Table 1.

Assay performance using Ct cutoff values between 34 and 40

Ct cutoff value	Se (%): RF (n = 50 fleas)	Se (%): HF (n = 50 fleas)	Sp (%)(n = 50 fleas)	PPV (%):RF vs UF	PPV (%):HF vs UF	% NTCs with no detectable vertebrate DNA (n = 110 wells)
40	84	90	88	100	≤88.2a	≥52.7b
39	84	90	90	100	≤90.0a	≥66.4b
38	84	90	90	100	≤90.0a	≥80.9b
37	78	76	92	100	≤90.5a	≥95.5b
36	74	64	94	100	91.4	100
35	72	40	96	100	90.9	100
34	68	20	96	100	83.3	100

Se, sensitivity, the percentage of rat blood-fed (RF) fleas or human blood-fed (HF) fleas with a detectable vertebrate blood meal; Sp, specificity, the percentage of unfed fleas without detectable vertebrate DNA; PPV, positive predictive value, the percentage of fleas with detectable rat DNA that had consumed rat blood (RF vs unfed [UF]) or the percentage of fleas with detectable human DNA that consumed human blood (HF vs UF); NTC, no template control.

^aSequencing indicated that all unfed flea DNA isolates with Ct ≤ 40 contained human DNA. We sequenced amplicons from fed flea samples only if they had a Ct value ≤ 36. We calculated PPV assuming that all rat blood-fed fleas and human blood-fed fleas with a detectable vertebrate blood meal and a Ct value >36 contained rat or human DNA, respectively. The true PPV for with Ct cutoff values > 36 could therefore be lower.

^bWe did not sequence amplicons from NTCs with Ct > 36. Sequencing could lead us to re-classify some false-positive NTCs as negative, increasing the percent of NTCs with no detectable vertebrate DNA.