Validation of fluorescence polarization assay (FPA) and comparison with other tests used for diagnosis of B. melitensis infection in sheep

doi:10.1016/j.vetmic.2005.10.009

Veterinary Microbiology

Volume 111, Issues 3–4, 20 December 2005, Pages 211-221

https://doi.org/10.1016/j.vetmic.2005.10.009 Get rights and content

Abstract

Fluorescence polarization assay (FPA) is a new test for the serological diagnosis of Brucella spp. infection in animals. The FPA is validated for the diagnosis of B. melitensis infection in sheep. For this purpose, 166 sera originated from natural infected sheep (verified by culture) and 851 sera originated from healthy animals (reared in areas where B. melitensis was never been isolated) were tested. The optimum cut-off value that offers the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 87 mP with the use of ROC analysis. The DSn and DSp of FPA using this cut-off value are calculated at 97.6 and 98.9% with a 95% confidence interval (CI) of 93.9–99.3% and 98.0–99.5%, respectively.

The DSn and DSp of FPA have been assessed also using as positive reference (n = 587), sera that gave positive results at least in two tests used for diagnosis of B. melitensis in sheep as Rose Bengal Test (RBT), modified Rose Bengal Test (m-RBT), complement fixation test (CFT), indirect Elisa (i-Elisa) and competition Elisa (c-Elisa) originated from animals reared in flocks infected by B. melitensis. The optimum cut-off value using the above panel of positive reference sera was the same offering a DSn of 95.9% with a 95% CI, 94.0–97.4%, since the DSp remains the same. The DSn and DSp as well as performance, accuracy and agreement of FPA's result were compared with those of other tests used. The accuracy of FPA is very high, similar with that of i-Elisa.

FPA is a promising assay, which offers a DSn and accuracy better that of those of the tests currently approved for the diagnosis of B. melitensis in sheep and goats. Due to its simplicity, the sort time that results can be obtained and its accuracy it can be used and improve the laboratory testing capacity as well as the efficacy of the eradication program based on test-and-slaughter policy.

Introduction

Brucellosis in sheep and goats, caused by B. melitensis, one of the most virulent species of Brucella, is responsible for important economic losses in sheep and goats farming. It is also a widespread zoonosis constituting a serious public health problem wherever the infectious agent is endemic, like in Mediterranean and Middle-East countries (Nicoletti, 1989, Corbel, 1989).

Diagnosis of Brucella spp. infection is mainly based on the detection of antibodies in serum by serological tests. The Rose Bengal Test (RBT) (Davies, 1971) and complement fixation test (CFT) (Alton et al., 1988) are the most accepted tests worldwide for this purpose (Garin-Bastuji and Blasco, 1997) and the only approved for certification of sheep and goats flocks due to brucellosis status in EU member states (ECD, 1991).

The RBT, due to its low sensitivity on sheep and goats sera, is suggested to be used only for identification of infected flocks (flock screening test) and not for individual animals (Blasco et al., 1994). Since CFT is regarded as more sensitive and specific, is used for individual testing of animals in infected flocks as well as confirmatory test (ECD, 1991; Nicoletti, 1969, MacMillan, 1990).

Although both tests when used singly or in combination (serial or parallel) are very effective as flock screening tests, they detect only 70% of the infected animals when these are tested individually. This makes the implementation of test-and-slaughter policy for brucellosis eradication in small ruminants not very effective (Nicoletti, 1969).

It is accepted that for serological diagnosis of brucellosis to be improved there is a need of other tests to be used more sensitive than the currently used (Blasco et al., 1994). For this purpose, indirect Elisa (i-Elisa), fluorescence polarization assay (FPA) and competition Elisa (c-Elisa) have been developed, which ability of identifying infected animals appears superior to RBT and CFT (Jaques et al., 1998, Nielsen and Gall, 2001).

The aim of the present study is the validation of FPA for the diagnosis of B. melitensis infection in sheep with the determination of cut-off offering the highest performance index (diagnostic sensitivity (DSn) + diagnostic specificity (DSp)) and the comparison of its performance with that of the other tests used for this purpose.

