times with PBST, and the plates then incubated for 1 h with anti-human IgG antibody conjugated with horseradish peroxidase (Dako, Copenhagen, Denmark), diluted 1 : 2000 with PBST-BSA. The plates were then washed three times with PBST and lOO@ tetramethylbenzidine (TMB, Dynatech Ltd, Surrey, UK) was added to each well. The colour reaction was stopped at 3.5 min with 150 pl 2M HzS04, and the optical density (OD) at 450 nm was measured using a Titertek Multiskan plane reader (Flow Labs, Herts, UK). The mean and standard deviation for each serum was calculated. Sera with a coefficient of variation (CV, so/mean) greater than 20% were discarded. The mean OD for each serum was converted into enzyme immunoassay units (EIU) using values from the pooled control sera in each plate. EIU values were used to normalize OD values between plates, and were calculated as: mean (test OD) - mean (pooled negative control OD) x 100 mean (pooled positive control OD) - mean (pooled negative control OD) The cutoff point between positive and negative sera was established as 2~ mean of all dye test negative sera. The sensitivity and specificity of the CR-ELISA was calculated as previously describedig, as well as the positive and negative predictive values for the assay. Briefly, sensitivity was calculated as the number of test positives (Tp) divided by the number of Tp plus false negatives, and specificity as the number of test negatives (Tn) divided by the number of Tn plus false positives. The positive predictive value was calculated as Tp divided by Tp plus false positives and the negative predictive value as Tn divided by Tn plus false negatives. Results

Human serum testing and analysis

A panel of 414 human sera was supplied by the PHLS Toxoplasma Reference Laboratory in Leeds, from

The CR-ELISA was initially set up using pooled positive and negative sera, then evaluated with a panel of 414 human sera. The results of this evaluation were

Fraser

et al.: Development

of sensitive

ELBA

for diagnosis

of toxoplasmosis

161

Table 1. Analysis of CR-EL&A assay performance comparison with the Sabin-Feldman dye test

80 70 f

60

i

30:

Sensitivity % I

CR-ELISA

IIF=

PPV,

positive

Specificity %

96.5 predictive

98.1 value;

NPV,

negative

predictive

by

PPV %

NPV %

99.3

91.0

value

lo 1-A / ~I I I I I7 011 0

Figure

10 20

30 40

50 60

70

80

90 100 110 120130

140

1

a 100 100

1

;; II,,, 1

10

100

1000

4y.

t..t

10000

100000

111r..

b 180

7

D 0

/

1

,,‘:

,,,,,

,,,,,,,,

10 hrrm~eelutlnrtlon

Figure

I

100

,,,,,,,

,,,,,,,,

1000 rmry

(,,,

10000 tttrer

100000

2

expressed as relative EIU, compared with their dye test titres and expressed as a frequency diagram (Figure 1). The cutoff point between positive and negative values, calculated as double the mean of dye test negative samples, was found to be 60 EIU. The CR-ELISA showed a clear separation between

negatives and positives, with the majority of the negative sera falling between 2&30 EIU. There was a clear association between dye test titre and EIU value. A number of sera (26) gave EIU values within 8% of the cutoff, and were considered equivocal, requiring re-testing for definitive diagnosis. A small number of positive sera fell below the cutoff point, giving false negative results (10 out of 285). These sera were also found to have low dye test titres, predominantly below a titre of 128, indicating a low level of specific antibody. IgM results for some of these sera were available, and it was interesting to note that one false negative and five of the equivocal samples had high IgM titres, indicating early acute infection. The specific IgG values obtained with this panel of sera using the CR-ELISA were compared with the dye test and haemagglutination assay data (Figure 2). There was a strong correlation between dye test titre and EIU value (Figure 2a) although the graph tends to plateau at high dye test titres, in excess of 2000. Further titration of these high titre sera may reveal a continuing rise in EIU values. The panel of sera was also tested using an in-house indirect haemagglutination assay (Figure 2b). This assay tends to give higher titres than the dye test and was 100% accurate for this panel when compared with the dye test. Figure 2b indicates that there is a strong relationship between the results of the two assays, although the range of IHAT titres is narrower than the dye test. The diagnostic accuracy of the assay can be expressed in terms of sensitivity, specificity and predictive values (Table 1). Sensitivity indicates the probability that the assay will give a positive result if the serum is from an infected patient, and was calculated as 96.5% (275 out of 285). Specificity indicates the probability of a negative result if no infection is present, shown as 98.1% (101 out of 103). The reliability of the assay is best expressed in terms of predictive values. The positive predictive value was calculated as 99.3%, and the negative predictive value as 91.0%. Plate to plate variation was also analysed and the CV was found to be 7.95% (+ 1.15% SEM). Discussion

