Evaluation of diagnostic tests for Trypanosoma evansi and their application in epidemiological studies in Indonesia

The diagnosis of Trypanosoma evansi infections is problematic because low, fluctuating parasitaemias are typical of sub-acute and chronic infections. Antigen-detection ELlSAs (Ag-ELISAs) that have been developed to detect trypanosomal antigens in serum are reported to be better indicators of current T. evansi infections than either parasitological or antibody-detection tests. Two T. evansi Ag-ELISAs based on different monoclonal antibodies (2G6 Ag-ELISA and Tr7 Ag-ELISA) were evaluated using buffaloes in Southeast Asia, where T. evansi is endemic and livestock are important for draught power, meat and investment. The two Ag-ELISAs were standardised in the UK, following international guidelines on data expression and quality assurance. Diagnostic sensitivities were estimated using buffaloes either experimentally infected (n=35) or naturally infected (n=l 39) with T. evansi and compared with estimates obtained for the microhaematocrit test (MHCT), mouse inoculation (MI), three antibody-detection tests (IgM ELISA, IgG ELISA and card agglutination test {CATT}). Diagnostic specificities were estimated with nonexposed British cattle (n=249) and Australian buffaloes (n=263), and positive and negative predictive values were calculated. Field studies were conducted in Central Java to estimate prevalence and true incidence rates of T. evansi infections in buffaloes. No previous studies have compared two T. evansi Ag-ELISAs, estimated the prevalence of T. evansi infections in multiple villages within a district or true incidence rates. The repeatability and robustness of the two Ag-ELISAs were shown to be high. Profiles of antigenaemia varied between individual buffaloes and between the two Ag-ELISAs. Antigen and antibody responses were first detected 7 to 42 days after infection, but in some buffaloes responses fluctuated below cut-off values during infection, whilst in other buffaloes antigen and antibody responses persisted after trypanocidal drug treatment. With the naturally-infected buffaloes, the diagnostic sensitivity estimate of the Tr7 Ag-ELISA (81%) was significantly higher than that of the 2G6 Ag-ELISA (71%), and the IgG ELISA sensitivity (89%) was significantly higher than either the IgM ELISA or CATT sensitivities (78%). The diagnostic specificity estimates obtained with the British cattle were 83% for the 2G6 Ag-ELISA and 78% for the Tr7 Ag-ELISA, and with the Australian buffaloes were 75% for the 2G6 Ag-ELISA, 78% for the Tr7 Ag-ELISA, 100% for the CATT, 89% for the IgM ELISA and 92% for the IgG ELISA. Only slight agreement was found between the two Ag-ELISAs (kappa = 0.20), but moderate agreement between the IgG ELISA and CATT (kappa = 0.58). Positive and negative predictive values ranged from 24% to 99% for prevalence values from 10% to 90%, and true prevalence was underestimated at higher test prevalence values and overestimated at lower test prevalence values. In Central Java, 2387 buffaloes were blood sampled in 59 villages, and estimates of test prevalence were 4% with the MHCT, 9% with MI, 58% with the 2G6 Ag-ELISA and 70% with the Tr7 Ag-ELISA, but prevalence values differed between districts and between villages. True incidence rates per animal-year at risk were 0.44 with the Tr7 Ag-ELISA and 0.22 with the 2G6 Ag-ELISA. Of 239 market buffaloes sampled, 10% were parasitaemic, 39% antigenaemic, 56% positive by IgG ELISA and 47% positive by CATT, representing an important source of T. evansi. The T. evansi Ag-ELISAs and antibody-detection tests used in this study have many advantages as screening tests over commonly used parasitological tests, in terms of their diagnostic sensitivity and ability to rapidly test large numbers of samples. The two T. evansi Ag-ELISAs could be applied in high prevalence areas, whilst antibody-detection tests (in particular, the IgG ELISA or CATT) would be more appropriate to test buffaloes in low prevalence areas or to confirm the negative-status of buffaloes prior to movement within Indonesia or export. Future work should aim to improve the specificities of the Ag-ELISAs, which were low in this study in contrast to previous reports. The CATT had a high positive predictive value even with low prevalence and could be adapted more readily to test individual buffaloes in the field. The selection of diagnostic tests for T. evansi depends not only on test validity parameters, but also on the prevalence of T. evansi in the test population, the principal testing objectives and practical considerations.