The study has been focused on FPA, since it is a relative new diagnostic test for brucellosis and there is limited published information about its performance in sheep sera (Nielsen et al., 2001, Nielsen et al., 2005). Additionally, due to its simplicity, the speed that results obtained and their objective interpretation, it can contribute significantly to improvement of eradication's program efficacy if its performance is superior to those of the tests used at the present.

Section snippets

Serological tests

All sera used in the study were tested with RBT, a modification of RBT (m-RBT), CFT, i-Elisa, c-Elisa and FPA in parallel.

FPA was conducted as it is described by Nielsen et al. (2001). Briefly, 1.0 ml of 0.01 M Tris buffer, pH 7.2, containing 0.15 M sodium chloride, 15 mM EDTA and 0.05% Igepal A-630, was placed in a 10 mm × 75 mm glass tube followed by 10 μl of serum. After mixing, a background reading was taken with the use of a portable Sentry Fluorescence polarization analyser (FPM Sentry, Diachemix

Results

The cut-off for FPA using as positive reference sera those originated from culture positive animals determined by ROC analysis at 87 mP. Any serum showing a value of exactly 87 mP units is considered as negative. The ROC diagram is presented in Fig. 1. The DSn and DSp of FPA using this cut-off determined at 97.6 and 98.9% with a 95% CI of 93.9–99.3% and 98.0–99.5%, respectively. The AUC calculated at 0.998 with a 95% standard error (S.E.) of 0.002 with positive and negative likehood ratio (+LR)

Discussion

The optimum cut-off value for FPA offering the highest “performance index” (sum of DSn and DSp) was determined by ROC analysis two ways, one using as positive reference, sera originated from culture positive animals (166 sera) and another using as positive reference, sera from animals reared in naturally infected flocks showing positive results simultaneously in two different tests conducted in parallel. Using the two different panels of positive reference sera, the accuracies of all tests were

Conclusions

FPA is a diagnostic test that can be performed easily and has many advantages compared with the other methods used for serological diagnosis of brucellosis in small ruminants. The results are obtained in a sort period of time, less than 5 min, the instrument calculates the mP value automatically and the interpretation is not subjective, anticomplementary or prozone effects are not observed and hemolysed sera can be tested with the same degree of accuracy. The test can be easily automated which

Acknowledgements

This study was supported in part by Diachemix Corp., Wisconsin, USA, which provided the antigens for the implementation of FPA.

References (26)

M. Greiner et al.
Epidemiologic issues in the validation of veterinary diagnostic tests
Prev. Vet. Med.
(2000)
M. Greiner et al.
Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests
Prev. Vet. Med.
(2000)
M. Lin et al.
Binding of Brucella abortus lipopolysaccharide O-chain fragment to a monoclonal anibody
Biol. Chem.
(1997)
K. Nielsen et al.
Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use
Vet. Microbiol.
(2001)
K. Nielsen et al.
Evaluation of serological tests for detection of caprine antibody to Brucella melitensis
Small Rumin. Res.
(2005)
F. Schoonjans et al.
Medcalc: a new computer program for medical statistics
Comp. Meth. Prog. Biomed.
(1995)
G.G. Alton et al.
Techniques for the Brucellosis Laboratory
(1988)
P. Armitage et al.
Statistical Methods in Medical Research
(1994)
J.M. Blasco et al.
Efficacy of different Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats
Vet. Rec.
(1994)
M.J. Corbel
Brucellosis epidemiology and prevalence worldwide

G. Davies

The Rose Bengal Test

Vet. Rec.

(1971)

European Council Directive, 1964. 64/432/EEC. On health problems affecting intra-community trade in bovine animals and...

European Council Directive, 1991. 91/68/EEC. On animal health conditions governing intra-community trade in ovine and...