A sensitive indirect human IgG ELISA for toxoplasmosis was developed using antigen derived from cotton rat exudates, as opposed to the more commonly used mouse-derived antigen, to determine whether this source

162

Serodiagn.

Immunother.

Infect. Disease

1994; 6: No 3

of parasite material was suitable for use in future diagnostic assays. When tested with a panel of 414 human sera, the CR-ELISA was found to give very good diagnostic accuracy, with high sensitivity, specificity and positive predictive values (99.3%). The negative predictive value proved slightly lower, possibly due to the smaller number of negative sera tested. Comparison of the CR-ELISA results with the other assays is problematical due to the variation in established benchmarks. However, where assays have been calibrated against the dye test14.*0,*l, comparison is possible. The CR-ELISA results compare very favourably with the PHLS in-house IgG ELISA system14, with only small differences in the sensitivity and specificity, but with a higher positive predictive value (99.3% CR-ELISA - 94.7% PHLS ELISA). The CR-ELISA also demonstrates increased sensitivity over the magnetic particle IgG ELISA system developed by Konishi and Takahashi?O (96.5% as compared to 94.9%) and markedly improved sensitivity and specificity over the Toxocell latex haemagglutination assay system2’ (94% and 97% respectively). In addition to high diagnostic accuracy, the CR-ELISA system has a number of other advantages. Unlike the dye test, IHAT and ILAT systems, the assay can deal with a large number of samples per plate, and does not rely upon subjective assessment of serum titre. The assay also avoids the need for a high level of manipulation*“, or the use of specialized equipment, such as automated plate systems22,23. At present, however, there exists no single test which can give the accuracy of the dye test. It has been suggested that direct agglutination or indirect immunofluorescence are the most likely successors of the dye test’?, though these still require the use of large numbers of Toxoplasma tachyzoites and rely upon subjective estimation of titres. If the hazardous dye test is to be superseded, we suggest that it needs to be replaced by a number of sensitive assays used in conjunction. The combination of IgG and IgM ELISA systems could give sufficient accuracy, though from this study, correlation of IgG and IgM values resulted in only a minor increase in the accuracy of the assay. The resolution of discrepant sera gave the assay a sensitivity of 96.8% (from 96.5%) and a negative predictive value of 91.8% (from 91.0%). The use of a third assay, such as the IHAT, in conjunction with the CR-ELISA and IgM assay, may give a diagnostic accuracy indistinguishable from the dye test and a valuable indication of the stage of the infection. The use of antigens derived from cotton rats has been shown to improve assay performance by comparison with mouse antigen studies. It would give a reduction in the cost of antigen preparation, both in terms of quantities produced, as well as in the number of animals used and may warrant further investigation. Acknowledgements

This work was supported by a grant from the Science and Engineering Council, and by a CASE award from

Amersham International Ltd. (Kodak Clinical Diagnostics Division). We gratefully acknowledge the help and advice of Mark Jones and Colin Crouch of Kodak and are indebted to Debra Evans for her technical assistance.

