Table 1.
List of pneumonia-causing pathogens.
Table 2.
List of target genes.
Figure 1.
(A) When pneumonia occurs, the numbers of both the causative pathogen and human inflammatory cells increase at the inflammation site. Meanwhile, the colonizing pathogen lags behind. The ratio of pathogen to human cells may be a good indicator for the differentiation of the causative pathogen from the colonizing pathogen. (B) The cell number ratio is measurable by quantitative PCR. The Ct (threshold cycle) is the PCR cycle at which a statistically significant fluorescent signal is first observed. Ctpathogen is the Ct for the pathogen-specific gene, Cthuman is the Ct for the human-specific gene, and both are log-proportional to the number of the cells (see Figure 2). Accordingly, ΔCtpathogen = −(Ctpathogen−Cthuman) is log-proportional to the ratio of pathogen to human cells. (C) Because ΔCtpathogen indicates the ratio of pathogen to human cells, we may be able to determine the ΔCtpathogen cutoff, a ΔCtpathogen value above which a pathogenic role of the pathogen in pneumonia is strongly suggested.
Figure 2.
Relationship between cell number and Ct.
Log-linear relationships between the copy number of pathogen-specific sequence and Ctpathogen and between the copy number of the human-specific sequence and Cthuman. (A) S. pneumoniae, H. influenzae, Pseudomonas spp., or M. catarrhalis was suspended in sputum. DNA was then purified from the suspension, and the target sequences specific to each organism were amplified by PCR. A log-linear relationship indicates that the sputum does not contain molecules that inhibit isolation of DNA or exponential amplification by PCR. Experiments were done in triplicate. A bar indicates standard deviation. (B) Human genomic DNA isolated from sputum was serially diluted, and a DNA sequence in the human SFTPC gene (arbitrarily selected from human genes, of which sequence is specific to human by BLAST search of GenBank database; Table 2) was amplified. A log-linear relationship indicates that the sputum does not contain molecules that inhibit isolation of DNA or exponential amplification by PCR.
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 Cthuman <27 (>7×103 human cells/µL of sputum; Figure 2B). Samples with M2–P3 appearance as well as a Cthuman <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. ΔCtpathogen 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 ΔCtpathogen cutoff (a red line) was defined as the smallest ΔCtpathogen for the blue circles. Sputum in which the pathogen was not detected and thus ΔCtpathogen was not assigned is shown at the bottom (labeled as “Not detected”). (C) Reproducibility of ΔCtpathogen 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 ΔCtpathogen. Both of the measurements provided ΔCtpathogen located on the same side (above or below) of the cutoff. Red line: the cutoff for each organism. (D) Temporal profile of ΔCtpathogen 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 ΔCtpathogen for S. pneumoniae > cutoff and 2 of pneumonia with ΔCtpathogen for H. influenzae > cutoff at day 1 were studied. ΔCtpathogen decreased to below the cutoff in the course of treatment.
Figure 4.
(A) ΔCtpathogen for each commensal organism (n = 153). Samples in which real-time PCR failed to detect the organism are shown at the bottom (“Not detected”). The ΔCtpathogen cutoff demarcated well the samples obtained from the patients in whom the likely causative pathogen was identified by criteria (1) – (4). (B) Interrelationship between the pathogens detected. Samples obtained from the patients in whom the other 3 commensal organisms were identified as a likely causative pathogen or samples in which a non-commensal organism was detected by real-time PCR are colored. Most of the colored circles are located below the cutoff line.
Table 3.
Protocol for the prospective observatory study.
Table 4.
Patients' characteristics.