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Brucellosis is a worldwide extended zoonosis with a heavy economic and public health impact. Cattle, sheep and goats are infected by smooth Brucella abortus and Brucella melitensis, and represent a common source of the human disease. Brucellosis diagnosis in these animals is largely based on detection of a specific immunoresponse. We review here the immunological tests used for the diagnosis of cattle brucellosis. First, we discuss how the diagnostic sensitivity (DSe) and specificity (DSp), balance should be adjusted for brucellosis diagnosis, and the difficulties that brucellosis tests specifically present for the estimation of DSe/DSp in frequentistic (gold standard) and Bayesian analyses. Then, we present a systematic review (PubMed, GoogleScholar and CABdirect) of works (154 out of 991; years 1960–August 2017) identified (by title and Abstract content) as DSe and DSp studies of smooth lipopolysaccharide, O-polysaccharide-core, native hapten and protein diagnostic tests. We summarize data of gold standard studies (n = 23) complying with strict inclusion and exclusion criteria with regards to test methodology and definition of the animals studied (infected and S19 or RB51 vaccinated cattle, and Brucella-free cattle affected or not by false positive serological reactions). We also discuss some studies (smooth lipopolysaccharide tests, protein antibody and delayed type hypersensitivity [skin] tests) that do not meet the criteria and yet fill some of the gaps in information. We review Bayesian studies (n = 5) and report that in most cases priors and assumptions on conditional dependence/independence are not coherent with the variable serological picture of the disease in different epidemiological scenarios and the bases (antigen, isotype and immunoglobulin properties involved) of brucellosis tests, practical experience and the results of gold standard studies. We conclude that very useful lipopolysaccharide (buffered plate antigen and indirect ELISA) and native hapten polysaccharide and soluble protein tests exist, provided they are applied taking into account the means available and the epidemiological contexts of this disease: i) mass vaccination; ii) elimination based on vaccination combined with test-and-slaughter; and iii) surveillance and existence of false positive serological reactions. We also conclude that the insistence in recent literature on the lack of usefulness of all smooth lipopolysaccharide or native hapten polysaccharide tests in areas where S19 vaccination is implemented is a misinterpretation that overlooks scientific and practical evidence.
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Citation Excerpt :
Theoretically hemolysis is not a problem and FPA can also be applied to blood or milk provided they are free from bacteria or other particulate matter that scatters light (Minas et al., 2005), including lipaemia (Lucero et al., 2003), and can be adapted to microtiter plates (Nielsen and Gall, 2001). Readings are obtained in a few seconds or minutes but, as auto-fluorescence increases with time, they are not completely stable (Nielsen and Gall, 2001), and the test is temperature-sensitive (Minas et al., 2005). These technical and biological variables explain why each labeled core-O-polysaccharide batch needs to be standardized in a fluorescence polarization analyzer and why FPA needs to be calibrated locally.
Bacteria of the genus Brucella cause brucellosis, the most common bacterial zoonosis worldwide. The diagnosis of Brucella abortus and Brucella melitensis ruminant brucellosis is based on bacteriological and immunological tests, the latter being routinely used in control and eradication and surveillance programs. Infections by smooth and rough Brucella spp., the use of smooth and rough vaccines, and the false-positive serological reactions caused by Yersinia enterocolitica O:9 and other cross-reacting bacteria represent the immunological contexts in which those tests are used. This complex context explains the large number of brucellosis tests that have been developed, and that vary in antigen type, antigen presentation, antibody and conditions for the reaction, the response detected and the sample required. This wealth of information and an imperfect understanding of Brucella antigens and of the peculiarities of the immunoresponse to Brucella has created confusion and led to several misconceptions on the usefulness and limitations of the brucellosis diagnostic tests. In this review, Brucella antigens are examined focusing on cellular topology, supramolecular properties, epitopic structure and lipopolysaccharide and protein cross-reactivity in the various contexts of the immune response in ruminants. Then, the significance of these features in diagnostic tests that use whole bacteria is discussed with respect to the activities of ruminant immunoglobulins, and the effect of pH on unspecific agglutinations, non-agglutinating and blocking antibodies, pseudo-prozones and complement activation. Similarly, the bacterial surface lipopolysaccharides and cognate polysaccharides are discussed with regards to topological effects, epitope exposure, ionic strength and antibody avidity in immunoprecipitation, immunosorbent and fluorescence polarization assays. Finally, the search for immunodominant protein antigens and their use in immunological tests is reviewed. Critical review of the existing information is necessary both to select optimal tests according to the logistical means available and the epidemiological context, and to focus the development of new tests.
Field evaluation of fluorescence polarization assay, and comparison with competitive ELISA for the detection of antibodies against Brucella melitensis in sheep in Sicily, Italy
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Citation Excerpt :