References 1 JacksonMH, Hutchinson WM. The prevalence and sourceof toxoplasmainfection in the environment. Adv Parasitol 1989; 28: 55-10.5 2 Ko RC, Wong FW, Todd D, Lam KC. Prevalenceof Toxoplasma gondii antibodiesin the Chinese population of Hong Kong. Tram R Sot Trop Med Hyg 1980;74: 3.514 3 Beverley JKA, Beattie CP, RosemanC. Human toxoplasmainfection. .I Hyg 1954;52: 37-46 4 Remington JS, Efron B, CavanaughE, Simon HJ. Trejos A. Studieson toxoplasmosisin El Salvador via dye test. Tram R Sot Trop Med Hyg 1970;64: 252-67 5 Wright WH. A summary of the newer knowledge of toxoplasmosis.Am J Clin Path01 1957;28: l-17 6 Remington JS, DesmontsG. Toxoplasmosis.In: Remington JS, Klein JO eds. Znfectious diseases of the fetus and newborn infant. WB Saunders:Philadelphia, 1990;89-195 7 Luft BJ, Remington JS. Toxoplasmic encephalitisin AIDS. Clin If Dis 1992;15: 211-22 8 Ambroise-ThomasP, Pelloux H. Toxoplasmosiscongenital and in immunocompromisedpatients: a parallel. Parasitol Today 1993;2: 61-3 9 Sabin AB, Feldman HA. Dyes asmicrochemical indicators of new immunity phenomenonaffecting a protozoan parasite (Toxoplasma). Science 1948;1081 660-3 10 Feldman HA, Lamb GA. A micromodification of the Toxoplasma dye test. J Parasitol 1966;52: 415 11 Holliman RE. The diagnosisof toxoplasmosis. Serodiagn

Immunother

Infect Dis 1990; 4: 83-93

12 JossAWL. Diagnosis.In: Ho-Yen D, JossAWJ eds. Human toxoplasmosis. Oxford: Oxford Medical Publications1992;79-118 13 Sutehall GM, Wreghitt TG. Falsepositive latex tests negative by ELISA for toxoplasmaIgG. J Clin Pathol 1989;42: 204-5 14 FrancisJM, Payne RA, Joynson DH. Rapid indirect enzyme-linked immunosorbentassay(ELISA) for detecting anti-toxoplasmaIgG: comparisonwith dye test. J C/in Path01 1988;41: 802-5 15 van Loon AM, van der Veen J. Enzyme-linked immunosorbentassayfor the quantitation of toxoplasma antibodiesin human sera.J Clin Path01 1980;33: 635-9 16 Payne RA, Isaac M, Francis JM. Enzyme-linked immunosorbentassay(ELISA) usingantibody class capture for the detection of anti-toxoplasmaIgM. J Clirz Path01 1982; 35: 892-6

17 Fraser A, Smith JE. Toxoplasma gondii: a comparison of productivity of antigen sources,and efficacy in diagnosticassay.Serodiagn Immunother Infect Dis 1994;6: 98-102 18 Hunter DA, Chadwick P, Balfour AH, Bridges JB. Examination of ovine foetal fluid for antibodiesto T. gondii by the dye test and an indirect immunofluorescencetest specific for IgM. Br Vet J 1982; 138:29-34 19 Tenter AM, JohnsonAM. Use of Toxoplasma gondii recombinant antigensfor diagnosisand seroepidemiology.In: Smith JE ed. Toxoplasmosis. NATO ASI series.Springer Verlag: Berlin 1992;209-15

Fraser

et al.: Development

of sensitive

20 Konishi E, TakahashiJ. Reproducibleenzyme-linked 1983; 17: 22.5-31

21 Gray JJ, Balfour AH, Wreghitt TG. Evaluation of a commerciallatex agglutination test for detecting antibodies to Toxoplasma gondii. Serodiagn Immunother Infect Dis 1990;4: 335-40 22 Safford JW, Abbott GG, Craine MC, MacDonald RG.

of toxoplasmosis

163

Automated microparticle enzyme immunoassays for IgG and IgM antibodies to Toxoplasma ,qondii. J Clin

immunosorbentassaywith a magnetic particle processingsystemfor diagnosisof toxoplasmosis.J Clin Micro&o1

ELISA for diagnosis

Pathol 23

1988; 44: 23842

Schaeffer LE. Dyke JW, Meglio FD, Murray PR. Crafts W, Niles AC. Evaluation of microparticle enzyme immunoassaysfor IgG and IgM to rubella virus and Toxoplasma gondii on the Abbott IMx automated analyser.J Clin Micro&o/ 1989: 27: 2410-13