Full title: An update on FIV and FeLV test performance using a Bayesian statistical approach

Full title: An update on FIV and FeLV test performance using a Bayesian statistical approach

Short title: FIV FeLV Bayesian

M.D.G. Pinches, G. Diesel, C. R. Helps, S. Tasker, K. Egan, T.J. Gruffydd-Jones

Mark D. G. Pinches BVSc MSc MRCVS

Gillian Diesel * BVSc MSc MRCVS

Christopher R. Helps BSc PhD

Sverine Tasker BVSc BSc PhD DSAM DipECVIM-CA MRCVS

Kathy Egan ART (Can)

Tim J Gruffydd-Jones BvetMed PhD DipECVIM-CA MRCVSUniversity of Bristol, Department of Clinical Veterinary Science, Langford House, Langford, BS40 5DU, UK

* The Royal Veterinary College, University of London, Hawkshead Lane, Hertfordshire, AL9 7TA

Keywords: FIV, FeLV, Bayesian analysis

Abstract

Background:

Screening tests for feline retroviruses are thought to have high sensitivity and specificity although previous studies that have evaluated these tests are slightly limited. Novel statistical approaches have been developed that allow estimation of sensitivity and specificity in situations where the true state of disease in individual animals cannot be assured.

Objective:

The purpose of this study was to evaluate the sensitivity and specificity of a variety of retrovirus tests, including some screening tests, in a population of cats potentially infected with either FeLV and/or FIV using a Bayesian statistical approach.

Methods

Four hundred and ninety blood samples from cats being evaluated for FIV infection were tested by two rapid immunomigration tests (Witness Single (WS), Witness Combi (WC); Synbiotics) and a plate based ELISA (Petcheck, IDEXX) for FIV antibody and by a newly designed real time PCR assay for FIV provirus. Four hundred and eighty four blood samples from cats being evaluated for FeLV infection were tested by two rapid immunomigration tests (Witness Single (WS), Witness Combi (WC); Synbiotics) and a plate based ELISA (Petcheck, IDEXX) for FeLV antigen and by a FeLV virus isolation technique. Results were then analysed using a Bayesian statistical method.

ResultsFor FIV tests, median sensitivity estimates were 0.98 for WS, 0.97 for WC, 0.98 for ELISA, and 0.92 for PCR. Median specificity estimates were for 0.96 for WS, 0.96 for WC, 0.93 for ELISA, and 0.99 for PCR. For FeLV tests median sensitivity estimates were 0.97 for WS, 0.97 for WC, 0.98 for ELISA, and 0.91 for VI. Median specificity estimates were 0.96 for WS, 0.96 for WC, 0.98 for ELISA, and 0.99 for VI.

Conclusions

The use of Bayesian statistical methods overcomes a variety of methodological problems associated with diagnostic test evaluations including lack of definitive reference test. The sensitivity and specificity of all six evaluated screening tests is high, however specificity estimates were slightly lower.

Introduction

Feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) are important retroviruses of cats. Accurate diagnosis of these diseases is important both for the diagnosis of retrovirus related disease and for the identification of infected cats in the control of both infections. A variety of different types of diagnostic tests are available including: rapid screening tests for antigen or antibody in plasma; virus isolation (VI) to detect infectious virus in the plasma; and a number of polymerase chain reaction (PCR) assays which detect proviral DNA in the blood. Immunoassays of differing types are most widely used in practice and these tests offer the advantage of speed and convenience. However, the information available about their performance is limited and somewhat conflicting 1,2,3. The previous studies that have evaluated FeLV and FIV immunoassay performance are limited to a degree, as all sample naturally infected populations, where true disease status is unknown. Further these studies also reference test only samples with positive results. Such an approach introduces selection bias and introduces some error into test performance calculations in particular estimates of test specificity 4. Also, the choice of reference test in retrovirus diagnosis is not without its difficulties, as there are questions as to which tests can be considered suitable as a reference test for infections in which virus integrates into the host genome. Virus isolation for example, the usual reference test for FeLV, has drawbacks as although this test carries a high specificity its sensitivity is potentially reduced in some situations 5,6,7,8,9,10. Often therefore, this makes it impossible to unambiguously state the disease status of tested animals 11. Thus misclassification of samples as true positive or true negative by the reference tests inevitably leads to errors in the sensitivity and specificity estimations of the evaluated tests 12,13 .However, there exist a variety of alternate statistical approaches which have been developed that allow estimation of test sensitivity and specificity when no definitive reference test is available 12,13,14 . One such approach is based upon Bayess theorem, where error probabilities (prior distributions) based upon previous knowledge of the evaluated tests, reference test and prevalence are introduced into the analysis. Using new data (likelihood) estimates of test sensitivity, test specificity and prevalence are updated (posterior distributions) using a Markov-chain Monte Carlo simulation. In essence this allows estimates of tests sensitivity and specificity in situations where analysis by traditional methods would have led to error 4,13,14 .

In this study, Bayesian analysis, a non gold standard based statistical approach to the test evaluation, is applied to the evaluation of a variety of tests for FeLV and FIV.

Materials and methods

The samples used in this study were consecutive diagnostic samples submitted to Langford Veterinary Diagnostics Laboratories by veterinarians for FIV and/or FeLV testing between September 2002 and June 2004. These were whole blood EDTA and/or heparin samples for which there was sufficient sample volume to perform all tests. Only samples from animals over 20 weeks of age were included in the study, to avoid problems related to the persistence of maternally derived antibody. All samples were split into two aliquots at submission. Plasma was used in the immunoassays whilst whole blood was used for FeLV Virus isolation (VI) and/or in the provirus FIV polymerase chain reaction assay (FIV PCR).

FIV

The FIV immunoassays that were evaluated were two rapid immunomigration (RI) tests, Witness single (WS) FIV (Witness; Synbiotics, distributed by Woodley

Equipment, Horwich, UK), Witness combi (WC) FIV/FeLV (Witness; Synbiotics) and a plate based FIV ELISA test (PetChek FIV, IDEXX Laboratories, Wrrstadt, Germany). A newly developed real-time provirus FIV polymerase chain reaction (FIV PCR) test was also used.

Four hundred and ninety samples were tested at submission by both RI tests and the ELISA according to the manufacturers instructions. Positive results were recorded when the intensity of colour change by visual assessment equalled or exceeded that of the positive control. Samples which gave colour change that was not as intense as the positive control were read by plate reader and results were recorded as positive if colour intensity was >50% and equivocal if colour intensity was