It was, subsequently, used for the diagnosis of brucellosis in goats and sheep, where Se and Sp were estimated as >95%. FPA was, therefore, judged as a promising diagnostic test, and further, field validation was suggested, especially in those areas where eradication of brucellosis in small ruminants is still to be achieved (Nielsen and Gall, 2001; Burriel et al., 2004; Minas et al., 2005, 2007). The objective of this study was to estimate accuracy of FPA by using a sample of sera from sheep populations from Southern Italy, where B. melitensis eradication and surveillance are currently implemented, and to obtain indications on the usefulness of FPA to increase Se and Sp of the testing procedure within ovine and caprine eradication programs.
The relative sensitivity of the fluorescence polarization assay (FPA) was estimated by testing blood sera from 1515 sheep, belonging to flocks infected with Brucella melitensis in Sicily, which were previously shown as positive to at least two of the following tests: rose bengal test (RBT), complement fixation test (CFT), competitive ELISA (c-ELISA). FPA specificity was estimated by testing sera from 719 sheep from B. melitensis-free flocks from Sicily and Sardinia. Based upon results of a receiver-operating-characteristic (ROC) curve, optimal FPA cut-off value in millipolarization units (mP) was chosen at the mean of negative controls tested in the same assay, plus 6, which corresponded to 96.0% sensitivity (95% confidence interval: 93.9, 97.3) and 100% (99.5, 100) specificity. Such a cut off value was the one yielding the maximum sum of sensitivity and specificity (196.0), and it was adopted for subsequent analyses. Maximum FPA sensitivity (98.8%; 91.7, 99.8) was obtained on sera which were positive to CFT and c-ELISA, but negative to RBT. Median FPA values (first, and third quartiles) (109.7, 53.7, 169.3) were greatest for sera which were positive to RBT, CFT, and c-ELISA. Sensitivity of c-ELISA, which was estimated as the proportion of positive results on RBT and CFT positive sera, was 99.8% (99.4, 100.0), whereas specificity was 100.0% (98.5, 100.0). Although FPA resulted as an accurate test, c-ELISA was characterized by a superior sensitivity and it is, therefore, proposed to be used, in parallel with RBT, to increase the testing sensitivity in regions and flocks which can be considered as at a great risk of infection.
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Swine brucellosis caused by Brucella suis biovar 2 is an emerging disease in Europe. Currently used diagnostic tests for swine brucellosis detect antibodies to the O-polysaccharide (O-PS) of Brucella smooth lipopolysaccharide (S-LPS) but their specificity is compromised by false-positive serological reactions (FPSRs) when bacteria carrying cross-reacting O-PS infect pigs. FPSRs occur throughout Europe, and the only tool available for a specific B. suis diagnosis is the intradermal test with Brucella protein extracts free of O-PS or S-LPS. Using sera of 162 sows naturally infected by B. suis biovar 2, 406 brucellosis-free sows, and 218 pigs of brucellosis-free farms affected by FPSR, we assessed the diagnostic performance of an indirect ELISA with rough LPS (thus devoid of O-PS) and of gel immunodiffusion, counterimmunoelectrophoresis, latex agglutination and indirect ELISA with O-PS free proteins in comparison with several S-LPS tests (Rose Bengal, complement fixation, gel immunodiffusion and indirect ELISA). When adjusted to 100% specificity, the sensitivity of the rough LPS ELISA was very low (30%), and adoption of other cut-offs resulted in poor specificity/sensitivity ratios. Although their specificity was 100%, the sensitivity of protein tests (ELISA, latex agglutination, counterimmunoelectrophoresis, and gel immunodiffusion) was only moderate (45, 58, 61 and 63%, respectively). Among S-LPS tests, gel immunodiffusion was the only test showing acceptable sensitivity/specificity (68 and 100%, respectively). Despite these shortcomings, and when the purpose is to screen out FPSR at herd level, gel immunodiffusion tests may offer a technically simple and practical alternative to intradermal testing.
Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep
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Citation Excerpt :
However, there is evidence that both tests are less sensitive and specific for the diagnosis of brucellosis in sheep and goats than in cattle (Blasco et al., 1994; Garin-Bastuji et al., 1998). Efforts to improve the serological detection of brucellosis in small ruminants have led to development of new assays, including indirect ELISAs (iELISAs), competitive ELISAs (cELISAs), the modified Rose Bengal test (MRBT) and the fluorescence polarisation assay (FPA) (Blasco et al., 1994; Díaz-Aparicio et al., 1994; Marín et al., 1999; Nielsen et al., 2004a; Minas et al., 2005, 2008; Garin-Bastuji et al., 2006). Although the sensitivity (Se) of most of these tests has been demonstrated as being equivalent to the EU-approved tests, their inclusion has not been accepted, because their specificity (Sp) has not been sufficiently documented under field conditions (EFSA, 2006).
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The true prevalence of brucellosis and diagnostic test characteristics of three conditionally dependent serological tests were estimated using the Bayesian approach in goats and sheep populations of Bangladesh. Serum samples from a random selection of 636 goats and 1044 sheep were tested in parallel by indirect ELISA (iELISA), Rose Bengal Test (RBT) and Slow Agglutination Test (SAT). The true prevalence of brucellosis in goats and sheep were estimated as 1% (95% credibility interval (CrI): 0.7–1.8) and 1.2% (95% CrI: 0.6–2.2) respectively. The sensitivity of iELISA was 92.9% in goats and 92.0% in sheep with corresponding specificities of 96.5% and 99.5% respectively. The sensitivity and specificity estimates of RBT were 80.2% and 99.6% in goats and 82.8% and 98.3% in sheep. The sensitivity and specificity of SAT were 57.1% and 99.3% in goats and 72.0% and 98.6% in sheep. In this study, three conditionally dependent serological tests for the diagnosis of small ruminant brucellosis in Bangladesh were validated. Considerable conditional dependence between IELISA and RBT and between RBT and SAT was observed among sheep. The influence of the priors on the model fit and estimated parameter values was checked using sensitivity analysis. In multiple test validation, conditional dependence should not be ignored when the tests are in fact conditionally dependent.

