Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Surveillance of African Trypanosomiasis in Eastern Zambia

African animal trypanosomiasis (AAT) control programs rely on active case detection through the screening of animals reared in disease endemic areas. This study compared the application of the polymerase chain reaction (PCR) and microscopy in the detection of trypanosomes in cattle blood in Mambwe, a rural district in eastern Zambia. Blood samples were collected from 227 cattle and tested for infection with trypanosomes using microscopy and Ribosomal RNA Internal Transcribed Spacers (ITS)-PCR. Microscopy on the buffy coat detected 17 cases, whilst thin and thick smears detected 26 cases and 28 cases, respectively. In total, microscopy detected 40 cases. ITS-PCR-filter paper (FP) on blood spots stored on FP detected 47 cases, and ITS-PCR-FTA on blood spots stored on Whatman FTA Classic cards detected 83 cases. Using microscopy as the gold standard, ITS-PCR-FTA had a better specificity (SP) and sensitivity (SE) (SP = 72.2%; SE = 77.5%; kappa = 0.35) than ITS-PCR-FP (SP = 88%; SE = 60%; kappa = 0.45). The prevalence of Trypanosoma brucei s.l. was higher on ITS-PCR-FTA (19/227) than on ITS-PCR-FP (0/227). Our results illustrate the complexities around trypanosomiasis surveillance in rural Africa and provide evidence of the impact that field conditions and staff training can have on diagnostic results, which in turn impact the success of tsetse and trypanosomiasis control programs in the region.