Figure 3. Determination of the ΔCt cutoff.

(A) Selection of purulent sputum. Sputum was classified by its gross appearance, with 50 samples studied for each classification. Purulent sputum had a Ct_human <27 (>7×10³ human cells/µL of sputum; Figure 2B ). Samples with M2–P3 appearance as well as a Ct_human <27 (enclosed by a dotted line) were studied further. Classification of the gross appearance of the sputum (M1, M2, P1, P2, and P3) are according to Miller and Jones [10]. (B) Determination of the ΔCt cutoff. ΔCt_pathogen was measured for 4 representative commensal organisms (n = 223). Samples from patients with pneumonia in which a likely causative pathogen was identified using criteria (1)–(4) (see Methods) are shown as blue circles, and samples from patients with pneumonia in which none of criteria (1) – (4) was fulfilled were shown as white circles. The ΔCt_pathogen cutoff (a red line) was defined as the smallest ΔCt_pathogen for the blue circles. Sputum in which the pathogen was not detected and thus ΔCt_pathogen was not assigned is shown at the bottom (labeled as “Not detected”). (C) Reproducibility of ΔCt_pathogen measurements. Duplicate samples were isolated from a single patient in a single day (n = 28), and each of the duplicate samples was independently measured for ΔCt_pathogen. Both of the measurements provided ΔCt_pathogen located on the same side (above or below) of the cutoff. Red line: the cutoff for each organism. (D) Temporal profile of ΔCt_pathogen during antibiotic treatment. A single sample set contains multiple sputum samples isolated from a single patient during antibiotic treatment. A total of 9 consecutive sample sets that included 7 of pneumonia with ΔCt_pathogen for S. pneumoniae > cutoff and 2 of pneumonia with ΔCt_pathogen for H. influenzae > cutoff at day 1 were studied. ΔCt_pathogen decreased to below the cutoff in the course of treatment.