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Veterinary Microbiology

Abstract

Introduction

Section snippets

Serological tests

Results

Discussion

Conclusions

Acknowledgements

References (26)

Epidemiologic issues in the validation of veterinary diagnostic tests

Prev. Vet. Med.

Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests

Prev. Vet. Med.

Binding of Brucella abortus lipopolysaccharide O-chain fragment to a monoclonal anibody

Biol. Chem.

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use

Vet. Microbiol.

Evaluation of serological tests for detection of caprine antibody to Brucella melitensis

Small Rumin. Res.

Medcalc: a new computer program for medical statistics

Comp. Meth. Prog. Biomed.

Techniques for the Brucellosis Laboratory

Statistical Methods in Medical Research

Efficacy of different Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats

Vet. Rec.

Brucellosis epidemiology and prevalence worldwide

The Rose Bengal Test

Vet. Rec.

Cited by (25)

A systematic review of current immunological tests for the diagnosis of cattle brucellosis

A review of the basis of the immunological diagnosis of ruminant brucellosis

Field evaluation of fluorescence polarization assay, and comparison with competitive ELISA for the detection of antibodies against Brucella melitensis in sheep in Sicily, Italy

Diagnostic performance of serological tests for swine brucellosis in the presence of false positive serological reactions

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep

Bayesian estimation of true prevalence, sensitivity and specificity of indirect ELISA, Rose Bengal Test and Slow Agglutination Test for the diagnosis of brucellosis in sheep and goats in Bangladesh