Comparison of reverse line blot and β-tubulin targeted nested pcr techniques in the detection of tick-borne heamoparasites of ruminants
Atuhaire, David Kalenzi
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In this study, Reverse Line Blot assay and Beta-tubulin targeted nested PCR were compared in detection of tick-borne piroplasms in areas around L.Mburo in Western Uganda. A total of 300 blood samples were collected from apparently normal cattle and examined for intra-erythrocytic forms of Theileria and Babesia parasites by Microscopy out of which 120 (40%) were positive for Theileria piroplasms. Eighty positive and 40 negative samples by microscopy were selected for comparison of the two tests. Out of the 80 samples positive by microscopy, 57 (71.25%) were positive for the individual species and 23 (28.75%) were negative by the RLB assay while 50 (62.5%) tested positive and 30 (37.5%) tested negative by beta-tubulin targeted nested PCR. Of the 57 samples positive by RLB assay, 8 (14.04%) were infected by Theileria mutans only, 47 (82.46%) were simultaneously infected with Theileria parva and Theileria spp (buffalo) but not with any other species, while one was simultaneously infected with Theileria parva, Theileria spp (buffalo) and Babesia bovis. Out of the 40 samples testing negative by microscopy, 17 (42.5%) samples were positive and 23 (57.5%) were negative by RLB assay while 11 (27.5%) were positive and 29 (72.5%) were negative by beta-tubulin nested PCR analysis. Eleven (64.7%) of the positive samples had mixed infection, while 6 (35.3%) had single infections. The RLB assay allowed the detection of individual species that simultaneously infected the cattle. However, it was not possible to identify the species with the beta-tubulin targeted nested PCR. The sensitivity of RLB technique for the detection of Theileria and Babesia spp was 71.25% (95% CI: 60.05 – 80.82%) and the specificity was 57.50% (95% CI: 40.89 - 72.96%) while the sensitivity of the beta-tubulin targeted nested PCR was 62.5% (95% CI: 50.96 – 73.08%) and the specificity was 72.5% (95% CI: 56.11 – 85.40). The Positive Predictive Value using RLB was 77.03 % and the Negative Predictive Value was 50 % while the Positive Predictive Value using beta-tubulin targeted nested PCR was 81.97 % and the Negative Predictive Value was 49.15%. The Kappa statistic for level of agreement in detection of tick-borne piroplasms between RLB assay and beta-tubulin targeted nested PCR was 0.7984 which indicated substantial agreement between the two tests. Sixty two of the samples positive by RLB were positive by beta-tubulin targeted nested PCR and 12 were negative, while none of the samples negative by RLB was detected as positive by beta-tubulin targeted nested PCR. The 46 samples negative by RLB were also negative by beta-tubulin targeted nested PCR. Although microscopy remains the most appropriate method for diagnosis in the field setting, its use as a gold standard may result into false negative result as observed in this study.