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. Author manuscript; available in PMC: 2015 Nov 12.
Published in final edited form as: J Med Entomol. 2012 Nov;49(6):1442–1452. doi: 10.1603/me12071

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.

a

Sequencing 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.

b

We 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.