Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for the detection of canine Leishmania infection

1 There is a need for standardization and simplification of the existing methods for molecular 2 detection of Leishmania infantum in the canine reservoir host. The commercially available 3 OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic 4 dipstick detection of PCR products, and is highly sensitive in humans, but not yet 5 independently validated in dogs. Here we compare the sensitivity of the OligoC-TesT with 6 that of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer-1 (ITS-1) 7 PCR, and a PCR/hybridization protocol, in longitudinal naturally infected canine bone 8 marrow samples for which parasite burdens were measured by real-time quantitative PCR 9 (qPCR). Sensitivity of the OligoC-TesT in infected dogs was 70% (95% C.I. 63-78%), 10 similar to that of kDNA PCR (72%; 95% C.I. 65-80%; P=0.69), but significantly greater than 11 PCR/hybridization (61%; 95% C.I. 53-69%; P=0.007) and ITS-1 nested PCR (54%; 95% C.I. 12 45-62%; P<0.001); real-time qPCR had the highest sensitivity (91%; 95% C.I. 85-95%; 13 P<0.001). OligoC-TesT sensitivity was greater in polysymptomatic and oligosymptomatic 14 dogs compared to asymptomatic dogs (93%, 74%, and 61%, respectively; P=0.005), a trend 15 also observed in the other qualitative PCR methods tested (P≤0.05). Test positivity increased 16 with increasing parasite burden measured by real-time qPCR: OligoC-TesT and kDNA PCR 17 detected 100% and 99% of positive samples when parasite burden exceeded 74 and 49 18 parasites/ml, respectively. The OligoC-TesT has high sensitivity for detection of canine 19 Leishmania infections; ease of operation and interpretation are further advantages for 20 veterinary diagnostic laboratories, and for large-scale survey work in developing countries. 21